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Infectious Diseases in Obstetrics and Gynecology I:1 (1993) (C) 1993 Wiley-Liss, Inc. Why a New Journal? Is there need for a journal of infectious diseases in obstetrics and gynecology? It is true that the number of articles related to infectious diseases in obstetrics and gynecology published in existing journals has increased significantly. Many of these articles contain important information on antibiotics or the microbiology and pathophysiology as well as the epidemiology of infections that the practitioner of obstetrics and gynecology needs for the prevention ofsuch diseases or diagnosis and treatment of patients affected by them. The primary care practitioner delivering health care to the female patient is also in need of this information. These articles are dispersed among various journals, however, and many of the journals are unrelated to the specialty of obstetrics and gynecology, making them not readily accessible to the obstetrician-gynecologist or the primary care physician. The conception ofthis journal is a result ofthe need for a single reference source on the most current information on infectious diseases associated with the obstetric and gynecologic patient. To fulfill this need, Infectious Diseases in Obstetrics and Gynecology will publish material encompassing not only infectious diseases affect- ing the female reproductive organs, but every aspect of infectious diseases associ- ated with the obstetric or gynecologic patient. Each issue will contain 8 to 12 articles divided among basic science, clinical research, and clinical practice as they relate to infectious diseases in the female patient. Included in each issue will be a review article on an infectious disease subject related to obstetrics and gynecology. This review article will contain a synopsis ofbackground information enabling the reader to develop a foundation of knowledge on the subject being discussed, information on current developments in the area, and management recommendations.

ew article on an infectious disease subject related to obstetrics and gynecology. This review article will contain a synopsis ofbackground information enabling the reader to develop a foundation of knowledge on the subject being discussed, information on current developments in the area, and management recommendations. Also included will be an obstetrical and a gynecological case presentation to serve as t stimulus for discussion on the management of problems commonly associated with our specialty, raising ques- tions and issues for further discussion. The case presentations should also offer the reader the most current diagnostic tools and management regimens. Finally, each issue will contain an editorial dealing with a pertinent topic. The intent of this editorial is to stimulate questions from the readers and answers from various authorities that will be published in subsequent issues. In summary, the purpose of this journal is to offer a forum for obtaining and disseminating information to clinicians and other health care providers, including researchers involved in the study or treatment of infectious diseases in the female patient as well as residents in training. Sebastian Faro, M.D., Ph.D. Editor-in-Chief Editorial

Infectious Diseases in Obstetrics and Gynecology 1:12-15 (1993) (C) 1993 Wiley-Liss, Inc. Pregnancy Outcome Following Pelvic Infection Miklos Toth, Anu Chaudhry, William J. Ledger, and Steven S. Witkin Department of Obstetrics and Gynecology, Cornell University Medical College, New York, NY ABSTRACT To determine whether a previous pelvic infection has an effect on the outcome of a subsequent pregnancy, we identified women with a diagnosis of pelvic inflammatory disease (PID), amnionitis, and postpartum or postabortal endometritis-salpingitis by a retrospective chart review ofall patients admitted to the Department of Obstetrics and Gynecology at The New York Hospital-Cornell Medical Center between 1975 and 1977 and between 1985 and 1988. Antimicrobial regimens effective against Chlamydia trachomatis were initiated in 1985. Controls were randomly selected patients presenting during the same time period for routine examinations who had normal Pap smears and no infections. Both groups were comparable for age, race, gravity, and parity. Differ- ences were evaluated by chi square analysis, using the Yates correction factor. We identified 183 women with a history of the above infections who subsequently conceived, and 82 controls. There were no differences in outcome between the two index groups. Term vaginal deliveries occurred in 14.2% ofthe women with a prior pelvic infection and in 56% ofthe controls (P < 0.001). Among the 97 women who had had PID, 21 (21.6%) had a spontaneous abortion in the subsequent pregnancy, as opposed to 6 (7.3%) ofthe controls (P 0.013). In addition, eight ofthe women with PID (but no controls) went into preterm labor (P 0.021). An increased incidence ofpreterm labor (P 0.001) was also observed in women with a history of amnionitis. A history of endometritis was not associated with an increased prevalence of abnormal outcome in subsequent pregnancies. PID and amnionitis may adversely affect the outcome of subsequent pregnancies. (C) 1993 WileyoLiss, Inc. KEY WORDS Pelvic inflammatory disease, amnionitis, spontaneous abortion, preterm labor elvic inflammatory disease (PID) occurs in about million cases annually in the United States.

outcome in subsequent pregnancies. PID and amnionitis may adversely affect the outcome of subsequent pregnancies. (C) 1993 WileyoLiss, Inc. KEY WORDS Pelvic inflammatory disease, amnionitis, spontaneous abortion, preterm labor elvic inflammatory disease (PID) occurs in about million cases annually in the United States. The inability to become pregnant after PID due to occlusion of the fallopian tube is well documented.2 However, aside from an increased prevalence of ectopic pregnancy following PID, 1,2 the effects of a previous and apparently effectively treated pelvic infection on the outcome of subse- quent pregnancies have been scarcely studied. Chlamydia trachomatis is a major cause ofPID in developed countries. 1'2 Recent studies have also documented an association between asymptomatic chlamydial infections, as diagnosed by the presence ofantichlamydial antibodies in women with no his- tory of a symptomatic chlamydial infection, and an increased prevalence of spontaneous abortions fol- lowing in vivo3'4 and in vitros'6 fertilization. Re- activation of latent chlamydial infections and/or sensitization of the immune system to antigens ex- pressed during pregnancy have been postulated to contribute to these first trimester pregnancy losses.4,6 To explore further the consequences of PID on subsequent pregnancies, we retrospectively exam- ined whether women with PID, as well as with amnionitis or postabortal or postpartum endometri- tis, had a higher prevalence of complications dur- ing future pregnancies than did other women. In Address correspondence/reprint requests to Dr. Miklos Toth, Ob/Gyn Department, Cornell University Medical College, 525 East 68th Street, New York, NY 10021. Received October 20, 1992 Clinical Study Accepted November 24, 1992 PELVIC INFECTIONS AND PREGNANCY TOTH ET AL. 1985, recognizing the role of C. trachomatis in the etiology of PID, the Centers for Disease Control (CDC) first recommended inclusion of antibiotic regimens highly effective against this organism in the treatment of women with pelvic infections. To examine whether this change in management im- proved the prevalence of a successful outcome of the first postinfection pregnancy, we compared pa- tients treated for pelvic infections both before and after 1985. The results suggest that women with PID had an increased prevalence of spontaneous abortion and preterm labor in the subsequent preg- nancy.

management im- proved the prevalence of a successful outcome of the first postinfection pregnancy, we compared pa- tients treated for pelvic infections both before and after 1985. The results suggest that women with PID had an increased prevalence of spontaneous abortion and preterm labor in the subsequent preg- nancy. No differences were found, however, be- tween groups ofwomen with PID who were treated prior to or after implementation ofthe CDC guide- lines for antibiotic coverage of C. trachomatis. MATERIALS AND METHODS All women, both ward and private cases, treated in the Department of Obstetrics and Gynecology at The New York Hospital-Cornell Medical Center between 1975 and 1989 were included. Charts from two groups ofwomen who had PID, amnion- itis, or postpartum or postabortal endometritis- salpingitis were selected from a computer database ofmedical records and reviewed for the first obstet- ric event following the infection. In the first group, admitted to the hospital and treated between 1975 and 1977, 1,100 charts were reviewed and 92 cases of subsequent pregnancies were identified. In the second group, composed of patients seen between 1985 and 1988, 1,320 charts were re- viewed and 91 cases were identified. The control group, consisting of 82 women, was obtained from a review of 980 charts of randomly selected patients who presented during 1975-1978 and 1985-1988 for a routine gynecological examination and who had a normal Pap smear and no infections prior to the first subsequent pregnancy. The three groups were comparable in age, race, gravity, and parity. A term (>37 weeks) vaginal delivery was con- sidered a normal outcome. Spontaneous abortion was defined as loss of pregnancy before completion ofthe 12th gestational week. No abortions occurred in our population between the 13th and 20th weeks of gestation. Two second trimester losses occurred in both the index and control groups, at 20 and 22 weeks and 20 and 23 weeks, respectively. These were not included in the spontaneous abortion group. Preterm labor was defined as the presence TABLE I. Pregnancy outcome following pelvic infections No. of women No with infection (%) Outcome infection 1975-1977 1985-1988 Term, vaginal delivery 46 (56.1%) 15 (16.3%) (I 1.5%) Complications 36 (43.9%) 77 (83.7%)* 85 (88.5%)* aComplications include spontaneous abortion, amnionitis, PROM, pre- term labor, preterm birth, and ectopic pregnancy. *P < 0.001 vs. the no infection group.

No with infection (%) Outcome infection 1975-1977 1985-1988 Term, vaginal delivery 46 (56.1%) 15 (16.3%) (I 1.5%) Complications 36 (43.9%) 77 (83.7%)* 85 (88.5%)* aComplications include spontaneous abortion, amnionitis, PROM, pre- term labor, preterm birth, and ectopic pregnancy. *P < 0.001 vs. the no infection group. of regular uterine contractions with progressive cervical dilation. Premature rupture of membranes (PROM) was defined as rupture ofthe membranes at least 12 hours before the onset ofuterine contrac- tions. Differences between groups were evaluated by chi square analysis, using the Yates correction factor. RESULTS The outcome of the first obstetric event subsequent to an upper reproductive tract infection, and the outcome in the controls, are shown in Table 1. Among the 92 women who were treated for infec- tions from 1975 to 1977, only 16.3 % experienced a term vaginal delivery in the first postinfection pregnancy. Similarly, only 1.5% of the 96 women treated for pelvic infection between 1985 and 1988 who subsequently conceived had a term pregnancy. Differences between these two groups were not significant. In marked contrast, 56% of the 82 control pregnancies resulted in a term vagi- nal delivery. This outcome was significantly differ- ent from both patient groups (P < 0.001). Since the two groups of patients with upper re- productive tract infections were similar in preg- nancy outcome, they were combined for subsequent analyses ofthe relationship between infectious diag- nosis and specific pregnancy complication. Results are shown in Table 2. Among the 183 patients there were 97 who had had PID, 64 with postpar- tum endometritis-salpingitis, 10 with postabortal endometritis-salpingitis, and 12 with amnionitis. Women with a past PID had a significantly (P < 0.013) increased prevalence of spontaneous abortion (21.7%) than did the controls (7.3%). Additionally, the prevalence of preterm labor was significantly greater in women with past PID (8.2%, P 0.021) and past amnionitis (25%, INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 13 PELVIC INFECTIONS AND PREGNANCY TOTH ET AL. TABLE 2. Effect of specific pelvic infections on subsequent adverse pregnancy outcome No. of women (%) Postpartum Postabortal Pregnancy PID endometritis endometritis outcome (n 97) (n 64) (n 10) Amnionitis None (n 12) (n 82) Spontaneous abortion 21 (21.6)* 6 (9.4) 2 (20.0) Preterm labor 8 (8.2)** 4 (6.3) (10.0) PROM 7 (7.2) 3 (4.7) (10.0) Preterm delivery 2 (2.

ns on subsequent adverse pregnancy outcome No. of women (%) Postpartum Postabortal Pregnancy PID endometritis endometritis outcome (n 97) (n 64) (n 10) Amnionitis None (n 12) (n 82) Spontaneous abortion 21 (21.6)* 6 (9.4) 2 (20.0) Preterm labor 8 (8.2)** 4 (6.3) (10.0) PROM 7 (7.2) 3 (4.7) (10.0) Preterm delivery 2 (2. 0 0 Ectopic pregnancy 9 (9.3) 3 (4.7) 0 Amnionitis (I.0) 0 0 0 6 (7.3) 3 (25.0)*** 0 2 (I 6.7) 2 (2.4) 0 0 0 4 (4.9) 0 0 *P 0.013 vs. control. **P 0.021 vs. control. ***P 0.001 vs. control. PID, pelvic inflammatory disease; PROM, premature rupture of membranes. P 0.001) than in the controls. The prevalence of pregnancy complications in women with postabor- tal and postpartum endometritis-salpingitis was not statistically different from the control group in this study. In each of the three women with previous am- nionitis who went into preterm labor in the subse- quent pregnancy, the amnionitis was detected dur- ing term, and not preterm, labor. This excludes the possibility that a prior preterm labor, rather than the amnionitis, was the risk factor for a subsequent preterm labor. DISCUSSION In the present study, women with a prior PID had a subsequent threefold higher prevalence of sponta- neous abortion than did women with no history of pelvic infection. In addition, the prevalence of pre- term labor was also significantly increased in women with a history of PID or amnionitis. These findings provide additional support to previous studies documenting an association between upper genital tract infection and subsequent adverse preg- nancy outcome. 7 Women with serological evidence of infection with C. trachomatis,3-6 a major cause ofboth PID and ectopic pregnancy, 1,2 had a signif- icantly higher occurrence of spontaneous abortion than did other women. A similar association between previous ectopic pregnancy and an increased preva- lence of spontaneous abortion has also been made. The mechanism relating prior pelvic infection to subsequent adverse pregnancy outcome remains to be established. In this regard, the failure to detect any improvement in pregnancy outcome following initiation of the CDC guidelines for the treatment of C. trachomatis suggests several mechanisms. Possible interpretations ofthese findings are that 1) C. trachomatb is not involved in the sequela; 2) the CDC guidelines were ineffective at completely elim- inating C.

etect any improvement in pregnancy outcome following initiation of the CDC guidelines for the treatment of C. trachomatis suggests several mechanisms. Possible interpretations ofthese findings are that 1) C. trachomatb is not involved in the sequela; 2) the CDC guidelines were ineffective at completely elim- inating C. trachomatis from the upper genital tract of women with PID and/or their sexual partner(s); and 3) mechanisms triggered by the initial infec- tion but no longer requiring the continued presence of bacteria may be involved. The first mechanism appears to be highly un- likely based on the above-mentioned associations between C. trachomatis and spontaneous abor- tions.3-6 There is evidence in the literature consis- tent with the second mechanism. The ability of C. trachomatis to persist in the upper genital tract fol- lowing standard antibiotic regimens for this organ- ism has been known since 1980. 9 Reactivation of these subclinical or latent chlamydial infections by the hormonal and immunological changes that ac- company pregnancy could lead to uterine inflam- mation and the subsequent expulsion of the fetus. The extensive immune system activation induced by C. trachomatis1' 11 could also alter immune reg- ulatory mechanisms that normally prevent expul- sion of the semi-allogeneic fetus. Furthermore, recent studies in experimental monkeys with trachoma have demonstrated that C. trachomatis RNA and DNA could be identified in lesions from inflammatory sites that were negative by culture for this organism. 12 This raises the pos- sibility that the persistence of C. trachomatis genetic material inside infected cells could lead to the syn- thesis and release of chlamydial antigens, even un- 14 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY PELVIC INFECTIONS AND PREGNANCY TOTH ET AL. der conditions in which viable organisms cannot be produced. Morrison et al. have demonstrated that the addition of penicillin to in vitro cultures of C. trachomatis prevented the formation of elementary bodies, but a 57 kD chlamydial protein continued to be synthesized and released from infected cells. 13 This 57 kD protein has been implicated in the induction of potent inflammatory responses in ani- mals previously sensitized to Chlamydia. 14 Evidence in support ofthe third proposed mech- anism is scant. We have previously suggested that the continued induction of interferon-/ by cells chronically infected with C.

. 13 This 57 kD protein has been implicated in the induction of potent inflammatory responses in ani- mals previously sensitized to Chlamydia. 14 Evidence in support ofthe third proposed mech- anism is scant. We have previously suggested that the continued induction of interferon-/ by cells chronically infected with C. trachomatis could in- duce the expression of major histocompatibility (MHC) class 2 antigens on the surface of fallopian tube epithelial cells. This would allow these cells to present epithelial antigens to lymphocytes, result- ing in immune system sensitization to epithelial antigens, is Recent evidence identifying the C. tra- chomatis 57 kD protein as a member of the 60 kD heat shock protein family, 13 with extensive amino acid sequence homology to the human 60 kD heat shock protein, suggests an additional mechanism. Sensitization of lymphocytes to conserved regions of the chlamydial 57 kD protein could eventually induce an autoimmune response to the human 60 kD heat shock protein. 16 We are aware that it is difficult to reach firm conclusions based on retrospective analyses. In the present investigation, however, we considered this approach to be acceptable because of our stable patient population and the ability to perform long- term follow-ups. In any event, the present data, in conjunction with earlier studies cited above, strongly suggest that the adverse effects of pelvic infection are not limited to tubal-based infertility and an increased risk of ectopic pregnancy but also influence the outcome of subsequent intrauterine pregnancies. These studies also emphasize the need for further investigations on the possible persis- tence of C. trachomatis in the genital tract following antibiotic treatment as well as for the initiation of prospective studies to assess the effect of antecedant upper genital tract infection by C. trachomatis and by other microorganisms on pregnancy outcome. REFERENCES 1. Expert committee on pelvic inflammatory disease: Pelvic inflammatory disease. Research directions for the 1990s. Sex Transm Dis 18:46-64, 1991. 2. Westrom L, Mardh PA: Acute pelvic inflammatory dis- ease (PID). In Holmes KK, Mardh PA, Sparling PF (eds): Sexually Transmitted Diseases. 2nd ed. New York: McGraw-Hill, pp 593-613, 1990. 3. Quinn P, Petric M, Barkin M: The prevalence of anti- body to Chlamydia trachomatis in spontaneous abortion and infertility. Am J Obstet Gynecol 156:291-296, 1987. 4.

nflammatory dis- ease (PID). In Holmes KK, Mardh PA, Sparling PF (eds): Sexually Transmitted Diseases. 2nd ed. New York: McGraw-Hill, pp 593-613, 1990. 3. Quinn P, Petric M, Barkin M: The prevalence of anti- body to Chlamydia trachomatis in spontaneous abortion and infertility. Am J Obstet Gynecol 156:291-296, 1987. 4. Witkin SS, Ledger WJ: Antibodies to Chlamydia tra- chomat# in sera of women with recurrent spontaneous abortions. Am J Obstet Gynecol 167:135-139, 1992. 5. Lunenfeld E, Shapiro BS, Sarov B, Sarov I, Insler V, Decherney A: The association between Chlamydia-specific IgG and IgM antibodies and pregnancy outcome in an in vitro fertilization program. J In Vitro Fertil Embryo Transfer 6:222-227, 1989. 6. Licciardi F, Grifo JA, Rosenwaks Z, Witkin SS: Rela- tion between antibodies to Chlamydia trachomatis and spontaneous abortion following in vitro fertilization. J Assisted Reprod Genet 9:207-210, 1992. 7. Toth M, Witkin SS, Ledger WJ, Thaler H: The role of infection in the etiology of preterm birth. Obstet Gynecol 71:723-726, 1988. 8. Fedele L, Acaia B, Parazzini F, Ricciardiello O, Candi- ani GB: Ectopic pregnancy and recurrent spontaneous abortion: Two associated reproductive failures. Obstet Gynecol 73:206-208, 1989. 9. Henry-Suchet J, Catalan F, Loffredo V, Serfaty D, Si- boulet A, Perol Y, Sanson MJ, Debache C, Pigeau F, Coppin R, DeBrux J, Poynard T: Microbiology of spec- imens obtained by laparoscopy from controls and from patients with pelvic inflammatory disease or infertility with tubal obstruction: Chlamydia trachomatis and Ure- aplasma urealyticum. Am J Obstet Gynecol 138:1022- 1025, 1980. 10 Witkin SS, Toth M, Jeremias j, Ledger WJ: Increased inducibility of inflammatory mediators from peripheral blood mononuclear cells ofwomen with salpingitis. Am J Obstet Gynecol 165:719-723, 1991. 11. Toth M, Jeremias J, Ledger WJ, Witkin SS: In vivo tumor necrosis factor production in women with salpingi- tis. Surg Gynecol Obstet 174:359-362, 1992. 12. Holland SM, Hudson AP, Bobo L, Whittum-Hudson JA, Viscidi RP, Quinn TC, Taylor HR: Demonstration of chlamydial RNA and DNA during a culture negative state. Infect Immun 60:2040-2047, 1992. 13. Morrison RP, Belland RJ, Lyng K, Caldwell HD: Chlamydial disease pathogenesis. The 57 kD chlamydial hypersensitivity antigen is a stress response protein. J Exp Med 170:1271-1283, 1989. 14. Morrison RP, Lyng K, Caldwell HD: Chlamydial disease pathogenesis. Ocular hypersensitivity elicited by a genus- specific 57-kD protein.

son RP, Belland RJ, Lyng K, Caldwell HD: Chlamydial disease pathogenesis. The 57 kD chlamydial hypersensitivity antigen is a stress response protein. J Exp Med 170:1271-1283, 1989. 14. Morrison RP, Lyng K, Caldwell HD: Chlamydial disease pathogenesis. Ocular hypersensitivity elicited by a genus- specific 57-kD protein. J Exp Med 169:663-675, 1989. 15. Grifo JA, JeremiasJ, Ledger WJ, Witkin SS: Interferon-/ in the diagnosis and pathogenesis of pelvic inflammatory disease. AmJ Obstet Gynecol 160:26-31, 1989. 16. Morrison RP, Manning DS, Caldwell I--ID: Immunol- ogy of Chlamydia trachomatis infections. In Quinn TC fed): Sexually Transmitted Diseases. New York: Raven Press, pp 57-84, 1992. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 15

Infectious Diseases in Obstetrics and Gynecology 1:16-22 (1993) (C) 1993 Wiley-Liss, Inc. Necrotizing Fasciitis: A Review of Management Guidelines in a Large Obstetrics and Gynecology Teaching Hospital C.D. Thompson, A.L. Brekken, and W.H. Kutteh Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX ABSTRACT Necrotizing fasciitis is a severe, life-threatening soft tissue infection that results in rapid and progres- sive destruction of the superficial fascia and subcutaneous tissue. Because of its varied clinical presentation and bacteriological make-up, it has been labelled with many other names such as acute streptococcal gangrene, gngrenous erysipelas, necrotizing erysipelas, hospital gangrene, and acute dermal gangrene. Although described by Hippocrates and Galen, it has received increasing attention in obstetrical and gynecological literature only within the last 20 years. This review includes two recent cases successfully managed at Parkland Memorial Hospital, Dallas, Texas. The first patient was a 50 year old, morbidly obese, diabetic woman who presented with a small, painful lesion on the vulva. After failing triple antibiotic therapy with ampicillin, clindamycin, and gentamicin, the diag- nosis of necrotizing fasciitis of the vulva was made, and she was taken to the operating room for extensive excision. She was discharged home on hospital day 29. The second patient was a 65 year old, obese, diabetic woman with risk factors for atherosclerosis who had a wound separation after an abdominal hysterectomy. Two days later a loss ofresistance to probing was noted in the subcutane- ous tissue. Necrotizing fasciitis was suspected, and she was taken to the operating room for resec- tion. The patient was discharged home on hospital day 27. The mortality rate after diagnosis of necrotizing fasciitis has been reported to be 30% to 60%. We review the literature and outline the guidelines used in a large Ob/Gyn teaching hospital to minimize the adverse outcome. Lectures on soft-tissue infections are included on a regular basis. The high-risk factors of age over 50, diabetes, and atherosclerosis are emphasized.

ciitis has been reported to be 30% to 60%. We review the literature and outline the guidelines used in a large Ob/Gyn teaching hospital to minimize the adverse outcome. Lectures on soft-tissue infections are included on a regular basis. The high-risk factors of age over 50, diabetes, and atherosclerosis are emphasized. The need for early diagnosis and surgical treatment within 48 hours is stressed, and any suspicious lesions or wound complications are reported to experienced senior house officers and staff. We use two recent eases to highlight the diagnostic clues and management strategies for this often fatal polymicrobial infection. (C) 1993 Wiley-Liss, Inc. KEY WORDS Soft tissue infections, gynecologic inections, obstetrical infections CASE REPORTS Case 50 year old white woman (G2,P2) originally presented to the Parkland Memorial Hospital Ob/Gyn Emergency Room complaining of a pain- ful "boil on my vagina." She had a cm follicular abscess on the left vulva at the intertriginous fold that would rub against her thigh as she walked. She denied fever, chills, or other systemic symptoms. Her past medical history was significant for a total abdominal hysterectomy with bilateral salpingo- oophorectomy and appendectomy in 1963, a ven- tral hernia repair in 1970, and a 20-year history of adult onset diabetes mellitus controlled with gly- buride 7.5 mg daily. Her serum glucose was 128 mg/dl. She had a 22 pack/year history of smoking and denied the use of alcohol. Physical examination revealed a morbidly obese (327 pounds) woman who was generally healthy, other than her present- Address correspondence/reprint requests to Dr. William H. Kutteh, Reproductive Endocrinology, Dept. of Ob/Gyn, 5323 Harry Hines Boulevard, Dallas, TX 75235-9032. Clinical Study Received July 10, 1992 Accepted December 15, 1992 NECROTIZING FASCHTIS MANAGEMENT GUIDELINES THOMPSON ET AL. Fig. I. Photographs of the vulvar area from case I..: Large area of involvement developed in 24 hours and was extensively dbrided. I: Continued tissue necrosis and loss of resistance to probing was noted at 36 hours and required repeat excision. 2: Granulation tissue and healing of wound bed 14 days after original surgery. ing complaint. The abscess was needle aspirated, and the patient was sent home on oral dicloxacillin 500 mg q6h. The following day, she returned to the emer- gency room complaining of increased pain and red- ness in the left groin area and subjective fever.

issue and healing of wound bed 14 days after original surgery. ing complaint. The abscess was needle aspirated, and the patient was sent home on oral dicloxacillin 500 mg q6h. The following day, she returned to the emer- gency room complaining of increased pain and red- ness in the left groin area and subjective fever. Exam was significant for a temperature of 38C, blood pressure 138/78, heart rate 88, respiratory rate 20. The left vulva was slightly edematous and erythe- matous. The left groin had an 8 10 cm erythe- matous area that extended down the inner thigh and was warm and tender to the touch. She had no clubbing or cyanosis of her extremities, nor did she have any pelvic masses or tenderness. She was alert and oriented and had a normal neurological exam. She was admitted to the hospital and placed on intravenous ampicillin (2 mg IV q4h), gentamicin (1 mg/kg IV qSh), and clindamycin (900 mg IV qSh). Laboratory evaluation revealed an elevated white blood cell count of 17,400/mm3 and a glu- cose of 168 mg/dl. The next day the area of inflam- mation had extended to 20 cm down the left inner thigh, and foul-smelling purulent material exuded from the aspirated lesion. The patient was taken to the operating room where exploration of the groin area revealed multiple loculated abscess cavities ex- tending from the left groin superior to the inguinal ligament and inferior to the femoral triangle and containing foul-smelling yellow-greenish pus. This material was sent for gram stain and aerobic and anaerobic cultures. Blood was sent for Clostridium toxin and aerobic and anaerobic cultures. The in- volved area was dissected underneath the subcuta- neous tissue to the inferior margin of the left vulva (Fig. 1A). The necrotic tissue was excised to the point of bleeding with no loss of resistance to prob- ing, and the area was irrigated with 6 liters ofsaline and packed. Post-operatively, she was managed with subcu- taneous insulin to maintain tight glucose control. Deep vein thrombosis (DVT) prophylaxis with a pneumatic compression hose on both legs and sub- cutaneous heparin 5,000 units twice daily was initi- ated in the operating room and continued post- operatively. Wound debridement and cleaning with one-quarter strength Dakin's solution (163 ml so- dium hypochlorite 5.25% plus 0.325g sodium bi- carbonate in 3,637 ml water)were performed twice daily, and sterile gauze was used for packing the wound. After 36 hours, further necrosis of the wound edges was apparent.

t- operatively. Wound debridement and cleaning with one-quarter strength Dakin's solution (163 ml so- dium hypochlorite 5.25% plus 0.325g sodium bi- carbonate in 3,637 ml water)were performed twice daily, and sterile gauze was used for packing the wound. After 36 hours, further necrosis of the wound edges was apparent. She was again taken to surgery, where blunt probing revealed loss of tis- sue resistance superiorlaterally close to the margin of the inguinal ligaments as well as medially past the inferior margin of the vulva and inner thigh (Fig. 1B). The skin, superficial fascia, and subcu- taneous tissue of the areas were excised to the point of bleeding and no loss of resistance to probing. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 17 NECROTIZING FASCHTIS MANAGEMENT GUIDELINES THOMPSON ET AL. Hemostasis was attained, and the wound was packed with sterile gauze. Ampicillin, clindamycin, and gentamicin were continued, as were sliding scale insulin, DVT pro- phylaxis, and twice daily wound ddbridement and dressing changes. Aztreonam (2 g IV q6h) was sub- stituted to reduce the risk of renal toxicity, which was significant in this patient. When no further signs of infection were present in the wound bed, one-quarter strength Dakin's solution was deleted, and sterile saline was substituted for wound care. The wound was cleaned with Biolex spray (a dilute aloe vera solution) and gel to promote granulation. Antibiotics were stopped after I0 additional days, after she had been afebrile for 48 hours. By this time, the wound was granulating well (Fig. C). Aerobic cultures demonstrated both gram-nega- tive (E. coli, Klebsiella pneumoniae) and gram-posi- tive rods (Corynebacterium sp). Anaerobic cultures showed gram-negative rods (Bacteroides sp). Blood cultures and C]ostridium toxin assay were negative. Pathological examination revealed necrotic skin with abscess and underlying fat necrosis. The pa- tient was discharged home on hospital day 25 on twice daily dressing changes, and was followed with weekly visits for 6 weeks. She was doing well at her 6-month follow-up. Case 2 A 65 year old black woman (G7PsA2) was admitted to Parkland Memorial Hospital for a hysterectomy after fractional dilatation and curettage revealed dysfunctional endometrium with cellular atypia.

daily dressing changes, and was followed with weekly visits for 6 weeks. She was doing well at her 6-month follow-up. Case 2 A 65 year old black woman (G7PsA2) was admitted to Parkland Memorial Hospital for a hysterectomy after fractional dilatation and curettage revealed dysfunctional endometrium with cellular atypia. Be- cause of her significant medical problems includ- ing hypertension, coronary artery disease, non-in- sulin dependent diabetes mellitus, degenerativejoint disease, and a history of a mild stroke, she was evaluated by the medicine department consult staff and cleared for surgery. She had a 30 pack/year history ofsmoking and denied alcohol use. Physical examination revealed an obese (235 pounds) woman, blood pressure 150/90, heart rate 90, res- piratory rate 20, temperature 37C. She underwent a total abdominal hysterectomy, bilateral salpingo- oophorectomy, and appendectomy without compli- cation. Pathology revealed multiple leiomyomata and no gross endometrial tumor. On post-operative day (POD 1), she had a maximum temperature of 38.5C. Her incision was draining a small amount of serosanguineous fluid but did not look infected. On POD 2 she had a maximum temperature of 38.5C, which was at- tributed to pulmonary atelectasis. She quickly defer- vesced and remained afebrile. On POD 4, a slight serosanguineous discharge was noted with wound separation apparent on probing. Her staples were removed to allow drainage, and the wound was packed with saline-moistened sterile gauze covered with dry sterile dressing and changed twice daily. She remained afebrile until POD 6, when her temperature spiked to 38.4C. At this time, her bandages contained greenish purulence, and the wound edges were necrotic and foul-smelling. When the wound was probed, loss of resistance of the subcutaneous tissue superficial to the rectal fas- cia extended 6 cm bilaterally. Only minimal skin erythema was present, but the wound margins were anesthetic. Importantly, the patient maintained nor- mal bowel function, and her fluid balance and elec- trolytes were well controlled. Necrotizing fasciitis was suspected, and the patient was placed on IV ampicillin (2 g IV q4h), gentamicin (1 mg/kg IV q8h), and clindamycin (900 mg IV qSh) and taken to the operating room that same day. The areas of necrosis were excised to the point at which no loss of resistance was encountered and brisk capillary bleeding was evident. Tissue was sent for aerobic and anaerobic cultures.

mpicillin (2 g IV q4h), gentamicin (1 mg/kg IV q8h), and clindamycin (900 mg IV qSh) and taken to the operating room that same day. The areas of necrosis were excised to the point at which no loss of resistance was encountered and brisk capillary bleeding was evident. Tissue was sent for aerobic and anaerobic cultures. The wound was irrigated with 3 liters ofsaline and packed with sterile gauze. Post-operatively, she was maintained with care- ful fluid management, and her triple antibiotics were continued. The wound was debrided and cleaned with one-quarter strength Dakin's solutin twice daily. The patient defervesced after 6 days, and granulation tissue became apparent. Hydro- therapy in a whirlpool bath was begun at this time. The wound occasionally required sharp ddbride- ment of necrotic debris, but otherwise it continued to granulate well. The wound culture revealed Pro- teus mirabilis. Pathology showed marked acute and chronic inflammation, necrosis, ulceration, and or- ganizing fat necrosis. The patient was discharged 27 days after her admission on twice daily dressing changes and weekly follow-up in clinic for 6 weeks. She was doing well 4 months post-operatively. DISCUSSION One of the earliest descriptions of hospital gan- grene was written in 1871 by Joseph Jones, a Con- 18 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY NECROTIZING FASCHTIS MAIVAGEMENT GUIDELINES THOMPSON ET AL. TABLE I. Risk factors for necrotizing fasciitis Age over 50 Arteriosclerosis Diabetes Obesity Smoking Previous radiation Operative trauma aMajor risk factors. federate Army surgeon: "a purple or blue spot is first perceived that is sometimes raised, and con- tains serum below. The skin in the affected spot may melt away in 24 hours, whilst a deep blue and purple, almost black, areola surrounding the dead mass, spreads rapidly in an ever increasing circle. This is witnessed most generally in the worst and fatal cases. ''4 The first large series was published by Meleney in 1924. 6 He studied 20 cases in China from which he cultured hemolytic streptococci and hence called the disease hemolytic streptococcal gan- grene. The term necrotizingfasciitis was coined in 1952 by Wilson. 6 The first vulvar cases were de- scribed in 1972 by Roberts.7 The exact incidence of necrotizing fasciitis is not known, but fortunately it is uncommon. It has been described in all ages and in almost every site of the body.

emolytic streptococcal gan- grene. The term necrotizingfasciitis was coined in 1952 by Wilson. 6 The first vulvar cases were de- scribed in 1972 by Roberts.7 The exact incidence of necrotizing fasciitis is not known, but fortunately it is uncommon. It has been described in all ages and in almost every site of the body. It is most commonly found in the lower extremities, followed by the upper extremities, ab- domen, perineum, groin, back, and buttocks.28 The initiating event is usually a minor penetrating injury and involves a surgical site less than half the time. In Rea's series of44 patients with necrotizing fasciitis, 80% of the cases originated outside the hospital with minor injuries such as abrasions, cuts, bruises, boils, and insect bites, and only 20% were post-surgical infections.2 In eight of the cases, no specific initiating factor could be found. Those at highest risk of necrotizing fasciitis are those whose repair mechanisms are compromised due to peripheral vascular damage. The most com- mon risk factors are summarizd in Table 1. The most frequent predisposing risk factors are ad- vanced age followed closely by arteriosclerosis and diabetes. Rea first noticed the association of these factors with necrotizing fasciitis in 1970. 2 Of 44 cases studied, 45.5% were over age 50, 22.2% had arteriosclerosis, and 18.2% had diabetes.2 In Rob- erts and Hester's and Roberts's collective cases of 22 patients with necrotizing fasciitis of the vulva, 68.1% were over age 50, and all but one, or 95.5%, were diabetic.7'9 He did not comment on patients with arteriosclerosis. Other factors thought to pre- dispose to necrotizing fasciitis are hypertension, vascular disease, obesity, renal failure, immuno- suppression, a history of radiation therapy, and operative trauma. 10, Necrotizing fasciitis is often misdiagnosed as cellulitis or a simple abscess because ofthe mislead- ing symptoms and signs of its victims. Early skin changes include erythema, tenderness, and edema extending beyond the area of erythema. These are usually accompanied by fever, malaise, and other systemic toxic signs. Early on, the skin may be intact. 11 Late signs include a purple or bluish dis- coloration, vesicles filled with red-black fluid, crep- itance, cutaneous anesthesia, and necrosis. Once these late signs appear, however, the area of under- lying destruction is usually large, and severe sys- temic toxicity develops.

igns. Early on, the skin may be intact. 11 Late signs include a purple or bluish dis- coloration, vesicles filled with red-black fluid, crep- itance, cutaneous anesthesia, and necrosis. Once these late signs appear, however, the area of under- lying destruction is usually large, and severe sys- temic toxicity develops. The pathognomonic sign present in 100% of the cases is subcutaneous and superficial fascial necro- sis.2 Classically, this extends along fascial planes beyond the area of skin involvement. When blunt dissection with a probe or even a finger is carried out, the superficial tissue is sheared away without resistance. Wilson admonished that when under- mining of this nature is demonstrated, the patient should receive immediate surgical therapy.6 The bacterial etiology of necrotizing fasciitis is polymicrobial (Table 2). In Meleney's series of 20 in 1924, the predominant organism was the he- molytic streptococcus, which was present in all cases, s Staphylococci was found in only 10%. Wil- son, in contrast, reported in 1952 that 88% of his cases were infected with staphylococci.6 Rea showed that streptococci and staphylococci were present in equal proportions, representing 43% each among his cases. 2 The majority of his cases grew out more than one organism, however. In a recent series by Stephenson the organisms most currently found were anaerobic Peptostreptococcus and Bacteroides. 12 These differences are most likely attributed to the improved techniques for culturing anaerobic or- ganisms in recent years. Other bacteria reported to be involved include E. coli, K. pneumoniae, Enter- obacter, Peptococcus, and Pseudomonas. 11 Vibrio sp. can be seen in wounds exposed to sea water. 13 Clostridium sp. are found less commonly in necro- tizing fasciitis, even in those cases with subcutane- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY NECROTIZING FASCHTIS MANAGEMENT GUIDELINES THOMPSON ET AL. TABLE 2. Polymicrobial etiology of necrotizing fasciitis Aerobes Anaerobes Gram-positive Cocci Group A, B, D streptococci Other -and /-streptococci Staphylococcus aureus Staphylococcus epidermidis Rods Lactobacilli Diphtheroids Gram-negative Cocci Neisseria gonorrhoeae Rods E. colP Klebsiella pneumoniae Enterobacter sp. P. mirabilis Pseudomonas aeruginosa Peptostreptococcus sp. Peptococcus sp. Clostridium sp. Propionibacterium sp. Eubacterium sp. Veillonella sp. Bacteroides fragilis group Other Bacteroides sp. Fusobacterium sp. aMost common isolates. TABLE 3.

ci Neisseria gonorrhoeae Rods E. colP Klebsiella pneumoniae Enterobacter sp. P. mirabilis Pseudomonas aeruginosa Peptostreptococcus sp. Peptococcus sp. Clostridium sp. Propionibacterium sp. Eubacterium sp. Veillonella sp. Bacteroides fragilis group Other Bacteroides sp. Fusobacterium sp. aMost common isolates. TABLE 3. Diagnostic clues for necrotizing fasciitis Cellulitis that fails to respond to antibiotic therapy Edema beyond the area of erythema Development of ecchymosis or vesicles over an area of cellulitis Presence of gas in the tissue as demonstrated by palpation (crepitus) ous emphysema. Subcutaneous emphysema is thought to occur by aerobic and anaerobic bacteria synergistically forming hydrogen and nitrogen gas. Gas formation is mostly associated with E. coli, mi- croaerophilic Streptococcus, and Bacteroides sp. Diagnosis of necrotizing fasciitis can be difficult and is often made after the disease is widespread. A high clinical suspicion must be maintained to en- able early and accurate diagnosis. Important diag- nostic clues that lead to high suspicion for necrotiz- ing fasciitis are listed in Table 3. Fisher emphasized the importance of radiological studies to demon- strate soft-tissue gas. 14 I-Ie showed that while crep- itus was found in 29% of patients with necrotizing fasciitis, soft-tissue gas was found by x-ray in 100%. Fisher also gave six clinical criteria to satisfy the diagnosis of necrotizing fasciitis: 1) extensive ne- crosis of supeficial fascia with widespread under- mining of surrounding tissue; 2) moderate to se- vere systemic toxic reaction with altered mental status; 3) absence of muscle involvement (vs. the prominent myonecrosis seen in certain clostridial infections and synergistic necrotizing cellulitis); 4) failure to demonstrate clostridia in wound and blood cultures; 5) absence of major vascular occlusion; and 6) pathological examination of ddbrided tissue showing intense leukocyte infiltration, focal necro- sis offascia and surrounding tissue, and thrombosis ofthe microvasculature. 14 Although clostridial spe- cies are typically not present in necrotizing fasciitis, it is important to rule out the presence ofthis patho- gen in the wound, as this may mean the deep fascia and muscle are involved (clostridial myonecrosis). At surgery, a frozen section can aid in this diagno- sis.

e microvasculature. 14 Although clostridial spe- cies are typically not present in necrotizing fasciitis, it is important to rule out the presence ofthis patho- gen in the wound, as this may mean the deep fascia and muscle are involved (clostridial myonecrosis). At surgery, a frozen section can aid in this diagno- sis. Classically, necrotizing fasciitis shows necrosis of the superficial fascia and subcutaneous tissue with an intense polymorphonuclear infiltration and presence of multiple microorganisms on gram stain. 11'13 A clostridial infection will be remark- able for the absence of an inflammatory infiltrate and the presence of many gram-positive rods. 13 If the latter is the case, close inspection of the deep fascia and underlying muscle is warranted. Stephen- son et al. 2 reported the presence of Clostridium tetani in one patient with necrotizing fasciitis ofthe vulva but indicated that no myonecrosis was present, as has been reported in cases with Clostridium per- fringens. Once suspicion is high for necrotizing fasciitis, the patient should be taken to the operating room for exploration and surgical d.bridement. Brewer and Meleney are credited with the first two success- ful surgical treatments of necrotizing fasciitis, then called progressive gangrenous infection of the skin and subcutaneous tissues, which occurred in and around abdominal incisions for operative care of acute perforative appendicitis. 15 In one case, an incision was made circumferentially around the in- fected area, down to the deep fascia, and the wound was packed. The enclosed area sloughed, but there was no progression of disease, so the wound ulti- mately granulated closed. The other case was suc- cessfully treated with excision of involved tissue to viable margins. If inspection shows the characteristic skin ecchy- mosis, it is likely that the area of undermining is great. It is important, however, to incise and dd- bride the entire extent of disease, until there is no further loss of resistance to blunt probing and until the tissue bleeds easily when cut. As Wilson stated, "to postpone surgery and use massive doses of anti- biotics is ineffective and, in addition, the incision 20 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY NECROTIZING FASCHTIS MANAGEMENT GUIDELINES THOMPSON ET AL. which must eventually be made must then be more extensive in a sicker patient. ''6 In such a patient, the post-operative care should be in an intensive care unit in isolation, much like that of a burn victim.

20 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY NECROTIZING FASCHTIS MANAGEMENT GUIDELINES THOMPSON ET AL. which must eventually be made must then be more extensive in a sicker patient. ''6 In such a patient, the post-operative care should be in an intensive care unit in isolation, much like that of a burn victim. Insensible fluid losses will be great, and the pros- pect of hypovolemia with hemoconcentration is high. 13 Aggressive fluid and electrolyte manage- ment is important, along with periodic blood trans- fusions to correct anemia due to red cell destruc- tion. Broad spectrum antibiotics (i.e., ampicillin, gentamicin, clindamycin) should be used until the identity and sensitivities of pathogens are known. We have found twice daily dressing changes and wound d4bridement to be sufficient for wound care. The wound is cleaned in this manner with 3% hydrogen peroxide or one-quarter strength Dakin's solution, and packed with sterile gauze. When the old dressings no longer have a foul odor, saline can be substituted for one-quarter strength Dakin's solu- tion for wound cleaning. We also use Biolex gel (an aloe vera-containing gel) to promote wound healing. Hyperbaric oxygen treatments have been cited as reducing edema, halting progression of tissue necrosis, and decreasing mortality, especially among wounds infected with clostridia. 13,16 Split- thickness skin grafting is an option for re-epithe- lializing large areas after adequate granulation. For- tunately, in our cases the extent of the disease was not so severe as to require disfiguring surgery, and the wounds were able to granulate in completely. Mortality rates for necrotizing fasciitis are gen- erally quoted as 30-60%, and antibiotics have not significantly changed this.2 Death is usually imme- diately attributed to overwhelming sepsis, compli- cations of diabetes (ketoacidosis), vascular insuffi- ciency, and hemodynamic collapse.3'8 Not only do old age and diabetes seem to be the most important predisposing factors for necrotizing fasciitis, but they are also the two leading factors for a grave prognosis.3 Rea showed the mortality rate in pa- tients over 50 years of age to be 67%, while that in diabetics to be 63 %.2 Roberts and Hester and Rob- erts had a mortality rate from his cases of 47% and 38%, respectively.7'9 Stephenson reported a 62% mortality in patients over age 50 and a 55% mortal- ity rate in diabetic patients.

showed the mortality rate in pa- tients over 50 years of age to be 67%, while that in diabetics to be 63 %.2 Roberts and Hester and Rob- erts had a mortality rate from his cases of 47% and 38%, respectively.7'9 Stephenson reported a 62% mortality in patients over age 50 and a 55% mortal- ity rate in diabetic patients. 12 Significantly, all of the fatalities had one or both of these significant high-risk factors. The most important factors for survival are rapid diagnosis, and rapid and ade- quate surgical treatment. Rea once again stresses this fact by showing that the average time from onset of disease to diagnosis and treatment of those who lived was 4 days, while that of those who died was 7 days.2 Stephenson found that 48 hours was a more significant time flame, after which the mor- tality rate was 75%. 12 The mortality rate in Wilson's collection was only 8.7%; this low num- ber may be attributed in part to the expertise of the house officers in the teaching program at Parkland Memorial Hospital, who had been trained to rec- ognize the clinical manifestations of the disease.6 SUMMARY We have presented two cases of necrotizing fasciitis recently managed at a large Ob/Gyn residency train- ing program. We have reviewed the patients' histo- ries, risk factors, signs and symptoms, bacteriol- ogy, pathology, therapy, and mortality factors associated with this life-threatening disease process. Our patients were both in a high mortality risk category for necrotizing fasciitis in that both were over 50 years old, had diabetes, and were obese. One patient had documented arteriosclerosis, and the other had risk factors for it (obesity, smoking, and diabetes). Despite these high risk factors, both of these patients did well. We agree with Wilson that high suspicion and prompt action on the part of the house officers involved in the care of these women were the keys to their successful outcome. 6 Some of the general guidelines for the management of necrotizing fasciitis at Parkland Memorial Hos- pital are listed in Table 4. In the first case, the main clinical clue was the failure ofthe presumed celluli- tis to respond to antibiotics. In the second, it was the loss of tissue resistance to blunt probing. Both were taken to surgery within 3 days of the present- ing problem. The first case showed the typical polymicrobial nature of necrotizing fasciitis, whereas in the sec- ond only one organism (P. mirabilis) was cultured.

s to respond to antibiotics. In the second, it was the loss of tissue resistance to blunt probing. Both were taken to surgery within 3 days of the present- ing problem. The first case showed the typical polymicrobial nature of necrotizing fasciitis, whereas in the sec- ond only one organism (P. mirabilis) was cultured. This may be due to the perioperative broad spec- trum antibiotic prophylaxis given to this patient at the time of her hysterectomy. Necrotizing fasciitis can occur in any surgical or nonsurgical insult. In the recent Ob/Gyn literature alone, there are cases reported from episiotomy, mini-laparotomy, diagnostic laparoscopy, and su- prapubic catheter placement. 16-19 There are also reports of patients without an obvious original ni- dus of infection, or patients on standard treatment INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY NECROTIZING FASCHTIS MANAGEMENT GUIDELINES THOMPSON ET AL. TABLE 4. Guidelines for managing necrotizing fasciitis at PMH I. Patients with suspicious lesions in the vulvar, groin, or thigh region with high-risk factors for necrotizing fasciitis are admitted for observation 2. Patients with presumed cellulitis who fail to respond to IV antibiotics are taken to surgery for wound exploration 3. Post-operative patients who have high-risk factors for necrotizing fasciitis and have wound separation with loss of tissue resistance are started on IV antibiotics and taken to the operating room for exploration 4. Junior house officers notify senior house officers and faculty experienced in the care of necrotizing fasciitis about any suspicious lesions or wound complications 5. Lectures on soft-tissue infections are included on a regular basis for residents and medical students 6. Wound care is managed by the same upper level resident on any service, and additional dbridement and/or return to the operating room is performed as needed 7. Survival depends on early recognition and immediate surgical dbridement to healthy tissue margins Remove all indurated, edematous, crepitant tissue Incise tissue to margins that bleed easily Change wound dressings frequently Initiate broad spectrum antibiotics regimens such as radiotherapy, chemotherapy, and anti-inflammatory drugs.2-22 It is often said that anyone who uses a certain kind of intervention or therapy should be prepared to deal with the consequences ofthat intervention or therapy.

leed easily Change wound dressings frequently Initiate broad spectrum antibiotics regimens such as radiotherapy, chemotherapy, and anti-inflammatory drugs.2-22 It is often said that anyone who uses a certain kind of intervention or therapy should be prepared to deal with the consequences ofthat intervention or therapy. Among the surgical specialties, obstetri- cians and gynecologists frequently deal with heavily contaminated body areas; therefore, knowledge of surgical complications is imperative, especially one as life-threatening as necrotizing fasciitis. ACKNOWLEDGMENTS The authors acknowledge the many hours of medi- cal care provided to these patients by the following physicians: Deborah Lenart, M.D., Stacy Travis, M.D., and Michael Bevers, M.D. We appreciate the skillful manuscript preparation by Ms. Maria McFarland. REFERENCES 1. Shy KK, Eschenbach DA: Fatal perineal cellulitis from an episiotomy site. Obstet Gynecol 54:292-298, 1979. 2. Rea WJ, Wyrick wJ, Jr: Necrotizing fasciitis. Ann Surg 172:957-964, 1970. 3. Addison NA, Livengood CH, Hill GB, Sutton GP, Fortier KJ: Necrotizing fasciitis of vulvar origin in dia- betic patients. Obstet Gynecol 63:473-479, 1984. 4. JonesJ: Investigations upon the nature, causes, and treat- ment of hospital gangrene as it prevailed in the Confeder- ate Armies 1861-65. New York, U.S. Sanitary Commis- sion, Surgical Memories of the War of Rebellion, 1871. As quoted in Wilson, B: Necrotizing fasciitis. Am Surg 18:416-431, 1952. 5. Meleney FL: Hemolytic streptococcus gangrene. Arch Surg 9:317-364, 1924. 6. Wilson B: Necrotizing fasciitis. Am Surg 18:416-431, 1952. 7. Roberts DB, Hester LL, Jr: Progressive synergistic bac- terial gangrene arising from abscesses of the vulva and Bartholin's gland duct. Am J Obstet Gynecol 114:285- 289, 1972. 8. Lee RA: Postoperative necrotizing fasciitis. In Nichols DH, Anderson GW (eds): Clinical Problems, Injuries, and Complications of Gynecologic Surgery. Baltimore: Williams & Wilkins, pp 1-4, 1988. 9. Roberts DB: Necrotizing fasciitis of the vulva. Am J Obstet Gynecol. 157:568-571, 1987. 10. Livengood CH, Soper JT, Clarke-Pearson DL, Addison WA: Necrotizing fasciitis in irradiated tissue from dia- betic women. J Reprod Med 36:455-458, 1991 11. Adelson MD, Joret DM, Gordon LP, Osborne NG: Recurrent necrotizing fasciitis of the vulva. J Reprod Med 36:818-822, 1991. 12. Stephenson H, Dotters DJ, Katz V, Droegmuller W: Necrotizing fasciitis of the vulva. Am J Obstet Gynecol 166:1324--1327, 1992. 13.

tissue from dia- betic women. J Reprod Med 36:455-458, 1991 11. Adelson MD, Joret DM, Gordon LP, Osborne NG: Recurrent necrotizing fasciitis of the vulva. J Reprod Med 36:818-822, 1991. 12. Stephenson H, Dotters DJ, Katz V, Droegmuller W: Necrotizing fasciitis of the vulva. Am J Obstet Gynecol 166:1324--1327, 1992. 13. Lewis RT: Soft tissue infections. In Wilmore DW (ed): Care of the Surgical Patient. New York: Scientific Amer- ican, pp 6.1-6.15, 1988. 14. Fisher JR, Conway MJ, Takeshita RT, Sandoval MR: Necrotizing fasciitis: Importance of roentgenographic studies for soft-tissue gas. JAMA 241:803-806, 1979. 15. Brewer GE, Meleney FJ: Progressive gangrenous infec- tion of the skin and subcutaneous tissues following opera- tion for acute perforative appendicitis. Ann Surg 84:438- 450, 1926. 16. Riseman JA, Zamboni WA, Curtis A, Graham DR, Kon- tad HR, Ross DS: Hyperbaric oxygen therapy for necro- tizing fasciitis reduces mortality and the need for d4bride- ments. Surgery 108:847-850, 1990. 17. Sotrel G, Hirsch E, Edelin KC: Necrotizing fasciitis following diagnostic laparoscopy. Obstet Gyneco162:675- 695, 1983. 18. Bearman DM, Livengood CH, Addison WA: Necrotiz- ing fasciitis arising from a suprapublic catheter site. J Reprod Med 33:411-413, 1988. 19. CedermaJP, Davies BW, Farkas SA, SontaJA, Swornio- wski T: Necrotizing fasciitis of the total abdominal wall after sterilization by partial salpingectomy. Am J Obstet Gynecol 162:138-139, 1990. 20. Fr6hlich EP, Schein M: Necrotizing fasciitis arising from Bartholin's abscess. Isr J Med Sci 25:644-647, 1989. 21. Hoffman MS, Turnquist D: Necrotizing fasciitis of the vulva during chemotherapy. Obstet Gynecol 74:483-484, 1989. 22. Smith RJ, Berk SC: Necrotizing fasciitis and nonsteroidal anti-inflammatory drugs. South Med J 84:785-787, 1991. 22 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:2-6 (I 993) (C) 1993 Wiley-Liss, Inc. Performance of the Syva Direct Fluorescent Antibody Assay for Chlamydia in a Low-Prevalence Population Mark B. Reedy, Patricia J. Sulak, William B. McCombs III, and Thomas J. Kuehl Departments of Obstetrics and Gynecology (M.B.R., P.J.S., T.J.K.), Microbiology (W.B.M.), Pathology (W.B.M., T.J.K.), and Medical Biochemistry and Genetics (T.J.K.), Scott & White Clinic and Memorial Hospital Texas A&M University College ofMedicine, Temple, TX ABSTRACT Chlamydia trachomatis is the most common reportable sexually transmitted disease (STD) in the United States. In the 1980s, rapid diagnostic tests for chlamydia began to replace more cumbersome tissue culture methods. Current data on rapid antigen detection assays demonstrate acceptable sensitivity, specificity, and predictive values in populations with a high prevalence of chlamydia. Few studies report the performance of these assays in a low-prevalence obstetric and gynecologic (Ob/Gyn) population. This study compares the most commonly used direct fluorescent antibody (DFA) assay (Syva Microtrak) with tissue culture (TC) in a low-prevalence population. Endocer- vical specimens (775) were tested from women at risk for chlamydia infection, and the prevalence was found to be 7.7%. The DFA assay demonstrated a sensitivity of 80% and a specificity of 97% compared with TC. The positive and negative predictive values were 72% and 98%, respectively. The results of this study indicate that the Syva DFA assay lacks the sensitivity and positive predictive value for routine use in Ob/Gyn populations with a low prevalence of C. trachomatis. (C) 1993 Wiley-Liss, Inc. KEY WORDS Rapid antigen detection assay, chlamydia trachomati,s, sexually transmitted disease hlamydia trachomatis is the most common re- portable sexually transmitted disease in the United States.

predictive value for routine use in Ob/Gyn populations with a low prevalence of C. trachomatis. (C) 1993 Wiley-Liss, Inc. KEY WORDS Rapid antigen detection assay, chlamydia trachomati,s, sexually transmitted disease hlamydia trachomatis is the most common re- portable sexually transmitted disease in the United States. l'2 Chlamydia is a major public health problem in the United States, with 3-5 mil- lion new cases estimated each year1-3 and an annual economic burden of more than $1 billion.4 The Centers for Disease Control recommends chla- mydia testing at the first prenatal visit and in the third trimester for high-risk patients. Clinical manifestations of the disease affect men, women, and children. Unfortunately, many genital chla- mydia infections are asymptomatic in women and can result in pelvic inflammatory disease, ectopic pregnancy, and infertility.3 The gold standard for detecting chlamydia is tissue culture (TC).2'3'5 Tissue culture is time consuming and expensive, and requires technical expertise. In the 1980s, rapid diagnostic tests for detecting chlamydia began replacing tissue culture methods. These antigen de- tection assays are simple to perform and offer rapid results. Current data on rapid antigen detection assays demonstrate acceptable sensitivity, specific- ity, and positive predictive value for routine use in high-prevalence populations. However, many of these assays are targeted at the private practitioner or clinic with a low prevalence of chlamydia infec- tion. Schachter6 has stated that nonculture methods for chlamydia antigen detection are best suited for high-prevalence populations or high-risk patients and that extrapolation of these test results to low- Presented at the April 27, 1992 Annual Meeting ofACOG at Las Vegas, Nevada. Address correspondence/reprint requests to Dr. Mark B. Reedy, Department of Obstetrics and Gynecology, Scott & White Clinic, 2401 S. 31st St., Temple, TX 76508. Received May 18, 1992 Clinical Study Accepted December 30, 1992 SYVA DFA ASSAY FOR CHLAMYDIA REEDY ET AL. prevalence populations must be made with caution. Recently, the American College of Obstetrics and Gynecology Update (Precis IV) stated that the use of rapid antigen detection assays in a low-preva- lence population has not been thoroughly investi- gated.7 This paper describes a prospective random- ized study comparing the Syva Microtrak direct fluorescent antibody (DFA) test against tissue cul- ture (TC) in a low prevalence population.

ecis IV) stated that the use of rapid antigen detection assays in a low-preva- lence population has not been thoroughly investi- gated.7 This paper describes a prospective random- ized study comparing the Syva Microtrak direct fluorescent antibody (DFA) test against tissue cul- ture (TC) in a low prevalence population. MATERIALS AND METHODS Study Population All specimens were collected at Scott & White Me- morial Hospital, Temple, Texas, from women at risk for chlamydia infection. Risk factors were: multiple sex partners, new sex partners, sexually active adolescents, pregnancy, history of other sex- ually transmitted disease, or signs of cervicitis. Samples were not collected from patients who re- ceived antibiotic therapy within 2 weeks of sam- pling. Previous studies at this hospital demon- strated the prevalence of chlamydia in this female population to be 8%. Collection of Specimens All specimens were collected from the endocervix per manufacturer's instructions. Dacron swabs (Medical Wire Equipment Co., Corsham, Wilts, U.K.) were used for TC. DFA Dacron swabs were supplied in the Microtrak DFA kit. Collection order was randomized by the last digit of the med- ical record number (MRN). Ifthe MRN ended in an odd number, the TC swab was collected first, followed by the DFA swab. Ifthe MRN was even, the order was reversed. If a gonorrhea specimen or a pap smear was requested, they were collected prior to the study swabs. Swabs were placed in their respective transport containers and were tested within 24 hours of collection simultaneously by the Syva DFA and TC isolation. Specimens were stored at 4C until tested. Laboratory Methods Cell Culture Methods Chlamydia cell culture swabs were used to inoculate McCoy cell monolayers followed by centrifugation for hour at 3,000g. One milliliter of Chlamydia Isolator Medium with cycloheximide (Bartels Immunodiagnostics, Inc., Bellevue, WA)was added to each culture, which was then incubated at 35-37C for 48-72 hours. A blind second passage was not performed. Cultures were stained with a C. trachomatis-specific fluorescent monoclonal anti- body (Chlamydia Culture Confirmation Kit, Syva Co., Palo Alto, CA). Positive results were deter- mined by identifying fluorescent-stained inclusion bodies. Results were entered in the main laboratory computer and retrieved after all testing was com- pleted. DFA Slides were processed and stained according to manufacturer's instructions (Microtrak, Syva Co.).

t, Syva Co., Palo Alto, CA). Positive results were deter- mined by identifying fluorescent-stained inclusion bodies. Results were entered in the main laboratory computer and retrieved after all testing was com- pleted. DFA Slides were processed and stained according to manufacturer's instructions (Microtrak, Syva Co.). Slides were screened with a Zeiss fluorescent microscope at a 400 magnification and con- firmed at 1,000. Slides were considered positive iften or more elemental bodies were identified. All slides were processed and read by one of four American Society of Clinical Pathologists (ASCP) Certified Medical Technologists experienced with the Syva DFA system. All technologists are regu- larly tested against the CAPS (Clinical Association of Pathologists) survey slides. The DFA results were interpreted and recorded before TC results were obtained. DFA specimens were considered inadequate if less than five endocervical cells were present on a smear. Statistical Methods Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated for all assays. Statistical significance was determined by chi square analysis. RESULTS A total of 775 endocervical specimens was tested by DFA and TC. Less than 3% of the original speci- mens were considered inadequate. These specimens were excluded from our final calculations. The prevalence of chlamydia based on TC results was 7.7%. The DFA demonstrated a sensitivity of 80% and a specificity of 97%. The positive and negative predictive values, were 72% and 98%, respectively (Fig. 1). There were 19 false positives and 12 false negatives. DISCUSSION Many rapid diagnostic tests are available for chlamydia detection. Tissue culture is still consid- INJ'ECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY SYVA DFA ASSAY FOR CHLAMYDIA REEDY ET AL. Results of DFA vs TC TC DFA sensitivity (a/a + e) 80% specificity (d/d + b) 97% PPV(a/a + b) 72% NPV(d/c + d) 98% Fig. I. The results of the Syva DFA for chlamydia detec- tion versus tissue culture isolation. ered the gold standard by which all other assays are judged. To date, no single rapid diagnostic test for chlamydia has proved ideal for routine screening, especially in a low-prevalence (<9%) popula- tion. 7-1 The most commonly used and studied DFA as- say is the Syva Microtrak C. trachomatis Direct Specimen Test. Therefore, we elected to study this test in our low-prevalence population.

o single rapid diagnostic test for chlamydia has proved ideal for routine screening, especially in a low-prevalence (<9%) popula- tion. 7-1 The most commonly used and studied DFA as- say is the Syva Microtrak C. trachomatis Direct Specimen Test. Therefore, we elected to study this test in our low-prevalence population. Despite published reports8' l, 12 that used less than ten ele- mental bodies per slide to signify a positive DFA test, we elected to follow the manufacturer's in- structions and use ten or more elemental bodies per slide for a positive test, since the majority of tests would be conducted in routine clinical laboratories and not under research protocols. Studies have shown that sensitivity can be increased by lowering the number of elemental bodies required for a pos- itive test, but this is at the expense of lowering the specificity and positive predictive value.9 The DFA demonstrated a sensitivity of 80%, specificity of 97%, and PPV of 72%. This is simi- lar to previously published reports (Table 1).9'13-18 Stamm l summarized 15 Syva DFA studies in high- and intermediate-prevalence popu- lations. In high-prevalence populations with a chlamydia prevalence of 15-26%, the cumulative sensitivity was 90% and specificity was 95%. The PPV was 90%. In populations with a prevalence of 9-1 1%, the sensitivity and PPV decreased to 77% and 79%, respectively. TABLE I. DFA (Microtrak) studies compared to TC on endocervical swabs in low-prevalence populations (< 9%) No. of Author patients Prevalence Sensitivity PPV Gannet al. 268 7 53 69 Lefebvre et al. 715 5 76 76 Phillips et al. 4 527 4 70 62 Forbes et al. s 642 7 60 74 Godfrey et al. 6 332 7 75 95 Graber et al. 7 187 8 100 65 Uyeda et al. e 401 7 96 93 aUsed less than ten elemental bodies for a positive test. The effect of a 3, 12, 24% prevalence on predictive values of an assay with an 80% sensitivity and a 97% specificity. A. 3% prevalence (n 1000) 24 29 PPV 45% 6 941 m'v 99% B. 12% prevalence (n 1000) 24 854 PPV 78% NPV 97% Co 24% prevalence (n 1000) 192 23 48 737 PPV 89% NPV 94% Fig. 2. How prevalence affects positive predictive value. Predictive values are influenced by prevalence rates in a population.2'6'11 Grimes19 stated that when screening for disease in a low-prevalence population, even with a high sensitivity and speci- ficity, the PPV would be low. Figure 2 illustrates how prevalence affects PPV. When interpreting these data, one must be aware that TC is not the perfect gold standard.

ce rates in a population.2'6'11 Grimes19 stated that when screening for disease in a low-prevalence population, even with a high sensitivity and speci- ficity, the PPV would be low. Figure 2 illustrates how prevalence affects PPV. When interpreting these data, one must be aware that TC is not the perfect gold standard. The sensi- tivity of isolating chlamydia from a single swab is estimated to be 70--80%.2,5,6,9 The use of immun- ofluorescent staining ofTC increases the sensitivity similar to that of a blind second passage, l0 Even with a blind second passage of fluorescent antibody staining, TC will not identify all infected patients. 4 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY SYVA DFA ASSAY FOR CHLAMYDIA REEDY ET AL. There were 19 false positives; six ofthese also tested positive by another antigen detection assay (TestPack Chlamydia Abbott Labs, Chicago, IL). Several authors8'11,12,20--22 have used a second an- tigen detection assay to demonstrate TC false nega- tives. If both antigen detection methods were posi- tive, they would consider the specimen as a true positive and the TC as a false negative. If these six specimens are considered true positives, the sensi- tivity ofthe Syva DFA increases from 80% to 82% and the specificity from 97% to 98%. The PPV increases from 72% to 80%. The original and ad- justed statistics are not significantly different (P > 0.0S). If TC has a sensitivity of 80% and the Syva DFA assay detects 80% of TC positives, then only 65% of infected patients are identified with the DFA assay. This appears to be an unsatisfactory detection record for a treatable infection that affects a broad spectrum of society. Also of concern is the low PPV. Between 20% and 30% of the Syva DFA positives will be false positives in this population. A false positive result on an STD test may have grave social implications. Thus, some authors advocate TC confirmation of positive DFA results when used in a low-preva- lence population.23 Antigen detection assays have demonstrated unequivocal false positive results in patients evaluated for sexual abuse, z4 The Centers for Disease Control (CDC) recommends only stan- dard tissue culture methods to identify C. tracho- marls in the evaluation of sexual abuse, Tissue culture isolation is still the most sensitive and spe- cific diagnostic test for chlamydia.

cal false positive results in patients evaluated for sexual abuse, z4 The Centers for Disease Control (CDC) recommends only stan- dard tissue culture methods to identify C. tracho- marls in the evaluation of sexual abuse, Tissue culture isolation is still the most sensitive and spe- cific diagnostic test for chlamydia. 9'1'22 Recent data show that DFA sensitivity may be increased by the use of the cytobrush to sample the endocervix.25'26 The cytobrush was not used in this study because the manufacturer did not recommend its use at the initiation of the study. The Syva Company still does not recommend the use of the cytobrush in pregnancy. The results of our study indicate that the Syva DFA assay lacks the sensitivity and PPV for rou- tine screening in Ob/Gyn populations with a low prevalence of C. trachomatis. With the advent of newer technologies for the detection of chlamydia (i.e., DNA probes and polymerase chain reaction), the private practitioner or clinic should critically analyze the performance of these assays in their targeted patient population before beginning wide- spread screening. REFERENCES 1. Centers for Disease Control: 1989 Sexually transmitted diseases treatment guideline. MMWR 38(S-8):1-43, 1989. 2. Sweet RL, Gibbs RS: Chlamydial infections. In: Infec- tious Diseases ofthe Female Genital Tract. 2nd ed. Balti- more: Williams & Wilkins, pp 45-74, 1990. 3. Martin DH: Chlamydial infections. Med Clin North Am 74:1367-1387, 1990. 4. Washington AE: Chlamydia: A major threat to reproduc- tive health. Research Highlights, Institute for Health Pol- icy Studies. University of California, San Francisco 2:1-3, 1984. 5. Holmes KK, Mardh P-A, Sparling PF, Wiesner PJ, Cates W, Jr, Lemon SM, Stamm WE: Chlamydia tra- chomatis. In: Holmes KK et al. (eds): Sexually Transmit- ted Diseases. 2nd ed. New York: McGraw-Hill, pp 917- 925, 1990. 6. Schachter J: Rapid diagnosis of sexually transmitted dis- easesmspeed has a price. Diagn Microbiol Infect Dis 4:185-189, 1986. 7. American College of Obstetrics and Gynecology: Precis IV ACOG, p 31, 1990. 8. Gann PH, Herrmann JE, Candib L, Hudson RW: Ac- curacy of Chlamydia trachomatis antigen detection meth- ods in a low-prevalence population in a primary care setting. J Clin Microbiol 28:1580-1585, 1990. 9. Barnes RC: Laboratory diagnosis of human chlamydial infections. Clin Microbiol Rev 2:119-136, 1989. 10. Stamm WE: Diagnosis of Chlamydia trachomatis geni- tourinary infections. Ann Intern Med 108:710-717, 1988. 11.

th- ods in a low-prevalence population in a primary care setting. J Clin Microbiol 28:1580-1585, 1990. 9. Barnes RC: Laboratory diagnosis of human chlamydial infections. Clin Microbiol Rev 2:119-136, 1989. 10. Stamm WE: Diagnosis of Chlamydia trachomatis geni- tourinary infections. Ann Intern Med 108:710-717, 1988. 11. Schwebke JR, Stature WE, Handsfield HH: Use of sequential enzyme immunoassay and direct fluorescent antibody tests for detection of Chlamydia trachomatis in- fections in women. J Clin Microbiol 28:2473-2476, 1990. 12. Baselski VS, McNeeley SG, Ryan G, Robison M: A comparison of nonculture-dependent methods for detec- tion of Chlamydia trachomatis infections in pregnant women. Obstet Gynecol 70:47-52, 1987. 13. Lefebvre J, Laperiere H, Rousseau H, Masse R: Com- parison of three techniques for detection of Chlamydia trachomatis in endocervical specimens from asymptomatic women. J Clin Microbiol 26:726-731, 1988. 14. Phillips RS, Hanff PA, Kauffman RS, Aronson MD: Use of a direct fluorescent antibody test for detecting Chlamydia trachomatis cervical infection in women seek- ing routine gynecologic care. J Infect Dis 156:576-581, 1987. 15. Forbes BA, Bartholoma N, McMillan J, Roefaro M, Weiner L, Welych L: Evaluation of a monoclonal anti- body test to detect chlamydia in cervical and urethral specimens. J Clin Microbiol 23:1136-1137, 1986. 16. Godfrey E, Winn W, Jr, Keathley JD. Performance of INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 5 SYVA DFA ASSAY FOR CHLAMYDIA REEDY ET AL. Microtrak direct test for Chlamydia trachomatis in a prev- alence study. Diagn Microbiol Infect 5:313-316, 1986. 17. Graber CD, Williamson O, Pike J, Valicenti j: Detection of Chlamydia trachomatis infection in endocervical speci- mens using direct immunofluorescence. Obstet Gynecol 66:727-730, 1985. 18. Uyeda CT, Welborn P, Ellison-Birang N, Shunk K, Tsaouse B: Rapid diagnosis of chlamydial infection with the MicroTrak direct test. J Clin Microbio120:948-950, 1984. 19. Grimes DA: Screening tests. What they are and what they aren't. Contemp OB/GYN 20:69-74, 1982. 20. Moncada J, Schachter J, et al: Confirmatory assay in- creases specificity of chlamydiazyme test for Chlamydia trachomatis infection of the cervix. J Clin Microbiol 28: 1770-1773, 1990. 21. Gaydos CA, Reichart CA, et al.: Evaluation of Syva enzyme immunoassay for detection of Chlamydia tracho- matis in genital specimens. J Clin Microbiol 28:1541- 1544, 1990. 22.

assay in- creases specificity of chlamydiazyme test for Chlamydia trachomatis infection of the cervix. J Clin Microbiol 28: 1770-1773, 1990. 21. Gaydos CA, Reichart CA, et al.: Evaluation of Syva enzyme immunoassay for detection of Chlamydia tracho- matis in genital specimens. J Clin Microbiol 28:1541- 1544, 1990. 22. Binn B, Williams JT, McDowell J, Brunham RT, Brun- ham RC: Screening for Chlamydia trachomatis infection in a pregnant counseling clinic. Am J Obstet Gynecol 15a: 1144-1149, 1988. 23. Martens MG: Office diagnosis of sexually transmitted diseases. Obstet Gynecol Clin North Am 16:659-677, 1989. 24. Dorian KJ, Holtzhauer F, et al.: False-positive results with the use of chlamydia tests in evaluation of suspected sexual abusemOhio, 1990. MMWR 39:932-935, 1991. 25. Weiland TL, Noller KL, Smith TF, Ory SJ: Compari- son of Dacron-tipped applicator and cytobrush for detec- tion ofchlamydial infections. J Clin Microbiol 26:2437- 2438, 1988. 26. Moncada J, Schacter J, Shipp M, Bolan G, Wilber J: Cytobrush in collection of cervical specimens for detec- tion of C. trachomatis. J Clin Microbiol 27:1863-1866, 1989. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:23-26 (1993) (C) 1993 Wiley-Liss, Inc. Cefotetan Susceptibility Testing Against Anaerobic Bacteria From Obstetrical and Gynecologic Sources: Comparison of Five Different Methods Maurizio Maccato, Gerald Riddle, and Sebastian Faro Section ofInfectious Diseases, Department of Obstetrics and Gynecology, Baylor College ofMedicine, Houston, TX ABSTRACT Five different antibiotic susceptibility methods were utilized to test the effectiveness of cefotetan against 200 anaerobic bacteria recovered from patients with obstetrical or gynecological infections. The object of this study was to determine if a more economical and rapid method for anaerobic susceptibility testing was as acceptable as the reference agar dilution method. The five methods were: 1) broth disk elution, 2) microbroth technique, 3) a commercially available microbroth tech- nique, 4) a commercially available spiral gradient technique, and 5) reference agar dilution. The minimal inhibitory concentrations (MICs) calculated from the spiral gradient technique were equal to or within one doubling dilution ofthe reference system in 99.5% of cases, while the percentage for the commercially available microbroth system was 96.8%, very similar to the microbroth technique used in our laboratory that yielded a percentage of 96.3. The disk elution method correlated to the reference agar dilution method in 95.3% cases. While the overall agreement between these tech- niques is good, especially for the spiral gradient system, clustering of certain organisms near the breakpoint ofthe antibiotic tested results in variability in the labeling ofthese organisms as suscepti- ble or resistant. This problem appears to be particularly significant for the disk elution method. Therefore, further refinements in these methods of susceptibility testing are needed in order to provide a more clinically useful assessment of the susceptibility or resistance of certain bacterial isolates. (C) 1993 Wiley-Liss, Inc.

tant. This problem appears to be particularly significant for the disk elution method. Therefore, further refinements in these methods of susceptibility testing are needed in order to provide a more clinically useful assessment of the susceptibility or resistance of certain bacterial isolates. (C) 1993 Wiley-Liss, Inc. KEY WORDS Agar dilution, spiral gradient, microbroth, disk elution ost soft tissue pelvic infections in obstetrics and gynecology are polymicrobial, with anaerobic bacteria often playing a significant role. Treatment of these infections is empiric and re- quires a broad-spectrum antimicrobial agent. Beta- lactam antibiotics, which have a broad spectrum of activity including aerobic, facultative, and obligate anaerobes, are suitable for the treatment ofobstetric and gynecologic infections, e.g., postpartum en- dometritis, pelvic inflammatory disease, and pelvic cellulitis. Cefotetan, a 7-methoxy beta-lactam anti- biotic, with a long serum half-life and a broad spectrum ofactivity, 1,2 is considered suitable for the empiric treatment of ob/gyn infections. Concerns have been raised about the apparent variability in in vitro activity of certain beta-lactam antibiotics against anaerobic organisms.3'4 There- fore, cefotetan was tested against anaerobic organ- isms isolated from patients with gynecologic or ob- stetrical soft tissue pelvic infections using five different antibiotic susceptibility testing methods, as described by the National Committee for Clini- cal Laboratory Standards (NCCLS).s'6 This was done in order to determine if a more economical or Address correspondence/reprint requests to Dr. Maurizio Maccato, Smith Tower, 6550 Fannin, Suite 701, Houston, TX 77030. Clinical Study Received November 3, 1992 Accepted December 15, 1992 CEFOTETAN SUSCEPTIBILITY TESTING FOR ANAEROBES MACCATO ET AL. rapid method for anaerobic sensitivity testing was as acceptable as the reference agar dilution method. MATERIALS AND METHODS The bacteria tested were isolated from patients who were treated for obstetrical or gynecologic pelvic infections. The organisms were frozen at -70C after being isolated and identified as described pre- viously.7 The bacteria were passed twice on solid media before being used for testing. The reference agar dilution testing was performed according to the NCCLS published method, s All media were prepared in the laboratory and pre-reduced shortly after preparation.

C after being isolated and identified as described pre- viously.7 The bacteria were passed twice on solid media before being used for testing. The reference agar dilution testing was performed according to the NCCLS published method, s All media were prepared in the laboratory and pre-reduced shortly after preparation. The broth disk dilution method was performed using thioglycolate broth according to the method described by the NCCLS.6 Like- wise, the microbroth method using pre-reduced Wilkins-Chalgren broth was performed according to the NCCLS publication.6 The Sceptor system (BBL, Cockeysville, MD) was used to test a com- mercially available microbroth system. Broth cul- tures of the isolates were adjusted to a turbidity equivalent of 0.5 MacFarland standard. One hun- dred microliters ofthis broth was added to 10 ml of pre-reduced Sceptor anaerobes minimal inhibitory concentration (MIC)-ID broth and vortexed. One hundred microliters per well of this Sceptor broth was then added to each of 84 wells in the Sceptor MIC panel and incubated in an anaerobic chamber for 48 hours at 36C. The Spiral system was used according to the instructions of Spiral System In- struments Inc. (Bethesda, MD). Antibiotic gradi- ents were made using the spiral platter on pre- reduced Wilkins-Chalgren agar. Sixty milliliters of agar were poured into 150 ml Petri dishes achieving an agar depth of 3.3 mm. The plates were allowed to sit in an anaerobic environment for hour before inoculation. Broth cultures (BHI plus vitamin K and hemin) of the isolates were adjusted to a turbidity equivalent to a 0.5 MacFar- land. A sterile Dacron swab was used to plate the isolates on the gradient plates. A spiral system tem- plate aided in the placement of 15 isolates for each gradient plate. Wilkins-Chalgren plates without drug were also inoculated as growth control. Plates were incubated for 48 hours at 36C in an anaero- bic chamber. Interpretation of the gradient plate results involved a measure in the length of each isolate using the spiral system template. The MIC values were calculated using the spiral gradient endpoint software. Bacteroidesfragilis ATCC252 8 5 was used as a control for all MIC procedures. This organism was tested with these systems to ensure that the MICs were within an acceptable range. Detailed descriptions of the methods can be found in the referenced NCCLS publications. 5'6 Ce- fotetan was provided by Stuart Pharmaceuticals (Wilmington, DE).

ATCC252 8 5 was used as a control for all MIC procedures. This organism was tested with these systems to ensure that the MICs were within an acceptable range. Detailed descriptions of the methods can be found in the referenced NCCLS publications. 5'6 Ce- fotetan was provided by Stuart Pharmaceuticals (Wilmington, DE). RESULTS Table summarizes the in vitro activity of ce- fotetan against the various anaerobes tested as deter- mined by the five different methods. Bacteroides capillosus, B. distasonis, B. thetaiotamicron, and Pre- votella ruminicola have relative resistance to ce- fotetan. The majority of organisms, mainly Por- phyromonas asaccharolyticus, P. bivius, P. intermedius, P. melaninogenicus, P. oralis, B. vulga- tus, B. fragilis, Fusobacterium, and Peptostreptococ- cus sp. are susceptible to cefotetan. With respect to the agar dilution method, the commercial spiral system identified MICs equal to or within one doubling dilution of the reference system in 99.5% oftested organisms. The commer- cial microbroth system identified 96.8% of MICs, while the microtube dilution identified 96.3% of MICs as equal to or within one doubling dilution of the reference system. The disk elution method breakpoint was in agreement with the susceptible or resistant determinations of the agar dilution refer- ence method in 95.3% of cases. However, among the 17 strains that were identified as resistant to cefotetan by agar dilution, the disk dilution tech- nique identified correctly as resistant only 8 isolates (47% agreement). Of the nine isolates that were identified as resistant with the agar dilution method and as susceptible by the disk elution technique, eight were at the breakpoint. The spiral gradient technique identified six isolates (65% agreement) as susceptible to cefotetan that the reference agar dilu- tion classified as resistant. The MICs for six of these isolates were of 64 Ixg/ml by agar dilution, and 32 bg/ml by the spiral gradient technique. Similar results were obtained with the commer- cially available microbroth system (71% agree- ment) and with the laboratory developed micro- broth system (65% agreement). In all cases, the 24 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY CEFOTETAN SUSCEPTIBILITY TESTING FOR ANAEROBES MACCATO ET AL. TABLE I. MIC90 and number of resistant isolates as identified by five different methods Organism Agar dilution Spiral (no. of strains) No. MICgo No. MICgo Method Sceptor microbroth No. MIC9o Laboratory Disk microbroth elution No.

IN OBSTETRICS AND GYNECOLOGY CEFOTETAN SUSCEPTIBILITY TESTING FOR ANAEROBES MACCATO ET AL. TABLE I. MIC90 and number of resistant isolates as identified by five different methods Organism Agar dilution Spiral (no. of strains) No. MICgo No. MICgo Method Sceptor microbroth No. MIC9o Laboratory Disk microbroth elution No. MICgo No. Porphyromonas asaccharolyticus (8) 0 32 0 16 0 16 0 16 0 Prevotella bivius (80) 32 32 32 32 0 Bacteroides fragilis (22) 3 64 2 16 2 16 2 16 2 P. intermedius (8) 0 32 0 16 0 16 0 16 P. melaninogenicus (18) 64 0 32 64 0 32 0 P. oralis (4) 0 16 0 16 0 16 0 16 0 B. vulgatus (3) 0 32 0 16 0 16 0 16 0 B. capillosus (12) 6 128 3 128 3 128 3 64 2 B. distasonis (3) 2 64 2 64 2 64 2 64 2 B. thetaiotamicron (I) 128 128 128 128 P. ruminicola (5) 2 128 2 128 2 128 2 128 Fusobacterium sp. (7) 0 8 0 8 0 4 0 4 0 Peptostreptococcus sp. (29) 16 0 8 0 8 0 4 0 aNumber of resistant isolates (MIC 64 Ig/ml). Minimal concentration to inhibit 90% growth (MICg0) is Ig/ml. MICs were higher by the agar dilution method than the other techniques. DISCUSSION While the role of anaerobic bacteria in the patho- genesis of infections in obstetrics and gynecology is well established, the need for susceptibility testing ofanaerobic organisms against antimicrobial agents is still subject to some controversy. 8 Initial antibi- otic therapy of female pelvic soft tissue polymicro- bial infections is empiric. Therefore, it is impor- tant to identify antimicrobial agents that have the necessary spectrum of activity to ensure adequate coverage of the suspected pathogens and to follow susceptibility patterns of anaerobes as different an- timicrobial agents are used. Unfortunately, the sus- ceptibility testing of antimicrobial agents against anaerobic organisms using the agar dilution method is technically complex and time consum- ing. Several alternative methods have, therefore, been proposed with the aim of reducing the cost in terms of time and material that such testing de- mands. Unfortunately, the use of different suscep- tibility testing methods has revealed the difficulty of standardizing different tests (or even the same test) among different laboratories. From our data, it appears that the spiral system, the microbroth, and the commercially available microbroth system are adequate methods to evaluate anaerobic suscep- tibility to cefotetan. The broth disk elution method did not provide the same degree of reliability as the other methods tested.

g different laboratories. From our data, it appears that the spiral system, the microbroth, and the commercially available microbroth system are adequate methods to evaluate anaerobic suscep- tibility to cefotetan. The broth disk elution method did not provide the same degree of reliability as the other methods tested. A recent report comparing the spiral gradient technique and the conventional agar dilution method concluded that the spiral technique is a reasonable alternative. 9 The data presented here confirm that the spiral method is as reliable as the reference method and in our laboratory was less tedious to use. We confirmed the report that the commercial broth microdilution system gives MICs lower than those of the agar dilution sys- 10 tern. The results published from other studies using the broth disk elution technique are consistent with our experience. 11 The broth disk elution method is inadequate for the clinical evaluation ofsusceptibil- ity of anaerobic organisms and has recently been eliminated as an approved method by the NC- CLSo 122 In conclusion, we believe that anaerobic suscep- tibility testing should be performed in major med- ical centers both to monitor the changing pattern.of antibiotic susceptibility and to help in the manage- ment ofclinically difficult cases. In our experience, the use of a less labor-intensive method may be an acceptable alternative if further refinements in the techniques will reduce the difficulties in the correct interpretation of MICs occurring near the break- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 25 CEFOTETAN SUSCEPTIBILITY TESTING FOR ANAEROBES MACCATO ET AL. point when anaerobic organisms are tested against cefotetan or other beta-lactam antibiotics. Close correlation of the in vitro results with the results of clinical therapy is obviously necessary to confirm the validity of in vitro testing of anaerobic bacteria as a tool toward better treatment of actual clinical cases. ACKNOWLEDGMENTS This work was supported in part by a grant from Stuart Pharmaceuticals, Division of ICI Americas, Inc. REFERENCES 1. Jones RN: Cefotetan: A review ofthe microbiologic prop- erties and antimicrobial spectrum. Am J Surg 155:16- 23, 1988. 2. Zabransky RJ, Bobey DG, Sheikh W: A multicenter study of the in vitro anti-anaerobic activity of cefotetan compared with other antimicrobial agents. 155:47-51, 1988. 3. Aldridge KE: Controversies in susceptibility testing of anaerobes. Clin Ther 10(Suppl A):2-11, 1987. 4.

robial spectrum. Am J Surg 155:16- 23, 1988. 2. Zabransky RJ, Bobey DG, Sheikh W: A multicenter study of the in vitro anti-anaerobic activity of cefotetan compared with other antimicrobial agents. 155:47-51, 1988. 3. Aldridge KE: Controversies in susceptibility testing of anaerobes. Clin Ther 10(Suppl A):2-11, 1987. 4. Wexler HM, Finegold SM: In vitro activity of selected antibiotics against anaerobes. Clin Ther 10(Suppl A): 12- 18, 1987. 5. National Committee for Clinical Laboratory Standards: Reference Agar Dilution Procedure for Antimicrobial Susceptibility Testing of Anaerobic Bacteria. Villanova, PA: NCCLA, MII-A, 1985. 6. National Committee for Clinical Laboratory Standards: Alternative Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria. Villanova, PA: NCCLA, M 17-P, 1985. 7. Faro S, Phillips LE, Marten MG: Perspective on the bacteriology of postpartum obstetrics-gynecolgic infec- tions. Am J Obstet Gynecol 158:694-700, 1988. 8. Finegold SM: Anaerobes: Problems and controversies in bacteriology, infections, and susceptibility testing. Rev Infect Dis 12(Suppl 2):$223-$230, 1990. 9. Wexler HM, Molitoris E, Jashnian F, Finegold SM: Comparison of spiral gradient and conventional agar di- lution for susceptibility testing of anaerobic bacteria. An- timicrob Agents Chemother 35:1196-1202, 1991. 10. Hussain Z, Lannigar R, Schieven BC, et al.: Comparison of susceptibility results of anaerobic organisms deter- mined by agar dilution method and Sceptor Anaerobic MIC/ID MicroBroth dilution panels. Diagn Microbiol Infect Dis 8:95-100, 1987. 11. Jones RN, Barry AL, Fuchs PC, Allen SD: Ceftizoxime and cefoxitin susceptibility testing against anaerobic bacte- ria: Comparison of results from three NCCLS methods and quality control recommendations for the reference agar dilution procedure. Diagn Microbiol Infect Dis 8:87-94, 1987. 12. National Committee for Clinical Laboratory Standards: Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria. 2nd ed. Villanova, PA: NCCLA, MII-A2, 1990. 26 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:27-31 (1993) (C) 1993 Wiley-Liss, Inc. Abdominal Wound Problems After Hysterectomy With Electrocauter vs. Scalpel Subcutaneous Incision David L. Hemsell, Patricia G. Hemsell, Brenda Nobles, Edward R. Johnson, Bertis B. Little, and Molly Heard Departments of Obstetrics and Gynecology and Anesthesia, The University of Texas Southwestern Medical Center, Parkland Memorial Hospital Dallas, TX ABSTRACT The purpose ofthis study was to evaluate the relationship between postoperative abdominal incision problems and opening subcutaneous tissues with electrocautery or scalpel. Women scheduled for elective abdominal hysterectomy who gave informed consent were randomly assigned to subcutane- ous abdominal wall tissue incision by electrocautery or scalpel. Postoperative abdominal wound problem diagnoses included seroma, hematoma, infection, or dehiscence without identifiable etiol- ogy. Fifteen of 380 women (3.9%) developed a wound problem; six had scalpel and nine had electrosurgical subcutaneous incisions (P 0.4). Thicker subcutaneous tissues (P 0.04) and con- current pelvic infection (P < 0.001) were significant risk factors for postoperative wound problems. Only two women (0.5%) developed an infection. We conclude that the method of subcutaneous tissue incision was unrelated to the development of postoperative abdominal incision problems in 380 women undergoing elective abdominal hysterectomy. (C) 1993 Wiley-Liss, Inc. KEY WORDS Postoperative, nonunion/infection, trees, plane, desert n electrosurgical knife was reported to be one of several variables contributing to postopera- tive abdominal wall incision infection. A random- ized clinical trial was designed to evaluate the rela- tionship between postoperative abdominal incision infection or separation due to any cause, and divid- ing subcutaneous abdominal wall tissues with either a scalpel or electrocautery prior to elective abdomi- nal hysterectomy. The incidence and type ofwound problems and risk factors would be determined.

luate the rela- tionship between postoperative abdominal incision infection or separation due to any cause, and divid- ing subcutaneous abdominal wall tissues with either a scalpel or electrocautery prior to elective abdomi- nal hysterectomy. The incidence and type ofwound problems and risk factors would be determined. MATERIALS AND METHODS Women scheduled to undergo elective abdominal hysterectomy for benign disease were invited to participate in this clinical trial and to sign an Insti- tutional Review Board-approved consent form. They were assigned to the scalpel or electrocautery group by a computer-generated randomization code. Exclusion criteria included pregnancy, pel- vic malignancy, allergy to cephalosporin or history of immediate hypersensitivity reaction to penicil- lin, requirement for other antimicrobials, and his- tory of receiving antibiotics during the preceding 7 days. A medical history was taken, a complete physical examination was performed, and routine laboratory tests were done. A povidone iodine shower and douche were taken by the patients the night prior to surgery. They were given a single g preoperative dose of cefazolin. The abdominal skin and vagina were prepped with povidone iodine in the operating room after general anesthesia was administered, and the surgi- Address correspondence/reprint requests to Dr. David L. Hemsell, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., G6.226, Dallas, TX 75235-9032. Received November 17, 1992 Clinical Study Accepted February II, 1993 ELECTROCAUTERY VS. SCALPEL INCISION HEMSELL ET AL. cal procedures were performed by third year house officers under faculty supervision. The abdominal skin was incised with a scalpel. Separate scalpel blades were used for skin and subcutaneous tissues in women in that arm of the study, cut vs. burn. Subcutaneous tissues were opened by either scalpel or electrocautery using the pure cut mode with an SSE2 L (Valleylab, Boulder, CO) electrosurgical generator. Physicians selected the power settings that were used. For the SSE2 L machine, settings were numbered, i.e., 3, 4, 5. Each setting deliv- ered a fixed amount of monopolar current at a 500 ohm load. For example, the setting for 3 was to deliver 75 watts, for 4 it was 125 watts, for 5 it was 160 watts, etc. Subcutaneous tissue thickness was measured at the maximal depth prior to opening the fascia. Hemostasis ofindividual bleeding sites could be accomplished with electrocautery or suture in either group.

ohm load. For example, the setting for 3 was to deliver 75 watts, for 4 it was 125 watts, for 5 it was 160 watts, etc. Subcutaneous tissue thickness was measured at the maximal depth prior to opening the fascia. Hemostasis ofindividual bleeding sites could be accomplished with electrocautery or suture in either group. The pelvis was irrigated with sterile saline following the hysterectomy, as were the sub- cutaneous tissues after fascial closure. For unifor- mity, subcutaneous tissues were not closed. Skin edges were approximated with staples. The study subjects were evaluated clinically twice daily dur- ing the immediate postoperative period, and were followed for up to 6 weeks after discharge from the hospital to detect the occurrence of both early and late wound problems. A wound culture was per- formed if incisional separation occurred irrespec- tive of etiology. Several categories of wound problems were in- cluded. Dehiscence was defined as separation of the subcutaneous tissues without seroma, hematoma, erythema, induration, unexpected tenderness, pu- rulent secretions, or a positive bacteriologic cul- ture. Seroma was the diagnosis when there was inci- sional dehiscence with abundant serous fluid. Hematoma was defined when it was discovered in the wound at incisional dehiscence. Volume of se- rous fluid was not measured nor were hematomas. Wound infection was diagnosed iferythema, indura- tion, and unexpected tenderness were present, if there was purulence at the time of dehiscence, or if there was a positive bacteriologic culture from that space. Quetelet's index [weight (kg)/heightZ(m)], was computed. Data were analyzed using Student's t test, Fish- er's exact probability test, Mann-Whitney U (Wilcoxon) statistic, Welch's approximation, or log likelihood Chi-square tests and by multiway analysis of variance. RESULTS Between July, 1987 and November, 1988, 380 women were enrolled in this clinical trial. The indications for surgery, similar in the two patient populations, were uterine leiomyoma (54%), chronic pelvic pain (16%)or abnormal bleeding (10%) unresponsive to medical management, stress urinary incontinence (8%), adnexal mass (7%), and cervical intraepithelial neoplasia plus abnormal uterine bleeding (5%). There was no significant correlation between the preoperative diagnosis and the development of a postoperative wound prob- lem. Sixty-two percent of the incisions were verti- cal, and the remainder were low transverse.

(8%), adnexal mass (7%), and cervical intraepithelial neoplasia plus abnormal uterine bleeding (5%). There was no significant correlation between the preoperative diagnosis and the development of a postoperative wound prob- lem. Sixty-two percent of the incisions were verti- cal, and the remainder were low transverse. The direction of abdominal wall incision was evenly distributed in women with each type of subcutane- ous incision. The clinical and surgical variables were similar in the two groups of women with the exception of race (Table 1). The distribution of wound prob- lems is presented in Table 2. Demographic, surgi- cal, and laboratory variables for these women are presented in Table 3 and are compared with those of women without wound problems. Race was not significantly related to the development ofa wound problem following surgery. There were no cases of fascial dehiscence. More women whose subcutane- ous tissues were opened with cautery did develop wound problems, but this difference was not statis- tically significant (P 0.4). Diabetes and asthma were not risk factors for wound problems, and no women were receiving steroids. Pelvic infection was a risk factor. Twenty- eight women (7.4%) developed a postoperative pel- vic infection requiring parenteral antimicrobial therapy; 13 of these had scalpel and 15 had electro- cautery subcutaneous entry (P 0.7). The mean hospital stay for these 28 women was 8.7 days, significantly (P < 0.001) prolonged over the en- tire group, as would be expected. Hospital stay associated with any type of wound problem was similar (P 0.8), and was 2 days longer than that observed for women with pelvic infection only (P < 0.001). Heavier women had deeper subcuta- neous tissues, which increased significantly (P 0.04) the likelihood of developing a postop- 28 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY ELECTROCAUTERY VS. SCALPEL INCISION HEMSELL ET AL. TABLE I. Clinical and surgical variables Clinical/surgical variables Subcutaneous incision (mean -+ SD) Scalpel (n 191) Electrocautery Significance (n 189) (P) Age (years) 39 -+ 8.8 Height (inches) 63 -+ 3.2 Weight (pounds) 169 -+ 45.4 Race Black 122 White 44 Hispanic 20 Other 5 Preoperative hemoglobin (g/dL) 12. 1.8 Postoperative hemoglobin (g/dL) 10.4 -+ 1.7 Operation duration (min) 137 -+ 42.4 Incision depth (cm) 3.5 1.7 Hospital stay (days) 4.7 1.9 39 -+ 8.3 1.0 63 -+ 3.3 1.0 168 41.3 0.8 0.04 101 66 21 12.2 _+ 1.5 0.1 10.5 -1.7 0.4 137 -39.9 1.0 3.6 +- 1.7 0.9 4.6 -+ 1.5 0.6 TABLE 2.

er 5 Preoperative hemoglobin (g/dL) 12. 1.8 Postoperative hemoglobin (g/dL) 10.4 -+ 1.7 Operation duration (min) 137 -+ 42.4 Incision depth (cm) 3.5 1.7 Hospital stay (days) 4.7 1.9 39 -+ 8.3 1.0 63 -+ 3.3 1.0 168 41.3 0.8 0.04 101 66 21 12.2 _+ 1.5 0.1 10.5 -1.7 0.4 137 -39.9 1.0 3.6 +- 1.7 0.9 4.6 -+ 1.5 0.6 TABLE 2. Wound problems Subcutaneous incision Problem Scalpel Electrocautery Infection 2 Seroma Hematoma 3 2 Dehiscence 3 4 erative wound problem. This applied to women with vertical and horizontal incisions. When eval- uated by multiway analysis ofvariance, with subcu- taneous depth as the dependent variable, increased age (P 0.01) and Quetelet's index (P 0.0001) were important, but the type ofincision (horizontal or vertical) was not related (P 0.2) to the devel- opment of a postoperative wound problem. DISCUSSION Although abdominal wall infection after hysterec- tomy has been addressed, the incidence of abdomi- nal incisional dehiscence without infection is gener- ally unreported. Dehiscence without infection was observed 6.5 times more frequently than was that associated with infection in this study, and the mean hospital stay was similar. The dose of prophylactic antibiotic administered to the women in this trial might theoretically lower the postoperative infec- tion rate in the abdominal wall, and decrease the potential for recovering bacteria from a clinically uninfected but colonized wound. The association between wound problems and pelvic infection is definite, but without established cause. It is possible that the number of bacterial colonies was low and was undetected by culture, but there were no clinical signs of infection. There was no obvious impairment of host-defense mecha- nisms in any of these women. Such impairment is more likely to occur among older and heavier women, however, and a higher incidence of pelvic infection may also be consistent with lowered host immunocompetence. None of the older, heavier women in this clinical trial had diabetes. Only one ofthese women had a seroma, the etiology ofwhich is also unknown. Although there was no evidence of subcutaneous bleeding prior to skin closure in any of the women, bleeding apparently occurred postoperatively in at least five women, and may be ascribed as the etiology for their postoperative wound separation.

Only one ofthese women had a seroma, the etiology ofwhich is also unknown. Although there was no evidence of subcutaneous bleeding prior to skin closure in any of the women, bleeding apparently occurred postoperatively in at least five women, and may be ascribed as the etiology for their postoperative wound separation. The largest group of women (seven) with a wound problem had neither an ex- cessive amount of serous fluid, hematoma forma- tion, clinical evidence of infection, nor positive bacteriologic culture. The role of the dose of pro- phylactic antibiotic in this scenario is unknown. Mean prolongation of the hospital stay by the de- velopment ofa wound problem was 5.2 days, more than a twofold increase, emphasizing the impor- tance of wound problems and their impact on health care costs. Unexpectedly, separation of the incision without infection had the same impact on hospital stay as did wound infection. Utilization of INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 29 ELECTROCAUTERY VS. SCALPEL INCISION HEMSELL ET AL. TABLE 3. Variables for women without and with postoperative abdominal incisional problems No wound problem Wound problem Significance Variable (n 365) (n 15) (P) Age (years) 39.6 -+ 8.6 39.6 8. 1.0 Height (inches) 63.6 -+ 3.0 65.5 _+ 3.6 0.02 Weight (pounds) 168.2 -+ 42.0 187.6 68.4 0.5 Quetelet's index (kg/m2) 29.4 -+ 7. 30.9 10.8 0.4 Race 0.8 Black 214 9 White 105 5 Hispanic 41 Other 5 Diabetes 27 0 0.3 Asthma 14 0.5 Preoperative hemoglobin (g/dL) 12.2 -+ 1.7 12.2 -+ 0.9 0.9 Postoperative hemoglobin (g/dL) 10.5 -+ 1.7 10.4 1.7 0.9 Electrocautery power setting 128.5 28.2 II 7.8 _+ 30.9 0.4 (watts @ 500 ohm load) Subcutaneous depth (cm) 3.5 1.7 4. -+ 2. 0.04 Operation duration (minutes) 136.7 _+ 41.0 154.0 -+ 41.3 0. Hospital stay (days) 4.5 I. 9.7 _+ 3.9 <0.001 Pelvic infection 22 6 <0.001 aData are mean -+ standard deviation. visiting nurses might shorten the hospital stay of women with wound problems. The SSE2 L unit has been replaced in our oper- ating rooms by a Force 2 (Valleylab) electrosurgical generator. Whereas the mean power setting of the SSE2 L observed in this clinical trial was over 117 watts, only 30 to 40 watts are required to achieve the same result with the new model. This is made possible by new software and hardware, alterna- tions in peak to peak voltage, altered rated load, more power at lower tissue impedance, and modi- fied power curves.

he SSE2 L observed in this clinical trial was over 117 watts, only 30 to 40 watts are required to achieve the same result with the new model. This is made possible by new software and hardware, alterna- tions in peak to peak voltage, altered rated load, more power at lower tissue impedance, and modi- fied power curves. Prior to this clinical investigation, the general practice in Parkland Memorial Hospital was to close the subcutaneous space with interrupted or running plain catgut suture after abdominal hyster- ectomy. A scalpel was used for the subcutaneous incision. Abdominal incision infection rates were as high as 6% in retrospective evaluations without prophylaxis and in prospective evaluations. For uniformity in this trial, subcutaneous suture was not placed, and the infection rate with prophylaxis was only 0.5%. That difference in infection rate, by historical comparison, is highly significant (P < 0.001), indicating that foreign bodies con- tribute to wound infection, as indicated by Ledger and many others. Cruse2 reported that use of the electrosurgical knife almost doubled the infection rate in all four categories of wound (clean, clean contaminated, contaminated, and dirty). When electrosurgical technology was new and techniques were unre- fined, large amounts of charred tissue were fre- quently left in the wound. This was believed to act as a foreign body, which enhanced wound infection when combined with necrotic tissue resulting from thermal injury. Cruse and Foord3 later reported that with operating room experience, and reduced generalized tissue destruction and char, the infec- tion rate in patients with clean wounds was the same whether or not the electrosurgical generator was used. Prophylactic antibiotic was not administered to those patients as it was in the women presented here. Johnson and Serpell4 reported no difference in wound inflammation or infection rates in 240 gen- eral surgical patients whose incisions were made with scalpel or electrocautery. Their patients also received prophylaxis, and patients with all catego- ries of wounds were included, in contrast to the present report. Thirty-five percent of patients in- cluded in that report had contaminated wounds, from which arose 71% of inflamed incisions and 81% of the wound infections, however. 30 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY ELECTROCAUTERY VS. SCALPEL INCISION HEMSELL ET AL.

were included, in contrast to the present report. Thirty-five percent of patients in- cluded in that report had contaminated wounds, from which arose 71% of inflamed incisions and 81% of the wound infections, however. 30 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY ELECTROCAUTERY VS. SCALPEL INCISION HEMSELL ET AL. There are animal model data documenting slower wound healing after electrosurgical incision when compared with scalpel incision. In such a report by Arnaud and Adloff,s there were compa- rable bursting strengths. In addition to the inci- sion, electrocautery results in adjacent tissue necro- sis. 6 Madden and coinvestigators7 reported an increased incidence of, and susceptibility to, infec- tion after electrocautery incision when compared with scalpel incision in an albino guinea pig model. It was correlated with histologic evidence of de- layed healing and tissue coagulation necrosis. The power of the data generated during this clinical trial did not detect a statistically significant difference in infection or other wound problems between women whose subcutaneous tissues were opened with an electrosurgical generator or a scal- pel. There were 50% more adverse wound out- comes in women whose tissues were opened with an electrosurgical generator, however. It would re- quire a sample size that was six times larger than that reported here, and a constant prevalence of wound problems in both groups, to detect a signif- icant (P 0.046) difference in wound complica- tions. The estimated number of abdominal hyster- ectomies performed in women in the United States for 1990 was 43 0,000.8 If one-half of those proce- dures included opening subcutaneous tissues with an electrosurgical instrument, and the same per- centage of women had wound problems, an antici- pated 18,000 additional hospital days would result from wound problems associated with electrosurgi- cal entry. In addition, Gatti and coworkers9 detected mu- tagenicity of electrocautery smoke collected during mammoplasty; the testing was performed by the Hazard Evaluations and Technical Assistance Branch of the National Institute of Occupational Safety and Health (NIOSH). The impact on oper- ating room personnel is unknown, but it does ap- pear that this surgical technique has the capacity for adverse reaction. Risks to the patient and operating room personnel may outweigh the hemostatic bene- fit of electrocautery incision. REFERENCES 1.

itute of Occupational Safety and Health (NIOSH). The impact on oper- ating room personnel is unknown, but it does ap- pear that this surgical technique has the capacity for adverse reaction. Risks to the patient and operating room personnel may outweigh the hemostatic bene- fit of electrocautery incision. REFERENCES 1. Ledger W: Prevention, diagnosis, and treatment of post- operative infections. Obstet Gynecol 55(Suppl 5):203S- 206S, 1980. 2. Cruse PJE: Some factors determining wound infection. A prospective study of30,000 wounds. In Polk HC Jr, Stone HH (eds): Hospital-Acquired Infections in Surgery. Bal- timore: University Park Press, pp 79-85, 1977. 3. Cruse PJE, Foord R: The epidemiology of wound infec- tion. A 10-year prospective study of 62,939 wounds. Surg Clin North Am 60:27-40, 1980. 4. Johnson CD, Serpell JW: Wound infection after abdomi- nal incision with scalpel or diathermy. Br J Surg 77:626- 627, 1990. 5. Arnaud JP, Adloff M: Electrosurgery and wound healing: An experimental study in rats. Eur Surg Res 12:439-443, 1980. 6. Mitchell JP, Lumb GN, Dobbie AK: A Handbook of Surgical Diathermy. Bristol: John Wright, pp 8-9, 1978. 7. Madden JE, Edlich RF, Custer JR, Panek PH, Thul J, Wangensteen OH: Studies in the management of the con- taminated wound. IV. Resistance to infection of surgical wounds made by knife, electrosurgery and laser. Am J Surg 119:219-222, 1970. 8. Detailed Diagnosis and Procedures National Hospital Dis- trict Survey, National Center for Health Statistics: Health, United States, 1990. DHHS Pub. No. (PHS) 91-1232. Hyattsville, MD: Public Health Service, 1991. 9. Gatti JE, Bryant CJ, Noone RB, MurphyJB: The mutage- nicity of electrocautery smoke. Plast Reconstr Surg 89: 781-784, 1992. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:32-36 (1993) (C) 1993 Wiley-Liss, Inc. Clinical Significance of Cred 's Prophylaxis in Germany at Present Udo B. Hoyme Department ofObstetrics and Gynaecology, University ofEssen, Essen, Germany ABSTRACT The introduction ofsilver nitrate for prophylaxis ofgonoc0ccal ophthalmia neonatorum is one ofthe milestones of preventive medicine. However, in our time an increasing necessity to review Credf's prophylaxis from both a human rights and a medical standpoint is required. The chairmen of the obstetrics and gynecology departments of the German university hospitals were questioned to learn about their policy and experience. Data were provided by 22 of 28 consulted institutions, represent- ing 31,700 annual deliveries seen over a mean period of5.5 years. Ocular prophylaxis was in use in 16 (73%) of the reporting hospitals (1% silver nitrate in 14 and gentamicin in 2). A nonspecific conjunctival reaction occurred in 5-10% of the newborns, but no major side effects were seen. Non-gonococcal ophthalmia neonatorum was observed in less than 0.1%; however, institutions without a preventive policy reported up to a 5% incidence of neonatal conjunctivitis, mostly due to Staphylococcus aureus, as well as Neisseria gonorrhoeae in two newborns. Application of silver nitrate is considered a necessary prophylactic measure and safe if it is properly administered. However, major efforts should be directed toward its replacement by alternative antiseptic substances as well as toward chlamydial screening and therapy in pregnancy. (C) 1993 Wiley-Liss, Inc. Kvx WORS Gonoblennorrhea in newborns, silver nitrate, treatment of chlamydial infection in pregnancy he establishment ofsilver nitrate prophylaxis of gonococcal ophthalmia neonatorum is one of the milestones of preventive medicine in the 19th century. In 1881 Carl Siegmund Franz CredO, one of the first editors and founder of the famous jour- nal Archivfiir Gyn#kologie, reported from Leipzig a decrease in the incidence of gonococcal oph- thalmia from 7.6% to 0.5% (Table 1) by the use of 2% silver nitrate.

of the milestones of preventive medicine in the 19th century. In 1881 Carl Siegmund Franz CredO, one of the first editors and founder of the famous jour- nal Archivfiir Gyn#kologie, reported from Leipzig a decrease in the incidence of gonococcal oph- thalmia from 7.6% to 0.5% (Table 1) by the use of 2% silver nitrate. In the following years prophy- laxis was established and required by law or by health department regulations in many countries all over the world. In Germany enforcement was ter- minated in 1986 by the proclamation of a new law (Gesetz tiber den Beruf der Hebamme und des Entbindungspflegers vom 4.6. 1986). As a result, an increasing necessity arose to review Cred's pro- phylaxis from both a human rights and a medical standpoint in the medical profession as well as in the government. This paper reports on recent prevention efforts in German university hospitals on the basis of a questionnaire. In addition, the literature is evalu- ated in an attempt to define the current standard of care according to the position of the German Fed- eral Health Department (Bundesgesundheitsamt) at Berlin. SUBJECTS AND METHODS In January 1992, 28 chairmen of the departments ofobstetrics and gynecology at the university hospi- tals in the former West Germany were sent the following questionnaire concerning prevention of ophthalmia neonatorum by Cred's procedure in their hospitals: Address correspondence/reprint requests to Dr. Udo B. Hoyme, Zentrum fir Frauenheilkunde, Universitiitsklinikum, Hufelandstrasse 55, D-4300 Essen, Germany. Clinical Study Received December 8, 1992 Accepted December 8, 1992 PREVENTION OF OPHTHALMIA NEONATORUM HOYME TABLE I. Incidence of ophthalmia neonatorum in Cred's original series before and after initiation of prophylaxis with 2% silver nitrate Deliveries Ophthalmia neonatorum Year (no.) (no.) (%) 1874 323 45 13.6 1875 287 37 12.9 1876 367 29 9.1 1877 360 30 8.3 1878 353 35 9.8 1879 389 36 9.2 1880 (to 5/3 I) 187 14 7.6 1880 (6/I-I 2/8) 200 0.5 aProphylaxis was not administered in this infant. 1. Do you perform neonatal ocular prophylaxis at your institution? Ifyes, by what method? 2. Did you observe significant or continuous adverse reactions following this prophylaxis? 3. Are you aware of any case of gonococcal con- junctivitis or of conjunctivitis of other etiol- ogy at your hospital? 4. How many deliveries and how many years do the figures include? 5.

your institution? Ifyes, by what method? 2. Did you observe significant or continuous adverse reactions following this prophylaxis? 3. Are you aware of any case of gonococcal con- junctivitis or of conjunctivitis of other etiol- ogy at your hospital? 4. How many deliveries and how many years do the figures include? 5. Do you agree to publication of the informa- tion provided concerning your hospital or do you prefer anonymity? 6. Do you want to be informed about the results and the progress of this inquiry? The last question refers in part to a similar in- quiry in the university and other major hospitals in the former East Germany. It was initiated by B. Viehweg at the same time and presented in April 1992. All data returned are analyzed and presented here, in some cases after a precise check-back with the providing institution. The data obtained were also taken into consideration in the decision-making process of the German Federal Health Depart- ment. RESULTS The data requested were provided by 22 of the 28 chairmen between January and March 1992. However, since three ofthem prefered anonymity, the participating institutions are listed by numbers (Table 2). The results reported represent a total of 31,700 annual deliveries seen over a mean period of 5.5 years (Table 3). Neonatal ocular prophylaxis was provided in 16 of 22 hospitals (73%). The use of 1% silver nitrate was mandatory in 12 hospitals. Occasional applica- tion because of certain indications or at parents' requests was reported by one institution each. Two hospitals performed prophylaxis with gentamicin, one on request and one as a routine procedure. Ophthalmia neonatorum but no gonococcal dis- ease was observed by those hospitals with prophy- laxis in less than 0.1%. On the other hand, institu- tions without routine preventive policy or with no prophylaxis reported up to 5% prevalence ofneona- tal conjunctivitis in general as well as two newborns with Neisseria gonorrhoeae. Staphylococcus aureus was specified as the major etiologic pathogen by some reporters, and in spite of all diagnostic efforts in the past, chlamydial infection was also demon- strated at four locations irrespective of prophylaxis policy. Nonspecific conjunctival reaction was reported by 6 of those 12 institutions using 1% silver nitrate routinely, with an estimated peak incidence of 5-10% on day 2. The other six hospitals did not observe this kind of reaction; however, it was also reported by one using gentamicin.

ns irrespective of prophylaxis policy. Nonspecific conjunctival reaction was reported by 6 of those 12 institutions using 1% silver nitrate routinely, with an estimated peak incidence of 5-10% on day 2. The other six hospitals did not observe this kind of reaction; however, it was also reported by one using gentamicin. Major, long- lasting, or permanent side effects were denied by all corresponding institutions, but discontinuation of prophylaxis was justified by one of them because of the severity of transient reactions. According to Viehwegs inquiry in the former East Germany, silver nitrate was used routinely in 19 of 23 re- sponding hospitals with no reported major side ef- fects or long-lasting damage in 860,300 cases. DISCUSSION Ophthalmia neonatorum is defined as the occur- rence of conjunctivitis within the first month of life. N. gonorrhoeae causes the most destructive form, 1-5 but other bacterial agents including Chlamydia trachomatis, Staphylococcus aureus, Strep- tococcus ssp., Haemophilus spp., and Pseudomonas spp. can also cause neonatal conjunctivitis3'4'6-9 (Table 4). In general, the incidence of severe oph- thalmia nenatorum parallels that of maternal gon- orrhea and chlamydial infection; however, other pathogens may also lead to conjunctivitis, with re- sulting septicemia, nasolacrimal duct obstruction, ulceration, scarring, and blindness in the new- born.4 This is in part due to the lack of sufficient INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 33 PREVENTION OF OPHTHALMIA NEONATORUM HOYME TABLE 2. Prophylaxis of ophthalmia neonatorum at German university hospitals: Results of an inquiry in January 1992 [response rate 79% (22/28)] Institution Deliveries/ Observation no. Method Side effects Ophthalmia year period (years) Cred None None 800 12 2 Cred 5% None 1,500 10 3 Cred Frequent, at day 2 O. I% C. trachomatis 4 Cred None None 1,000 5 Cred None None 1,800 5 6 Cred 10% C. trachomatis 800 5 7 Cred Frequent 0.1% S. aureus 1,200 5 8 Cred None None 1,600 3 9 Cred None None 1,000 0 Cred None None 2,000 Cred Unspecific Unspecific I, 100 13 12 Cred Unspecific None 500 5 13 Cred None C. trachomatis, some S. aureus 1,000 2 14 Cred None None 1,600 4 15 Gentamicin Unspecific 0.05% 1,800 16 Gentamicin None Rare 1,900 17 None (Unspecific) 2 N. gonorrhoeae, others 2,400 10 18 None 3-5% S. aureus 1,600 S 19 None (Significant) None 1,700 8 20 None (Unspecific)d None 1,600 21 None (Unspecific) Some C. trachornatis, S.

tis, some S. aureus 1,000 2 14 Cred None None 1,600 4 15 Gentamicin Unspecific 0.05% 1,800 16 Gentamicin None Rare 1,900 17 None (Unspecific) 2 N. gonorrhoeae, others 2,400 10 18 None 3-5% S. aureus 1,600 S 19 None (Significant) None 1,700 8 20 None (Unspecific)d None 1,600 21 None (Unspecific) Some C. trachornatis, S. aureus 1,000 3 22 None (Unspecific)d Unspecific 2,400 6 aNo gonococcal infection. bprophylaxis administered occasionally. cProphylaxis on parents request. dBefore prophylaxis was abandoned. amounts of lysozyme, IgA, and lacrimal fluid on the newborn cornea. 3 First introduced as prophylaxis against gonococ- cal ophthalmia neonatorum by Carl Siegmund Franz Cred in 1880, silver nitrate remained the standard of care in many countries worldwide; it is or has been required or recommended by state laws or health department regulations or officials. 12'13 In addition, it is listed by the World Health Orga- nization as one ofthe essential drugs. 14 The current discussion focusses on some of the problems with silver nitrate2'15: 1) it does not provide perfect protection against neonatal (gonococcal) oph- thalmia; 2) it often causes chemical irritation of the conjunctiva; and 3) its use may be superfluous in some populations. To consider these issues a com- mittee of the German Federal Health Department evaluated the recent situation in Germany and re- viewed the arguments for and against prophylaxis of ophthalmia neonatorum in order to establish a recommendation for the standard of care in Ger- many. The arguments will be discussed here. TABLE 3. Prophylaxis of ophthalmia neonatorum at German university hospitals: summary of an inquiry in January 1992 [response rate 79% (22/28)] Deliveries/year Number 31,700 Range 500-2,400/hospital Observation period (years) Mean 5.5 Range I-I 3 Any prophylaxis 16/22 (73%) I% Silver nitrate 12 I% Silver nitrate occasionally I% Silver nitrate on request Gentamicin Gentamicin on request Prophylaxis "Controls" 0-10% Unspecific reactions, occasionally S. aureus, O. I% C. trachomatis, no reported gonococcal conjunctivitis 0-5% S. aureus, 2 cases of gonococcal conjunctivitis, some C. trachomatis, others 34 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY PREVENTION OF OPHTHALMIA NEONATORUM HOYME TABLE 4. Causes of ophthalmia neonatorum in the United States Source Approximate Time of frequency onset (%) (days) Chemical conjunctivitis 7 6-24 hours Chlamydia trachomatis 3-13 5-14 Escherichia coli 5-9 5-21 Haemophilus spp.

4 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY PREVENTION OF OPHTHALMIA NEONATORUM HOYME TABLE 4. Causes of ophthalmia neonatorum in the United States Source Approximate Time of frequency onset (%) (days) Chemical conjunctivitis 7 6-24 hours Chlamydia trachomatis 3-13 5-14 Escherichia coli 5-9 5-21 Haemophilus spp. 5 5-21 Herpes simplex virus Rare 2-14 Neisseria gonorrhoeae 3 2-5 Staphylococcus aureus 5-30 5-21 Streptococcus viridans 4-16 5-21 For data, see Dinsmoor4. The incidence of N. gonorrhoeae in pregnant women in Germany is estimated to be as low as 0.1%, or approximately 700cases/year. On the other hand, neonatal gonococcal conjunctivitis is a severe, potentially blinding disease, that can be prevented only ifearly diagnosis and adequate ther- apy is available.2'4' 12 The abandonment of prophy- laxis would without doubt lead to an increase of ophthalmia neonatorum and its sequela, as this in- quiry has shown (Tables 2 and 3). Prophylaxis against gonococcal conjunctivitis could be terminated if a sufficient, cost-effective screening system for pregnant women (until term) were available. However, there is no such system. Selective application ofdiagnostic procedures is not an alternative or solution, since gonoccoccal coloni- zation in pregnancy is usually asymptomatic, the definition of certain risk groups is almost impossi- ble, 16 and furthermore such testing imposes an unacceptable stigma. In addition, selective screen- ing does not solve the problem of neonatal gonococ- cal disease for those who are not examined. Routine prophylaxis involves expenses in drugs, organization, and personnel. However, these ef- forts can be kept low and safety can be maintained by the use of standardized, commercially available disposable applicators. All prophylactic procedures cause corneal irrita- tion (Table 2) and also pain and discomfort. 7'8 However, there is no well-documented case of per- manent or severe damage following adequate silver nitrate application, t9-21 Psychological aspects have been considered by several authors, but there is also no evidence that the degree of "eye openness" ofthe newborn significantly affects the attention of the mother immediately postpartum. The infant has other behaviors and attributes such as cry, body texture, movement, odor, and nursing behavior that facilitate bonding.22'23 Like many preventive methods, neonatal ocular prophylaxis does not provide complete protection, as was seen in the inquiry.

ects the attention of the mother immediately postpartum. The infant has other behaviors and attributes such as cry, body texture, movement, odor, and nursing behavior that facilitate bonding.22'23 Like many preventive methods, neonatal ocular prophylaxis does not provide complete protection, as was seen in the inquiry. This holds true for both gonococcal conjunctivitis and other bacterial infec- tions (Table 4), especially chlamydial disease.24'25 However, the antimicrobial activity of silver ni- trate covers a broad spectrum of causative agents regardless of bacterial resistance and without the other inherent characteristic limitations of antibiot- ics.4'9'12'15'18'22'24-27 The exception, C. trachom- atis, should be of no relevance in the future, since the German Society of Gynecology and Obstetrics and the German Society of Perinatal Medicine de- cided in 1992 to recommend routine screening and therapy for chlamydial infection as a standard of care in pregnancy.28 In brief, the guidelines em- phasize diagnosis as early as possible in pregnancy by a direct test and treatment by erythromycin eth- ylsuccinate 500 mg q.i.d, for 10 days after the 14th gestational week; partner therapy is mandatory; Cred6's prophylaxis with 1% silver nitrate is also recommended by the guidelines, since eye infec- tions of other etiologies may still occur, and no well-studied alternative exists. The enforcement of Cred6's prophylaxis in Ger- many by law was discontinued in 1986. A restora- tion of the old status is not desirable, since contro- versial human rights and legal aspects are of increasing importance. On the other hand, negli- gence and/or abolition of routine prophylaxis that result in damage to the newborn could probably create another legal issue, the charge of having neglected the current standard of care. This state- ment accords with the results of this and Viehweg's inquiries and is supported as well by the literature and by several national and international authori- 10,11,14 ties, e.g., the World Health Organization, the Centers for Disease Control, 3 and a committee of the German Federal Health Department. In Germany it was also supported by at least 80% of ophthalmologists consulted at university hospitals (A.A. Bialasiewicz, personal communication).

l and international authori- 10,11,14 ties, e.g., the World Health Organization, the Centers for Disease Control, 3 and a committee of the German Federal Health Department. In Germany it was also supported by at least 80% of ophthalmologists consulted at university hospitals (A.A. Bialasiewicz, personal communication). Evaluation of the arguments and the literature and the results of this inquiry appear to warrant the INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 35 PREVENTION OF OPHTHALMIA NEONATORUM HOYME following conclusions for Germany, as drawn by the Federal Health Department: gonococcal infec- tion in pregnancy is a rare disease; however, the identification and treatment ofinfected mothers can not be counted on. 12'16 Therapy of the infected infant appears to involve an unjustifiable risk. Thus neonatal ocular prophylaxis against gonococ- cal and other infections still appears to represent the standard of care 1'13 and is used in the majority of German hospitals questioned. Silver nitrate 1% aqueous solution is considered a necessary prophy- lactic measure, safe if properly adminis- tered.4'12'17'19'21'27 A new law is not warranted in Germany for several reasons. The routine adminis- tration of such antibiotics as penicillins, aminogly- cosides, erythromycin, or tetracyclines cannot be recommended as an alternative since serious unto- ward reactions (due to poor stability and applicabil- ity of the drug, a limited bacteriological spectrum, selection of resistant strains, and possible allerge- nicity for the newborn) must be taken into ac- count. 9'15'18'25'26 The data on their efficacy are also not superior or convincing enough to justify their routine use in prevention of bacterial oph- thalmia neonatorum, particularly if chlamydial in- 25,26 fection is detected and treated in pregnancy. However, major efforts should be directed toward conducting scientific studies on the replacement of silver nitrate by alternative antiseptic substances (e.g., octenidin or biguanides) in the future. REFERENCES 1. Cred6 CSF: Die Verhfitung der Augenentzfindung der Neugeborenen. Arch Gynikol 17:50-53, 1881. 2. Rothenberg R: Ophthalmia neonatorum due to Neisseria gonorrhoeae: Prevention and treatment. Sex Transm Dis 6:187-191, 1979. 3. Eder W: Cred6-Prophylaxe mit Silbernitrat beim Neuge- borenen? Gyn/kol Prax 11:627-6.30, 1987. 4. Dinsmoor MJ: Ophthalmia neonatorum. Contemp Ob- stet Gynecol 37"112-114, 1992. 5.

-53, 1881. 2. Rothenberg R: Ophthalmia neonatorum due to Neisseria gonorrhoeae: Prevention and treatment. Sex Transm Dis 6:187-191, 1979. 3. Eder W: Cred6-Prophylaxe mit Silbernitrat beim Neuge- borenen? Gyn/kol Prax 11:627-6.30, 1987. 4. Dinsmoor MJ: Ophthalmia neonatorum. Contemp Ob- stet Gynecol 37"112-114, 1992. 5. Laga M, Meheus A, Piot P: Epidemiology and control of gonococcal ophthalmia neonatorum. Bull WHO 67:471- 478, 1989. 6. Fransen L, Van den Berghe P, Mertens A, Van Brussel K, Clara R, Piot P: Incidence and bacterial aetiology of neonatal conjunctivitis. Eur J Pediatr 146" 152-155, 1987. 7. Huber-Spitzy V, Arocker W, Schmidt C: Ophthalmia neonatorum. Klin Monatsbl Augenheilkd 191:341-343, 1987. 8. Isenberg S: Source of conjunctival bacterial flora at birth and implications for ophthalmia neonatorum prophylaxis. Am J Ophthalmol 106:458-462, 1988. 9. Hedberg K, Ristinen TL, SolerJT, White KE, Hedberg CW, Osterholm MT, MacDonald KL: Outbreak of erythromycin-resistant staphylococcal conjunctivitis in a newborn nursery. Pediatr Infect Dis J 9:268-273, 1990. 10. WHO: Prevention and treatment of conjunctivitis in newborn at the primary level. PBL/84.4 1-23. 11. WHO: Conjunctivitis of the Newborn. Geneva: WHO, 1986. 12. Hammerschlag MR: Neonatal ocular prophylaxis. Pedi- atr Infect Dis J 7:81-82, 1988. 13. CDC: 1989 Sexually Transmitted Diseases Treatment Guidelines. MMWR 38:1-43, 1989. 14. WHO: Unentbehrliche Arzneimittel. Ffinfte Model- liste. Frankfurt/Main, Medico Internat, 1989. 15. Chandler JW: Controversies in ocular prophylaxis of newborns. Arch Ophthalmol 107:814-815, 1989. 16. Brunham RC, Holmes KK, Embree JE: Sexually trans- mitted diseases in pregnancy. In Holmes KK, Mardh P-A, Sparling PF, Wiesner PJ (eds): Sexually Transmit- ted Diseases, 2nd ed. New York: McGraw-Hill, pp 771- 801, 1989. 17. Nishida H, Risemberg HM: Silver nitrate solution and chemical conjunctivitis. Pediatrics 56:368-373, 1975. 18. Christian JR: Comparison ofocular reactions with the use ofsilver nitrate and erythromycin ointment in ophthalmia neonatorum prophylaxis. J Pediatr 37:55-60, 1960. 19. Giffin RB: Eye damage in newborns from use of strong silver nitrate solutions. Cal Med 107:178-181, 1967. 20. Hornblass A: Severe silver nitrate ocular damage. NY J Med 1875-1878, October 1976. 21. Schirner G, Schrage NF, Salla S, Teping Ch, Reim M, Burchard W-G, Schwab B: Corneal silver deposits fol- lowing Cred6's prophylaxis. Lens Eye Toxic Res 7:445- 457, 1990. 22.

g silver nitrate solutions. Cal Med 107:178-181, 1967. 20. Hornblass A: Severe silver nitrate ocular damage. NY J Med 1875-1878, October 1976. 21. Schirner G, Schrage NF, Salla S, Teping Ch, Reim M, Burchard W-G, Schwab B: Corneal silver deposits fol- lowing Cred6's prophylaxis. Lens Eye Toxic Res 7:445- 457, 1990. 22. Bernstein GA, Davis JP, Katcher ML: Prophylaxis of neonatal conjunctivitis. Clin Pediatr 21:545-550, 1982. 23. Butterfield PM, Emde RN, Svejda MJ: Does the early application of silver nitrate impair maternal attachment? Pediatrics 67:737-738, 1981. 24. Bryant B: Unit dose erythromycin ophthalmic ointment for neonatal ocular prophylaxis. JOGN 13:83-87, 1984. 25. Black-Payne C, Bocchini JA, Cedotal C: Failure oferyth- romycin ointment for postnatal ocular prophylaxis of chlamydial conjunctivitis. Pediatr Infect Dis J 8:491- 498, 1989. 26. Latif A, Mason P, Marowa E, Paraiwa E, Dhamu F, Tambo J, Gwanzura L, Mapeta D, Jongeling G: Man- agement of gonococcal ophthalmia neonatorum with sin- gle-dose kanamycin and ocular irrigation with saline. Sex Transm Dis 15:108-109, 1988. 27. Lund RJ, Kibel MA, Knight GJ, Van der Elst C: Pro- phylaxis against gonococcal ophthalmia neonatorum. S Afr Med J 72:620-622, 1987. 28. Hoyme UB: Chlamydia trachomatis-Infektionen in der Schwangerschaft. Perinatal Med 4:53-56, 1992. 6 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:37-39 (1993) (C) 1993 Wiley-Liss, Inc. Sheathing of the Endovaginal Ultrasound Probe: Is It Adequate? Ronald Jimenez and Patrick Duff Division ofMaternal-Fetal Medicine, University ofFlorida College ofMedicine, Gainesville, FL ABSTRACT The purpose of this prospective investigation was to compare two methods for sheathing of the endovaginal ultrasound probe. The study was conducted over a 7-month period in 1991-1992. In the first half of the investigation, latex examination gloves were used to sheath the endovaginal probe; during the second half of the investigation, latex condoms were used. Following the ultrasound examination, the probes were inspected for gross contamination by the ultrasonographer. The sheaths were then tested for perforations by filling them with water to twice their usual volume and observing for leaks. Fifty unused gloves and condoms were similarly tested to determine the preva- lence of preexisting defects. One hundred twenty-eight gloves and 102 condoms from patients were tested. Four gloves (3.1%, 95% C.I. 1.6-4.6%) and seven condoms (6.9%, 95% C.I. 4.4-9.4%) had perforations (NS). When the probe was covered by a glove, one instance of visible contamination occurred (0.78%, 95% C.I. 0-1.6%) compared with eight instances when the probe was covered with a condom (7.8%, 95% C.I. 5.2-10.4%, P < .007). The prevalance of preexisting defects in the 50 unused gloves was 2%, which is not significantly different from the prevalence in used gloves. There were no defects in the 50 unused condoms compared with 7 in the used condoms (P .057). Visible contamination of the endovaginal probe with blood or genital tract secretions is more likely when condoms are used as sheaths. However, even gloves provide imperfect coverage of the probe, illustrating the need for thorough decontamination ofthe endovaginal instrument after each use. (C) 1993 Wiley-Liss, Inc. KEY WORDS Contamination of ultrasound equipment, infection control n recent years the medical community and public have become justifiably concerned about the risk oftransmitting and acquiring certain infectious dis- eases during medical procedures.

ofthe endovaginal instrument after each use. (C) 1993 Wiley-Liss, Inc. KEY WORDS Contamination of ultrasound equipment, infection control n recent years the medical community and public have become justifiably concerned about the risk oftransmitting and acquiring certain infectious dis- eases during medical procedures. Most investiga- tions have focused on disease transmission as a re- sult of "invasive" surgical procedures. The risk of other less traumatic manipulations, such as endo- vaginal ultrasound, has not been clearly defined. The first objective of the present investigation was to determine the frequency of manufacturing defects in two devices commonly used to cover the endovaginal probe: latex gloves and condoms. The second objective was to determine the subsequent frequency of perforation ofthese devices when they were stretched over the endovaginal ultrasound probe. The third objective was to determine the frequency with which the probe becomes visibly or grossly contaminated by blood and/or genital tract secretions as a result of an endovaginal scan. MATERIALS AND METHODS This prospective study was conducted from Octo- ber 1, 1991 to April 30, 1992, at Shands Hospital, University ofFlorida. During the study period, all endovaginal ultrasound examinations were per- formed by one of three attending physicians and Address correspondence to Dr. Patrick Duff, Department of Obstetrics and Gynecology, University of Florida College of Medicine, P.O. Box 100294, Gainesville, FL 32610-0294. Received December 30, 1992 Clinical Study Accepted March II, 1993 SHEATHING OF ENDOVAGINAL PROBE JIMENEZ AND DUFF four senior residents. For the first halfofthe inves- tigation, the endovaginal probe (5 mHz, General Electric Medical Systems, Milwaukee, WI) was covered with a latex examination glove (Aladan Corp., Dothan, AL). Ultrasound gel was applied to the probe prior to placement of the probe in the second or third glove finger. Gel also was applied to the outside of the covered probe prior to its insertion into the vagina. The tip of the probe measured 2 2 5 cm. The total length of the probe and handle was 21 cm. The diameter and length of the opened, but unstretched glove finger, were 2.5 cm and 8.5 cm, respectively. Midway through the study, we became aware that physicans working in a separate facility in our department were using condoms rather than gloves to cover the endovaginal probe.

th of the probe and handle was 21 cm. The diameter and length of the opened, but unstretched glove finger, were 2.5 cm and 8.5 cm, respectively. Midway through the study, we became aware that physicans working in a separate facility in our department were using condoms rather than gloves to cover the endovaginal probe. Subsequent to this discovery, we decided to complete the investigation using a different probe cover and, thereby, provide comparative data regarding the adequacy of the different sheaths. Accordingly, for the last half of the investigation, the probe was covered with a latex condom (Trojans, Carter-Wallace, NY). Gel was applied as previously described. The outer di- ameter of the rim of the condom was 3.7 cm. The length of the opened but unstreteched condom was 5 cm. At the time of this study, a fastener was not available to secure the upper end of the condom to the shaft of the probe. At the conclusion ofthe ultrasound examination, the sheaths were placed in plastic bags. A label was affixed to each bag, and the sonographer was asked to indicate whether the sheath had an obvious per- foration and whether there were blood or vaginal secretions on the probe. The probe was then cleansed with a dilute solution ofsodium hypochlorite. The gloves and condoms were tested for perfora- tion by filling them with water to approximately twice their ususal volume and examining them for leakage of water. Fifty unused gloves and 50 un- used condoms were similarly tested to determine the frequency of preexisting perforations. All tests of patency were performed by one of the authors (R.J.). Our results are initially reported with descrip- tive statistics. Fisher's exact probability test was used to compare observed differences in frequency of" perforation. P < 05 was considered statistically significant. Ninety-five percent confidence inter- vals also are reported, when appropriate. R$UkT$ Four of 128 used gloves (3.1%, 95% C.I. 1.6- 4.6%) had perforations. Seven of 102 used con- doms (6.9%, 95% C.I. 4.4-9.4%) had perfora- tions. This difference is not statistically significant (P .16). When the probe was covered by a glove, one instance of visible contamination occurred (0.78%, 95% C.I. 0-1.6%). In this case, there was no perforation in the glove, and contamination of the shaft of the probe apparently resulted from leakage of fluid around the cuff of the glove because of slippage ofthe glove during the examination.

was covered by a glove, one instance of visible contamination occurred (0.78%, 95% C.I. 0-1.6%). In this case, there was no perforation in the glove, and contamination of the shaft of the probe apparently resulted from leakage of fluid around the cuff of the glove because of slippage ofthe glove during the examination. When a condom was used to cover the probe, eight cases of obvious contamination occurred (7.8%, 95% C.I. 5.2-10.4%). In six of these cases, the visible contamination coincided with the site of perforation in the condom. In two additional cases, there was no perforation in the condom, and leakage occurred around the open end ofthe condom onto the shaft of the probe. In one other instance in which a small perforation was present in the condom, no visible contamination was evident. The observed differ- ence in frequency of visible contamination was highly significant (P < .007). There was no clus- tering of perforations or contamination in speci- mens submitted by any single sonographer. The prevalence of defects in unused gloves was 2%, which is not significantly different from ob- served prevalence in used gloves. There were no preexisting defects in unused condoms compared with 6.9% in used condoms (P .057). DISCUSSION In recent years, physicians have become increas- ingly concerned about the risk of acquiring an in- fectious disease as a result of an occupational in- jury. 1,2 While this issue is a valid concern, health care workers also must be aware of their potential role in transmitting infection from themselves to their patients or from one patient to another. Con- taminated medical equipment certainly is a possible mechanism for horizontal transmission of infection such as viral hepatitis, HIV infection, gonorrhea, chlamydia, and trichomoniasis. 3 Although ultrasound imaging is not usually re- garded as an invasive procedure, endovaginal scan- ning may pose some risk of infection to patients. In infected women, genital tract secretions may con- tain a high inoculum of infectious organisms. Ifthe INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY SHEATHING OF ENDOVAGINAL PROBE JIMENEZ AND DUFF endovaginal ultrasound probe is not appropriately decontaminated after each procedure, and if breaks in the sheath used to cover the probe occur, poten- tially virulent organisms may be inoculated into a previously uninfected patient. Once they are inocu- lated onto the mucosal membrane, microbes may then gain access to the systemic circulation.

e is not appropriately decontaminated after each procedure, and if breaks in the sheath used to cover the probe occur, poten- tially virulent organisms may be inoculated into a previously uninfected patient. Once they are inocu- lated onto the mucosal membrane, microbes may then gain access to the systemic circulation. Previ- ous investigations of surgical glove perforations have demonstrated that manufacturing defects in unused gloves may be present and that breaks in the glove may occur during surgical manipulation.4-8 Similarly, while latex condoms are clearly more effective barriers for transmission of infection than condoms made of natural substances, they do not provide perfect protection against breakage and sub- sequent disease transmission. Our investigation demonstrates that perforations occur in approximately 3% (95% C.I. 1.6-4.6%) of latex gloves and 7% (95% C.I. 4.4-9.4%)of latex condoms used to sheath the endovaginal probe. Visible contamination of the probe with blood or genital tract secretions is more likely to occur when condoms are used as a sheath (7.8% vs. 0.78%, P < .007). This effect appears to be the result of three factors: a slightly higher rate of perforations in condoms, larger perforations in condoms, and additional leakage of genital secretions around the open end ofthe condom. Condoms are shorter than gloves and are not as easy to maintain in taut appli- cation against the shaft of the endovaginal probe without the aid of a fastener. We recognize that our findings may be applica- ble only to certain types of gloves and condoms and to probes with the specific configuration used in this study. We also acknowledge that our study design has the potential for bias because the com- parative portion ofthe investigation was introduced belatedly, and patients were not randomly assigned to the different types of sheaths. Nevertheless, we did not approach the second half of the investiga- tion with any preconceived notion about which sheath was superior. There were no changes in study personnel, and the reporting of end points was uniform. Moreover, all examinations for per- forations were performed by a single observer us- ing a well established methodology. Therefore, until additional information is pub- lished, we recommend the use oflatex gloves rather than condoms to sheathe the vaginal probe. Even when gloves are used, some breakage and contami- nation may occur.

xaminations for per- forations were performed by a single observer us- ing a well established methodology. Therefore, until additional information is pub- lished, we recommend the use oflatex gloves rather than condoms to sheathe the vaginal probe. Even when gloves are used, some breakage and contami- nation may occur. Accordingly, the probe should be cleansed with an appropriate disinfectant, such as a dilute hypochlorite solution, after each use. REFERENCES 1. Recommendations for preventing transmission of human immunodeficiency virus and hepatitis B virus to patients during exposure-prone invasive procedures. MMWR 40: 1-9, 1991. 2. Welch J, Tilzey AJ, Webster M, Noah ND, BaratvalaJE. Hepatitis B infections after gynaecological surgery. Lancet 1:205-207, 1989. 3. Possible transmission ofhuman immunodeficiency virus to a patient during an invasive dental procedure. MMWR 39:489-493, 1990. 4. Rhoton-Vlasak A, Duff P. Glove perforations and blood contact associated with manipulation of the fetal scalp elec- trode. Obstet Gynecol 81:224-226, 1993. 5. Bennett B, Duff P. The effect of double gloving on fre- quency of glove perforations. Obstet Gynecol 78:1019- 1022, 1991. 6. Chapman S, Duff P. Frequency of glove perforations and subsequent blood contact in association with selected obstet- ric surgical procedures. Am J Obstet Gynecol (in press). 7. Dodds RD, Guy PJ, Peacock AM, Duffy SR, Barker SGE, Thomas MH. Surgical glove perforation. BrJ Surg 75:968, 1988. 8. Dodds RD, Barker SGE, Morgan NH, Donaldson DR, Thomas MH. Self-protection in surgery: The use of dou- ble gloves. BrJ Surg 77:219-220, 1990. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:40-45 (1993) (C) 1993 Wiley-Liss, Inc. In Vitro Susceptibility Testing of Clinical Isolates of Chlamydia trachomatis Mark G. Martens, Sebastian Faro, Maurizio Maccato, Gerald Riddle, Hunter Hammill, and Y. Wang Section ofInfectious Diseases, Department of Obstetrics and Gynecology, Baylor College ofMedicine, Houston, TX ABSTRACT Penicillin class antibiotics have demonstrated varying degrees of in vivo and in vitro success when tested against Chlamydia trachomatis. The activity of ampicillin-sulbactam, an agent commonly utilized in the treatment ofpelvic infections, was tested to ascertain if any antichlamydial activity is present. Up to six endocervical isolates of C. trachomatis were tested against each offive antibiotics including doxycycline, erythromycin, clindamycin, ampicillin/sulbactam, and sulbactam alone. Mc- Coy cell monolayers were inoculated with high inclusion counts of 10,000-30,000 inclusion-forming units (IFU) per coverslip, and exposed to each antibiotic. Up to nine subsequent antibiotic free culture passes were performed to assess the viability of abnormal inclusions. Doxycycline, erythro- mycin, and clindamycin achieved 100% eradication of inclusions at concentrations of 4.0, 2.0, and 1.0 g/mL. Exposure to ampicillin/sulbactam resulted in a greater than 99% reduction in the inclu- sion count at 32.0 g/mL, while sulbactam by itself demonstrated considerably less activity. Abnor- mal inclusions were noted only in the ampicillin/sulbactam exposed cells, and these, plus all inclu- sions remaining following sublethal exposure to the other antibiotics, resulted in regrowth to control levels in subsequent passes. Doxycycline and erythromycin demonstrated excellent activity. Clin- damycin and ampicillin/sulbactam also significantly reduced inclusion formation, and therefore may provide adequate C. trachomatis coverage in patients receiving these antibiotics for pelvic infections. (C) 1993 Wiley-Liss, Inc. KEY WORDS Chlamydia trachomatis, ampicillin-sulbactam, penicillins hlamydia trachomatis is one of the most preva- lent sexually transmitted organisms of the fe- male genital tract.

efore may provide adequate C. trachomatis coverage in patients receiving these antibiotics for pelvic infections. (C) 1993 Wiley-Liss, Inc. KEY WORDS Chlamydia trachomatis, ampicillin-sulbactam, penicillins hlamydia trachomatis is one of the most preva- lent sexually transmitted organisms of the fe- male genital tract. Infection may result in serious sequelae such as infertility, ectopic pregnancy, and pelvic inflammatory disease. Treatment regimens usually consist oftetracycline or erythromycin; how- ever, sulfa drugs and clindamycin have demon- strated good activity,z'3 Penicillin-based antibiotics have demonstrated efficacy in some in vitro studies,4'5 and in fact have recently been recommended as an alternative ther- apy to erythromycin in the treatment of chlamydial infection in pregnancy. 6 However, due to the elon- gated life cycle of C. trachomatis, single dose ther- apy with penicillin G, tetracycline, or erythromy- cin does not cure cases of urethritis caused by C. trachomatis. 6 Penicillins have also been noted to produce ab- normal inclusions, which may revert to normal at a later time.7 This observation may have possibly discouraged the study of newer broad spectrum penicillins for treatment of C. trachomat# infec- tions despite early, although limited, clinical suc- cess. Penicillins have been reported to eradicate C. Address correspondence/reprint requests to M.G. Martens, Division of Infectious Diseases, Department of Obstetrics and Gynecology, The University ofTexas Medical Branch, 3.108 Old Children's Hospital, Galveston, TX 77550. Clinical Study Received January 15, 1993 Accepted January 21, 1993 SUSCEPTIBILITY TESTING OF CHLAMYDIA TRACHOMATIS MARTENS ET AL. trachomatis in eight of ten women treated with a 21-day course of ampicillin for cervical and/or ure- thral infection. 8 Bowie et al. reported the microbi- ological cure in six of six men with urethritis when treated with a 10-day regimen of amoxicillin. 9 Martin et al. reported the microbiological cure of C. trachomatis infection in seven patients treated with ticarcillin/clavulanic acid, whereas three pa- tients treated with cephalosporins remained culture positive after treatment. 5 These results correlated well with their in vitro susceptibility results of 19 isolates of C. trachomatis against broad spectrum semi-synthetic penicillin and cephalosporins. Ticarcillin, mezlocillin, and piperacillin exhibited good in vitro susceptibility results in 19 isolates of C.

ulture positive after treatment. 5 These results correlated well with their in vitro susceptibility results of 19 isolates of C. trachomatis against broad spectrum semi-synthetic penicillin and cephalosporins. Ticarcillin, mezlocillin, and piperacillin exhibited good in vitro susceptibility results in 19 isolates of C. trachomatis, while cefoxitin, cefotaxime, and cefamandole exhibited poor in vitro activity. Also, despite excellent in vitro activity, treat- ment failures with the current drugs of choice, tetracycline and erythromycin, have also been re- ported.6-1 Similarly, in vitro resistance of eryth- romycin has been reported, but is rare. Several investigators have attempted to predict clinical efficacy of antibiotic regimens by perform- ing in vitro susceptibility testing. However, be- cause of the variety of in vitro susceptibility testing techniques that have been utilized, clinical correla- 2,4,7,8,12--17 tions do not always agree. The present investigation provides additional information concerning the susceptibility pattern for various antibiotics, including ampicillin/ sulbactam, which may be clinically useful in the treatment of chlamydial infections. SUBJECTS AND METHODS Source of Isolates Endocervical specimens were collected from two patient populations: 1) nonpregnant adolescents at- tending a teen health clinic; and 2) postpartum patients who were evaluated for endometritis. Processing of Specimens A Dacron swab was inserted in the endocervix after the portion of the cervix had been cleansed with a cotton-tipped applicator. The swab was rotated sev- eral times to collect cellular material and was then placed in Chlamydia transport medium. The trans- port tube was refrigerated (2-8C) and kept on ice until delivery to the laboratory (within 24 hours). Upon receipt in the laboratory, the transport tubes were vortexed for 30 seconds to remove cellu- lar material from the fiber matrix. Excess moisture was removed from the swab by pressing it against the side of the tube as it was withdrawn, and the swab was then discarded. The tubes were centri- fuged at 200g for 5 minutes to remove debris. The supernatant (0.3 mL) was used to inoculate McCoy cell monolayers grown on coverslips contained in 1-dram vials after the overlay medium had been removed. All transport tubes were then frozen at -70C. The inoculated vials were centrifuged for hour at 3,000g at 35C for hour.

ed at 200g for 5 minutes to remove debris. The supernatant (0.3 mL) was used to inoculate McCoy cell monolayers grown on coverslips contained in 1-dram vials after the overlay medium had been removed. All transport tubes were then frozen at -70C. The inoculated vials were centrifuged for hour at 3,000g at 35C for hour. Fresh isola- tion medium was added, and the vials were incu- bated for an additional 48-72 hours before stain- ing. The Chlamydia transport media, McCoy cell monolayers, and isolation medium were obtained from Bartels Immunodiagnostics (Bellevue, WA). The McCoy cell monolayers were stained with Jones' iodine or with Chlamydia-specific fluores- cent antibody (FA) reagent (Ortho Diagnostic, Raritan, NJ). Susceptibility Testing Stock solutions (100 x highest concentration tested) were prepared in minimal essential medium (MEM). Aliquots were frozen at -70C for no longer than 30 days before use. The concentrations (Ixg/mL) of the antibiotics tested were as follows: clindamycin 0.25, 0.50, and 1.0; doxycycline 4.0, 8.0, and 16.0; and sulbactam 3, 6, and 12. Slightly higher concentrations were utilized due to the high Chlamydia inoculum used. Ampicillin/sulbactam (Roerig, Groton, CT), were tested at a 2:1 ratio of ampicillin (6, 12, and 24 Ixg/mL) to sulbactam. The earlier penicillins have been tested extensively and therefore ampicillin was not included in this investigation.2'4'5 Frozen transport media was thawed and used to inoculate five vials of McCoy cells. After 48-72 hours, McCoy cell vials were prepared for passage by first centrifuging at 3,000g for 30 minutes to reassociate elementary bodies with the monolayer. The overlay medium was then decanted and re- placed with 0.4 mL 2 sucrose phosphate medium (2SP). The McCoy cell vials were frozen twice at -70C, thawed, and sonicated in an ultrasonic bath for 2 minutes. The sonicated suspensions were passed through a sterile 1.2-1xm syringe tip filter to INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 41 SUSCEPTIBILITY TESTING OF CHLAMYDIA TRACHOMATIS MARTENS ET AL. TABLE I. Activity of antibiotics vs. Chlamydia trachomatis No. of Antibiotic isolates Concentration (lg/mL) 0.25 0.5 1.0 Sulbactam 5 Ampicillin/sulbactam 5 Clindamycin 6 99 99 100 Doxycycline S Erythromycin 5 2.0 4.0 8.0 16.0 32.0 0 64 68 95 95 99 I00 I00 I00 I00 I00 I00 not tested. bConcentrations utilized for sulbactam: 3, 6, and 12 Ig/mL. CPercentage of inclusions inhibited at each concentration of antibiotic. remove cell debris.

1.0 Sulbactam 5 Ampicillin/sulbactam 5 Clindamycin 6 99 99 100 Doxycycline S Erythromycin 5 2.0 4.0 8.0 16.0 32.0 0 64 68 95 95 99 I00 I00 I00 I00 I00 I00 not tested. bConcentrations utilized for sulbactam: 3, 6, and 12 Ig/mL. CPercentage of inclusions inhibited at each concentration of antibiotic. remove cell debris. A 0.1 mL portion of the sus- pension was used to inoculate new vials. At least three passes were performed using MEM overlay media with antibiotics. An additional three passes were performed using MEM overlay media with- out antibiotics and with 5% heat inactivated fetal calf serum. McCoy cells were also maintained in MEM without antibiotics for 3 days prior to inoc- ulation. An inoculum containing 10,000-30,000 IFUs per coverslip was obtained with this method. A 0.1-mL portion of the final inoculum was added to each of four McCoy cell monolayers at each antibiotic concentration and centrifuged for hour at 3,000g, 30C. The monolayers were incu- bated for hour at 35C. Feeding MEM contain- ing the desired concentrations of antibiotics tested was then added to the monolayer. The monolayers were incubated for 48 hours. Two of the monolay- ers were stained, and two were used for a passage into four new vials. Between the first and second passes, monolayers were washed with antibiotic- free MEM to remove residual antibiotic. For pas- sage, 2 mL of 2SP media were added per vial. The cell layer was then disrupted with a Pasteur pipette, frozen at -70C, thawed, refrozen, and sonicated. Additional passes were performed for each isolate until regrowth of initial inoculum size or no growth occurred up to a total of ten passes. This procedure produced an inoculum containing ele- mentary bodies without cell debris. Antibiotic-free medium was added to triplicate inoculated McCoy cells as control. Monolayers were stained with a fluorescent antibody reagent, (Ortho Diagnostics). Layers were then examined microscopically, and the number ofnormal inclusions, ifany, was deter- mined. RESULTS Five clinically recovered isolates of C. trachomatis were tested against sulbactam, ampicillin/ sulbactam, doxycycline, and erythromycin. Six iso- lates were utilized in the clindamycin group. Good activity against C. trachomatis was demonstrated by all antibiotics at normally achievable serum levels with the exception of sulbactam. Percent inhibi- tions at various antibiotic concentrations are listed in Table I.

sulbactam, doxycycline, and erythromycin. Six iso- lates were utilized in the clindamycin group. Good activity against C. trachomatis was demonstrated by all antibiotics at normally achievable serum levels with the exception of sulbactam. Percent inhibi- tions at various antibiotic concentrations are listed in Table I. Minimal concentrations to inhibit 90% growth (MIC90) were <0.25 Ig/mL for clin- damycin, <2.0 Ig/mL for erythromycin, <4.0 Ig/mL for doxycycline, <8.0 Ig/mL for ampicillin/sulbactam, and >64 Ig/mL for sulbac- tam alone. Normal inclusions were noted following antibi- otic exposure to sulbactam, clindamycin, doxycy- cline, and erythromycin as well as the control groups, and were regular in shape and stained uni- formly in brilliant green (Fig. 1). Abnormal inclu- sions were smaller, misshaped, appeared hollow, and stained faintly (Fig. 2), and were noted only following exposure to ampicillin/sulbactam. Maximum inhibition occurred after the antibi- otic-treated pass (first pass), and regrowth of inclu- sions to pretreated levels occurred with ampicillin/ sulbactam, sulbactam alone, and clindamycin. Regrowth to control levels occurred after a mean of six passes. No regrowth of C. trachomatis occurred with doxycycline or erythromycin at any for the ten passes performed following exposure to the initial concentrations of antibiotic. DISCUSSION Several antibiotics have demonstrated varying lev- els of" activity against C. trachomatis. However, the 42 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY SUSCEPTIBILITY TESTING OF CHLAMYDIA TRACHOMATIS MARTENS ET AL. Fig. I. Normal inclusion. Fluorescent antibody stain, 400. Fig. 2. Abnormal inclusion. Fluorescent antibody stain, 400. tetracyclines and erythromycins, as well as their derivatives, have consistently demonstrated excel- lent in vitro and in vivo activity. Contraindications in pregnant patients, compliance, or tolerance problems have prompted the investigation of other antimicrobial agents. Penicillin class antibiotics are believed to be generally safe in pregnancy and are often better tolerated than erythromycin. Several investigations have demonstrated excellent in vitro and in vivo activity, as mentioned earlier. How- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 43 SUSCEPTIBILITY TESTING OF CHLAMYDIA TRACHOMATIS MARTENS ET AL. ever, susceptibility testing of C.

n pregnancy and are often better tolerated than erythromycin. Several investigations have demonstrated excellent in vitro and in vivo activity, as mentioned earlier. How- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 43 SUSCEPTIBILITY TESTING OF CHLAMYDIA TRACHOMATIS MARTENS ET AL. ever, susceptibility testing of C. trachomatis to pen- icillin is complicated by the production of abnor- mal inclusion bodies and the difficulty oflong-term clinical follow-up necessary for correlation of in vivo and in vitro findings. The abnormal inclusions are readily detectable by FA staining (Figs. 1, 2), and this perhaps should be the method of choice for detecting inclu- sions during C. trachomatis susceptibility testing with the penicillins. However, the use of FA stain- ing and the detection of abnormal inclusions com- promises some investigators' definition of MIC and MBCs. Heretofore, the MIC values repre- sented the concentration of antibiotic that inhibited the development of inclusions in McCoy cells. The MBC value represented the concentration of anti- biotics that prevented regrowth of inclusions in antibiotic-free McCoy cells. Other investigators have utilized a variety of means to compare the activity of penicillin and other antibiotics. Bowie utilized MIC, MBC, and >99% decrease in inclu- sion formation.4 Martin et al. utilized percentage inhibition at various antibiotic concentrations. 5 However, until the viability and importance of abnormal inclusions are better understood, great variability in the reporting of anti-chlamydial ac- tivity will probably continue. In this investigation, ampicillin/sulbactam, clin- damycin, doxycycline, and erythromycin demon- strated good in vitro activity at concentrations readily achievable in human serum. Only doxycy- cline and erythromycin eliminated all inclusions consistently, with no regrowth of inclusions, de- spite ten passes. While the penicillins failed to eradicate inclusion formation entirely, their activ- ity was comparable to clindamycin when examined at the extremely high inoculum utilized in these tests. Therefore, in patients being treated for pelvic or other infections with ampicillin/sulbactam or clindamycin, additional coverage of chlamydial in- fections with erythromycin and doxycycline may not be necessary. Perhaps reculturing ofthe patient can demonstrate eradication of the chlamydia, and save the patient the additional expense and compli- cations of these antibiotics.

nfections with ampicillin/sulbactam or clindamycin, additional coverage of chlamydial in- fections with erythromycin and doxycycline may not be necessary. Perhaps reculturing ofthe patient can demonstrate eradication of the chlamydia, and save the patient the additional expense and compli- cations of these antibiotics. The regrowth of the clindamycin-treated cells can be explained by the incomplete eradication of C. trachomatis at lower doses of clindamycin with subsequent regrowth of few, but viable, remaining inclusions. However, one sample demonstrated re- growth after four passes in which no inclusions were noted after antibiotic exposure, perhaps signi- fying elementary bodies in the absence of formed inclusions. Thus, the cause of inclusion regrowth with ampicillin/sulbactam may not necessarily be due to the abnormal inclusions, but to a similar regrowth of low numbers of inclusions or individ- ual elementary bodies. Regardless ofthe etiology of regrowth of inclusions after subsequent antibiotic- free passes in vitro, it is difficult to utilize these results to predict in vivo efficacy. Sulbactam, by itself, demonstrated poor activity against C. trachomatis, similar to previously pub- lished reports with other [3-1actamase inhibitors.4 However, the combination ampicillin/sulbactam demonstrated good activity, with a reduction in inclusion counts similar to clindamycin at achiev- able serum levels. Clindamycin has been recom- mended (or the treatment of C. trachomatis by the Centers for Disease Control, 18 although it is not their primary agent due to doxycycline's excellent activity. Penicillin's activity in clinical trials is en- couraging, if limited. In vivo, both antibiotics' activities may be augmented by host defenses that combine with the antibiotic's activity to eradicate the infection. However, because ofthe regrowth of inclusions after initial inhibition, adequate clinical trials need to be completed. Emphasis on long-term follow-up is critical in order to assess the possible regrowth of previously inhibited, but not eradi- cated inclusions. The high inoculum utilized in this investigation is artificially achieved, and probably not similar to actual clinical infections. However, it confirms the excellent activity of the tetracyclines and erythro- mycins, while still highlighting good activity for those antibiotics that may be bacteriostatic, if not bacteriocidal. Thus, in summary, in vitro susceptibility testing of C.

y achieved, and probably not similar to actual clinical infections. However, it confirms the excellent activity of the tetracyclines and erythro- mycins, while still highlighting good activity for those antibiotics that may be bacteriostatic, if not bacteriocidal. Thus, in summary, in vitro susceptibility testing of C. trachomatis confirmed the excellent activity of doxycycline and erythromycin. Clindamycin and ampicillin/sulbactam demonstrated good activity, with greater than 99% reduction of inclusion counts at achievable serum concentrations. Sulbac- tam demonstrated limited activity, consistent with other [3-1actamase inhibitors. While clindamycin and ampicillin/sulbactam cannot at this time be rec- ommended as the primary treatment for chlamydial 44 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY SUSCEPTIBILITY TESTING OF CHLAMYDIA TRACHOMATIS MARTENS ET AL. infections, those patients receiving these antibiotics for other indications, in which a chlamydial culture is reported positive, may not necessarily need addi- tional, automatic treatment with doxycycline or erythromycin. However, documentation of eradi- cation of Chlamydia by repeat tissue culture is rec- ommended. REFERENCES 1. Schacter J, Grossman M: Chlamydia infections. Annu Rev Med 32:45-61, 1981. 2. Kuo CC, Wang SP, Grayston JT: Antimicrobial activity of several antibiotics and sulfonamide against Chlamydia trachomatis organisms in cell culture. Antibicrob Agents Chemother 12:80-83, 1977. 3. Walsh M, Kappus EW, Quinn TC: In vitro evaluation of CP-62,933 erythromycin, clindamycin and tetracy- cline against Chlamydia trachomatis. Antibicrob Agents Chemother 31:811-812, 1987. 4. Bowie WR: In vitro activity ofthe clavulanic acid, amox- icillin and ticarcillin against Chlamydia trachomatis. An- tibicrob Agents Chemother 29:713-715, 1986. 5. Martin DH, Pastorek JG, Faro S: In vitro and in vivo activity of parenterally administered [3-1actam antibiotics against Chlamydia trachomatis. Sex Transm Dis 13:81- 87, 1986. 6. Noguera X, Ferrer M, Ortola E, Lopez-Marin L: Eval- uation of doxycycline in the treatment of urethritis and cervicitis caused by Chlamydia trachomatis. Clin Ther 9(Suppl A):33-37, 1986. 7. Lee CK, Bowie WR, Alexander ER: In vitro assays ofthe efficacy of antimicrobial agents in controlling Chlamydia trachomatis propagation. Antimicrob Agents Chemother 13:441-445, 1978. 8. Bowie WR: Lack of in vitro activity of cefoxitin, cefa- mandole, cefuroxime and piperacillin against Chlamydia trachomatis.

, 1986. 7. Lee CK, Bowie WR, Alexander ER: In vitro assays ofthe efficacy of antimicrobial agents in controlling Chlamydia trachomatis propagation. Antimicrob Agents Chemother 13:441-445, 1978. 8. Bowie WR: Lack of in vitro activity of cefoxitin, cefa- mandole, cefuroxime and piperacillin against Chlamydia trachomatis. Antibicrob Agents Chemother 21:339-340, 1982. 9. Bowie WR, Alexander ER, Holmes KK: Eradication of Chlamydia trachomatis from the urethras of men with nongonococcal urethritis by treatment with amoxicillin. Sex Transm Dis 8:79-81, 1981. 10. Bowie WR, Manzon LM, Borrie-Hume CJ, Fawcett A, Jones PK: Efficacy of treatment regimens for lower uro- genital Chlamydia trachomatis infection in women. Am J Obstet Gynecol 142:125-129, 1982. 11. Mourad A, Sweet RL, Sugg N, Schacter J: Relative resistance to erythromycin in Chlamydia trachomatis. An- timicrob Agents Chemother 18:696-698, 1980. 12. Bailey JMG, Heppleston C, Richmond SJ: Comparison of the in vitro activities of ofloxacin and tetracycline against Chlamydia trachomatis. Antibicrob Agents Chemother 26:13-16, 1984. 13. Hammerschlag MR, Gleyzer A: In vitro activity of a group of broad spectrum cephalosporins and other [3-1ac- tam antibiotics against Chlamydia trachomatis. Antibicrob Agents Chemother 23:493-494, 1983. 14. Hobson D, Lee N, Bushell AC, Withana N: Activity of [3-1actam antibiotics against Chlamydia trachomatis in Mc- Coy cell cultures In Mardh PA, Holmes KK, Oriel JD, Piot P, Schachter J (eds): Chlamydial Infections. Amster- dam: Elsevier Biomedical Press, p 249, 1982. 15. Ridgway GL, Owen JM, Oriel JD: The antimicrobial susceptibility of Chlamydia trachomatis in cell culture. Br J Vener Dis 54:103-106, 1978. 16. Segreti J, Kessler HA, Kapell KS, Trenholem GM: In vitro activity of A-56268 (TE-031) and four other anti- microbial agents against Chlamydia trachomatis. Antibi- crob Agents Chemother 31:100-101, 1987. 17 Martens MG, Faro S: 3-1actam antibiotics and Chlamy- dia trachomatis. Adv Ther 5:113-118, 1988. 18. Centers for Disease Control: STD treatment guidelines. MMWR 34:1-4S, 1985. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:49-50 (1993) (C) 1993 Wiley-Liss, Inc. Development of Pelvic Abscess Following Water-Skiing Injury Mark D. Pearlman and Lauren Zoschnick University ofMichigan Medical Center, Ann Arbor, MI ABSTRACT Several descriptions ofhydrostatic injuries while water-skiing have been described, including lacer- ations of the perineum, vagina, and cervix. Salpingitis or pelvic abscess resulting from water-skiing injuries are rare but important complications. A case of a pelvic abscess following a fall while water-skiing is described. The abscess was drained laparoscopically, resulting in a good clinical outcome. The mechanism ofinjury and recommendations for prevention are also presented. Upper genital tract infection may result from water-skiing injuries due to hydrostatic pressure forcing bacteria and water through the vagina and cervix into the endometrium, fallopian tube, and perito- neal cavity. While an uncommon complication, physicians and other practitioners caring for women should be aware ofthis potential complication from water-skiing. (C) 1993 Wiley-Liss, Inc. KF.Y WORDS Wounds and injuries, salpingitis, douche CASE REPORT 29-year-old woman (GO) was transported to the University of Michigan Emergency Cen- ter approximately 2 hours after losing her balance and falling backwards while water skiing. Her ini- tial complaints included nausea, vomiting, and lower back pain. On admission, she was conscious with stable vital signs, and a temperature of 99. IF. The abdomen was soft with minimal ten- derness in both lower quadrants, but no peritoneal signs. No vaginal bleeding or lacerations were noted. Pelvic examination revealed minimal lower abdominal tenderness, but no significant adnexal tenderness or masses were detected. The uterus was normal in size, anteverted, and nontender. Her past medical history was significant for a 3 year history of primary infertility. Laparoscopy per- formed approximately 2 months before her injury revealed stage II endometriosis and a right ovarian luteoma. The endometriosis was treated with lap- aroscopic laser ablation, and she recovered un- eventfully.

ntender. Her past medical history was significant for a 3 year history of primary infertility. Laparoscopy per- formed approximately 2 months before her injury revealed stage II endometriosis and a right ovarian luteoma. The endometriosis was treated with lap- aroscopic laser ablation, and she recovered un- eventfully. Abdominal radiographs made at the time of ad- mission revealed a nonspecific bowel gas pattern. A computerized tomography (CT) scan of the abdo- men and pelvis in the emergency center was normal without evidence of free fluid in the abdomen. She was admitted overnight for observation and dis- charged the next day in stable condition. Five days following discharge, the patient was seen by her family practitioner for a febrile illness, and "strep pharyngitis" was diagnosed. She was treated with penicillin V tablets, 250 mg for 7 days. Three days following initiation of this treatment, she returned to her family practitioner with persis- tent fever, and increasing left lower abdominal pain. Pelvic inflammatory disease was diagnosed and she was started on oral metronidazole and dox- ycycline. Two days later she was seen at the Uni- versity of Michigan Hospital with the complaint of increasingly severe bilateral lower abdominal pain. She denied being sexually active since, or for 2 weeks prior to the injury. On this admission her vital signs were stable with a temperature of Address correspondence/reprint requests to Dr. Mark D. Pearlman, 1500 E. Medical Center Dr., D2202 MPB; Box 0718, Ann Arbor, MI 48109. Received December 8, 1992 Gynecological Case Report Accepted February 26, 1993 PELVIC ABSCESS FOLLOWING WATER-SKIING INJURY PEARLMAN AND ZOSCHNICK 99.9F. Tenderness and guarding in both lower quadrants with rebound tenderness were found on physical examination. Bimanual examination of the pelvis revealed a tender 9 cm left adnexal mass. The admitting WBC count was 14,900/mm with a hematocrit of 36.1%, a Westegren sedimentation rate of 58 mm/sec, and a normal urinalysis. Chlamydia and gonorrhea cultures of the cervix were negative. A pelvic ultrasound revealed a 9 6 6 cm complex cystic mass in the left ad- nexa. She was admitted with a presumed left pelvic abscess and was begun on parenteral ampicillin, gentamicin, and metronidazole. The following day, a laparoscopic examination revealed a left pel- vic abscess involving the ovary, left fallopian tube, bowel, and omentum. The abscess was drained and the pelvis was thoroughly irrigated through the laparoscope.

left pelvic abscess and was begun on parenteral ampicillin, gentamicin, and metronidazole. The following day, a laparoscopic examination revealed a left pel- vic abscess involving the ovary, left fallopian tube, bowel, and omentum. The abscess was drained and the pelvis was thoroughly irrigated through the laparoscope. A closed drainage system was placed in the region and brought out through the abdomi- nal wall. Parenteral antibiotic therapy was contin- ued for a total of 8 days, and was discontinued after the patient had been afebrile for 48 hours. The WBC count on discharge was 10,300/ram3. Oral amoxicillin/clavulinic acid, 500 mg TID, was pre- scribed for 10 days. Aerobic and anaerobic cultures of the purulent fluid at laparoscopy revealed only a moderate quantity ofGroup C streptococci. She has done well since discharge. DISCUSSION Hydrostatic injury ofthe perineum, vagina, vulva, bladder, rectum, and descending colon have been described. (]-4 Most vaginal injuries resulting from water-skiing accidents involve the mechanism of falling backwards and striking the perineum on the water, which may force water through the va- gina and cervix into the upper genital tract or peri- toneal cavity. While a few prior descriptions of salpingitis following water-skiing injury have been presented, we can find no report of a resultant pelvic abscess.(5-7 The hydrostatic pressure of this water likely forces bacteria present both in the water and in the vagina and endocervix into the upper genital tract. This douche of lake water under high pressure probably traversed the lower and upper genital tract into the peritoneal cavity, causing the immedi- ate onset of symptoms. The initial event was fol- lowed by multiplication of bacteria in the region of inoculation, eventually resulting in pelvic abscess. The actual microbiology of this abscess was proba- bly altered by the antibiotics administered before laparoscopy, as it is reasonable to assume that this was initially a polymicrobial infection. It is unlikely that this was a preexisting abscess or that it developed from a sexually transmitted infection following the injury, as there was docu- mentation of a normal left adnexa on laparoscopy 2 months prior, a normal pelvic CT scan on the day of the injury, and self-described abstinence since the injury. While not common, douche-induced water-ski- ing injuries of the perineum, including laceration of the lower genital tract, rectum, and descending colon, have been described.

ormal left adnexa on laparoscopy 2 months prior, a normal pelvic CT scan on the day of the injury, and self-described abstinence since the injury. While not common, douche-induced water-ski- ing injuries of the perineum, including laceration of the lower genital tract, rectum, and descending colon, have been described. These accidents may also result in infections of the upper genital tract or pelvis. The risk of injury could be substantially reduced by wearing rubber wet suits to prevent direct hydrostatic injury. It would seem prudent to recommend the use of protective garments of this type for all water-skiers. Manufacturers of water skis could provide this information in an insert provided with the purchase of all new skis, or a small sticker on each ski suggesting the use of a rubber wet suit while skiing, particularly in women. Gynecologists, emergency medicine physi- cians, surgeons, and other providers of health care for women should be aware of potential water pres- sure injuries following water-skiing. REFERENCES 1. Niv J, Lessing JB, Hartuv J, Peyser MR: Vaginal injury from sliding down a water chute. Am J Obstet Gynecol 166:930-931, 1992. 2. Tweedle PG: Gynecologic hazards of water-skiing. CMA 108:20-21, 1973. 3. Edington RF: Vaginal injuries due to water-skiing. CMA 119:310-311, 1978. 4. Ramey JR: Intrarectal tear with bleeding from water-ski- ing accident. J Fla Med Assoc 61:162, 1974. 5. Planner D: Salpingitis and water-skiing. Med J Aust I:320, 1964. 6. Morton DC: Gynaecological complications of water-ski- ing. Med J Aust II:1256-1257, 1970. 7. Brisbane gynaecologist: Dangers of water-skiing. Med J Aust I:646, 1968. 50 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:51-57 (1993) (C) 1993 Wiley-Liss, Inc. Infection and Infertility Sebastian Faro Department ofGynecology and Obstetrics, University ofKansas School ofMedicine, Kansas City, KS ABSTRACT Asymptomatic infection appears to be a common cause offallopian tube damage resulting in ectopic pregnancy or infertility. (C) 1993 Wiley-Liss, Inc. KEY WORDS Bacterial vaginosis, Mycoplasma, Ureaplasma, asymptomatic infection he role of serious pelvic infection in female infertility is well recognized. However, the role of asymptomatic infection caused by Neisseria gonorrhoeae and Chlamydia trachomatis, although of great concern, has not been well established. Sig- nificant research efforts have been made to define these asymptomatic sexually transmitted diseases (STDs) and their role in infertility. In conjunction with much of the theorizing on the infectious etiol- ogy of infertility, the role ofthe vaginal microflora has also been implicated in these infections. Cur- rently, the initial infection is thought to be due to the gonococcus and/or chlamydia. This initial in- fection is then followed by ascension of potentially destructive microbes endogenous to the lower fe- male genital tract. THE VAGINAL MICROFLORA The lower female genital tract is a delicate ecosys- tem maintained in dynamic equilibrium. This bal- ance can be tipped by any number of factors, both endogenous and exogenous. The bacteriologic make-up of the lower genital tract includes both synergistic bacteria and antagonistic organisms. The healthy vaginal ecosystem is characterized by a pI--I of 3.8 to 4.2; a slate-grey to white, odorless dis- charge; and the presence of other gram-positive commensal bacteria, with the dominant bacterium being Lactobacillus. Also present are many other bacteria, gram-positive aerobes, facultative gram- negative bacteria, anaerobic gram-negative bac- teria, and gram-positive bacteria. The presence of Lactobacillus appears to be pivotal in maintaining the equilibrium and preventing potentially patho- genic bacteria from gaining dominance.

s. Also present are many other bacteria, gram-positive aerobes, facultative gram- negative bacteria, anaerobic gram-negative bac- teria, and gram-positive bacteria. The presence of Lactobacillus appears to be pivotal in maintaining the equilibrium and preventing potentially patho- genic bacteria from gaining dominance. The cur- rent theory is that the production of lactic acid by lactobacilli maintains the appropriate pH. In addi- tion, these bacteria produce hydrogen peroxide that is toxic to anaerobic bacteria, which do not produce peroxidase. When the balance is upset, the com- mensal bacteria decrease in number, reducing the hydrogen ion concentration and hydrogen peroxide and allowing the growth of facultative and anaero- bic bacteria, thereby resulting in an unhealthy state. Important to remember is that, despite the many forms of vaginitis, certain types are more conduc- tive to the production of" upper genital tract infec- tion, such as endometritis and salpingitis. The most common types of vaginitis are bacterial vaginitis (30% to 35% ofcases), yeast vaginitis (20%-25%), and trichomoniasis (10%-15%).2-4 Trichomonia- sis should serve as a marker organism for the possi- ble existence of other infections, as it is commonly found in association with gonorrhea and chlamy- dia. 5-7 The unhealthy or abnormal vaginal flora is char- acterized by a pH >4.5. A discharge is also char- acteristic; it is usually dirty-grey but may be colors Address reprint requests to Sebastian Faro, M.D., Ph.D., Department of Gynecology and Obstetrics, The University of Kansas School of Medicine, 3901 Rainbow Blvd., Kansas City, KS 66160-7316. Review Article Received January 20, 1993 Accepted April 7, 1993 INFECTION AND INFERTILITY FARO other than white. It may or may not be frothy and may or may not be homogenous. Typically, it emits an amine odor when mixed with 10% potassium hydroxide (KOH). Microscopic examination usu- ally reveals the squamous epithelial cells to be cov- ered by adherent bacteria, obliterating the cytoplas- mic membrane and nucleus. In the case of bacterial vaginosis (BV), white blood cells (WBCs) are usu- ally absent, thus the use of the suffix "-osis" to indicate the absence of an inflammatory reaction, but numerous individually free-floating bacteria are present. Microbiologic evaluation of this type of lower genital tract will reveal the presence of large num- bers of facultative and obligate anaerobes. With BV, the patient is more likely to have an STD.

x "-osis" to indicate the absence of an inflammatory reaction, but numerous individually free-floating bacteria are present. Microbiologic evaluation of this type of lower genital tract will reveal the presence of large num- bers of facultative and obligate anaerobes. With BV, the patient is more likely to have an STD. In one study, 54% of the women with BV reported having had an STD. Other bacterial derangements of the vaginal microflora may exist, especially in patients who have been repeatedly treated for vaginitis but whose flora has not regained the com- position of a healthy state. Gardnerella vaginalis vaginitis, originally described by Gardner and Dukes, consists of a vaginal discharge resembling that of BV, but differing in that the free-floating bacteria found in the vaginal fluid are not individ- ually distributed but in aggregates. Rarely, if ever, are WBCs present. 9 Some patients present with bacterial vaginitis consisting predominantly of Gardnerella vaginalis with few, if any, other types of bacteria and, rarely, anaerobes. Whereas other types ofvaginitis may have clinical parameters sim- ilar to those found with BV, 30% of patients with a discharge that liberates a foul odor will have BV and 17% ofpatients with clue cells but no dominant anaerobic bacteria will have BV. 1 In one study, patients with persistent vaginitis who had been re- peatedly treated with antibiotics were enrolled. 1' All patients had a vaginal pH of > 4.5 and the liberation of amines when the discharge was mixed with 10% KOI-I. These patients were divided into two groups. The first group consisted of 48 pa- tients. Microscopic analysis of the vaginal fluid of all patients in the first group demonstrated numer- ous individually free-floating bacteria and numer- ous WBCs. Half of the patients in this first group did not demonstrate Gardnerella vaginalis as part of the microflora, and half demonstrated Gardnerella vaginalis in counts of <103 colony-forming units per ml (cfu/ml) ofvaginal fluid. The second group of patients all demonstrated Gardnerella vaginalis, but in high colony counts of 3 l0s to 9 109 cfu/ml of vaginal fluid. The vaginal fluid of the patients in this second group revealed clue cells, rare WBCs, and bacteria floating in clumps, not individually.

rming units per ml (cfu/ml) ofvaginal fluid. The second group of patients all demonstrated Gardnerella vaginalis, but in high colony counts of 3 l0s to 9 109 cfu/ml of vaginal fluid. The vaginal fluid of the patients in this second group revealed clue cells, rare WBCs, and bacteria floating in clumps, not individually. The finding of varieties of bacterial infections raises the concern regarding not the par- ticular type of vaginitis, but the possibility that the bacteria present may ascend to the upper genital tract and act synergistically in causing a progressive infection. Important to remember is that the individual with bacterial vaginitis or BV is at greater risk for having an accompanying STD. This epidemiologic information is important for two reasons. First, it implies that the patient's behavior pattern may cur- rently place her at risk for upper genital tract infec- tion. Second, it encourages the physician to obtain a detailed sexual history to determine if the patient has been at risk for a prolonged time, making the possibility of" a past or present upper genital tract infection more of a reality. EVALUATION OF THE PATIENT FOR INFECTION The physician should encounter little difficulty in evaluating the patient with an acute symptomatic genital tract infection. However, the goal should be the prevention of damage of the fallopian tubes which might result in abnormal function or infer- tility. The annual ectopic pregnancy rate from 1970 through 1989 has risen four-fold from 4.5 to 16.1/ 1000 pregnancies. 12 Hospital admissions for ec- topic pregnancy in 1991 were 88,400, a 10% in- crease over 1988. This rise in the ectopic pregnancy rate is believed to be due to the rise in the number of cases of pelvic inflammatory disease (PID). Al- though obtaining data with regard to infertility and infection is difficult, the estimation has been made that, after a single episode of PID, approximately 12% of the women will be infertile; after two epi- sodes, 25% will be infertile; and, after three or more episodes, more than 50% will be infertile. 3 The Centers for Disease Control has estimated that four million initial visits to physicians for PID were made in 1991, generating approximately 250,000 hospital admissions. 14 To prevent fallopian tube damage, the physician must recognize the earliest stage of the disease pro- cess. The initial site of infection in the female pa- 2 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY INFECTION AND INFERTILITY FARO tient is the cervix.

991, generating approximately 250,000 hospital admissions. 14 To prevent fallopian tube damage, the physician must recognize the earliest stage of the disease pro- cess. The initial site of infection in the female pa- 2 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY INFECTION AND INFERTILITY FARO tient is the cervix. However, the difficulty lies in the fact that the greatest percentage of infected women are asymptomatic. The physician may be clued in by asking the patient the appropriate ques- tions indicating historical evidence for the possible existence of an STD: 1. Have you noticed a change in your vaginal discharge? 2. Do you have spotting following sexual inter- course? 3. Have you noticed a sudden onset ofpain with sexual intercourse? Close attention should be paid to the endocervi- cal epithelium, specifically noting the presence of hypertrophy. A dacron swab should be passed into the endocervical canal and rotated for 5 to 10 sec- onds to determine if any endocervical mucus is present. Typical findings suggesting cervical infec- tion, namely Chlamydia trachomatis and Neisseria gonorrhoeae, are that (1) the endocervical tissue bleeds easily when the pap smear is taken and (2) the patient's pap smear reveals inflammation. 15,16 The physician should remember that patients do not present with complaints of cervicitis and that cervical infectionbyN. gonorrhoeaeand/or C. tracho- matis is frequently asymptomatic. 16 Therefore, to prevent significant infection, the physician must be able to recognize early asymptomatic infection. Infection of the fallopian tubes is thought to occur by progressive upward migration from the endocervix along the columnar epithelium lining the endocervix and endometrium. Patients who de- velop endometritis may not have overt signs or symptoms of infection. The patient may develop vague lower abdominal cramping-like pain, recent onset of dyspareunia or dysmenorrhea, break- through bleeding if she is utilizing oral contracep- tive pills (OCPs) for birth control, or irregular bleeding if she is not using hormonal contracep- tion. 17 Thus, the patient who is suspected ofhaving endometritis should be thoroughly evaluated. Spec- imens for culture of N. gonorrhoeae and C. tra- chomatis should be obtained from the endocervix. Then, the portio ofthe cervix should be thoroughly cleansed. If Betadine is used, the cervix should be gently wiped off before an endometrial sample is taken.

cted ofhaving endometritis should be thoroughly evaluated. Spec- imens for culture of N. gonorrhoeae and C. tra- chomatis should be obtained from the endocervix. Then, the portio ofthe cervix should be thoroughly cleansed. If Betadine is used, the cervix should be gently wiped off before an endometrial sample is taken. The endometrial sample is best obtained by inserting a pipette through the endocervical canal to the uterine fundus and obtaining a tissue specimen. The pipette should be carefully withdrawn without touching the vaginal walls. The specimen should be divided into two portions. One should be sent for histologic evaluation and the other one sent in an anaerobic transport vessel for the culture of N. gonorrhoeae, C. trachomatis, aerobic, facultative, and obligate anaerobic bacteria. The presence of plasma cells, especially if C. trachomatis is the in- fecting organism, is highly correlated with salpin- gitis. 18,19 Endometritis, frequently asymptomatic, has been reported to occur in approximately 40% of women with cervicitis. 18 It has also been reported that women clinically diagnosed as having salpingi- tis, but laparoscopically found to have normal fallo- pian tubes, had endometritis by biopsy.2 RISK FACTORS The first line of intervention is prevention; how- ever, the physician rarely has the opportunity to teach prevention. Therefore, the existence of infec- tion of the cervix or endometrium should be con- sidered as the second line of intervention. The sec- ond opportunity to prevent serious tubal damage requires recognizing the patient at risk. The physi- cian must be comfortable with taking a detailed sexual history from a young patient who is sexually active, especially one utilizing OCPs, Norplant (Wyeth-Ayerst Laboratories, P.O. Box 8299, Phil- adelphia, PA 19101), or other methods of birth control. The age of first sexual intercourse is a risk factor. Young, sexually experienced teenagers are three times more likely to have had PID.21 The reason for this risk factor is not understood, but may be related to the fact that the younger patient is more likely to have multiple sexual partners, expo- sure to a larger pool of STDs, and larger areas of exposed endocervical epithelium.22'23 Thus, it is extremely important to obtain an accurate and de- tailed sexual history from the patient, ideally when the patient is seen at the initial visit.

the younger patient is more likely to have multiple sexual partners, expo- sure to a larger pool of STDs, and larger areas of exposed endocervical epithelium.22'23 Thus, it is extremely important to obtain an accurate and de- tailed sexual history from the patient, ideally when the patient is seen at the initial visit. If the patient states that she is not sexually active at the present time, the physician has an excellent opportunity to begin the educational process on transmission of STDs and the consequences of infection. Patients older than 25 years who acquire a C. trachomatis or N. gonorrhoeae infection that leads to PID have a greater chance of resulting infertility than patients younger than 25 years.24 However, the younger patient is more likely to experience INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY INFECTION AND INFERTILITY FARO repeated infection and episodes ofPID. The patient who develops PID is at risk for tubal damage, depending upon the severity or degree of inflam- mation she experiences. Women with mild disease are unlikely to develop tubal damage, whereas women with severe disease are five times more likely to develop significant tubal damage.25 Once the patient has been given the facts con- cerning the transmission and consequences of STDs, the discussion should turn to contraceptive methods. Contraception should also be discussed with the patient, especially if sexual intercourse is occurring or is about to commence. Most individ- uals will have some knowledge of OCPs and per- haps Norplant. However, they will not be know- ledgeable about the relationship of hormonal contraception and the acquisition of STDs. A look at the available data indicates that the use of oral contraceptives is associated with increased detection of cervical infection by C. trachomatis. However, oral contraceptives appear to be associated with a decreased risk of PID.26-29 Although these find- ings are difficult to reconcile, they may be attribut- able to behavioral characteristics among individu- als who use OCPs or to mild, undetected forms of PID among OCP users. There is also evidence that estrogen facilitates the adherence of C. trachomatis to endometrial cells. However, when estrogen is combined with high doses of progesterone, adher- ence is not favored.3 Animal studies have not helped to clarify this issue. Oral contraceptives fed to guinea pigs seem to encourage both lower and upper genital tract infection by C. trachomatis.

dherence of C. trachomatis to endometrial cells. However, when estrogen is combined with high doses of progesterone, adher- ence is not favored.3 Animal studies have not helped to clarify this issue. Oral contraceptives fed to guinea pigs seem to encourage both lower and upper genital tract infection by C. trachomatis. 1-34 In one study, the use ofhigh-dose estrogen-contain- ing pills appeared to be associated with a higher incidence of PID.s Barrier contraception, including condoms and spermicides, offers enhanced protection against tu- bal infertility secondary to infection. The use of condoms alone provides only marginal protection. However, combining spermicides and condoms of- fers a two-fold increase in protection against tubal infertility.s This point should be emphasized to young women who are engaging in sexual inter- course but do not have reliable partners or whose partners have multiple partners. Another risk factor, one which is poorly under- stood, is the relationship between cigarette smoking and the risk of tubal infertility. This relationship appears to be statistically stronger in nulliparous TABLE I. Microbial flora of the healthy vagina Lactobacillus Streptococcus Staphylococcus Corynebacterium Diphtheroides Enterococcus Escherichia Proteus Klebsiella Enterobacter Morganella Peptostreptococcus Bacteroides Fusobacterium Veillonella Eubacterium women and dependent upon the intensity of smok- ing. It does not appear to be related to behavioral patterns.6-8 One hypothesis as to the mechanism by which smoking may be associated with tubal infertility is that smoking may interfere with the host immune response, thereby allowing the infect- ing organisms to gain entrance to the fallopian tube.39 The relationship between exogenous, be- havioral, and endogenous factors and the risk of tubal infertility is still unclear and remains to be unraveled. MICROBIOLOGY AND INFERTILITY The preceding discussion has alluded to the more common STDs and their role in infertility. The role of abnormal bacteriology of the lower genital tract has also been mentioned. The normal or healthy vaginal microflora is dominated by Lacto- bacillus, but numerous other bacteria make up the normal flora (Table 1). These bacteria exist in a dynamic relationship that is commensal, synergis- tic, and antagonistic. The bacteria also exist in a dynamic equilibrium with their environment, a balance that is constantly challenged by external and internal factors.

bacillus, but numerous other bacteria make up the normal flora (Table 1). These bacteria exist in a dynamic relationship that is commensal, synergis- tic, and antagonistic. The bacteria also exist in a dynamic equilibrium with their environment, a balance that is constantly challenged by external and internal factors. The relationship of these bacteria is dependent upon dominance which, in turn, is determined by bacterial metabolic products as well as environmen- tal factors. In the healthy state, commensal bac- teria, lactobacilli, corynebacteria, and diphtheroids are present in large numbers, with lactobacilli be- ing present in concentrations of >105 cfu/cc of vaginal fluid. When the environmental conditions of the vagina become altered, the characteristics of 54 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY INFECTION AND INFERTILITY FARO the microbial flora are altered to favor the patho- genic organisms. This can happen when the pH is altered by environmental factors or by the intro- duction of exogenous pathogens. The most com- mon organisms, e.g., N. gonorrhoeae and C. tra- chomatis, are introduced through sexual intercourse. However, other conditions not directly related to sexual activity may also have a bearing, for exam- ple, BV in which T. vaginalis and other organisms make up the microflora. Bacteria such as G. vaginalis occupy a perplex- ing position in vaginitis. There is little doubt that this organism can play a pathogenic role under the appropriate set of circumstances. While there is considerable discussion over its role in vaginitis, there are no data to suggest that it plays a significant role in PID or infertility. Bacteria such as Myco- plasma and Ureaplasma occupy a significant contro- versial role in infertility. Although the role of Mycoplasma and Urea- plasma as causative agents in infertility has not been established, they have been implicated. These bac- teria have been associated with a variety ofobstetri- cal and gynecologic infections, such as postpartum endometritis, postpartum bacteremia, post-cesar- ean abdominal wound infection, and early preg- nancy loss.40-44 Mycoplasmal organisms are con- sistently found in 20% to 40% ofthe healthy vaginal microbial flora, while colonization of the lower genital tract is directly related to the age of first coitus and the number of sexual partners.4s Infertility attributed to Mycoplasma is a subject that has waxed and waned over the years.

Mycoplasmal organisms are con- sistently found in 20% to 40% ofthe healthy vaginal microbial flora, while colonization of the lower genital tract is directly related to the age of first coitus and the number of sexual partners.4s Infertility attributed to Mycoplasma is a subject that has waxed and waned over the years. In a study published in 1979, Gnarpe and Friberg divided 55 couples into four groups.46 Group A consisted of 36 couples with unexplained infertility who had Mycoplasma colonization rates of 25% and Urea- plasma colonization rates o 92%. Group B con- tained 19 couples in which the females all had antisperm antibodies, 15% had Mycoplasma, and all had Ureaplasma. Group C was made up of 40 pregnant patients, of whom 7.5% had Mycoplasma and 22.5% had Ureaplasma. Group D consisted of 23 men with pregnant wives, none of whom had Mycoplasma but 26% of whom had Ureaplasma. This study suggested that Mycoplasma may play a role in infertility. Graber and colleagues studied 25 women with unexplained infertilityand found Urea- plasma in 80% of these women compared to a 36% colonization rate in 25 fertile controls (p < 0.001).47 Although this study did show a statistical correla- tion between Mycoplasma and infertility, a direct cause-and-effect relationship was not demonstrated. DeLouvois et al, in 1974, studied 120 infertile couples and 92 pregnant patients and found no statistical difference with regard to colonization rates ofMycoplasma or Ureaplasma.45 These studies encourage investigators to con- sider that, if Mycoplasma is involved in infertility, then treatment directed specifically against these bacteria should result in improved fertility rates. Friberg and Gnarpe sampled couples for the pres- ence ofMycoplasma.48 Ifthe patients were positive, they were not treated for three months. If they did not achieve pregnancy within this time, both were treated with doxycycline, 100 mg twice daily for 7 to 16 days. They found a 25% pregnancy rate in 16 women having antisperm antibodies and a 29% pregnancy rate in 3 8 women not having antisperm antibodies. Although this study suggested that there may be a relationship between colonization and fertility and that treatment may be of" benefit, there was no control group. Stray-Pedersen et al, in 197 8, studied three groups of women.49 Group A was made up of 46 women with a history of habitual abortion, 13 of whom were positive for Urea- plasma.

uggested that there may be a relationship between colonization and fertility and that treatment may be of" benefit, there was no control group. Stray-Pedersen et al, in 197 8, studied three groups of women.49 Group A was made up of 46 women with a history of habitual abortion, 13 of whom were positive for Urea- plasma. All were treated with doxycycline and 13 who had positive cultures became pregnant and 10 delivered term infants. Group B consisted of 18 women with unexplained infertility, 50% of whom were colonized with Ureaplasma. Group C con- tained 45 fertile women, of whom only 7% were found to be colonized. In 1983, Toth et al reported on 161 infertile couples in whom only the males were found to be colonized with Ureaplasma. s They were treated with doxycycline, 100 mg twice daily for 28 days. Follow-up cultures showed that the organism had been eradicated in 129 patients. Sixty percent of the couples who were culture-neg- ative achieved pregnancy, whereas only 5% of the couples in whom the male remained positive achieved pregnancy. Several investigators have been unable to con- firm the relationship of treating Mycoplasma and Ureaplasma colonization and achieving fertility. In 1975, Harrison et al. studied 88 couples and di- vided them into three groups, s Group A contained 3 0 couples with colonization rates of23 % for Myco- plasma and 70% for Ureaplasma. Group B con- tained 28 couples, of whom 14% were positive for INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY INFECTION AND INFERTILITY FARO Mycoplasma and 64% for Ureaplasma. Group C consisted of 30 couples, of whom 26% were found to have Mycoplasma and 57% had Ureaplasma. Pa- tients in group A were treated with doxycycline for 28 days and 16% achieved pregnancy. Individuals assigned to group B received a placebo, and 14% became pregnant. Group C received no treatment, neither doxycycline nor placebo, and 16% achieved pregnancy. Idriss et al, in 1978, studied 200 infer- tile couples and found that 42% of the treatment group became pregnant, whereas 37% of the non- 52 treatment group became pregnant. Thus, the data regarding Mycoplasma and Urea- plasma are at best confusing. The studies that have been conducted lack a precise scientific approach. Perhaps studies should be carried out using quanti- tative bacteriology, serology, and DNA probes of the tissue in question.

f the non- 52 treatment group became pregnant. Thus, the data regarding Mycoplasma and Urea- plasma are at best confusing. The studies that have been conducted lack a precise scientific approach. Perhaps studies should be carried out using quanti- tative bacteriology, serology, and DNA probes of the tissue in question. A patient with unexplained infertility is particularly perplexing because an ex- planation cannot be offered to her, creating a deeper sense of frustration in the patient. However, the development ofnew technology in the area ofinfec- tious disease is providing a better understanding of the subtleties of subclinical infection, which should permit a more scientific approach to studying the pathophysiology oftubal disease secondary to infec- tion. This knowledge, in turn, will allow the clini- cian to prescribe a directed plan of management. The most important aspect of preventing infer- tility or tubal damage secondary to infection is not the design of more potent treatment agents, but the prevention and recognition of the earliest signs of infection. Prevention of tubal damage and infertil- ity does not begin with the treatment of salpingitis. Rather, the physician must begin the educational process on the patient's first visit. Dialogue should begin at this point and continue with each visit to reinforce what has been presented at earlier visits and keep the patient current on the latest informa- tion regarding transmission and protective mea- sures. The second opportunity to prevent tubal dam- age and infertility is the recognition of the early signs ofinfection in the asymptomatic patient. These signs should be used to develop a significant history and to determine at what degree of risk the particu- lar patient has placed herself. REFERENCES 1. Eschenback DA, Davick PR, Williams BL, et al: Preva- lence of hydrogen peroxide-producing Lactobacillus spe- 6 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY cies in normal women and women with bacterial vagino- sis. J Clin Microbiol 27:251-256, 1989. 2. Berg AO, Heidrich FE, Fihn SN, et al: Establishing the cause of genitourinary symptoms in women in a family practice: Comparison ofclinical examination and compre- hensive microbiology. JAMA 251:620-625, 1984. 3. Fleury FJ: Adult vaginitis. Clin Obstet Gyneco124:407- 438, 1981. 4. Karnaky KJ: Diagnosis and treatment ofvaginitis (letter). Am J Obstet Gynecol 129:929-930, 1977. 5.

of genitourinary symptoms in women in a family practice: Comparison ofclinical examination and compre- hensive microbiology. JAMA 251:620-625, 1984. 3. Fleury FJ: Adult vaginitis. Clin Obstet Gyneco124:407- 438, 1981. 4. Karnaky KJ: Diagnosis and treatment ofvaginitis (letter). Am J Obstet Gynecol 129:929-930, 1977. 5. Fouts AC, Kraus SJ: Trichomonas vaginalis: Reevaluation of its clinical presentation and laboratory diagnosis. J Infect Dis 14:137-143, 1980. 6. Judson FN: The importance of coexisting syphilitic, chlamydial, mycoplasmal and trichomonal infection in the treatment ofgonorrhea. Sex Transm Dis 6:112-119, 1979. 7. Wright RA, Judson FN: Relative and seasonal incidences of the sexually transmitted diseases. A two-year statistical review. Br J Vener Dis 54:433-440, 1978. 8. Lossick JG: The descriptive epidemiology of vaginal tri- chomoniasis and bacterial vaginosis. In Horowitz BJ, Mardh P-A (eds): Vaginitis and Vaginosis. New York, Wiley-Liss, p775, 1991. 9. Gardner I-IL, Dukes CD: Haemophilus vaginalis vagini- tis. Am J Obstet Gynecol 69, 1955. 10. Faro S, Phillips LE: Non-specific vaginitis or vaginitis of undetermined aetiology. Int J Tissue React 9:173-177, 1987. 11. Faro S: Persistent vaginitis and vaginosis. In Horowitz BJ, Mardh P-A (eds): Vaginitis and Vaginosis. New York, Wiley-Liss, p 237, 1991. 12. Centers for Disease Control: Ectopic pregnancymUnited States, 1988-1989. MMWR 41:591-594, 1992. 13. Cates W Jr, Rolls RT Jr, Aral SO: Sexually transmitted diseases, pelvic inflammatory disease and infertility: An epidemiologic update. Epidemiol Rev 12:199-220, 1990. 14. Centers for Disease Control: Sexually transmitted disease surveillance. MMWR 1991. 15. Brunham RC, PaavonenJ, Stevens CE, et al: Mucopuru- lent cervicitis: The ignored counterpart in women of ure- thritis in men. N Engl J Med 311:1-6, 1984. 16. Harrison HR, Costin M, Meder JB, et al: Cervical Chlamydia trachomatis infection in university women: Re- lationship to history, contraception, ectopy and cervicitis. Am J Obstet Gynecol 153:244-251, 1985. 17. Wasserheit JN, Bell TA, Kiviat NB, et al: Microbial causes of proven pelvic inflammatory disease and efficacy of clindamycin and tobramycin. Ann Intern Med 104: 187-193, 1986. 18. Paavonen J, Kiviat NB, Brunham RC, et al: Prevalence and manifestations of endometritis among women with cervicitis. Am J Obstet Gynecol 152:280-286, 1985. 19.

TA, Kiviat NB, et al: Microbial causes of proven pelvic inflammatory disease and efficacy of clindamycin and tobramycin. Ann Intern Med 104: 187-193, 1986. 18. Paavonen J, Kiviat NB, Brunham RC, et al: Prevalence and manifestations of endometritis among women with cervicitis. Am J Obstet Gynecol 152:280-286, 1985. 19. Kiviat NB, Wolner-Hanssen P, Eschenbach DA, et al: Endometrial histopathology in patients with culture- proven upper genital tract infection and laparoscopically acute salpingitis. Am J Surg Pathol 14:167-175, 1990. 20. Paavonen J, Aine R, Teisala K, Heinonen PK, Punnonen R: Comparison ofendometrial biopsy and peritoneal fluid cytologic testing with laparoscopy in the diagnosis ofacute INFECTION AND INFERTILITY FARO pelvic inflammatory disease. Am J Obstet Gynecol 151: 645-650, 1985. 21. Bell TA, Holmes KK: Age specific risks of syphilis, gonorrhea and hsopitalized pelvic inflammatory disease in sexually experienced U.S. women. Sex Transm Dis 11: 291-295, 1984. 22. Wasserheit JN: Pelvic inflammatory disease and infertil- ity. Md Med J 36:58-63, 1987. 23. O'Reilly KR, Aral SO: Adolescents and sexual behavior: Trends and implications for STD. J Adolesc Health Care 6:262-270, 1985. 24. Westrom K, Mardh P-A: Acute pelvic inflammatory dis- ease (PID). In Holmes KK, Mardh P-A, Sparling PF, et al. (eds): Sexually transmitted diseases. 2nd ed. New York: McGraw-Hill, p 593, 1990. 25. Westrom L: Effect of acute pelvic inflammatory disease on fertility. Am J Obstet Gynecol 121:707-713, 1975. 26. Washington AE, Gove S, Schachter J, et al: Oral contra- ceptives, Chlamydia trachomatis infection, and pelvic in- flammatory disease. JAMA 253:2246-2250, 1985. 27. Senanayake P, Kramer DG: Contraception and the etiol- ogy of pelvic inflammatory disease: New perspectives. Am J Obstet Gynecol 138:852-860, 1980. 28. Svensson L, Mardh P-A, Westrom L: Infertility after acute salpingitis with special reference to Chlamydia tra- chomatis. Fertil Steril 40:322-329, 1983. 29. Wolner-Hanssen P, Eschenbach DA, Paavonen J, et al: Decreased risk of symptomatic chlamydial pelvic inflam- matory disease associated with oral contraceptive use. JAMA 251:2553, 1984. 30. Maslow AS, Davis CH, ChoongJ, Wyrick PB: Estrogen enhances attachment of Chlamydia trachomatis to human endometrial epithelial cells in vitro. Am J Obstet Gynecol 159:1006-1014, 1988. 31. Barron AL, Pasley JN, Rank RG, White HJ, Mrak RE: Chlamydia salpingitis in female guinea pigs receiving oral contraceptives.

84. 30. Maslow AS, Davis CH, ChoongJ, Wyrick PB: Estrogen enhances attachment of Chlamydia trachomatis to human endometrial epithelial cells in vitro. Am J Obstet Gynecol 159:1006-1014, 1988. 31. Barron AL, Pasley JN, Rank RG, White HJ, Mrak RE: Chlamydia salpingitis in female guinea pigs receiving oral contraceptives. Sex Transm Dis 15:169-173, 1988. 32. Rank RG, White HJ, Hough AJ, PasleyJN, Barron AL: Effect of estradiol on Chlamydial infection in female guinea pigs. Infect Immun 38:699-705, 1982. 33. Rank RG, Barron AL: Specific effects of estradiol on genital mucosal antibody response in chlamydial ocular and genital infections. Infect Immun 55:2317, 1987. 34. Pasley JN, Rank RG, Hough AJ, et al: Absence of pro- gesterone effects on chlamydial genital infection in female guinea pigs. Sex Transm Dis 12:155-158, 1985. 35. Cramer DW, Goldman MB, Schiff I, et al: The relation- ship of tubal infertility to barrier method and oral contra- ceptive use. JAMA 257:2446-2450, 1987. 36. DalingJR, Weiss NS, Spadoni LR, et al: Cigarette smok- ing and primary tubal infertility. In Rosenbert MJ (ed): Smoking and reproductive health. Littleton MA: PSG Publishing, p 40, 1987. 37. Phipps WR, Cramer DW, Schiff I, et al: The association between smoking and female infertility as influenced by cause of the infertility. Fertil Steril 48:377-382, 1987. 38. Marchbanks PA, Lee NC, Peterson HB: Cigarette smok- ing as a risk factor for pelvic inflammatory disease. Am J Obstet Gynecol 162:639-644, 1990. 39. Hershey P, Pendergast D, Edwards A: Effects of ciga- rette smoking on the immune system: Follow-up studies in normal subjects after cessation of smoking. Med J Aust 2:425, 1983. 40. Phillips LE, Faro S, Pokorny SF, et al: Postcesarean wound infection by Mycoplasma hominis in a patient with persistent postpartum fever. Diag Microbiol Infect Dis 7:193-197, 1987. 41. Faro S, Martens M, Hammill H, Phillips LE, Smith D, Riddle G: Ticarcillin/clavulanic acid versus clindamycin and gentamicin in the treatment of post-cesarean endo- metritis following antibiotic prophylaxis. Obstet Gynecol 73:808-812, 1989. 42. Maccato M, Faro S, Summers KL: Wound infections after cesarean section with Mycoplasma hominis and Urea- plasma urealyticum: A report ofthree cases. Diagn Micro- biol Infect Dis 13:363-365, 1990. 43. Roberts S, Maccato M, Faro S, Pinell P: The microbiol- ogy of post-cesarean wound morbidity. Obstet Gynecol 81:383-386, 1993. 44.

, Faro S, Summers KL: Wound infections after cesarean section with Mycoplasma hominis and Urea- plasma urealyticum: A report ofthree cases. Diagn Micro- biol Infect Dis 13:363-365, 1990. 43. Roberts S, Maccato M, Faro S, Pinell P: The microbiol- ogy of post-cesarean wound morbidity. Obstet Gynecol 81:383-386, 1993. 44. Kundsin RB, Driscoll SG, Ming PL: Strain of Myco- plasma associated with human reproductive failure. Sci- ence 157:1573-1574, 1967. 45. Louvois J de, Blades M, Harrison RF, Hurley R, Stan- ley VC: Frequency of Mycoplasma in infertile and fertile couples. Lancet 1:1073-1075, 1974. 46. Gnarpe H, Friberg J: Mycoplasma and human reproduc- tive failure. I. The occurrence of different mycoplasmas in couples with reproductive failure. Am J Obstet Gyne- col 114:727-731, 1972. 47. Graber CD, Creticos P, Valicdenti J, Williamson HO: T-mycoplasma in human reproductive failure. Obstet Gy- necol 54:558, 1979. 48. Friberg J, Gnarpe H: Mycoplasma and human reproduc- tion. III. Pregnancy in infertile couples treated with doxy- cycline for T-mycoplasma. Am J Obstet Gynecol 116:23, 1973. 49. Stray-Pedersen B, EngJ, Reikvam TM: Uterine T-myco- plasma colonization in reproductive failure. Am J Obstet Gynecol 130:307-311, 1978. 50. Toth A, Lesser WL, Brooks C, Labriola D: Subsequent pregnancy in 161 couples treated for T-mycoplasma geni- tal tract infection. N Engl J Med 308:505-507, 1983. 51. Harrison RF, Blades M, DeLouvois J, Hurley R: Doxy- cycline treatment and human fertility. Lancet 1:605, 1975. 52. Idriss WM, Patton WC, Taymor MC: On the etiologic role of Ureaplasma urealyticum (T-mycoplasma) infection in infertility. Fertil Steril 30:293-296, 1978. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:59 (1993) (C) 1993 Wiley-Liss, Inc. Streptococcus agalactiae Streptococcus agalactiae, a unique bacterium commonly referred to as group B streptococcus (GBS), is evolving to become a major pathogen that causes a wide variety of problems across a broad spectrum of individuals. The greatest controversy over this bacterium involves the obstetrical patient. Lawyers have made this organism a centerpiece, leading their clients to believe that the presence of GBS should always be sought and detected, thereby allowing the physician the opportunity to prevent the diseases it causes. Their position has been supported by our pediatric colleagues, who recommend universal screening of all pregnant patients. These proponents of universal screening appear not to under- stand that inaccuracies surround the detection of this bacterium or that the infant may be infected prior to birth. The fact remains that universal screening cannot be implemented until an inexpensive but highly accurate method has been developed to accomplish such screening. At present, the gold standard is the use of enrichment techniques in obtaining cultures of the organism from the lower genital tract. However, no agreement has been reached as to when in pregnancy the patient should be screened. This dilemma is due both to our inability to detect extremely low numbers of colonizing bacteria and to the transitional state that characterizes some colonized patients. Rapid tests have proved to be unreliable because they are associated with a significant false- negative rate. False-positive tests are also of concern because antibiotics would be administered to individuals not needing therapy, placing them at risk for adverse reactions, some of which may be serious and even life-threatening. The recommendation of administering antibiotic therapy to all positively colo- nized laboring patients has evolved from the earlier recommendation of treating high-risk patients. In the meantime, legal pressure has created a debate on whether or not patients should be screened and all colonized patients should be treated.

recommendation of administering antibiotic therapy to all positively colo- nized laboring patients has evolved from the earlier recommendation of treating high-risk patients. In the meantime, legal pressure has created a debate on whether or not patients should be screened and all colonized patients should be treated. At the same time, large randomized, blinded studies on these specific issues are lacking. There is no doubt that this is an emotional issue from the patient's point ofview, nor is there any doubt that this bacterium should be prevented from causing infection not only in the newborn and mother, but also in the gynecologic patient scheduled for pelvic surgery. Not only has the legal profession not helped to find a scientific solution, it has caused many physicians to undertake management pro- grams that are not beneficial or safe or are not effective or cost-effective. Although we agree that the patient at risk should be managed aggressively, recommendations for mass screening should be held in abeyance until more specific, sensitive, and economical tests have been developed and proved to be reliable. Sebastian Faro Editor-in-Chief Editorial

Infectious Diseases in Obstetrics and Gynecology 1:60-64 (1993) (C) 1993 Wiley-Liss, Inc. Intrauterine Pressure Catheter in Labor: Associated Microbiology Phillip Pinell, Sebastian Faro, Scott Roberts, Sy Le, Maurizio Maccato, and Hunter Hammill Department of Obstetrics and Gynecology, Baylor College ofMedicine, Houston (P.P., M.M., H.H.), Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas (S.R.), TX, Department of Gynecology and Obstetrics, University ofKansas School ofMedicine, Kansas City (S.F.), KS, and Department ofObstetrics and Gynecology, Group Medical Health Associates, Tucson (S.L.), AZ ABSTRACT Objective: The purpose of this study was to determine if bacterial growth occurred in the amniotic fluid of laboring women. Twenty patients who required an intrauterine pressure catheter (IUPC) during labor were studied. Amniotic fluid samples were aspirated during labor and at the time of delivery. Methods: IUPCs were placed in laboring patients for a variety ofreasons. Cervical cultures were taken prior to insertion ofan IUPC. After the IUPC was placed, amniotic fluid cultures were taken both at the time ofplacement and 30 minutes prior to delivery. These cultures were sent for aerobic, anaerobic, Mycoplasma, and Ureaplasma cultures. Results: The increase in bacterial concentration from the initial sample to the final sample was statistically significant (P < 0.01) for both aerobes and anaerobes. Amniotic fluid samples demon- strated a median of 0 bacterial species per patient on initial collection and 2 bacterial species per patient in final collection. The mean count of cfu for aerobes in the initial amniotic samples was 3.5 104, compared to that ofthe second samples, which was 1.4 105. The mean count ofcfu for anaerobes in the initial amniotic fluid samples was 4.1 102, compared to that of the second samples, which was 8.0 103. Only 3 of20 patients developed chorioamnionitis, with only I patient having an increased number ofbacterial species significantly higher than the median. Although 80% ofpatients had a colony count >102 cfu/cc, only 19% of this group developed chorioamnionitis.

compared to that of the second samples, which was 8.0 103. Only 3 of20 patients developed chorioamnionitis, with only I patient having an increased number ofbacterial species significantly higher than the median. Although 80% ofpatients had a colony count >102 cfu/cc, only 19% of this group developed chorioamnionitis. Conclusions: The number of bacterial species and colony counts increased significantly during labor, but this factor alone was not enough to cause chorioamnionitis in a significant number of patients. (C) 1993 Wiley-Liss, Inc. KEY woPs intrauterine pressure catheter, bacterial colonization, amniotic fluid, chorioamnionitis, endomyometritis ntrauterine pressure catheters (IUPCs) are fre- quently employed to assess labor by monitoring the intensity and frequency of uterine contractions. Recently, the IUPC has been utilized to perform amnioinfusion in situations where the amniotic fluid is significantly decreased to prevent cord compres- sion during uterine contractions. The IUPC may also provide a potential route for endogenous bacte- ria of the lower genital tract to gain access to the uterine cavity. 2'3 Miller et al.4 found no differ- Address correspondence/reprint requests to Dr. Phillip Pinell, Department of Obstetrics and Gynecology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Received October 2, 1992 Clinical Study Accepted July 6, 1993 IUPC IN LABOR AND MICROBIOLOGY PINELL ET AL. TABLE I. Demographics of patient population (N 20) Race Black 3 Hispanic 13 White 3 Pakistani Age Mean 26.5 years Mode 24 years Range 14-40 years Gravidity Mean 3.2 Mode 2 Range I-9 Parity Mean 1.2 Mode 0 Range 0-6 Vaginal exams Mean 6.5 Mode 6 Range 3-10 Delivery Vaginal (spontaneous) 12 Vaginal (forceps) 4 Cesarean section 4 No. of hours of rupture of membranes Mean II hours 38 minutes Mode 6.5 hours Range 2 hours 10 minutes- 33 hours 29 minutes ences among culture results of amniotic fluid sam- ples obtained by aspiration through an IUPC, am- niocentesis, and collection at the time of cesarean section. However, being a qualitative bacteriologic study, it did not reveal the dynamic relationship that may occur between the bacteria and amniotic fluid with respect to time. The relationship between bacterial colonization of the intrauterine environment and bacterial growth during labor in nulliparous women has been previously described.

a qualitative bacteriologic study, it did not reveal the dynamic relationship that may occur between the bacteria and amniotic fluid with respect to time. The relationship between bacterial colonization of the intrauterine environment and bacterial growth during labor in nulliparous women has been previously described. 5 This study was designed to identify and quantitate the bacterial flora of the intrauterine cavity during labor in patients requir- ing an IUPC and to examine the effect of time on bacterial colonization. MATERIALS AND METHODS This study was conducted on Baylor College of Medicine's Obstetrics Service at Ben Taub General Hospital, a county hospital that serves the indigent and lower socioeconomic population of Harris County, TX. IUPCs were placed in laboring pa- tients for one of the following reasons: dysfunc- tional labor (50% of patients), trial of labor with history of cesarean section (25%), amnioinfusion on the basis of frequent occurrence of variable de- celerations of the fetal heart rate (15%), or the inability to adequately monitor uterine contractions (10%). Prior to placement ofthe IUPC, a cervical spec- imen was collected with a sterile cotton-tipped ap- plicator and placed in anaerobic brain-heart infu- sion broth transport media. After the IUPC was in place, amniotic fluid was aspirated for the culture of bacteria. The initial 5 cc of amniotic fluid aspi- rated was discarded. Then, an additional 3 cc was aspirated and placed in anaerobic brain-heart infu- sion broth transport media. A second specimen was obtained 30 minutes prior to delivery. All speci- mens were stored at 4C immediately after collec- tion and processed within 24 hours. Remel blood agar, chocolate agar, and McConkey's medium were inoculated for the isolation ofaerobic bacteria. The following media were inoculated for the isola- tion of anaerobic bacteria: CDC blood, KVKDK, and PEACDC agar. A-7 medium was inoculated in an attempt to isolate Mycoplasma and Ureaplasma. Qualitative and quantitative bacteriology were per- formed on all specimens except Mycoplasma and Ureaplasma as previously described.6'7 The number of vaginal examinations were corded, commencing at the time of IUPC place- ment until the time of the second amniotic fluid collection. For the purposes of this study, chorioamnionitis was defined as an infection of the chorioamniotic membranes and the amniotic cavity clinically man- ifested by maternal fever >38C, uterine tender- ness, and/or foul-smelling amniotic fluid.

time of IUPC place- ment until the time of the second amniotic fluid collection. For the purposes of this study, chorioamnionitis was defined as an infection of the chorioamniotic membranes and the amniotic cavity clinically man- ifested by maternal fever >38C, uterine tender- ness, and/or foul-smelling amniotic fluid. Postpar- tum endomyometritis was considered to be an infection of the endometrium or decidua with ex- tension to the myometrium with clinical findings of 2 readings of maternal temperature elevation >38C after the first 24-hour postpartum period, uterine tenderness, and leukocytosis [white blood cell counts (WBCs) > 15,000]. Statistics The number of bacterial isolates was expressed as means -+ standard deviation, and the median num- ber of isolates for each group, aerobes and anaer- obes, was determined. The Student's t-test was used INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 61 IUPC IN LABOR AND MICROBIOLOGY PINELL ET AL. TABLE 2. Cervical cultures at time of IUPC insertion Aerobes No. positive Species cultures % Anaerobes Species No. positive cultures % Staphylococcus not aureus 14 23.7 Diphtheroids 14 23.7 Lactobacillus 6 I0. S. aureus 2 3.4 S. agalactiae 3 5. Enterococcus faecalis 4 6.8 Gardnerella vaginalis 13 22. Pseudomonas aeruginosae 1.7 Klebsiella pneumoniae 1.7 Acinetobacter 1.7 Totals 59 Peptostreptococcus anaerobius 7.7 S. morbillorum 3 23 P. magnus 7.7 Propionibacterium 7.7 Clostridium sp. 7.7 Bacteroides bivius 7.7 B. intermedius 7.7 B. oralis 2 15.4 B. melaninogenicus 7.7 Fusobacterium 7.7 13 to compare mean numbers of bacterial isolates and concentrations between the initial and second sam- ples. A two-tailed test was employed with P < 0.05 considered significant. The median numbers ofiso- lates were compared between the initial and second samples using the Wilcoxon rank-sum analysis. RESULTS The demographic data of the 20 patients in this study are presented in Table 1. Twelve patients (60%) had a spontaneous vaginal delivery, 4 (20%) had forceps vaginal delivery, and 4 (20%) required cesarean section. Two of the patients who were delivered by cesarean section and one who was de- livered by low forceps developed chorioamnionitis (3/20 or 15%). All 3 ofthese patients had oral body temperatures >38C, elevated WBCs, and uterine fundal tenderness. The WBCs on admission for patient nos. 5, 10, and 14 were 9,200, 11,300, and 9,700, respectively. The WBCs at the time chorio- amnionitis was diagnosed were 32,300, 17,300, and 12,000, respectively.

itis (3/20 or 15%). All 3 ofthese patients had oral body temperatures >38C, elevated WBCs, and uterine fundal tenderness. The WBCs on admission for patient nos. 5, 10, and 14 were 9,200, 11,300, and 9,700, respectively. The WBCs at the time chorio- amnionitis was diagnosed were 32,300, 17,300, and 12,000, respectively. No patient developed postpartum endomyometritis. The spectrum of bacterial species from the cer- vical cultures at the time of IUPC insertion is shown in Table 2. The bacterial flora of the amni- otic fluid collected at the beginning and near the time of delivery are listed in Table 3. Quantitative results from amniotic fluid samples at the two col- lecting times are summarized in Table 4. Ureaplasma was isolated from the cervix of 80% (16/20) of the patients. Forty percent (8/20) of the TABLE 3. Amniotic fluid cultures No. of positive cultures At time of IUPC placement No. Within 30 minutes of delivery No. Aerobes Lactobacillus 4 Diphtheroids 3 Staphylococcus 3 not oureus Enterococcus faecalis S. agalactiae Klebsiella pneumoniae Gardnerella vaginalis 2 otals 15 Anaerobes S. morbillorum 2 Bacteroides intermedius Totals 3 Lactobacillus 7 Diphtheroids 6 Staphylococcus 4 not oureus S. oureus E. foecolis K. pneumonioe G. vaginalis 6 Escherichia coli 2 28 S. morbillorum 2 S. subterminale B. oralis B. urealyticus B. bivius Fusobacterium Veillonella sp. 8 amniotic fluid cultures from the initial collection grew Ureap]asma, compared to 45% (9/20) of the final fluid samples. Mycoplasma was isolated from the cervix of 15% (3/20) of patients. Five percent (1/20) of the amniotic fluid samples at the initial collection and final collection had positive Myco- plasma cultures. The number of different aerobic and anaerobic species per patient from the first and second amni- 62 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY IUPC IN LABOR AND MICROBIOLOGY PINELL ET AL. TABLE 4. Quantitative analysis of amniotic fluid bacteria Patient Initial collection no.

had positive Myco- plasma cultures. The number of different aerobic and anaerobic species per patient from the first and second amni- 62 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY IUPC IN LABOR AND MICROBIOLOGY PINELL ET AL. TABLE 4. Quantitative analysis of amniotic fluid bacteria Patient Initial collection no. Aerobes Anaerobes Mycoplasma Ureaplasma Aerobes Final collection Anaerobes Mycoplasma Ureaplasma 5x 10zLB 2 102BI 2 No growth (+) 3 to GV (+) 3.5 103 SNA 2 103 DIP 4 No growth (+) 5 No growth 6 4 103KP 7 2 104LB 104 DIP 7 x 103 SNA 8 No growth 9 5 x l0 LB 1.2 103 GBBS 10 3 X 104 DIP 6 103 GV IX 104 LB 4 103 EF 12 4 x 103 SNA 13 No growth 14 No growth 16 No growth 17 No growth 18 No growth 19 No growth 20 No growth 2.4 x 103 LB No growth 3 102 LB 1.2 X 104 SM (+) 2.2 x 103 DIP 6 x 102 SNA 5 x 103 GV ('+') J0 GV 2 X 10 V (-J-) (--) 5 x 102 SNA 2 x 102 DIP 5 X 104 LB IX 104 LB (+) l0 GV 10 GV l0 F (+) 5 X 104 KP 2.4 104 LB IX 104 SNA No growth 8x 103LB 5 l0 GBBS 7x 103SM (+) 3 103 DIP 2x 103SM (+) 1.2 x 104 GV x 103LB (+) 2 x 103 EF x 104 BIV (+) 3 x 103 DIP No growth (+) 8 x 10 LB (+) Ix I0 DIP 5 No growth (+) 2 x 103 SA 9 x 103 CS (+) 1.4 x 104 DIP 5 x 103 GV No growth 2 x 103 BU x 103 SM (+) 2 x 103 SNA 5 x 103 BO (+) Ix 103 GBBS No growth Ix 10 EC aBI Bacteroides intermedius; BIV B. bivius; BO B. oralis; BU B. urealyticus; CS Clostridium sp.; DIP Diphtheroids; EC Enterococcus; EF E. faecalis; F Fusobacterium; GBBS group B beta-strep; G Gardnerella vaginalis; KP Klebsiella pneumoniae; LB Lactobacillus; SA Staphylococcus aureus; SM S. morbillorum; SNA Staphylococcus not aureus; V Veillonella sp. otic fluid samples varied from 0 to 4 species (Table 4). The mean number of bacterial species in the initial fluid samples was 0.9 and the median was 0. The mean number of bacterial species in the final fluid samples was 1.8 and the median was 2. The mean count ofcfu for aerobes in the initial amniotic fluid samples was 3.5 104, compared to that of the second sample, which was 1.4 l0s. This increase in bacterial count was statistically signifi- cant (P < 0.01). Likewise, the mean count of anaerobic cfu from the first collection was 4.1 102, compared to that of the second sample, which was 8.0 x 103. This increase was also statis- tically significant (P < 0.01). DISCUSSION IUPCs are used in laboring patients for monitor- ing purposes and for performing amnioinfusions.

< 0.01). Likewise, the mean count of anaerobic cfu from the first collection was 4.1 102, compared to that of the second sample, which was 8.0 x 103. This increase was also statis- tically significant (P < 0.01). DISCUSSION IUPCs are used in laboring patients for monitor- ing purposes and for performing amnioinfusions. Because they are placed transvaginally, the possibil- ity exists ofintroducing vaginal flora into the amni- otic cavity. The lower genital tract, especially the vagina, represents a unique microsphere of micro- biological life. Surveying the microbiology of the genital tract during labor is important to determine INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 63 IUPC IN LABOR AND MICROBIOLOGY PINELL ET AL. the dynamics of the microbiological ecology of am- niotic fluid, e.g., which organisms become domi- nant and their relationship to the development of chorioamnionitis and endomyometritis. In patients with acute chorioamnionitis, a polymicrobial con- tamination of the amniotic cavity occurs following rupture ofmernbranes and labor. Gilstrap and Cun- ningham9 reported a mean per patient of 2.5 mi- croorganisms, 72% of which were gram-positive cocci. In this study, a mean of 0.9 bacterial species was noted, and final samples demonstrated a mean of 1.8 bacterial species per patient. Of the 3 of 20 patients who developed chorioamnionitis in this study, only patient had bacterial species numbers significantly higher than the means noted. Inoculum size is a significant factor in the devel- opment of chorioamnionitis and postpartum endo- myometritis. Both Miller et al.4 and Gibbs et al. 10 demonstrated that intraamniotic infection occurred with greater than 102 c(u. While 80% of the pa- tients in our study had colony counts >10z cfu, only 19% of this group developed chorioamnioni- tis. Thus, colonization alone appears not to be the only significant factor. According to Larsen et al., 1,12 the amniotic fluid contains an active bacterial in- hibitor. It was noted in our study that growth oc- curred despite this amniotic fluid inhibitor. Al- though bacterial growth occurs, clinical evidence of an infection does not necessarily ensue. Clinical infection may depend on a number of issues, such as pathogenicity (virulence) and tissue invasion. Concentration may also play a role in this process. In conclusion, our study demonstrated that both the number of bacterial species and the quantitative cfu increase significantly during labor.

necessarily ensue. Clinical infection may depend on a number of issues, such as pathogenicity (virulence) and tissue invasion. Concentration may also play a role in this process. In conclusion, our study demonstrated that both the number of bacterial species and the quantitative cfu increase significantly during labor. This factor alone was not enough to result in chorioamnionitis or postpartum endomyometritis in all patients. REFERENCES 1. Miyazola FS, Taylor NA: Saline amnioinfusion for relief of variable or prolonged decelerations. Am J Obstet Gy- necol 146:670-685, 1983. 2. Levison ME, Corman LC, Carrington ER, Kaye D: Quantitative microflora of the vagina. Am J Obstet Gy- necol 127:80-85, 1977. 3. Bartlett, JG, Moon NE, Goldstein PR, Goren B, Onder- donk AB, Polk BF: Cervical and vaginal flora: Ecologic niches in the lower female genital tract. Am J Obstet Gynecol 130:658-661, 1978. 4. Miller JM Jr, Hill GB, Welt SI, Pupkin MJ: Bacterial colonization of amniotic fluid in the presence of ruptured membranes. Am J Obstet Gynecol 137:451-458, 1980. 5. Silver RK, Gibbs RS, Castillo M: Effect of amniotic fluid bacteria on the course oflabor in nulliparous women at term. Obstet Gynecol 68:587-592, 1986. 6. Phillips LE, Faro S, Martens MG, et al.: Postcesarean microbiology of high risk patients treated for endometri- tis. Curr Ther Res Clin Exp 42:1157-1165, 1987. 7. Faro S, Phillips LE, Baker JL, Goodrich KH, Turner RM, Riddle GD: Comparative efficacy and safety of mezlocillin versus cefoxitin versus clindamycin plus gen- tamicin in the treatment of patients with postpartum en- dometritis. Obstet Gynecol 69:760-766, 1987. 8. Gilstrap LC, Cunningham FG: Microflora of the genital tract. In Gilstrap LC, Faro S (eds): Infections in Preg- nancy. New York: Wiley-Liss, pp 1-5, 1990. 9. Gilstrap LC, Cunningham FG: Single drug regimen proves effective in postcesarean infection. Contemp Ob/ Gyn 14:68-75, 1979. 10. Gibbs RS, Blanco JC, St Clair PJ, Castaneda YS: Quan- titative bacteriology of amniotic fluid from patients with clinical intraamniotic infection at term. J Infect Dis 145: 1-8, 1982. 11. Larsen B, Snyder IS, Galask RP: Bacterial growth inhi- bition by amniotic fluid. I. In vitro evidence for bacterial growth inhibiting activity. Am J Obstet Gynecol 119: 492, 1974. 12. Larsen B, Snyder IS, Galask RP: Bacterial growth inhi- bition by amniotic fluid. II. Reversal of amniotic fluid bacterial growth inhibition by addition of a chemically defined medium.

h inhi- bition by amniotic fluid. I. In vitro evidence for bacterial growth inhibiting activity. Am J Obstet Gynecol 119: 492, 1974. 12. Larsen B, Snyder IS, Galask RP: Bacterial growth inhi- bition by amniotic fluid. II. Reversal of amniotic fluid bacterial growth inhibition by addition of a chemically defined medium. Am J Obstet Gynecol 119:497, 1974. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:65-70 (1993) (C) 1993 Wiley-Liss, Inc. Does Method of Placental Removal or Site of Uterine Incision Repair Alter Endometritis After Cesarean Delivery? Everett F. Magann, Mark K. Dodson, Robert L. Harris, Randall C. Floyd, James N. Martin, Jr., and John C. Morrison Department ofObstetrics and Gynecology, University ofMississippi Medical Center, Jackson, MS ABSTRACT Objective: his investigation was undertaken to evaluate the relationship between postcesarean en- dometritis and (1) method of placental removal and (2) site for uterine repair. Methods: This prospective, randomized study included 120 patients who underwent primary or repeat abdominal delivery for arrest of progress in labor, fetal distress, or breech presentation. Parturients were divided into four groups: Imspontaneous placental detachment, in situ uterine repair; IImspontaneous placental detachment, exteriorized uterine repair; IIImanual placental removal, in situ uterine repair; and IVmmanual placental removal, exteriorized uterine repair. Prophylactic antibiotics were not used. Results: Endometritis was significantly increased in the manual removal/exteriorized uterine repair group versus all the other groups including the spontaneous removal in situ (group I, P 0.012), the spontaneous removal/exteriorized repair group (group II, P 0.034), and the man- ual removal/in situ repair group (group III, P 0.043). Comparison ofgroup IV (manual removal/ exteriorized repair) with the combined groups I, II, and III (spontaneous removal/in situ repair, spontaneous removal/exteriorized repair, and manual removal/in situ repair) was significantly dif- ferent (P 0.005). Prior to delivery, use of an internal monitoring system, skill of the operating surgeon, and type of anesthesia were similar among groups. Conclusions: The findings of this investigation suggest that, when other known causes of infec- tious morbidity are constant, manual placental removal in association with exteriorization for uterine repair significantly increases postcesarean endometritis. (C) 1993 Wiley-Liss, Inc.

anesthesia were similar among groups. Conclusions: The findings of this investigation suggest that, when other known causes of infec- tious morbidity are constant, manual placental removal in association with exteriorization for uterine repair significantly increases postcesarean endometritis. (C) 1993 Wiley-Liss, Inc. KEY woP,S Spontaneous expulsion, manual extraction, uterine position }ostpartum endometritis, defined as a maternal temperature of > 38C on two separate occa- sions 6 hours apart after the first 24 hours with uterine tenderness and/or foul-smelling lochia, is a common postoperative complication ofcesarean sur- gery. The incidence ofthis complication in an indi- gent patient population ranges between 20% and 85%. A number of operative and obstetric factors are related to the development of infection follow- ing cesarean birth. Obstetric factors that are thought to contribute to the development of postoperative endometritis include: (1) the duration of labor2'3; (2) rupture of the membranes and the length of time between membrane rupture and operative de- livery4'5; (3) the number of vaginal examinations6; (4) the use of internal fetal scalp and uterine pres- sure monitoring devices7; and (5) indigent patients regardless of race. Operatives factors that have an impact on postcesarean infectious morbidity in- clude: (1) the skill of the operating surgeon6; (2) Address correspondence/reprint requests to Dr. Everett F. Magann, Department ofObstetrics and Gynecology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216-4505. Clinical Study Received November 18, 1992 Accepted May 5, 1993 INFECTIOUS MORBIDITY FOLLOWING CESAREAN SECTION MAGANN ET AL. procedure length > hour9; (3) type of anesthe- sia10; (4) blood loss > 800 m19; and (5) maternal obesity. 11 There is a diversity of published opinion in re- gard to a recommended method for removal of the placenta during cesarean surgery. Should it be ex- pelled spontaneously12 or manually extracted?3 Similarly, two methods of uterine incision repair are recommended, one, in situ tissue approxima- tion to avoid trauma to the adnexa, 14 or two, uter- ine exteriorization to facilitate wound closure. s Hershey and Quilligan6 demonstrated no increase in the incidence ofpostoperative infectious morbid- ity following cesarean delivery with external uter- ine repair. However, the associated method of pla- cental removal was not addressed.

to the adnexa, 14 or two, uter- ine exteriorization to facilitate wound closure. s Hershey and Quilligan6 demonstrated no increase in the incidence ofpostoperative infectious morbid- ity following cesarean delivery with external uter- ine repair. However, the associated method of pla- cental removal was not addressed. The purpose ofthis investigation was to evaluate the impact of the method of placental removal (spontaneous or manual) and the site of uterine repair (in situ or exteriorized) on the incidence of infectious morbidity following cesarean birth. MATERIALS AND METHODS This prospective, randomized study included 120 consecutive patients who had a cesarean delivery for obstetric reasons between October 1991 and Janu- ary 1992. Exclusion factors were the presence of chorioamnionitis at the time of cesarean birth, pa- tient refusal to participate following informed con- sent, and antenatal treatment with steroids or insu- lin. A sample size and power analysis were done prior to initiating this study. It was calculated that, to reduce the rate of endometritis from 40% to 20%, 42 women would be needed in each arm of the study. After 30 women had been evaluated in each arm, an interim analysis was performed. Be- cause a statistically significant difference was al- ready evident, no further patients were enrolled into this study. All patients in this study agreed to participate and signed an informed consent form that was ap- proved by the Committee on Human Investigation at the University of Mississippi Medical Center. The informed consent made patients aware that prophylactic antibiotics would not be used so that the unique relationship between placental manage- ment and site ofuterine repair could be targeted for evaluation.. Study subjects were placed into one of four groups: Ispontaneous placental detach- ment/in situ uterine repair; IImspontaneous pla- cental detachment/exteriorized uterine repair; III manual placental removal/in situ uterine repair; or IV--manual placental removal/exteriorized uterine repair. Random group assignment was ensured by card selection from sealed opaque envelopes with group appointment derived from a random num- ber table. The population cared for by the obstetric service at the University of Mississippi Medical Center is predominantly an indigent population (79% below the 125 percentile of poverty level).

up assignment was ensured by card selection from sealed opaque envelopes with group appointment derived from a random num- ber table. The population cared for by the obstetric service at the University of Mississippi Medical Center is predominantly an indigent population (79% below the 125 percentile of poverty level). The profile of patients was not significantly different between those who underwent repeat cesarean delivery without trial of labor and those who labored prior to ab- dominal surgery. The incidence of postcesarean en- dometritis was approximately 33 % in this popula- tion. The indications for operative delivery were fetal distress, arrest of progress in labor, repeat cesarean surgery, breech presentation, and severe preeclamp- sia without thrombocytopenia or coagulopathy. No prophylactic antibiotics were used, none of the pa- tients were on antibiotics for urinary tract infection or group B streptococcus therapy, and the 12 pa- tients with chorioamnionitis on antibiotics at the time of cesarean birth were excluded. Chromic su- ture material was employed on the uterus, polygly- colic suture on the fascia, and skin clips on each operation procedure. After delivery of the infant and according to previously determined group as- signment, the placenta was either manually removed or allowed to detach spontaneously with gentle cord traction and uterine massage, and then the uterus was left in situ or exteriorized for uterine incision closure. All patients had their uterus cleansed with a gauze sponge after the placenta had been removed. All uterine incisions were closed with a double- layer closure using chromic suture. The pelvis was irrigated with saline prior to abdominal wound closure in all patients. The diagnosis of infectious morbidity was made by (1) an elevation of the maternal temperature of > 38C on two separate occasions 6 hours apart the first 24 hours with (2) uterine tenderness, and/or (3) a foul-smelling lochia. Once febrile morbidity was identified, complete physical examination in- cluding a pelvic examination was performed. After urine and blood but not endometrial cultures were 66 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY INFECTIOUS MORBIDITY FOLLOWING CESAREAN SECTION MAGANN ET AL. TABLE I. Demographic characteristics Group Age Race Nulliparas/multiparas Length of Type of cesarean procedure (primary/repeat) (min) I. Spontaneous in situ 24.6 -+ 4.4 19/7/4 14/I 6 II. Sponraneous exteriorized 22.6 -+ 4.7 22/7/I 13/I 7 III.

AND GYNECOLOGY INFECTIOUS MORBIDITY FOLLOWING CESAREAN SECTION MAGANN ET AL. TABLE I. Demographic characteristics Group Age Race Nulliparas/multiparas Length of Type of cesarean procedure (primary/repeat) (min) I. Spontaneous in situ 24.6 -+ 4.4 19/7/4 14/I 6 II. Sponraneous exteriorized 22.6 -+ 4.7 22/7/I 13/I 7 III. Manual in situ 25.2 6.9 21/6/3 16/14 IV. Manual exteriorized 22.5 -+ 8.5 23/4/3 15/15 P value NS NS NS 22/8 34.6 __II 24/6 32.5 _15 23/7 38.2 8.5 22/8 34.8 _I0 NS NS Black/Caucasian/American Indian. NS, not significant. obtained, patients with suspected endometritis were treated with intravenously administered triple anti- biotics consisting of ampicillin, 2 g every 6 hours; gentamicin (Garamycin, Schering Corporation, Ke- nilworth, NJ), 1.5 mg/kg every 8 hours; and clin- damycin, 900 mg every 8 hours. Endometrial cul- tures were not obtained because they yielded inconclusive results associated with contaminated specimens obtained transcervically. All patients were placed on triple antibiotics, and uterine cul- tures did not alter antibiotic management. Surgery was primarily performed by a second- year Ob-Gyn resident with the assistance ofeither a fourth-year Ob-Gyn resident or maternal-fetal med- icine fellow. Labor and delivery personnel acted as the scrub and circulating nurses on all surgeries. All operative procedures were performed between 0700 and 2300 hours in order that one of the two authors (E.F.M. or M.K.D.) could be present to calculate blood loss. The duration of labor, length of rupture of the membranes prior to delivery, number of vaginal examinations, and use of an internal monitoring system were recorded for all patients. The internal monitoring system consisted of a fetal scalp electrode and an open-ended, fluid- filled catheter inserted through the cervix and at- tached to a strain-gauge transducer. The types of abdominal and uterine incision were recorded for each operative case. Each operative procedure was assessed for length of surgery, type of anesthesia used, and amount of blood loss. The blood loss was determined by a modification of the volumetric technique ofWangensteen, 17 by which blood in the suction apparatus prior to irrigation is measured and the laparotomy pads and sponges are weighed. Statistical analysis was performed by corrected chi-square, Fisher's exact test, and Wilcoxon test. A P value of < 0.05 was considered statistically significant.

olumetric technique ofWangensteen, 17 by which blood in the suction apparatus prior to irrigation is measured and the laparotomy pads and sponges are weighed. Statistical analysis was performed by corrected chi-square, Fisher's exact test, and Wilcoxon test. A P value of < 0.05 was considered statistically significant. RESULTS One hundred twenty women were enrolled in this study, with 30 women assigned to each of the four arms. Due to the findings of elevated maternal temperature, leukocytosis with a left shift, and foul- smelling amniotic fluid at the time of cesarean sur- gery with subsequent culture confirmation of infec- tion done at the time of cesarean section, the investigators excluded several patients from the study. These included three women from the spon- taneous removal/in situ repair group (group I), four from the spontaneous removal/extea'iorized re- pair group (group II), two from the manual removal/in situ repair group (group III), and three from the manual removal/exteriorized repair group (group IV). The demographics of age, race, gra- vidity, operative time, indications for cesarean sec- tion, and number ofrepeat cesarean operations with concurrent bilateral tubal ligation were similar among groups (Table 1). The incidence of postce- sarean endometritis was 33% (36/108) overall. En- dometritis occurred in repeat cesarean deliveries without labor in 15.8% (3/19), in contrast to 40% (4/10) in repeat cesarean delivery patients who ex- perienced labor. Other deliveries without labor in- cluded 14 women with breech presentations, 3 (21.4%) of whom developed postpartum endo- metritis. Excluding women with chorioamnionitis (3, 4, 2, 3, respectively, in groups I-IV) and comparing infected and noninfected women in groups I-IV by chi-square, we calculated a statistically significant difference (P 0.04) with three degrees of free- dom. Comparison ofgroup IV (15 infected, 12 not INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 67 INFECTIOUS MORBIDITY FOLLOWING CESAREAN SECTION MAGANN ET AL. Infection No Infection / 25 / 10 0 Spon-In Situ Spon Ext Man in Situ Man Ext Group Group II Group III Group IV Fig. I. Infectious morbidity in each of the four patients groups (group 22%, group II 27%, group III 29%, and group IV 55%). Patients with chorioamnionitis were excluded from the study population. P < 0.005 for groups I, II, and III compared with group IV. Spon, spontaneous; Man, manual; Ext, exteriorized.

III Group IV Fig. I. Infectious morbidity in each of the four patients groups (group 22%, group II 27%, group III 29%, and group IV 55%). Patients with chorioamnionitis were excluded from the study population. P < 0.005 for groups I, II, and III compared with group IV. Spon, spontaneous; Man, manual; Ext, exteriorized. infected), group I (6 infected, 21 not infected), and group III (8 infected, 20 not infected) produced P values of 0.012, 0.034, and 0.043, respectively. Comparison of similar data in groups I, II, and III by chi-square reflected a P value of 0.9. Since there was no difference among women in those three groups, they can be combined and compared with women in group IV. Comparison ofgroup IV with the combined groups I, II, and III, showed a sig- nificant difference between the two populations (rel- ative risk 3.6; 95% confidence interval 1.3-9.7, P < 0.005) (Fig. 1). The duration oflabor, length ofrupture ofmem- branes prior to delivery, use of an internal moni- toring system, and number ofvaginal examinations were similar among groups (Table 2). The. type of uterine and abdominal incision, the choice of anes- thesia, and the expertise of the operating surgeon were not significantly different among the four categories. Blood loss between groups was significantly in- creased in the groups with manual removal of the placenta (groups III and IV; 1,342 --+ 549 cc and 1,146 280 cc, respectively), compared with the groups with spontaneous expulsion of the placenta (groups I and II; 640 234 cc and 644 235 cc, respectively) (P < 0.001). No patient was trans- fused with any blood products. Blood cultures were positive in 6% of the pa- tients with postcesarean infectious morbidity and were not significantly different among groups. Two patients did not respond to triple antibiotic therapy and, with a negative CT scan to rule out a pelvic abscess, were started on heparin therapy for sus- pected septic pelvic thrombophlebitis with resolu- tion of the febrile morbidity. DISCUSSION The most significant finding ofthis investigation is the demonstration ofan increased frequency ofpost- operative endometritis in women in whom the pla- centa was manually removed and the uterus exteri- orized for repair. Manual placental removal has been shown to increase the rate of postcesarean en- 68 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY INFECTIOUS MORBIDITY FOLLOWING CESAREAN SECTION MAGANN ET AL. TABLE 2.

perative endometritis in women in whom the pla- centa was manually removed and the uterus exteri- orized for repair. Manual placental removal has been shown to increase the rate of postcesarean en- 68 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY INFECTIOUS MORBIDITY FOLLOWING CESAREAN SECTION MAGANN ET AL. TABLE 2. Known factors for infectious morbidity Group Mean duration Mean Mean duration of rupture Internal Mean number maternal weight of labor of membranes monitoring of vaginal (kg) (hr) (hr) (yes/no) examinations I. Spontaneous in situ 87.09 -+ 19.96 13.8 -+ 9.4 II. Spontaneous exteriorized 88.45 -12.25 13.3 -+ 8.7 III. Manual in situ 92.99 -+ 4.08 16.2 _+ 8.5 IV. Manual exteriorized 9 I. 17 +- 4.54 19.8 -+ 17.9 P value NS NS 22.7 -15.5 13/9 5.0 -+ 3.5 14.8 -+ 8.9 I/I 4.7 -+ 2.2 15.3 -+ 7.7 I/I 5.6 -+ 2.8 18.0 -+ 17.2 12/8 4.5 -+ 1.4 NS NS NS NS, not significant. dometritis even when prophylactic antibiotics were administered. 18 Because cesarean delivery6 is the most important cause ofpostpartum infectious mor- bidity, these findings could influence the reduction of postcesarean infections. Because the number of patients delivered abdominally in this country con- tinues to range between 20% and 25%, a large population of patients with infectious morbidity will have prolonged hospital stays, exposure to an- tibiotics with potentially serious side effects, and possible long-term consequences on overall mater- nal health. Many factors contribute to cesarean- related infectious morbidity, including: socioeco- nomic status of the patient, length of the operative procedure, number of vaginal examinations, ma- ternal weight, amount of operative blood loss, and 2--10 skill of the operating surgeon. The present study comprises a group of women predominantly of low socioeconomic status. The maternal weight, duration oflabor, time ofrupture ofthe membranes prior to operative delivery, num- ber of vaginal examinations, type of uterine and abdominal incisions, and length of the operative procedure were similar among all four groups. However, blood loss was significantly increased in association with manual removal of the placenta (groups III and IV) in comparison with spontane- ous placental removal with gentle cord traction (groups I and II, P < 0.001), although no patient received blood transfusion.

procedure were similar among all four groups. However, blood loss was significantly increased in association with manual removal of the placenta (groups III and IV) in comparison with spontane- ous placental removal with gentle cord traction (groups I and II, P < 0.001), although no patient received blood transfusion. Operative blood loss in excess of 800 ml is associated with an increased frequency of postpartum infections.9 None of our 120 patients received a blood transfusion, and the group with the greatest mean blood loss (group III) in this study did not have a significantly increased infection rate. Instead, it was the manual removal/ exteriorized repair group (group IV), with an aver- age blood loss 200 ml less than that of group III, who had a significant increase in the frequency of postcesarean endometritis. In contrast to the findings in this investigation, Hershey and Quilligan16 did not report increased infectious morbidity in women in whom the uterus was exteriorized for uterine incisional repair. Since the method of placental removal was not addressed by those authors, it is unclear whether that was a factor in their findings. We also did not observe increased infectious morbidity unless the placenta was removed manually in addition to exterioriza- tion of the uterus for repair. Which ofthe factors ofplacental removal method or location of uterine repair are most important toward the development of infectious morbidity? If uterine exteriorization alone increases the rate of infection, then the group in which the placenta was spontaneously expelled and the uterus was exterior- ized for repair (group II) would be expected to have an increased rate. If manual removal of the placenta alone caused an increased rate of infection, then the rates would have been increased in group III (manual removal of the placenta, in situ uterine repair). We observed increased endometritis only when the placenta was removed manually in combi- nation with uterine exteriorization for repair. Thus, the findings of this investigation suggest that, when other known causes of endometritis are equal among groups, manual removal of the pla- centa in association with exteriorization ofthe uterus for repair significantly increases postcesarean sec- tion endometritis. ACKNOWLEDGMENT This work was supported in part by the Vicksburg Hospital Medical Foundation. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 69 INFECTIOUS MORBIDITY FOLLOWING CESAREAN SECTION MAGANN ET AL. REFERENCES 1.

xteriorization ofthe uterus for repair significantly increases postcesarean sec- tion endometritis. ACKNOWLEDGMENT This work was supported in part by the Vicksburg Hospital Medical Foundation. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 69 INFECTIOUS MORBIDITY FOLLOWING CESAREAN SECTION MAGANN ET AL. REFERENCES 1. Faro S: Infectious disease relations to cesarean section. Obstet Gynecol Clin North Am 15:685-695, 1988. 2. D'Angelo LJ, Sokol RJ: Time related peripartum deter- minates of postpartum morbidity. Obstet Gynecol 55: 319-323, 1980. 3. Gibbs RS: Clinical risk factors for puerperal infection. Obstet Gynecol 55:178-183, 1980. 4. Gilstrap LC, Cunningham FG: The bacterial pathogene- sis ofinfection following cesarean section. Obstet Gynecol 53:545-549, 1979. 5. Page FO, Howard PR, Martin JN, Martin RW, Rivlin ME, Morrison JC: High risk factors for cesarean febrile morbidity. J Miss State Med Assoc 28:27-29, 1987. 6. Rehu M, Nilsson CG: Risk factors for febrile morbidity associated with cesarean section. Obstet Gynecol 56:269- 273, 1980. 7. Hagen D: Maternal febrile morbidity associated with fetal monitoring and cesarean section. Obstet Gynecol 46:260-262, 1975. 8. Gibbs RS: Infection after cesarean section. Clin Obstet Gynecol 28:697-710, 1985. 9. Haaglund L, Christensen KK, Christensen P: Risk fac- tors in cesarean section infection. Obstet Gynecol 62: 145-150, 1983. 10. Anstey JT, Sheldon GW, Blythe JG: Infectious morbid- ity after primary cesarean sections in a private institution. Am J Obstet Gynecol 136:205-210, 1980. 11. Nielson TF, Hokegard KH: Postoperative cesarean sec- tion morbidity: A prospective study. Am J Obstet Gyne- col 146:911-916, 1983. 12. Phelan JP, Clark SL: Cesarean Delivery. New York: Elsevier, pp 201-218, 1988. 13. Cunningham FG, MacDonald PC, Gant NF (eds): Ce- sarean section and cesarean hysterectomy. In: Williams Obstetrics. 18th ed. New York: Appleton & Lange, pp 451-459, 1989. 14. Dunn LJ: Cesarean section and other obstetric operations. In Scott JR, Disaia PJ, Hammond CB, Spellacy WN (eds): Danforth's Obstetrics and Gynecology. 6th ed. Phil- adelphia: JB Lippincott, pp 639-658, 1990. 15. Depp R: Cesarean delivery and other surgical procedures. In Gabbe SG, Niebyl JR, Simpson JL (eds): Obstetrics: Normal and Problem Pregnancy, 2nd ed. New York: Churchill Livingstone, pp 635-693, 1991. 16, Hershey DW, Quilligan EJ: Extraabdominal uterine ex- teriorization at cesarean section. Obstet Gynecol 52:189- 192, 1978. 17.

esarean delivery and other surgical procedures. In Gabbe SG, Niebyl JR, Simpson JL (eds): Obstetrics: Normal and Problem Pregnancy, 2nd ed. New York: Churchill Livingstone, pp 635-693, 1991. 16, Hershey DW, Quilligan EJ: Extraabdominal uterine ex- teriorization at cesarean section. Obstet Gynecol 52:189- 192, 1978. 17. Wagensteen GH: The controlled administration of fluid to surgical patients. Minn Med J 25:783-789, 1942. 18. McCurdy CM, Magann EF, McCurdy CJ, Saltzman AK: The effect ofplacental management ofcesarean deliv- ery on operative blood loss. Am J Obstet Gynecol 167: 1363-1367, 1992. 70 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:7-11 (1993) (C) 1993 Wiley-Liss, Inc. Intravenous Penicillin for Antenatal Syphilotherapy Henry L. Galan, Paul M. Yandell, and Alfred B. Knight Department of Obstetrics and Gynecology, Texas A&M University Health Science Center, Scott and White Clinic and Memorial Hospital, Temple, TX ABSTRACT A 21 year old woman (G2 P0101) of24 weeks gestation presented with syphilis ofunknown duration. Sonography revealed fetal hydrops and placental thickening. Weekly intramuscular injections of2.4 million U Bicillin for 3 weeks was initiated as recommended by the Centers for Disease Control. Repeat sonogram 1 week after starting treatment revealed increased ascites and a new pericardial effusion. Due to the worsening fetal condition, therapy was altered and the patient was admitted for IV penicillin. She received a continuous infusion of 18 million U penicillin G daily for 10 days. Serial sonograms showed improvement offetal ascites and pericardial effusion with 10 days ofIV therapy, and complete resolution of hydrops was noted within 3 weeks. The fetus was born at term with no stigmata ofcongenital syphilis on newborn exam, and laboratory tests suggested adequate treatment in utero. (C) 1993 Wiley-Liss, Inc. KEy WORIS Fetal hydrops, congenital syphilis, T. pallidum n 1987 the incidence of syphilis reached its high- est peak since 1950. This increase was greater in women than men, regardless of ethnic group. Furthermore, the increase in congenital syphilis paralleled the rise in women. 2 Benzathine penicil- lin G is considered the treatment of choice for maternal infection by the Centers for Disease Con- trol (CDC); it prevents neonatal infection in 97- 100% of cases. 3-5 However, if there is reinfection, severe infection, or treatment late in pregnancy (second or third trimester), treatment failure can occur.

ne penicil- lin G is considered the treatment of choice for maternal infection by the Centers for Disease Con- trol (CDC); it prevents neonatal infection in 97- 100% of cases. 3-5 However, if there is reinfection, severe infection, or treatment late in pregnancy (second or third trimester), treatment failure can occur. 5'6 The CDC has reported that these failures are responsible for 35% of neonatal infections.2'7 In the past 20 years, no prospective, randomized study evaluating fetal syphilotherapy has been pub- lished, s,7 Left untreated, congenital syphilis has signifi- cant perinatal morbidity and mortality, with re- ported mortality rates of 40-54%. 1,8,9 Treatment failure in severe fetal infection may result in fetal death.3'5 In a recent case report, Hallak et al. at- tempted to treat a hydropic fetus with high-dose IV penicillin. 10 They were unable to complete therapy due to fetal distress during fetal blood transfusion that resulted in an emergent cesarean section. We describe a pregnancy with a severely affected fetus that had complete resolution of fetal hydrops and placental thickening 3 weeks after initiation of ma- ternal IV infusion of penicillin G with no clinical stigmata of congenital syphilis identified at birth. CASE REPORT A 21 year old black woman (G2P0101) at 24 weeks gestation by menstrual and ultrasound dating was diagnosed with syphilis ofunknown duration at her initial prenatal visit with the local health depart- ment. There was no prior history of syphilis. Her obstetrical history was complicated by prior pre- term delivery at 36 weeks gestation in which serol- ogy testing for syphilis was negative. She had had multiple sexual contacts in the past. Her rapid plasma reagin (RPR) was 1:64 and microhemag- Address correspondence/reprint requests to Dr. Alfred B. Knight, Dept. of Ob/Gyn, Scott & White Clinic, 2401 S. 31st St., Temple, TX 76508. Clinical Study Received June 10, 1992 Accepted December 19, 1992 IV PENICILLIN FOR ANTENATAL SYPHILOTHERAPY GALAN ET AL. Fig. I. Fetal sonograms prior to high-dose intravenous penicillin. .: Disproportion of fetal abdo- men to thorax. Echodensities in the bowel (arrows). Ascites noted in periphery of abdomen. ! Dilated loops of small bowel within marked ascites. C: Thickened placenta measuring 5-7 cm. D: Pericardial effusion (arrows). glutination assay for antibodies to Treponema palli- dum (MHATP) was reactive. Spinal tap was not performed because of a normal neurologic exami- nation.

Ascites noted in periphery of abdomen. ! Dilated loops of small bowel within marked ascites. C: Thickened placenta measuring 5-7 cm. D: Pericardial effusion (arrows). glutination assay for antibodies to Treponema palli- dum (MHATP) was reactive. Spinal tap was not performed because of a normal neurologic exami- nation. She was started on weekly injections of 2.4 million U of Bicillin for 3 weeks as recommended by the CDC for syphilis of unknown duration. An ultrasound performed at the start of therapy re- vealed fetal hydrops and placental thickening, an enlarged liver, intestinal echodensities, and di- lated loops of small bowel. Parvovirus B-19 titers showed immunity. A repeat ultrasound 8 days after initiation of therapy revealed a pericardial effusion and progression of the ascites (Fig. 1). Due to worsening fetal conditions despite week of Bicillin treatment, the patient was admitted to the antepartum service for aggressive IV penicillin G therapy with doses routinely used to treat adult neurosyphilis. Daily penicillin G at 18 million U continuous IV infusion was initiated and continued for 10 days. The patient tolerated treatment well and remained afebrile throughout her hospitaliza- tion. She did not experience the Jarisch-Herxhe- imer reaction other than a few uterine contractions requiring no tocolysis. No fetal heart rate abnor- malities were noted during episodic monitoring. Serial ultrasound was performed throughout the hospitalization. After 4 days of therapy the ascites and pericardial effusion were unchanged. Six days after therapy there was slight improvement of the ascites. On day 8 of therapy significant improve- 8 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY IV PENICILLIN FOR ANTENATAL SYPHILOTHERAPY GALAN ET AL. Fig. 2. Fetal sonograms 3 weeks following initiation of high-dose intravenous penicillin. A: Resolu- tion of ascites. I: Normal-appearing placenta. ment of both ascites and pericardial effusion were noted. On the day of discharge, following 10 days of IV penicillin, ultrasound revealed even less as- cites and the pericardial effusion was just detect- able. Three weeks after initiation of treatment, fol- low-up ultrasound revealed complete resolution of fetal hydrops and placental thickening (Fig. 2). There was good interval growth, and the placenta was no longer hydropic. Ultrasound was per- formed at 2-4 week intervals until delivery, and all remained normal. The patient delivered at 37+ weeks gestation after the onset of spontaneous labor.

ed complete resolution of fetal hydrops and placental thickening (Fig. 2). There was good interval growth, and the placenta was no longer hydropic. Ultrasound was per- formed at 2-4 week intervals until delivery, and all remained normal. The patient delivered at 37+ weeks gestation after the onset of spontaneous labor. No stigmata of congenital syphilis were evident on the newborn examination. Complete blood count showed no ev- idence of anemia or thrombocytopenia. Liver func- tion tests were normal. Umbilical cord Venereal Disease Research Laboratory (VDRL) titer was 1:2. Fluorescent treponemal antibody-absorption (FTA-ABS) on serum was 3 + reactive. Cerebral spinal fluid (CSF) VDRL was non-reactive, FTA- ABS was 3+ reactive, and FTA-ABS IgM was negative. CSF gram stain and cultures were nega- tive. The CSF protein was elevated at 194 mg/dl (normal 15-60), glucose was 37 mg/dl (normal 50-80), and the cell count was unremarkable. At the 4- week check, the infant was doing well and had surpassed its birth weight. Repeat VDRL was weakly reactive with 0 dilutions. Repeat CSF stud- ies showed an improved, yet persistantly elevated protein of 123 mg/dl. At the 2 month check, the infant was progressing well and was neurologically intact. VDRL was nonreactive and FTA-ABS was 3+ reactive. It was felt that the infant had been treated adequately in utero and that the decreasing levels of CSF protein represented a normalizing trend. However, because the consequences of early childhood neurosyphilis are tragic and there was concern about the ability to follow this child closely over a longer period of time, the infectious disease consult service chose to treat the child again with 200,000 U of aqueous penicillin G IM twice daily for 14 days. DISCUSSION With the decline in incidence of Rh isoimmuniza- tion, fetal hydrops is more commonly of infectious origin. A review of the literature by Hallak et al. revealed only 14 cases of nonimmune hydrops due to syphilis. Other infectious etiologies of nonim- mune hydrops sited were cytomegalovirus, bacte- ria, toxoplasmosis, rubella, herpes, Listeria, and Chlam2dia. o Although fetal hydrops due to syphi- lis is rare, it is likely to increase along with the increasing incidence of congenital syphilis. The current CDC recommended treatment for syphilis in the pregnant individual is similar to that of the nonpregnant individual.

oxoplasmosis, rubella, herpes, Listeria, and Chlam2dia. o Although fetal hydrops due to syphi- lis is rare, it is likely to increase along with the increasing incidence of congenital syphilis. The current CDC recommended treatment for syphilis in the pregnant individual is similar to that of the nonpregnant individual. 9 The literature re- flects uncertainty regarding the efficacy of this treatment, as shown by the number of studies re- porting treatment failures, which account for 3 5% of neonatal infections.2's'6'2' A well-controlled evaluation of the current CDC-recommended treatment for syphilis in pregnancy has not been reported, in part because of our inability to mea- sure the response of the disease to therapy.'s'6 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 9 IV PENICILLIN FOR ANTENATAL SYPHILOTHERAPY GALAN ET AL. However, sonography is a mechanism by which the response to therapy of the severely infected fetus can be monitored. In a 1988 publication, Wendel notes that women treated in the late second or third trimester of pregnancy may deliver affected infants prema- turely or experience fetal demise soon after therapy, the latter possibly occurring secondary to the Jarisch-Herxheimer reaction. He also states that benzathine penicillin may prevent congenital syph- ilis, but that it may not alter the course of severe fetal disease. Several reports have documented high perinatal mortality with fetal hydrops due to syphi- lis. 14-17 The hydropic fetus has a poor prognosis in spite of having a treatable infection, hence the deci- sion for aggressive parenteral therapy of the mother. Newborns with congenital syphilis can present with overwhelming, multisystem infection or be entirely asymptomatic, with the only diagnostic test a positive serology. The severely infected syphilitic fetus demonstrates hepatosplenomegaly, skin and scalp edema, ascites, pleural effusion and fibrosis, cardiac effusion, gastrointestinal adhesions, and he- matopoietic changes. 18 Ultrasound can detect and monitor placental thickening, hepatomegaly, as- cites, hydrops, and small bowel dilatation. 14,19--22 Cordocentesis has been used in a small number of cases. These infected fetuses demonstrate anemia, thrombocytopenia, and elevated liver enzymes. A recent study by Wendel et al. described a hydropic fetus treated by current CDC recommen- dations that had resolution ofhydrops 4 weeks after treatment.

Infectious Diseases in Obstetrics and Gynecology I: 134-136 (1993) (C) 1993 VViley-Liss, Inc. Evaluating the Concentration of a Candida albicans Suspension Acficio Rodrigues, C. Pina Vaz, A. Freitas da Fonseca, and J. Martinez-de-Oliveira Departments ofMicrobiology (A.R., C.P.V., A.F.d.F.) and Gynecology (J.M.-d.-O.), Porto Faculty ofMedicine, University ofPorto, Porto, Portugal ABSTRACT Objective: The objective of this study was to develop a reproducible method of establishing the concentration ofyeast cells per milliliter of solution. Methods: Three methods of determining the number of yeast cells in solution were compared: Neubauer's counting chamber, spectrophotometry, and nephelometry. Results: All three methods were comparable and reproducible. The following formulas were highly effective in determining the number of yeast cells in solution: chamber ( 103/ ml) 64.3 / 8,206 spectrophotometry (absorbance); and chamber ( 103/ml) -0.2 / 64 nephelometry (volt). Conclusions: Utilization of spectrophotometry or nephelometry and the appropriate formula allow for the precise determination, which is easily reproducible, ofthe concentration ofyeast cells in solution. This will facilitate experimentation involving precise inocula or requirement for specific concentrations ofyeast cells for various experiments. (C) 1993 Wiley-Liss, Inc. KEg WORDS Candida albicans, spectrophotometry, nephelometry, hemocytometer, Neubauer's chamber aginal candidosis, first described in 1849, is one of the most common lower genital tract infections. 1-3 Since Candida albicans can be found in up to 40% of healthy women, its diagnosis and treatment can be problematic. Vaginal culture continues to be the most sensi- tive method for the detection ofvaginal yeast infec- tion. Culture of Candida species is definitely supe- rior to direct microscopic detection and latex agglutination methods.4,s Isolation of a yeast (with growth) in culture from a vaginal specimen is con- sidered to be definitive proofofits presence and the "gold standard" in clinical microbiology and re- search. However, the true sensitivity ofculture has not yet been determined.

ilatation. 14,19--22 Cordocentesis has been used in a small number of cases. These infected fetuses demonstrate anemia, thrombocytopenia, and elevated liver enzymes. A recent study by Wendel et al. described a hydropic fetus treated by current CDC recommen- dations that had resolution ofhydrops 4 weeks after treatment. This fetus had mild hepatosplenomegaly at birth and required additional treatment in the neonatal period.21 We describe placental thicken- ing and progressive fetal hydrops that showed sono- graphic evidence of improvement during high- dose IV penicillin treatment with subsequent complete resolution of hydrops 3 weeks after initia- tion of therapy. The neonate was asymptomatic and had biochemical evidence of treated syphilis infec- tion. Penicillin G carries a category B risk factor and is commonly used in obstetrical infections. Prior to 1950, adverse fetal outcome due to maternal peni- cillin G administration was reported, but was likely due to impurities of the preparation. No evidence of birth defects induced by penicillin G has been documented. It passes rapidly into the fetal circula- tion and amniotic fluid in therapeutic levels, except in the first trimester, when levels may be subthera- peutic in the amniotic fluid. Parenteral penicillin G reaches equal levels in maternal serum and arnni- otic fluid within 60-90 minutes. 23 Therefore, doses that are normally used to treat adult neuro- syphilis may attain serum and amniotic fluid levels high enough to treat severe infections in utero. The rapid response to high-dose penicillin G therapy and lack of syphilis stigmata at birth sug- gest that this means oftreatment may serve a role in the severely affected fetus, particularly late in preg- nancy. The availability of high-resolution sonogra- phy provides a means to closely monitor the re- sponse of the disease to treatment closely. Further investigation is warranted to evaluate the efficacy of high-dose IV penicillin versus current CDC rec- ommended treatments for the severely syphilitic fetus. REFERENCES 1. Centers for Disease Control: Syphilis and congenital syphilis, United States, 1985-1988. MMWR 37:486- 489, 1988. 2. Centers for Disease Control: Congenital syphilis, United States, 1983-1985. MMWR35:625-628, 1986. 3. Thompson SW: Treatment of syphilis in pregnancy. J Am Vener Dis Assoc 3:159-167, 1976. 4. Nelson NA, Struve VR: Prevention ofcongenital syphilis by treatment of syphilis in pregnancy. JAMA 161:869- 872, 1956. 5.

988. 2. Centers for Disease Control: Congenital syphilis, United States, 1983-1985. MMWR35:625-628, 1986. 3. Thompson SW: Treatment of syphilis in pregnancy. J Am Vener Dis Assoc 3:159-167, 1976. 4. Nelson NA, Struve VR: Prevention ofcongenital syphilis by treatment of syphilis in pregnancy. JAMA 161:869- 872, 1956. 5. Wendel GD: Gestational and congenital syphilis. Clin Perinatol 15:287-303, 1988. 6. Mamunes P, Cave UG, Budell JW, et al.: Early diagno- sis of neonatal syphilis: Evaluation of a gamma M-fluo- rescent treponemal antibody test. Am J Dis Child 120: 17-21, 1970. 7. Centers for Disease Control: Guidelines for the preven- tion and control of congenital syphilis. MMWR 37:s-1, 1988. 8. Hira SK, Bhat GJ, Patel JB, et al.: Early congenital syphilis: Clinicoradiographic features in 202 patients. Sex Transm Dis 12:177-183, 1985. 9. Chapel TA: Congenital syphilis. Compr Ther 14:25-28, 1988. 10. Hallak M, Peipert JF, Ludomirsky A, Byers J: Nonim- mune hydrops fetalis and fetal congenital syphilisMa case report. J Reprod Med 37:173-176, 1992. 11. Watson J, Campbell S: Antenatal evaluation and manage- ment in nonimmune hydrops fetalis. Obstet Gynecol 67: 589-592, 1986. 12. Zenker PN, Rolls RT: Treatment of syphilis. Rev Infect Dis 12(6):s590-603, 1989. 13. Ricci JM, Fojaco RM, O'Sullivan MJ: Congenital syph- ilis: The University of Miami/Jackson Memorial Medi- O INt,'ECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY IV PENICILLIN FOR ANTENATAL SYPHILOTHERAPY GALAN ET AL. cal Center experience, 1986-1988. Obstet Gynecol 74: 687-693, 1989. 14. Hill LM, Maloney JB: An unusual constellation of sono- graphic findings associated with congenital syphilis. Ob- stet Gynecol 78:895-897, 1991. 15. Chawla V, Pandit PB, Nkrumah FK: Congenital syphilis in the newborn. Arch Dis Child 63:1393-1394, 1988. 16. Chiang W, Wei P: Immunoglobulins in hydrops fetalis. AmJ Obstet Gynecol 114:816-818, 1972. 17. Bulova SI, Schwartz E, Harrer WV: Hydrops fetalis and congenital syphilis. Pediatrics 42:285-287, 1972. 18. Ingall D, Musher D: Syphilis. In: Infectious Diseases of the Fetus and Newborn infant. Klein JO, Remington JS, ed., Saunders, Philadelphia, pp 335-374, 1991. 19. Lucas MJ, Theriot SK, Wendel GD: Doppler diastolic and systolic ratios in pregnancies complicated by syphilis. Obstet Gynecol 77:217-222, 1991. 20. Yiu-Chiu V, Chiu L: Sonographic features of placental complications in pregnancy. AJR 138:879-885, 1982. 21.

emington JS, ed., Saunders, Philadelphia, pp 335-374, 1991. 19. Lucas MJ, Theriot SK, Wendel GD: Doppler diastolic and systolic ratios in pregnancies complicated by syphilis. Obstet Gynecol 77:217-222, 1991. 20. Yiu-Chiu V, Chiu L: Sonographic features of placental complications in pregnancy. AJR 138:879-885, 1982. 21. Wendel GD Jr, Sanchez PJ, Peters MT, Harstad TW, Potter LL, Norgard MV: Identification of Treponema pallidum in amniotic fluid and fetal blood from pregnan- cies complicated by congenital syphilis. Obstet Gynecol 78:890-894, 1991. 22. Satin AJ, Twickler DM, Wendel GD: Congenital syphi- lis associated with dilation of fetal small bowel. J Ultra- sound Med 11:49-52, 1992. 23. Briggs GG, Freeman RK, Yaffe SJ: Drugs in Pregnancy and Lactation. Williams and Wilkins, Baltimore, pp 482-485, 1990. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:71-75 (1993) (C) 1993 Wiley-Liss, Inc. Development of a High Performance Liquid Chromatographic Assay for Measuring Mezlocillin in Serum or Tissue Sandra Mya, Ron E. Gain, and Bryan Larsen Departments of Obstetrics and Gynecology (S.M.), Biological Science (R.E.G.), and Microbiology and Obstetrics and Gynecology (B.L.), Marshall University School ofMedicine, Huntington, WV ABSTRACT Objective: This study evaluated the blood and uterine tissue concentration of mezlocillin, a broad- spectrum penicillin. Methods: We adapted a liquid chromatographic method to measure mezlocillin in serum and tissue. Mezlocillin reference standard was diluted in water, chromatographed on a reversed phase C18 column eluted at 1.5 ml/min with acetonitrile and phosphate buffer (1:3 v:v), and detected spectrophotometrically at 210 nm. Mezlocillin was administered to 14 premenopausal women sched- uled to undergo vaginal hysterectomy. Each patient received a 4 g IV infusion of the drug 30 to 60 min prior to surgery. During surgery, tissue was removed from the uterine cervix and blood was obtained for assay ofmezlocillin content. Results: Chromatography of the mezlocillin standard furnished a discrete peak with a retention time of 2.4 min. The sensitivity of the assay was 0.1 pg/ml with a linear response up to 100 g/ml. The correlation coefficient for the standard curve was 0.9997. When reference standard was diluted in pooled human serum, the assay was complicated by interfering compounds. These were removed by ether extraction. The sensitivity of the assay performed in serum was 3 Cg/ml. Serum samples contained from 81.2 to 358 pg ofmezlocillin/ml with an average serum concentration of207.5 g/ml. When serum containing a known amount of mezlocillin was homogenized for a period of time similar to that required to homogenize tissue samples, a detectable loss of drug was observed and was applied as a correction factor to the measured tissue levels. After correction, the average tissue level was 117.2 g/ml and ranged from 27% to 98% ofthe serum levels.

of mezlocillin was homogenized for a period of time similar to that required to homogenize tissue samples, a detectable loss of drug was observed and was applied as a correction factor to the measured tissue levels. After correction, the average tissue level was 117.2 g/ml and ranged from 27% to 98% ofthe serum levels. Conclusions: The serum concentration ofmezlocillin after IV infusion of4 g was greater than that required to inhibit the majority of the most significant organisms responsible for post-hysterectomy sepsis. Although tissue levels appeared to be consistently lower than serum levels, they could be expected to provide an inhibitory effect against many of the bacterial strains that contaminate the surgical site. (C) 1993 Wiley-Liss, Inc. KEY WORDS Antibiotics, pharmacodynamics, ureidopenicillin otent broad-spectrum congeners of penicillin are finding increasing use in a variety ofclinical settings. The rational use ofsuch antibacterial agents requires knowledge of the pharmacokinetics and pharmacodynamics of these drugs. Sensitive meth- ods for assaying drugs in serum or tissue are needed for developing such information, and high perfor- mance liquid chromatography (HPLC) has become a useful technique for obtaining such information. The present study adapted an HPLC method for analysis of serum and tissue samples from patients treated with rnezlocillin. HPLC methods for de- Address correspondence/reprint requests to Dr. Bryan Larsen, Department of Microbiology, Marshall University School of Medicine, Huntington, WV 25701. Clinical Study Received December 23, 1992 Accepted August 12, 1993 MEZLOCILLIN IN TISSUE MYA ET AL. termination of the related compounds mezlocillin and azlocillin in plasma have been reported, but these methods have not been tailored for use with tissue. The methods available for measuring plasma levels were evaluated and adapted for use with se- rum and tissue specimens. MATERIALS AND METHODS Mezlocillin as the sodium salt with a potency of 920 Ixg/mg was provided by Miles Pharmaceuticals (New Haven, CT). The drug was reconstituted with distilled water to make a stock solution of 100.28 txg/ml. Stock solutions were stored at -74C. The 14 patients studied were scheduled to un- dergo vaginal hysterectomy and were given preop- erative prophylaxis consisting of 4 g mezlocillin dissolved in 500 ml of normal saline by IV infu- sion. The antibiotic infusion was started within hr of the time the surgical procedure began.

k solutions were stored at -74C. The 14 patients studied were scheduled to un- dergo vaginal hysterectomy and were given preop- erative prophylaxis consisting of 4 g mezlocillin dissolved in 500 ml of normal saline by IV infu- sion. The antibiotic infusion was started within hr of the time the surgical procedure began. During the surgical procedure, when the uterus was being removed, a blood sample was drawn from the antecubital vein, and the blood was al- lowed to clot. A section was cut from the uterine cervix, and both blood and tissue were transported to the laboratory, where the serum was removed from the clot and serum and tissue stored at -74C until analysis. The method for tissue and serum analysis was established experimentally (see Results). Prior to assay, tissue and blood samples were thawed at room temperature. Approximately 0.25 g was cut from the tissue sample, weighed to the nearest 0.1 mg, minced, and placed in a tube containing 3 ml of water. These samples were kept on ice until the tissue was liquified with a Tekmar tissue homoge- nizer (Tekmar, Cincinnati, OH). The homoge- nate was deproteinized by the addition of 5 ml acetonitrile, followed by centrifugation for 15 min at 2,000g. The supernatant fluid was removed and extracted 3 times with 3 ml of diethyl ether. The aqueous phase was then injected onto the chromato- graph. The serum samples were deproteinized with equal volumes ofacetonitrile, centrifuged at 2,000g for 15 min, and extracted 3 times with 2.5 volumes of diethyl ether. The aqueous phase was chromato- graphed. Samples were analyzed by I--IPLC on a 4.6 220 mm octadecyl silyl, 5 Im particle size column with a guard column ofidentical composition. Sam- ples of 20 Ixl were injected and eluted with 1:3 acetonitrile:0.01 M potassium dihydrogen phos- phate buffer, pH 7, at a flow rate of 1.5 ml/min. The column effluent was monitored spectrophoto- metrically at 210 nm. Except where specifically noted, all measurements were made by triplicate determinations on each sample, and the identity of mezlocillin was based on retention time while quan- titation was based on peak height. Dilutions of mezlocillin standard were made in water or pooled human serum to determine linear- ity, repeatability, and sensitivity ofthe assays. Stan- dard curves made in serum were used to establish drug levels in the patient specimens. A control to determine whether drug may have been lost during the process oftissue disruption was prepared.

tandard were made in water or pooled human serum to determine linear- ity, repeatability, and sensitivity ofthe assays. Stan- dard curves made in serum were used to establish drug levels in the patient specimens. A control to determine whether drug may have been lost during the process oftissue disruption was prepared. Serum containing a known amount of mezlocillin was homogenized for a period of time equal to the time required to disrupt the tissue samples. This control was then subjected to depro- teinization and ether extraction as for other sam- ples. The peak height obtained from this sample was compared with the result obtained from a se- rum sample with an identical concentration of me- zlocillin that had not undergone homogenization. This provided a correction factor that was applied to tissue concentrations to correct for drug loss due to tissue homogenization. RESULTS Figure shows that the mezlocillin standard di- luted in water produced a symmetrical peak with a retention time of 2.4 min. Thus, the chromato- graphic method used was deemed acceptable for detecting the pure compound. A series ofdilutions ofmezlocillin standard from 0.1 Ixg/ml to 100.28 Ixg/ml were made in water to allow determination of the sensitivity and linearity of the assay method. The smallest concentration detected was 0.1 Ig/ml, which furnished an aver- age peak height of 0.00083 absorbance units at 210 nm. The assay was linear over the full range of concentrations and provided a correlation coeffi- cient (r2) Of 0.9997. Ten replicate chromatograms of a standard con- taining 100.28 Ixg mezlocillin standard/ml were evaluated to determine the repeatability of peak 72 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY MEZLOCILLIN IN TISSUE MYA ET AL. lpg MEZ= 0.0079 TIME Fig. I. Mezlocillin (MEZ) reference standard diluted in wa- ter chromatographed on a reversed phase column under conditions described in the Materials and Methods section. A single symmetrical peak representing the pure compound was observed. Chromatography of a larger concentration revealed a linear relationship between peak height and con- centration. height and retention time. The average retention time was 2.40 + 0.024 rain (SD). The average peak height was 0.774 + 0.0118 absorbance units (SD). The coefficient ofvariation (relative SD) for retention time was 1% and for peak height was 1.5%. The procedure was therefore considered to be highly repeatable.

entration. height and retention time. The average retention time was 2.40 + 0.024 rain (SD). The average peak height was 0.774 + 0.0118 absorbance units (SD). The coefficient ofvariation (relative SD) for retention time was 1% and for peak height was 1.5%. The procedure was therefore considered to be highly repeatable. The methods for preparation of serum samples for chromatographic determination of mezlocillin were evaluated. A known concentration ofmezlocil- lin was added to a pool of human serum, and vari- ous methods were used to prepare aliquots of that serum for analysis. The first method involved ad- dition of ml of 5% perchloric acid to ml of the serum sample followed by centrifugation. This pro- vided a protein-free sample for chromatography. As illustrated in Figure 2, this method of prepara- tion was unsuitable because exposure to perchloric acid appeared to generate at least two new peaks, which may have been degradation products of me- zlocillin. Furthermore, it was not possible to iden- tify the mezlocillin peak unequivocally in the acid- treated serum. The second method of sample preparation em- ployed the addition of ml ofacetonitrile to ml of serum to precipitate the protein from the sample. It was believed that the acetonitrile would be less likely to cause degradation of the mezlocillin than would perchloric acid. However, the protein-free super- natant fluid from acetonitrile-treated serum yielded a chromatogram similar to that obtained when se- rum without mezlocillin was treated with acetoni- trile. This finding suggested that the mezlocillin was being masked by some component ofthe serum. It was subsequently found that diethyl ether ex- traction removed the interfering compounds from the acetonitrile-precipitated samples, as shown in Figure 3. In addition, the acetonitrile entered the ether phase, which eliminated the dilution of the sample due to the deproteinization step. Consequently, the acetonitrile precipitation followed by ether extrac- tion was adopted for sample preparation. When standards prepared in water, treated with acetonitrile, and extracted with ether were com- pared with standards diluted in water, no apprecia- ble loss of drug due to the extraction procedure was observed. Recoveries ranged from 97.7% to 106% for the concentrations of mezlocillin observed in the patient samples. Based on these findings, stan- dard curves for mezlocillin assay were prepared in pooled serum, and patient samples were compared with these standards.

e loss of drug due to the extraction procedure was observed. Recoveries ranged from 97.7% to 106% for the concentrations of mezlocillin observed in the patient samples. Based on these findings, stan- dard curves for mezlocillin assay were prepared in pooled serum, and patient samples were compared with these standards. Tissue samples were also compared with a stan- dard curve prepared in serum. However, it was determined that the tissue homogenization was ex- pected to have reduced the amount of mezlocillin detectable by 37.5% based on homogenizing serum samples with added mezlocillin. As a consequence of this finding, the observed tissue values were increased by a factor of 0.375. The serum and tissue levels observed in the 14 patients studied are summarized in Table 1. The levels observed in serum were consistently higher than those found in tissue. The highest serum levels tended to be associated with the highest tissue lev- els, but the relationship of serum to tissue levels revealed an r2 of 0.58. This study was not designed to be a pharmaco- kinetic study, but rather an evaluation of the serum and tissue levels obtained simultaneously after IV infusion. Therefore, the time after infusion that the tissue sample was removed and the serum sample was taken was not identical in each case and varied from 5 rain to 130 min. In the limited number of samples available in the study, no linear correlation between the time after infusion and tissue or serum levels was apparent. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 73 MEZLOCILLIN IN TISSUE MYA ET AL. E MEZ MEZ $ S C A B 2.5 min 2.5 min 2.$ min Fig. 2. Evaluation by chromatograms of perchloric acid precipitation for preparation of serum samples. A: Mezlocillin (MEZ) standard (6.25 Ig/ml, final concentration) diluted in water with a retention time of approximately 2.4 min. B: Mezlocillin, 6.25 Ig/ml, with 2.5% perchloric acid. The height of the mezlocillin peak was diminished (compared with A), and multiple new peaks appeared, suggesting degradation products' (2: Mezlocillin, 6.25 Ig/ml (final concentration), in serum deprotein- ized with perchloric acid, showing that mezlocilin is obscured by serum components. S: Start of each chromatographic run. DISCUSSION The use ofantimicrobial prophylaxis in such surgi- cal procedures as vaginal or abdominal hysterec- tomy is so common that one may be tempted to consider studies involving prophylaxis as trivial.

h perchloric acid, showing that mezlocilin is obscured by serum components. S: Start of each chromatographic run. DISCUSSION The use ofantimicrobial prophylaxis in such surgi- cal procedures as vaginal or abdominal hysterec- tomy is so common that one may be tempted to consider studies involving prophylaxis as trivial. Despite the frequent application ofprophylaxis, the fundamental reasons why this type of drug use is effective is still a matter ofconjecture. It is assumed that antibiotics administered preoperatively affect microorganisms that contaminate the operative site, but it is not clear whether any of the organisms are killed, prevented from replicating, or simply pre- vented from producing virulence factors. It is like- wise not clear what relevance in vitro minimal in- hibitory concentrations have for these tissues. The length of time and concentration of drug required to be present in the tissue for effective prophylaxis TIME Fig. 3. A: Pattern obtained when ether-extracted serum is chromatographed. B: Results when the serum is spiked with mezlocillin (M) standard and extracted with ether. The me- zlocillin produces a resolved and quantifiable peak. 74 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY MEZLOCILLIN IN TISSUE MYA ET AL. TABLE I. Observed concentrations of mezlocillin in serum and uterine cervix from women given 4 g of mezlocillin prior to vaginal hysterectomy Concentration (lg/g) Ratio Tissue Serum (tissue/serum) Mean 117.2 207.5 0.567 Standard 66.9 93.0 0.233 deviation Range 27.5-228. 81.2-358.0 0.268-0.982 are also open to question. Analysis oftissue levels of various antimicrobials as well as serum levels rep- resents a necessary tool for enhancing our under- standing of the effects of prophylaxis at the opera- tive site. The work presented here adapted a chromato- graphic procedure for use in determining the se- rum and tissue levels of mezlocillin. An attempt was made to take into account the possibility that interaction ofthe drug with tissue or serum compo- nents may reduce the amount of drug apparent in our assays. It cannot be claimed that subjecting mezlocillin-spiked serum to the homogenization step provided a true .control for possible loss in tissue samples, although the data suggest that such an observation is not irrelevant.

ofthe drug with tissue or serum compo- nents may reduce the amount of drug apparent in our assays. It cannot be claimed that subjecting mezlocillin-spiked serum to the homogenization step provided a true .control for possible loss in tissue samples, although the data suggest that such an observation is not irrelevant. An ideal study would have added drug to tissue from a patient not treated with antibiotic and processed the tissue as described; however, obtaining tissue samples from patients who had not received the drug was outside the bounds of the protocol approved by the IRB, leaving the serum homogenization test as the sub- optimal alternative. Certainly, any future applica- tion ofthis method could be improved by including a control using tissue from a patient not exposed to antibiotic. The question of why the homogenizing proce- dure made a portion of the drug unrecoverable is not explained by the studies presented. Oxidative damage to the drug or some irreversible reaction with protein or other constituents of the biological matrix are potential explanations. The possibility that drug was incorporated into micelles during the homogenization process may be less likely, since the solvent extraction steps should destroy such structures. In view of the above considerations, it may be stated that in all cases, some mezlocillin was present in uterine tissue at the time of incision, and the tissue concentration was at least one quarter that present in serum if one accepts the correction fac- tor. A more conservative estimate (without the cor- rection factor) of tissue concentrations ranges from 17.2 to 142.5 txg/g and would average 35% of the simultaneous tissue values. For therapy of established infections, it is desir- able to achieve serum and tissue concentrations greater (generally by 2-4 times) than the minimal inhibitory concentrations of the etiologic agents. Somewhat less certain are the serum or tissue levels required to achieve prophylaxis against infection of the operative site. It is not clear whether it is neces- sary to achieve minimal inhibitory concentrations against microorganisms at the surgical site. The levels of mezlocillin observed in serum and tissue are well above the minimal inhibitory concentra- tions reported for some ofthe microorganisms com- monly involved in infections after hysterectomy.2 For example, 90% ofBacteroidesfragilis strains are inhibited by 32 Ig of mezlocillin/ml and 90% of Escherichia coli strains are inhibited by 64 Ig/ml.

in serum and tissue are well above the minimal inhibitory concentra- tions reported for some ofthe microorganisms com- monly involved in infections after hysterectomy.2 For example, 90% ofBacteroidesfragilis strains are inhibited by 32 Ig of mezlocillin/ml and 90% of Escherichia coli strains are inhibited by 64 Ig/ml. It should be noted, however, that minimal inhibi- tory concentrations are determined in vitro with a constant concentration of drug and with the test organism exposed to that drug for many hours. In the patient, the concentration of the drug is con- stantly changing. The present study, not being a pharmacokinetic study, did not sample tissue and serum at identical times after infusion. Thus, while significant levels of antibiotic were detected in the patients studied, future work will need to elucidate the relevance of brief exposures of microorganisms to these concentrations of mezlocillin. This chro- matographic technique has proved an appropriate tool in establishing the concentration of mezlocillin to be studied in future investigations. REFERENCES 1. Rouan MC: Antibiotic monitoring in body fluids. J Chro- matogr 340:361-400, 1985. 2. Parry MF, Pancoast SJ: Antipseudomonal penicillins. In Cunha B, Ristuccia A (eds): Antimicrobial Therapy. New. York: Raven Press, pp 197-208, 1984. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 75

Infectious Diseases in Obstetrics and Gynecology 1:104-107 (1993) (C) 1993 Wiley-Liss, Inc. Treatment of Cervical Chlamydial Infection With Amoxicillin/Clavulanate Potassium Melinda S. Mann, Sebastian Faro, Maurizio L. Maccato, and Raymond H. Kaufman Private practice, Brooklyn, NY (M.S.M.), Department of Gynecology and Obstetrics, University of Kansas School ofMedicine, Kansas City, KS (S.F.), and Department of Obstetrics and Gynecology, Baylor College ofMedicine, Houston, TX (M.L.M., R.H.K.) ABSTRACT Objective: To determine if amoxicillin/clavulanate potassium is effective in the treatment of Chlamy- dia trachomatis endocervicitis. Methods: Thirty-two patients with culture-proven endocervical infection were treated with amoxicillin/clavulanate potassium, 500 mg orally 3 times a day for 10 days. Post-treatment endocer- vical specimens were obtained at 2, 4, and 6 weeks for culture ofC. trachomatis. Male partners were treated with doxycycline, 100 mg orally twice daily for 10 days. The couples were provided condoms and asked to use them throughout the duration ofthe study. Results: All patients treated with amoxicillin/clavulanate potassium were cured of signs of cervi- citis. All were found to be free of C. trachomatis at their follow-up visits. Conclusions: Amoxicillin/clavulanate potassium is effective in eradicating C. trachomatis. (C) 1993 WileyoLiss, Inc. KEY WORDS Penicillin-class, cerviitis, bacteria hlamydia trachomatis is considered to be the most common sexually transmitted bacterium in the United States. It is estimated that 4 million infec- tions occur per year. In the female, C. trachomatis is one etiologic agent responsible for cervicitis, ure- thritis, endometritis, and salpingitis.2-s C. tra- chomatis has been associated with abortion, prema- ture rupture of amniotic membranes, premature labor, chorioamnionitis, and postpartum endo- metritis. 6-1 Like other sexually transmitted dis- eases, C. trachomatis can be found in association with other bacteria, e.g., Neisseria gonorrhoeae in 60% of cases. 11 Standard treatment has been tetra- cycline in the non-pregnant patient and doxycycline or erythromycin in the pregnant patient.

, and postpartum endo- metritis. 6-1 Like other sexually transmitted dis- eases, C. trachomatis can be found in association with other bacteria, e.g., Neisseria gonorrhoeae in 60% of cases. 11 Standard treatment has been tetra- cycline in the non-pregnant patient and doxycycline or erythromycin in the pregnant patient. Recently, clindamycin has been shown to be effective and synergistic when combined with gentamicin. 12 Var- ious penicillins have been shown to be effective in vitro and in vivo. 13-16 The present study was undertaken to determine if the combination of amoxicillin plus clavulanate potassium (Augmentin, Beecham Laboratories, Bristol, TN) is effective in eradicating C. trachom- at#. This agent was chosen for its broad spectrum of activity. In vitro studies have shown that it is effective against Gardnerella vaginalis (S. Faro, per- sonal communication), a significant advantage be- cause patients with C. trachomatis are commonly found to be positive for G. vaginalis as well. In addition, the presence of G. vaginalis may indicate an abnormal vaginal flora dominated by anaerobic bacteria, e.g., bacterial vaginosis. Augmentin is Address correspondence/reprint requests to Dr. Sebastian Faro, Department of Gynecology and Obstetrics, University of Kansas School ofMedicine, 3901 Rainbow Blvd., Kansas City, KS 66160-7316. Clinical Study Received June 16, 1993 Accepted August 12, 1993 AUGMENTIN IN CERVICAL CHLAMYDIA MANN ET AL. suitable not only against anaerobes but also against facultative anaerobes and gram-positive aerobes. Augmentin has also been shown to be effective against [3-1actamase-producing N. gonorrhoeae. For these reasons, it has the potential for being suitable as a single agent for the treatment of C. trachomatis, N. gonorrhoeae, and bacterial vaginosis. SUBJECTS AND METHODS Study subjects were chosen from the patients seen at the colposcopy clinic of the Department of Obstet- rics and Gynecology at Baylor College of Medi- cine. Patients 18 years ofage or older were enrolled if they had chlamydia-positive endocervical cul- tures, had not used antibiotics within the previous 30 days, and had given written informed consent. If otherwise eligible, patients younger than 18 but older than 13 years of age were enrolled if written informed consent and parental written consent had been obtained. Patients who were allergic to or suspected of being allergic to penicillin or who admitted to having more than one sexual partner were excluded from the study.

wise eligible, patients younger than 18 but older than 13 years of age were enrolled if written informed consent and parental written consent had been obtained. Patients who were allergic to or suspected of being allergic to penicillin or who admitted to having more than one sexual partner were excluded from the study. The male partners of all patients enrolled in the study were treated with doxycycline, 100 mg orally twice daily for 10 days. An ample supply of con- doms was given to each patient for the entire dura- tion of the study. Patients who failed to use the condoms during sexual intercourse were dropped from the study. All female patients enrolled in the study were treated with Augmentin, 500 mg orally 3 times a day for 10 days. Patients were instructed not to take any other antibiotics during the study period. Patients were asked to return at 2, 4, and 6 weeks after completion of therapy for test-of-cure cultures. Specimens for the isolation and culture of C. trachomat# were obtained from the endocervix us- ing a sterile Dacron swab mounted on a plastic shaft. The portio of the cervix was first cleansed by gently wiping away with a large cotton swab any vaginal discharge that may have been present. The sterile Dacron swab was placed deep into the endo- cervical canal and rotated in a clockwise manner for 15 to 30 sec. The swab was then placed into Bartel's transport medium (Bartel, Bellevue, WA) and taken immediately o the laboratory for process- ing. 17 C. trachomatis was isolated on cyclohexim- ide-treated McCoy cells (Viromed Laboratories, Minnetonka, MN). C. trachomatis inclusions were TABLE I. Results of treating Chlamydia trachomatis with Augmentin No. of No. of Culture patients weeks results 4 0 Not done 5 2 Negative 4 2, 4 Negative 18 2, 4, 6 Negative identified by staining with monoclonal immunoflu- orescent antibody (Ortho Diagnostics, Raritan, NJ). All specimens were passed blindly twice in McCoy tissue cultures. RESULTS Thirty-five patients were enrolled in the study and 32 (91%) completed the study by having at least one follow-up post-treatment culture. Three pa- tients were excluded from the study: 1) one patient became dizzy while taking Augmentin and volun- tarily discontinued the medication; 2) one was seen by another physician and given doxycycline; and 3) one failed to use condoms. Nineteen patients were black (59%), nine were Hispanic (29%), and four were Caucasian (12.5%). The ages ranged from 14 to 66 years old, with a mean of 24 years.

e taking Augmentin and volun- tarily discontinued the medication; 2) one was seen by another physician and given doxycycline; and 3) one failed to use condoms. Nineteen patients were black (59%), nine were Hispanic (29%), and four were Caucasian (12.5%). The ages ranged from 14 to 66 years old, with a mean of 24 years. The population may reflect the fact that the colposcopy clinic serves primarily an indigent population made up largely of black and Hispanic patients. Augmentin was well tolerated, with minimal adverse effects. Two patients developed nausea and vomiting. However, when they took the medica- tion after meals, they had no further problems and completed a full course of therapy. Two patients developed vaginal discharge and itching. Examina- tion at their initial follow-up evaluations revealed the presence offungal pseudohyphae, and a diagno- sis of Candida vaginitis was made. Both patients were treated successfully with butoconazole. Thirty-two patients completed a 10-day course ofAugmentin. Nine patients returned only for their initial 2-week visit. Five patients returned for their 2- and 4-week visits, and 18 patients returned for their 2-, 4-, and 6-week visits (Table 1). Thirty-one patients had negative follow-up cul- tures of the endocervix. However, one patient found to be negative at the 2-week visit was positive at the 4-week visit. She admitted to having had INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 105 AUGMENTIN IN CERVICAL CHLAMYDIA MANN ET AL. unprotected intercourse after the 2-week post-ther- apy evaluation. She was re-treated with Augmentin and subsequently found to be negative at the next post-therapy evaluation. One 66-year-old patient was particularly inter- esting. She had been a widow for 11 years and stated that she had not engaged in sexual activity since her husband died. She was found to harbor C. trachomatis in her endocervical canal. This patient's culture was strongly positive, that is, there were numerous inclusion bodies present. Therefore, the possibility of it being a false-positive culture could be unlikely. Augmentin was successful in eradicat- ing C. trachomatis, as she was found to have nega- tive cultures at 2, 4, and 6 weeks post-therapy. DISCUSSION The current recommended therapy for cervical chlamydial infection in the non-pregnant patient is doxycycline, 100 mg orally twice daily for 7 to 10 days. A main disadvantage of doxycycline is its relatively narrow spectrum of antimicrobial activ- ity.

ve nega- tive cultures at 2, 4, and 6 weeks post-therapy. DISCUSSION The current recommended therapy for cervical chlamydial infection in the non-pregnant patient is doxycycline, 100 mg orally twice daily for 7 to 10 days. A main disadvantage of doxycycline is its relatively narrow spectrum of antimicrobial activ- ity. Thus, it is frequently prescribed as an "add-on" antibiotic for patients in whom pelvic inflamma- tory disease is suspected or diagnosed. Chlamydial infection in the pregnant patient is primarily treated with erythromycin, 500 mg orally 4 times a day for 7 to 10 days. However, erythro- mycin frequently causes gastrointestinal distur- bances, which, in pregnancy, represent a common complication. Therefore, in the pregnant patient treated with erythromycin, compliance is a signifi- cant problem. Azithromycin, a new macrolide, is approved for the treatment of C. trachomatis in a single daily dose. However, this agent has not been approved for use in pregnancy nor in the treatment ogonor- rhea. Ofloxacin has been shown to be effective and has been approved for the treatment of both N. gonorrhoeae and C. trachomatis. However, quinolo- nes are contraindicated in pregnant patients, breast feeding patients, and children under the age of 16. In addition, ofloxacin has a limited spectrum of activity against anaerobes; therefore, its use in those patients with concomitant bacterial vaginosis would be questionable. Hospitalized patients with acute salpingitis are currently treated with cefoxitin, ceftizoxime, ce- fotetan plus doxycycline, or clindamycin plus gen- tamicin. Ambulatory treatment of pelvic inflam- matory disease mainly relies on ceftriaxone plus doxycycline. Ceftriaxone does not provide adequate coverage against anaerobes, and one must question the logic of using a single dose of ceftriaxone, al- though it has a long half-life, to prevent progres- sive salpingitis. The goal oftreating acute salpingi- tis is to prevent damage to the fallopian tube, thereby reducing the risk of ectopic pregnancy and/or infertility. Despite the reported side effect ofdiarrhea, which might limit its use,5 Augmentin would seem a more logical choice in treating the ambulatory patient because of its broader spectrum of activity. Recently, amoxicillin has been shown to have moderate in vitro anti-chlamydial activity. Clinical trials with amoxicillin used in different dosing reg- imens have demonstrated efficacy in treating chlamydial infection.

a more logical choice in treating the ambulatory patient because of its broader spectrum of activity. Recently, amoxicillin has been shown to have moderate in vitro anti-chlamydial activity. Clinical trials with amoxicillin used in different dosing reg- imens have demonstrated efficacy in treating chlamydial infection. 8'16 Other penicillins have been shown to have anti-chlamydial activity, namely, mezlocillin, ticarcillin, timentin, and pip- eracillin. 14--17 Augmentin was chosen for study because it is active against penicillinase-producing strains of N. gonorrhoeae, G. vaginalis, and gram-positive or- ganisms, e.g., Streptococcus agalactiae, Staphylococ- cus aureaus, and Enterococcusfaecalis. Augmentin is active against many members of the Enterobacteri- ace.ae and anaerobes. The presence of clavulanate potassium, a [3-1actamase inhibitor, increases the spectrum of activity of amoxicillin, especially against [3-1actamase-producing bacteria. Our study showed that this antibiotic is effective therapy for C. trachomatis infections. Augmentin presents the opportunity of using a single agent for the treatment ofN. gonorrhoeae and C. trachomatis cervicitis. Augmentin also affords the opportunity to treat bacterial vaginitis in the non-pregnant and pregnant patient who is not aller- gic to penicillin. Additional studies are needed to confirm its activity in the treatment of bacterial vaginosis. Its spectrum ofactivity makes it a poten- tial agent for the treatment of pelvic inflammatory disease in an ambulatory setting. Although the activity of Augmentin is probably no better than ampicillin or amoxicillin against C. trachomatis, it does offer advantages, as already stated, over these other penicillins. However, ampi- cillin or amoxicillin could be used for the treatment of C. trachomatis cervicitis in pregnancy. In the non-pregnant patient, Augmentin would be prefer- 106 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY AUGMENTIN IN CERVICAL CHLAMYDIA MANN ET AL. able to the other oral penicillins because of its broader spectrum ofactivity, thus playing a greater preventive role with regard to fallopian tube dam- age. In this instance, the additional cost of Aug- mentin may be justified. However, further investi- gation of the role of this agent in treating female pelvic infection is warranted. Augmentin, in the present study, proved to be 100% effective in eradicating C. trachomatis from the cervix.

gard to fallopian tube dam- age. In this instance, the additional cost of Aug- mentin may be justified. However, further investi- gation of the role of this agent in treating female pelvic infection is warranted. Augmentin, in the present study, proved to be 100% effective in eradicating C. trachomatis from the cervix. One patient was initially treated and found to be cured, but was subsequently found to be positive, re-treated, and cured. Thus, Augmen- tin appears to be a suitable agent for the treatment of chlamydial cervicitis in the pregnant and non- pregnant patient. REFERENCES 1. Centers for Disease Control: Chlamydia trachomatis infec- tions: Policy guidelines for prevention and control. MMWR 34(Suppl 3s):53s-74s, 1985. 2. Thompson SE, Washington AE: Epidemiology of sexu- ally transmitted Chlamydia trachomatis infections. Epide- miol Rev 5:96-123, 1983. 3. Martin DH, Pastorek JG, Faro S: Risk factors for Chlamydia trachomatis in a high-risk population of preg- nant women. In Oriel D, Ridgway G, Schachter J, et al. (eds): Chlamydial Infections. Cambridge: Cambridge University Press, p 189, 1986. 4. Martin DH, Koutsky L, Eschenbach DA, et al.: Prema- turity and perinatal mortality in pregnancies complicated by maternal Chlamydia trachomat# infections. JAMA247: 1585-1589, 1982. 5. Wolner-Hanssen P, Paavonen J, Kiviat N, et al.: Ambu- latory treatment of suspected pelvic inflammatory disease with Augmentin, with or without doxycycline. Am J Ob- stet Gynecol 158(3 pt 1):577-579, 1988. 6. Centers for Disease Control: Sexually transmitted disease treatment and guidelines, 1985. MMWR 34(Suppl 4s): 75s-108s, 1985. 7. Bowie WR, Alexander ER, Holmes KK: Eradication of Chlamydia trachomatis from the urethras of men with nongonococcal urethritis by treatment with amoxicillin. Sex Transm Dis 8:79-81, 1981. 8. Csango PA, Gundersen T, Martinsen IM: Effect of amoxicillin on simultaneous Chlamydia trachomatis in men with gonococcal urethritis: Comparison of three dosage regimens. Sex Transm Dis 12:93-96, 1985. 9. Platt MS: Neonatal Haemophilus vaginalis infection. Clin Pediatr 10:513, 1971. 10. Sanders LL, Jr, Harrison HR, Washington AE: Treat- ment ofsexually transmitted chlamydial infections. JAMA 255:1750-1756, 1986. 11. Toomey KE, Barnes RC: Treatment of Chlamydia tra- chomatis genital infection. Rev Infect Dis 12(Suppl 6): s645-s655, 1990. 12. Pearlman MD, Faro S, Riddle GD, Tortolero G: In vitro synergy of clindamycin and aminoglycosides against Chlamydia trachomatis.

transmitted chlamydial infections. JAMA 255:1750-1756, 1986. 11. Toomey KE, Barnes RC: Treatment of Chlamydia tra- chomatis genital infection. Rev Infect Dis 12(Suppl 6): s645-s655, 1990. 12. Pearlman MD, Faro S, Riddle GD, Tortolero G: In vitro synergy of clindamycin and aminoglycosides against Chlamydia trachomatis. Antimicrob Agents Chemother 34: 1399-1402, 1990. 13. Martens MG, Faro S, Maccato M, Riddle G, Hammill H, Wang Y: In vitro susceptibility testing of clinical isolates ofChlamydia trachomatis. Infect Dis Obstet Gyne- col 1:40-45, 1993. 14. Martin DH, Pastorek JG, Faro S: In vitro and in vivo activity of parenterally administered [3-1actam antibiotics against Chlamydia trachomatis. Sex Trans Dis 13:81-87, 1981. 15. Bowie WR: In vitro activity ofthe clavulanic acid, amox- icillin, and ticarcillin against Chlamydia trachomatis. An- timicrob Agents Chemother 29:713-715, 1986. 16. Martens MG, Faro S: [3-1actam antibiotics and Chlamy- dia trachomat#. Adv Ther 5:113-118, 1988. 17. Kirshon B, Faro S, Phillips LE, Pruett K: Correlation of ultrasonography and bacteriology of the endocervix and posterior cul-de-sac of patients with severe pelvic inflam- matory disease. Sex Transm Dis 15:103-107, 1988. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:108-113 (1993) (C) 1993 Wiley-Liss, Inc. Quinolones for the Treatment of Neisseria gonorrhoeae and Chlamydia trachomatis Sebastian Faro Department ofGynecology and Obstetrics, The University ofKansas School ofMedicine, Kansas City, KS ABSTRACT The most commonly sexually transmitted bacteria are Neisseria gonorrhoeae and Chlamydia tracho. matis. The quinolones ofloxacin and ciprofloxacin have been shown to have activity against both of these bacteria in vitro and in vivo. Ofloxacin is particularly well suited for the treatment of N. gonorrhoeae and C. trachomatis cervical infection, which can be considered the earliest manifestation of pelvic inflammatory disease (PID). Not only can ofloxacin be effectively used as a single agent, it is also useful in treating urinary tract infections caused by Enterobacteriaceae. Although it has moderate activity against anaerobes in general, ofloxacin does have activity against the anaerobes commonly isolated from female patients with soft tissue pelvic infections. Thus, ofloxacin has the potential for being utilized to treat early salpingitis. (C) 1993 Wiley-Liss, Inc. KEY WORIS Ofloxacin, bacterial infections, sexually transmitted diseases Ieisseria gonorrhoeae and Chlamydia trachomatis are the two most common sexually transmitted bacteria. They are responsible for the majority of diseases involving the female reproductive tract that can result in devastating sequelae. The spectrum of diseases secondary to infection with either or both of these bacteria encompasses relatively simple in- fections such as urethritis and cervicitis and more complex infections such as Bartholin's gland and Skene's gland abscesses, endometritis, salpingitis, disseminated gonococcal disease, and gonococcal ar- thritis. -6 Infection of the upper genital tract can and often does result in infertility.

es relatively simple in- fections such as urethritis and cervicitis and more complex infections such as Bartholin's gland and Skene's gland abscesses, endometritis, salpingitis, disseminated gonococcal disease, and gonococcal ar- thritis. -6 Infection of the upper genital tract can and often does result in infertility. Even if the patient is able to achieve pregnancy, she is at con- siderable risk for the development of an ectopic pregnancy.7-1 In addition, an individual who has had salpingitis is often left with pelvic adhesions and chronic pelvic pain or develops a pyosalpinx that can result in a hydrosalpinx or tubo-ovarian abscess.7'1-13 Such an individual must often un- dergo a hysterectomy with removal of her ovaries, requiring subsequent hormonal replacement. Therefore, the best defense against the patient's developing advanced disease is early recognition of the initial infection along with appropriate treat- ment and follow-up management. Early gonococcal infection, i.e., urethritis and cervicitis, is currently treated with ceftriaxone (Rocephin, Roche Laboratories, Nutley, NJ), 150 mg IM, followed by doxycycline, 100 mg orally twice daily for 7 days, to eradicate any chlamydial infection. This treatment regimen, recommended by the Centers for Disease Control (CDC), 4 is a logical one because these two bacteria commonly coinfect individuals. Of women with gonococcal cervicitis, approximately 26% will be coinfected with C. trachomat#. 15 Since most patients are treated prior to receipt of the culture results, it is important that antibiotic therapy be effective against both N. gonorrhoeae (including penicillinase-pro- Address correspondence/reprint requests to Dr. Sebastian Faro, Department of Gynecology and Obstetrics, The University ofKansas School ofMedicine, 3901 Rainbow Blvd., Kansas City, KS 66160-7316. Review Article Received August 18, 1993 Accepted August 19, 1993 QU1NOLONE TREATMENT OF GONORRHEA AND CHLAMYDIA FARO TABLE I. Risk factors for sexually transmitted disease 15 to 25 years of age (age group most vulnerable) Use of oral contraceptives Presence of an IUD Multiple sex partners Exposure to an individual with an STD Presence of an STD History of an STD History of PID ducing strains) and C. trachomatis. In addition, patients seen in sexually transmitted disease (STD) clinics and emergency rooms are not usually com- pliant in their treatment regimen.

ives Presence of an IUD Multiple sex partners Exposure to an individual with an STD Presence of an STD History of an STD History of PID ducing strains) and C. trachomatis. In addition, patients seen in sexually transmitted disease (STD) clinics and emergency rooms are not usually com- pliant in their treatment regimen. If they are not treated at the time of their initial examination but are asked to return for the administration of appro- priate treatment when the laboratory data are known, they are not likely to return. On the other hand, the administration of two antibiotics, one injectable and one oral, is costly and time consum- ing. Therefore, an effective single antibiotic agent for the treatment of both infections would be less costly, more effective, and more convenient. PATIENT EVALUATION Appropriate treatment of the patient with an STD begins with an understanding of the profile of the individual at risk. The first indication that an indi- vidual may be harboring N. gonorrhoeae or C. tra- chomatis is the presence of another sexually trans- mitted organism such as Trichomonas vaginalis, herpes virus, or the human papillomavirus. Demo- graphic characteristics ofpatients at risk for acquir- ing an STD are given in Table 1. A detailed sexual history is important in treating the sexually active patient. Germane to the treatment and follow-up of such a patient is determining whether specimens for the culture ofN. gonorrhoeae and C. trachomatis should also be obtained from the oropharynx and rectum. The clinician should also ascertain whether the patient or her sexual partner has multiple part- ners. Questions should be asked with regard to the presence of genital lesions, past or present, and the existence of lower abdominal pain, no matter how mild or vague. The patient should be further que- ried regarding the use of OCPs and any abnormal vaginal bleeding, i.e., irregular menstrual bleed- ing, breakthrough bleeding, or postcoital bleed- ing. TABLE 2. Ulcerative lesions of the genital tract Syphilis Chancroid Lymphogranuloma venereum Granuloma inguinale Herpes The evaluation begins with a physical examina- tion, reserving the pelvic assessment until last. The pelvic examination should include a thorough as- sessment of the external genitalia with retraction of the clitoral hood and inspection between the crural folds. A colposcopic examination will facilitate the detection of vulvar lesions.

gins with a physical examina- tion, reserving the pelvic assessment until last. The pelvic examination should include a thorough as- sessment of the external genitalia with retraction of the clitoral hood and inspection between the crural folds. A colposcopic examination will facilitate the detection of vulvar lesions. The labia should be thoroughly painted with 5% acetic acid to highlight abnormal tissue and discern acetowhite epithelium. If an ulcerative lesion is found, it should be evalu- ated for the STDs listed in Table 2. Ulcerative lesions may or may not be differenti- ated on physical examination. The chancre of syph- ilis is typically a clean-appearing, painless lesion, whereas the lesions of the other bacterial STDs are painful, with ragged borders, and have an overall "dirty" appearance. Herpetic ulcers vary in size from pinpoint lesions to large, crater-like ulcers and are typically very painful. Appropriate speci- mens should be obtained for Gram stain, Giemsa stain, dark-field examination, and culture. The lab- oratory should be consulted for the precise method for obtaining, transporting, and labeling the speci- mens. In this way, the specimens will be processed appropriately for retrieval of the desired organ- isms. The vestibule of the vagina should be inspected for the presence of a purulent discharge. The ure- thra and Skene's and Bartholin's glands should be palpated to determine if a purulent exudate is present. Ifan exudate is present, a specimen should be obtained for Gram stain and culture for the isolation of N. gonorrhoeae, C. trachomatis, Myco- plasma hominis, and Ureaplasma urealyticum. Again, the entire genitalia should be examined for ulcer- ative or abnormal growths. A biopsy specimen should be taken of any lesion that is suspicious for malignancy. The vagina should be inspected for the charac- teristics of any discharge: color, consistency, pH, and odor. A healthy vaginal discharge has a pH of 3.8 to 4.2. A pI-I value between 4.2 and 4.5 may INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 109 QUINOLONE TREATMENT OF GONORRHEA AND CHLAMYDIA FARO indicate an abnormality, whereas a pI--I > 4.5 defi- nitely indicates an abnormal vaginal microflora. Lower hydrogen ion concentrations (pH > 4.5) indicate the presence of high counts of anaerobic bacteria or T. vaginalis. The evaluation of the cervix begins with an as- sessment of the endocervical tissue: is it hyper- trophic, erythematous, necrotic, or ulcerated?

defi- nitely indicates an abnormal vaginal microflora. Lower hydrogen ion concentrations (pH > 4.5) indicate the presence of high counts of anaerobic bacteria or T. vaginalis. The evaluation of the cervix begins with an as- sessment of the endocervical tissue: is it hyper- trophic, erythematous, necrotic, or ulcerated? Is an endocervical mucopurulent discharge present? It should also be noted whether the cervix bleeds briskly when gently touched with a cotton-tipped applicator. The aforementioned characteristics are all indications that an infection may be present. An endocervical specimen should be obtained with a Dacron-tipped applicator with a plastic or metal shaft. Cultures should be obtained for C. tracho- matis and N. gonorrhoeae. A Gram stain of the endocervical discharge is helpful. If the specimen does not reveal the presence of squamous epithelial cells, Candida albicans, or T. vaginalis, it can be considered a valid specimen not containing a vagi- nal discharge. WBCs with gram-negative diplo- cocci may indicate N. gonorrhoeae. However, if there are WBCs but no gram-stainable bacteria, a tentative diagnosis of C. trachomatis infection can be made, although an M. hominis or U. urealyticum infection cannot be excluded. The Gram stain should not be performed in lieu of obtaining speci- mens for the isolation of N. gonorrhoeae, C. tra- chomatis, or other indicated STDs. A pap smear should be obtained if several months have elapsed since the patient's last pap smear or if she has evi- dence of cervicitis. The pap smear will help to establish the presence ofan inflammatory condition or, ifall culture data are negative, a viral infection. In addition to cultures for N. gonorrhoeae and C. trachomat#, specimens should be obtained for M. hominis and U. urealyticum. Next, a bimanual examination should be per- formed. Although the patient may be suspected of having an uncomplicated lower genital tract infec- tion, it is crucial to the preservation of her fertility and prevention of an ectopic pregnancy that upper genital tract involvement be ruled out. The presen- tation of a patient with pelvic inflammatory disease (PID) is subtle, in contrast to the presentation ofan individual with obvious salpingitis, who has a pu- rulent discharge, cervical motion and adnexal ten- derness, fever, and an elevated WBC count. The essence of the examination is the detection of ten- TABLE 3.

en- tation of a patient with pelvic inflammatory disease (PID) is subtle, in contrast to the presentation ofan individual with obvious salpingitis, who has a pu- rulent discharge, cervical motion and adnexal ten- derness, fever, and an elevated WBC count. The essence of the examination is the detection of ten- TABLE 3. Fluoroquinolones Amifloxacin Ciprofloxacin Difloxain Enoxacin Fleroxain Lomefloxacin Norfloxacin Ofloxacin Pefloxacin Temafloxacin Tosufloxacin derness on motion or palpation ofthe cervix, uterus, or adnexa. Even though the patient is afebrile with a normal WBC count, she may have vague lower abdominal pain that she does not relate to the physi- cian or is not aware of until a pelvic examination is performed. TREATMENT As mentioned previously, the CDC's recommenda- tion for patients with suspected or proven uncom- plicated gonococcal or chlamydial infection is the administration of ceftriaxone, 250 mg IM, fol- lowed by doxycycline, 100 gm orally twice daily for 7 days. 14 However, this treatment is costly and time-consuming, and the IM injection may be pain- ful to the patient. The new quinolone ofloxacin offers the opportunity to treat both gonococcal and chlamydial infections with a single oral agent. The fluoroquinolones are derived from nalidixic acid, which was placed in clinical use in 1962 pri- marily for the treatment of gram-negative urinary traction infections. 16 The new quinolones differ from nalidixic acid in having a fluorine atom and piperazyl moiety (Table 3). Although the fluoro- quinolones all possess the basic quinolone struc- turea fluorine atom at the 6 position and a piper- azine ring at the 7 positionmthey differ from one another by substitutions at the position. Ofloxacin is unique among the fluoroquinolones in that it has a tricyclic ring structure. The tricyclic ring results in the molecule's having increased gram-positive and anaerobic activity and decreased metabolism. Ofloxacin has a broad spectrum of activity that includes gram-positive aerobic and facultative anaerobes (Table 4). Bacteroidesfragilis, Bacteroides melaninogenicus, Fusobacterium, Peptostreptococcus, and Clostridium difficile are all sensitive to ofloxa- cin. 17-20 However, its efficacy in the treatment of I0 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY QUINOLONE TREATMENT OF GONORRHEA AND CHLAMYDIA FARO TABLE 4.

bes (Table 4). Bacteroidesfragilis, Bacteroides melaninogenicus, Fusobacterium, Peptostreptococcus, and Clostridium difficile are all sensitive to ofloxa- cin. 17-20 However, its efficacy in the treatment of I0 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY QUINOLONE TREATMENT OF GONORRHEA AND CHLAMYDIA FARO TABLE 4. Bacterial spectrum of activity of ofloxacin Enterobacteriaceae STB Citrobacter Hemophilus ducreyi Enterobacter Chlamydia trachomatis Escherichia Neisseria gonorrhoeae Hafnia Ureaplasma urealyticum Klebsiella Pseudomonas Morganella Gram-positive organisms Proteus Staphylococcus aureus Providencia Streptococcus agalactiae Salmonella Streptococcus pneumoniae Serratia Streptococcus pyogenes Yersinia Enterococcus faecalis asexually transmitted bacteria. blncludes both penicillin-sensitive and penicillin-resistant strains. infections involving anaerobic bacteria has not been established. Until sufficient data are available, an agent with a proven anaerobic spectrum should be included in the treatment regimen. The antimicrobial spectrum ofactivity exhibited by ofloxacin makes it useful for a variety of infec- tions, e.g., skin and soft tissue, respiratory, uri- nary tract, gonococcal, and chlamydial infec- tions.21-24 The susceptible minimal inhibitory concentration (MIC) breakpoint is 2 Ixg/ml, and the MIC for resistance is > 8 g/ml.25'26 In vitro and in vivo studies have shown that ofloxacin is as effective as amoxicillin or ampicillin plus probene- cid and doxycycline for the treatment ofgonococcal and chlamydial infection. In a multicenter study, a single 400-mg dose ofofloxacin was compared with a single 3.0-g dose ofamoxicillin or a 4.5-g dose of ampicillin plus g of probenecid. Ofloxacin erad- icated the gonococcus in 98% of the patients, whereas the amoxicillin-probenecid regimen eradi- cated 95%. 24 In a comparative study of the treat- ment of chlamydia, 533 patients were randomly assigned to a 7-day course of either ofloxacin, 300 mg every 12 hours, or doxycycline, 100 mg every 12 hours. The two agents were reported to be equally effective, achieving cure rates of 99% and 95%, respectively.27-3 Although ciprofloxacin has been shown to have activity against N. gonorrhoeae and C. trachomatis, patients treated for the latter have relapsed. There- fore, while ciprofloxacin appears to be quite ade- quate for the treatment of N. gonorrhoeae, it is not reliable for the treatment of C. trachomatis. Nor- floxacin also has activity against N.

as been shown to have activity against N. gonorrhoeae and C. trachomatis, patients treated for the latter have relapsed. There- fore, while ciprofloxacin appears to be quite ade- quate for the treatment of N. gonorrhoeae, it is not reliable for the treatment of C. trachomatis. Nor- floxacin also has activity against N. gonorrhoeae but not against C. trachomatis. The use of norfloxacin to treat cervical infection caused by N. gonorrhoeae is appropriate but requires the addition of an agent effective against C. trachomatis. To be cost-effec- tive and therapeutically sound, the treatment of cervicitis suspected of being caused by either or both of these sexually transmitted bacteria should be accomplished with a single effective agent such as ofloxacin. METABOLISM Ofloxacin, unlike other fluoroquinolones, is not metabolized to compounds with lesser antimicro- bial activity. All fluoroquinolones except ofloxacin are metabolized or produce an oxoquinolone me- tabolite that interferes with the metabolism of theo- phylline and caffeine.31,2 Urinary recovery ofme- tabolites of ofloxacin accounts for approximately 5% of the drug. The dosage of ofloxacin must be adjusted in the face of renal impairment, that is, a creatinine clearance of < 50 ml/min. The half-life of ofloxacin is 9 hours. After the administration of 500 mg ofofloxacin twice a day and achievement of a plasma steady state, the peak level exceeding 5 Ixg/ml and trough level of p.g/ml are obtained. TOXICITY Approximately 3,200 patients in clinical trials in the United States have received ofloxacin for 3 to 10 days. The most frequent adverse reactions were nausea (3.5%), insomnia (1.8%), headache (1.4%), and dizziness (1.2%). Animal studies revealed that arthropathy, char- acterized by blisters, erosion, and an increase in synovial fluid, occurred in rats and dogs. The ef- fects were age and dose dependent.4 These effects are characteristics of all quinolones and, although arthropathy has not been demonstrated in humans, children and pregnant women should not receive fluoroquinolone therapy. CONCLUSIONS Of the quinolones currently available, only ofloxa- cin presents the physician with the opportunity of treating the two most common sexually transmitted bacterial infections with a single agent. The CDC recommended in 1985 that any treatment for un- complicated gonorrhea include treatment for chlamydial infection.

IONS Of the quinolones currently available, only ofloxa- cin presents the physician with the opportunity of treating the two most common sexually transmitted bacterial infections with a single agent. The CDC recommended in 1985 that any treatment for un- complicated gonorrhea include treatment for chlamydial infection. 14 This recommendation was deemed necessary because ofthe decrease in efficacy INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY QUINOLONE TREATMENT OF GONORRHEA AND CHLAMYDIA FARO of penicillin in treating gonococcal infection and the increase in the chromosomally mediated resis- tance of N. gonorrhoeae to tetracycline as well as penicillin. 15 Ofloxacin offers the benefits of not being affected by enzymes that afford the gonococ- cus resistance to tetracycline or penicillins and of being effective as a single dose. The antibacterial activity of the quinolones resides in their ability to interfere with DNA synthesis by inhibiting the bacterial DNA gyrase enzyme that is responsible for the spiral structure producing supercoils, which are characteristic of DNA.3s'36 The fact that only bacteria possess DNA gyrase increases the safety of fluoroquinolones. Since ofloxacin is taken orally and has excellent absorption via the gastrointestinal tract, therapy is facilitated in busy clinical settings in comparison with IM injection ofceftriaxone or 3 to 4.5 g of amoxicillin or ampicillin. The spectrum ofactivity of ofloxacin makes this agent potentially suited for the treatment of PID. Initial studies (unpublished) have shown that oflox- acin is as effective as cefoxitin in the treatment of early, uncomplicated PID. The major concern is the decreased activity of quinolones against gram- negative obligate anaerobes. However, an individ- ual experiencing a first episode ofPID is primarily infected with N. gonorrhoeae, or C. trachomatis, or both and is not likely to have a complex infection involving both aerobic and anaerobic bacteria. If there is a concern over a possible mixed aerobic and anaerobic infection, ofloxacin, which provides ex- cellent coverage against N. gonorrhoeae, C. tra- chomatis, Enterobacteriaceae, and gram-positive aerobic cocci, could be administered with an agent such as metronidazole. Additional studies are needed to determine if ofloxacin is indicated in the treatment of PIE). There is no doubt that ofloxacin is well suited for the treatment of uncomplicated gonococcal and chlamydial infection.

bacteriaceae, and gram-positive aerobic cocci, could be administered with an agent such as metronidazole. Additional studies are needed to determine if ofloxacin is indicated in the treatment of PIE). There is no doubt that ofloxacin is well suited for the treatment of uncomplicated gonococcal and chlamydial infection. Although ofloxacin has been associated with low toxicity, arthropathic effects have been demonstrated in rats and dogs. There- fore, ofloxacin, like other quinolones, should not be administered to children younger than 18 years ofage or to pregnant or breastfeeding women. REFERENCES 1. Faro S: Chlamydia trachomatis infection in women. J Re- prod Med 30:273-278, 1985. 2. McCormack WM, Rosner B, McComb DE, et al.: In- fection with Chlamydia trachomatis in female college stu- dents. AmJ Epidemiol 121:107-115, 1985. 3. Paavonen J, Roberts PL, Stevens CE, et al.: Randomized treatment of mucopurulent cervicitis with doxycycline or amoxicillin. Am J Obstet Gynecol 161 128-135, 1989. 4. Phillips RS, Safran C, Aronson MD, Taylor WC: Should women be tested for gonococcal infection of the cervix during routine gynecologic visits? An economic appraisal. Am J Med 86:297-302, 1989. 5. Backman M, Ruden A-K, Bygdeman SM, et al.: Gono- coccal serovar distribution in Stockholm with special ref- erence to multiple infections and infected partners. Acta Pathol Microbiol Immunol Scand Sect B 93:225-232, 1985. 6. Covino JM, Cummings M, Smith B, et al.: Comparison ofofloxacin and ceftriaxone in the treatment ofuncompli- cated gonorrhea caused by penicillinase-producing and non-penicillinase-producing strains. Antimicrob Agents Chemother 34:148-149, 1990. 7. Kirshon B, Faro S, Phillips LE, Pruett K: Correlation of ultrasonography and bacteriology of the endocervix and posterior cul-de-sac of patients with severe pelvic inflam- matory disease. Sex Transm Dis 15:103-107, 1988. 8. Washington AE, Cates W, Zaidi AA: Hospitalization for pelvic inflammatory disease. JAMA 25:2529-2533, 1984. 9. Chaim W, Sarov B, Sarov I, Piura B, Cohen A, Insler V: Serum IgG and IgA antibodies to chlamydia in ectopic pregnancies. Contraception 40:59-71, 1989. 10. Conway D, Glazener CM, Caul EO, et al.: Chlamydial serology in fertile and infertile women. Lancet 1:191- 193, 1984. 11. Dodson MG, Faro S, Gentry LO: Treatment of acute pelvic inflammatory disease with aztreonam, a new mono- cyclic beta-lactam antibiotic, and clindamycin. Obstet Gy- necol 67:657-662, 1986. 12.

89. 10. Conway D, Glazener CM, Caul EO, et al.: Chlamydial serology in fertile and infertile women. Lancet 1:191- 193, 1984. 11. Dodson MG, Faro S, Gentry LO: Treatment of acute pelvic inflammatory disease with aztreonam, a new mono- cyclic beta-lactam antibiotic, and clindamycin. Obstet Gy- necol 67:657-662, 1986. 12. Ginsberg K, Faro S: Management ofpelvic inflammatory disease. Infect Surg 6:562-567, 1987. 13. Svensson L, Mardh PA, Westrom L: Infertility after acute salpingitis with special reference to Chlamydia tra- chomatis. Fertil Steril 40:322-329, 1983. 14. Centers for Disease Control: 1985 STD treatment guide- lines. MMWR 34(Supp145):755-1085, 1985. 15. Rice RJ, Thompson SE: Treatment of uncomplicated in- fections due to Neisseria gonorrhoeae. A review of clinical efficacy and in vitro susceptibility studies from 1982 through I985. JAMA 255:1739-1746, 1986. 16. Lesher GY, Froelich ED, Gruet MD, et al: 1,8 Naph- thyridine derivatives. A new class of chemotherapeutic agents. J Med Pharm Chem 5:1063, 1962. 17. Gruneberg RN, Felmingham D, O'Hare MD, et al.: The comparative in vitro activity of ofloxacin. J Antimi- crob Chemother 22(Suppl C):9-19, 1988. 18. van Caekenberghe DL, Pattyn SR: In vitro activity of ciprofloxacin compared with those ofnew fluorinated pip- erazinyl-substituted quinolone derivatives. Antimicrob Agents Chemother 25:518-521, 1984. 19. King A, Phillips I: The comparative in vitro activity of eight newer quinolones and nalidixic acid. J Antimicrob Chemother 18(Suppl D): 1-20, 1986. 112 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY QUINOLONE TREATMENT OF GONORRHEA AND CHLAMYDIA FARO 20. Delmee M, Avesani V: Comparative in vitro activity of seven quinolones against 100 isolates of Clostridium diffi- cile. Antimicrob Agents Chemother 29:374-375, 1986. 21. Gentry LO, Rodriguez-Gomez G, Zeluff BJ, Khoshdel A, Price M: A comparative evaluation of oral ofloxacin versus intravenous cefotaxime therapy for serious skin and skin structure infections. Am J Med 87(Suppl 6C): 57S-60S, 1989. 22. Cox CE: Ofloxacin in the management of complicated urinary tract infections, including prostatitis. Am J Med 87(Suppl 6C):61S-68S, 1989. 23. Stocks JM, Wallace RJ, Griffith DE, Garcia JG, Kohler RB: Ofloxacin in community-acquired lower respiratory infections. A comparison with amoxicillin or erythromy- cin. Am J Med 87(Suppl 6C):52S-56S, 1989. 24. Lutz FB: Single-dose efficacy of ofloxacin in uncompli- cated gonorrhea. Am J Med 87(Suppl 6C):69S-74S, 1989. 25.

cks JM, Wallace RJ, Griffith DE, Garcia JG, Kohler RB: Ofloxacin in community-acquired lower respiratory infections. A comparison with amoxicillin or erythromy- cin. Am J Med 87(Suppl 6C):52S-56S, 1989. 24. Lutz FB: Single-dose efficacy of ofloxacin in uncompli- cated gonorrhea. Am J Med 87(Suppl 6C):69S-74S, 1989. 25. Fuchs PC, Barry AL, Jones RN, Thornsberry C: Pro- posed disk diffusion susceptibility criteria for ofloxacin. J Clin Microbiol 22:310-311, 1985. 26. National Committee for Clinical Laboratory Standards: Performance Standards for Antimicrobial Susceptibility Testing; Second Informational Supplement. Villanova, PA: NCCLS, M 100-$2, 1987. 27. Schachter J, Moncada JV: In vitro activity of ofloxacin against Chlamydia trachomatis. Am J Med 87(Suppl 6C): 14S-16S, 1989. 28. Batteiger BE, Jones RB, White A: Efficacy and safety of ofloxacin in the treatment ofnongonococcal sexually trans- mitted disease. AmJ Med 87(Suppl 6C):75S-77S, 1989. 29. judson FN, Beals BS, Tack KJ: Clinical experience with ofloxacin in sexually transmitted disease. Infection 14(Suppl 4):$309-$310, 1986. 30. Fransen L, Avonts D, Piot P: Treatment of genital chlamydial infection with ofloxacin. Infection 14(Suppl 4):$318-$320, 1986. 31. Edwards DJ, Bowles SK, Svensson CK, Rybak MJ: Inhi- bition of drug metabolism of quinolone antibiotics. Clin Pharmacokinet 15:194-204, 1988. 32. Wijnands WJ, Vree TB, van Herwaarden CL: The influ- ence of quinolone derivatives on theophylline clearance. Br J. Clin Pharmacol 22:677-683, 1986. 33. Flor S: Pharmacokinetics of ofloxacin: An overview. Am J Med 87(Suppl 6C):24S-30S, 1988. 34. Davis GJ, McKenzie BE: Toxicologic evaluation ofoflox- acin. Am J Med 87(Suppl 6C):43S-46S, 1989. 35. Gellert M, Mizuuchi K, O'Dea MH, Nash HA: DNA gyrase: An enzyme that introduces superhelical turns into DNA. Proc Natl Acad Sci USA 73:3872-3876, 1976. 36. Cook TM, Brown KG, Guss WA: Bactericidal action of nalidixic acid on B. subtilis. J Bacteriol 92:1510-1514, 1966. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:76-81 (1993) (C) 1993 Wiley-Liss, Inc. Prematurity, Subclinical Intraamniotic Infection, and Fetal Biophysical Parameters: Is There a Correlation? Susan M. Cox, Scott Roberts, Percilis Roussis, Berry A. Campbell, and Thomas E. Curry, Jr. Department ofObstetrics and Gynecology, University ofKentucky, Lexington, KY ABSTRACT Objective: This prospective study was undertaken to examine the effects ofsubclinical intraamniotic infection on fetal behavioral patterns. Methods: Amniotic fluid was obtained from four groups of patients (n 99): group 1, patients with preterm premature rupture of the fetal membranes (PPROM) without infection; group 2, patients with PPROM and infection; group 3, patients with preterm labor (PTL) and without infection; and group 4, patients with PTL and infection. Fetal biophysical profiles were obtained on admission to the labor suite. Amniotic fluid was analyzed for the presence of microorganisms and endotoxin to confirm intraamniotic infection; cytokines interleukin (IL)-113, IL-6, and IL-8 were also assayed. Results: We found no association between low scores for biophysical parameters and subclinical infection in patients with PPROM or PTL. Conclusions: We could not demonstrate that upon a patient's admission to the labor hall absent fetal breathing and absent fetal movement, as well as reactivity, correlate with subclinical intraam- niotic infection. Elevated cytokines, i.e. IL-113, IL-6, and IL-8 were associated with subclinical chorioamnionitis. (C) 1993 Wiley-Liss, Inc. KEY WORDS Preterm labor, PROM, cytokines, interleukin uring the last several years a body of evidence has accumulated suggesting that preterm labor (PTL) and premature rupture of the fetal mem- branes (PROM) are causally related to subclinical infection.

ated with subclinical chorioamnionitis. (C) 1993 Wiley-Liss, Inc. KEY WORDS Preterm labor, PROM, cytokines, interleukin uring the last several years a body of evidence has accumulated suggesting that preterm labor (PTL) and premature rupture of the fetal mem- branes (PROM) are causally related to subclinical infection. The findings of such studies supportive ofthis premise include the isolation of microorgan- isms from amniotic fluid (AF) in 15% of preterm labor pregnancies and the presence of bacterial endotoxin in amniotic fluid in approximately 30% of preterm labor pregnancies.2'3 Additionally, the inflammatory microbial products associated with subclinical infection, such as cytokines, are elevated in amniotic fluid of preterm pregnancies. For example, interleukin (IL)-I[3 is found in AF of approximately 50% of PTL pregnancies4; second, high levels of IL-6 are present in AF of approximately 50% of PTL pregnancies5; third, increased concentrations of IL-8 are found in AF of PTL pregnancies associated with intraamniotic infection (IAI)6; fourth, tumor necrosis factor (TNF)-e is present in AF of some PTL pregnan- cies7'8; and, finally, prostaglandins are identified in AF of infection-associated preterm labor.9' 10 Analysis of fetal behavior has recently been used to evaluate patients with PROM. The findings of several studies suggest that the infected fetus be- haves differently from the noninfected fetus. 11-14 In particular, a decrease in the amount of fetal Address correspondence/reprint requests to Dr. Susan M. Cox, at her present address, of University ofTexas Southwestern Medical Center, Dept. ofOB/GYN, 5323 Harry Hines Boulevard, Dallas, TX 75235-9032. Clinical Study Received January 19, 1993 Accepted May II, 1993 INFECTION AND BIOPHYSICAL PROFILE COX ET AL. TABLE I. Selected biophysical parameters of patients with preterm premature rupture of membranes (PPROM) and preterm labor (PTL) with intact fetal membranes according to presence or absence of intraamniotic infection PPROM (n 30) PTL (n 69) Infected Noninfected Infected Noninfected (n 6) (n 24) (n 8) (n 61) Absent fetal breathing (17) 5 (21) 2 (25) 7 (I I) Absent fetal movement (17) 0 (I 3) 0 Nonreactivity (17) 2 (8) (I 3) 5 (8) No significant differences detected. Number, percent in parentheses. breathing and fetal movement, as well as a nonreac- tive nonstress test (NST), has been observed in the presence of infection.

Absent fetal breathing (17) 5 (21) 2 (25) 7 (I I) Absent fetal movement (17) 0 (I 3) 0 Nonreactivity (17) 2 (8) (I 3) 5 (8) No significant differences detected. Number, percent in parentheses. breathing and fetal movement, as well as a nonreac- tive nonstress test (NST), has been observed in the presence of infection. The purpose of this study was to determine pro- spectively the incidence of subclinical IAI in preg- nancies complicated by PTL and PROM. Addi- tionally, we sought to determine if absent fetal breathing, fetal body movement, or nonreactivity on admission to the labor hall could serve as an indicator of subclinical infection. SUBJECTS AND METHODS The University of Kentucky (UK) Chandler Med- ical Center is a Level III regional referral center serving eastern and central Kentucky. The majority of patients entering this study were transferred to UK from nearby facilities, and all patients received magnesium sulfate tocolysis for transfer. Entry criteria for this prospective study in- cluded: 1) either documented PTL (cervix > 2 cm and 80% effaced or documented cervical change) or documented PPROM (nitrazine, pooling, and ferning tests); 2) singleton of fewer than 34 com- pleted weeks of gestation; and 3) no other medical or obstetric complications. All patients were admit- ted to the labor suite for evaluation. After the pa- tient had given informed consent, amniotic fluid was retrieved by transabdominal amniocentesis. The fluid was placed on ice and transported to the labo- ratory. After centrifugation at 300g for 10 minutes at 4C, the supernatant was stored in polypropylene tubes at -70C until cytokine analysis. A small amount of fluid was stored at -20C in endotoxin free tubes until the Limulus amoebocyte lysate (LAL) test was performed for endotoxin detection. Amniotic fluid for microbiologic analysis was trans- ported to the hospital laboratory in a capped plastic syringe and plated immediately for aerobic and anaerobic culture. Intraamniotic infection was de- fined as the presence of bacteria or endotoxin in the amniotic fluid. The biophysical profile score was obtained in all patients by means of a real time ultrasonographic machine equipped with a 3.5 MHz curvilinear- array transducer.

d plated immediately for aerobic and anaerobic culture. Intraamniotic infection was de- fined as the presence of bacteria or endotoxin in the amniotic fluid. The biophysical profile score was obtained in all patients by means of a real time ultrasonographic machine equipped with a 3.5 MHz curvilinear- array transducer. Scanning consisted ofa 30 minute observation period and a biophysical profile score assigned as per the criteria of Manning and co- workers, is An NST was considered reactive if 2 episodes of fetal heart rate acceleration greater than 15 beats for 15 seconds were observed in 20 min- utes. An NST was considered nonreactive if there were no accelerations in 40 minutes. Amniotic fluid was analyzed for endotoxin as previously described by Cox and colleagues.2 Quan- tification of cytokines in the AF was measured by sensitive and specific enzyme-linked immunosor- bent assays (ELISA) for IL-113 (Cistron Biotech- nologies, Pinebrook, NJ), IL-6 (Genzyme Corpo- ration, Cambridge, MA), and IL-8 (R and D Systems, Minneapolis, MN). These assays were validated for use in AF by determining nonspecific binding and parallelism of the assay. Analysis ofcategorical data was performed using 2 2 contingency tables with the two-tail Fisher's exact test. Continuous data were analyzed using the Mann-Whitney two-sample statistic. A P value less than 0.05 was considered significant. RESULTS The outcome of the patients in the study is summa- rized in Table 1. Amniotic fluid was retrieved from all 99 patients. No patients met the criteria for clinical chorioamnionitis, such as the presence of fever, maternal tachycardia, fetal tachycardia, and uterine tenderness. Of the 30 patients with PPROM on admission, 20% had laboratory signs INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 77 INFECTION AND BIOPHYSICAL PROFILE COX ET AL. TABLE 2. Amniotic fluid analysis and outcomes in patients with premature rupture of the fetal membranesa IAI (+) IAI (-) (n 6)b (n 24)b Culture positive [no. (%)] 3 (50) 0 0.005 LPS positive [no. (%)] 4 (67) 0 <.001 Mean IL-113 (ng/mL) 18.6 -+ 7. 0.027 Mean IL-6 (ng/mL) 10.6 2.4 0.26 -+ 0.01 0.008 Mean IL-8 (ng/mL) 90. -+ 1.7 1.6 -0.06 0.004 Fetal breathing absent [no. (%)] (17) 4 (16) NS Fetal movement absent [no. (%)] (17) 0 NS Nonreactivity [no. (%)] (17) 2 (8) NS Discharged undelivered [no. (%)] 0 3 (I 3) NS Mean time to delivery (d) 5.5 1.0 6.9 -1.8 NS alL, interleukin; LPS, lipopolysaccharide.

+ 0.01 0.008 Mean IL-8 (ng/mL) 90. -+ 1.7 1.6 -0.06 0.004 Fetal breathing absent [no. (%)] (17) 4 (16) NS Fetal movement absent [no. (%)] (17) 0 NS Nonreactivity [no. (%)] (17) 2 (8) NS Discharged undelivered [no. (%)] 0 3 (I 3) NS Mean time to delivery (d) 5.5 1.0 6.9 -1.8 NS alL, interleukin; LPS, lipopolysaccharide. IAI (+), intraamniotic infection present; IAI (-), IAI absent;--, not,detectable; NS, not significant. TABLE 3. Amniotic fluid analysis and outcomes in pregnancies complicated by preterm labor (intact membranes) IAI (+) IAI (-) (n 8)b (n 61)b Culture positive [no. (%)] 3 (38) 0 0.001 LPS positive [no. (%)] 5 (62) 0 <0.001 Mean IL-113 (ng/mL) 3.2 -+ 0.5 <0.001 Mean IL-6 (ng/mL) 9.0 1.6 0.3 <0.001 Mean IL-8 (ng/mL) 89.8 -13.3 1.9 -+ 0.17 <0.006 Fetal breathing absent [no. (%)] (I 3) II (18) NS Fetal movement absent [no. (%)] 0 0 Nonreactivity [no. (%)] (I 3) 4 (7) NS Discharged undelivered [no. (%)] (I 3) 45 (74) 0.001 IL, interleukin; LPS, lipopolysaccharide. lAI (+), intraamniotic infection present; IAI (-), IAI absent;--, not detectable. of infection (6 of 3 0 patients), while only 11.6% of the PTL group had signs of infection (8 of 69 patients). The biophysical profile was similar in the PROM and PTL infected groups (Table 1). The outcome of the PROM study patients is summarized in Table 2. Three of the 30 patients (10%) had a positive AF culture, and 2 of these patients were also lipopolysaccharide (LPS) posi- tive. Ofthe culture-negative AF(s), two were LPS- positive. An additional patient who was both cul- ture-negative and LPS-negative had high levels of cytokines (i.e., IL- [3, IL-6, and IL-8) and there- fore was considered in the IAI group. Of the six patients who had IAI, the mean levels ofinflamma- tory cytokines, i.e., IL- [3, IL-6, and IL-8, were 18.6 ng/mL (range 1.1 to 97.8 ng/mL), 10.6 ng/mL (range 1.0 to 33.2 ng/mL), and 90.1 ng/mL (range 1.1 to 230.8 ng/mL), respectively. One of the pregnancies complicated by infection had absent fetal breathing, one had absent fetal movement, and one had no reactivity on a nonstress test. On the other hand, the group with no evidence of intraamniotic infection had AF levels of inflam- matory cytokines that were undetectable (IL- [3) or at the lower limits of assay sensitivity (IL-6 and IL-8). In patients with no evidence of IAI, how- ever, four ofthe fetuses lacked fetal breathing move- ments, and two had nonreactive nonstress tests (Table 3). The mean time to delivery in the infected vs.

of inflam- matory cytokines that were undetectable (IL- [3) or at the lower limits of assay sensitivity (IL-6 and IL-8). In patients with no evidence of IAI, how- ever, four ofthe fetuses lacked fetal breathing move- ments, and two had nonreactive nonstress tests (Table 3). The mean time to delivery in the infected vs. noninfected group was 5.5 and 6.9 days, respectively (range 0.75 to 14 days in infected group vs. <1 to >30 days in the noninfected group). The outcome of the preterm labor pregnancies with intact fetal membranes is summarized in Ta- ble 3. Eight of the 69 patients (11.6%) had evi- dence of IAI: 3 of these patients were culture- positive, and an additional 5 patients who were culture negative had endotoxin in the amniotic 78 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY INFECTION AND BIOPHYSICAL PROFILE COX ET AL. TABLE 4. Results in those pregnancies that delivered within 48 hours of amniocentesis and biophysical profile PPROM (n 10) Preterm labor (n 17) AI (/) IAI (-) IAI + IAI (-) (2) (8) P (4) (13) P Culture positive (no.) 0 NS 0 NS LPS positive (no.) 0 NS 3 0 0.006 IL-II3 (ng/mL) 5.6 _+ 4.6 0.037 4.8 _+ 1.2 0.003 IL-6 (ng/mL) 23.7 _+ 9.4 0.037 13.3 _+ 4.4 0.42 0.003 IL-8 (ng/mL) 148.2 82.6 0.96 0.037 154.7 +- 28.2 5.2 +- 1.0 0.005 Fetal breathing absent [no. (%)] 0 2 (25) NS 0 3 (23) NS Fetal movement absent [no. (%)] (S0) 0 NS 0 0 NS Nonreactivity [no. (%)] 1(50) 0 NS 1(25) 2 (15) NS IAI (+), intraamniotic infection present; IAI (-), IAI absent; L, interleukin; LPS, lipopolysaccharide; PPROM, preterm premature rupture of membranes; not detectable; NS, not significant. fluid. In the infection-positive group, the mean levels of IL-I[3 and IL-6 were 3.2 ng/ml (range 0.1 to 8.9 ng/mL)and 9.0 ng/mL (range .07 to 34.7 ng/mL), respectively. The mean level ofIL-8 in the infected group was 89.8 ng/mL (range 0.4 to 240.4 ng/mL). In the noninfected group, IL-I[3 was not detected in the AF samples analyzed. With respect to IL-6, the majority of AF samples (75.4%) had no IL-6 detected; however, there was a subset of patients (n 15) in whom low levels of IL-6 were identified (range 0.23 to 1.5 ng/mL). In two patients ofthis subset, cervical cultures were positive for group B streptococcus, and the in- traamniotic IL-6 was 1.5 ng/mL in both samples. In patients who were negative for cervical group B streptococcus (n 13), IL-6 was 0.2 ng/mL to 0.9 ng/mL.

om low levels of IL-6 were identified (range 0.23 to 1.5 ng/mL). In two patients ofthis subset, cervical cultures were positive for group B streptococcus, and the in- traamniotic IL-6 was 1.5 ng/mL in both samples. In patients who were negative for cervical group B streptococcus (n 13), IL-6 was 0.2 ng/mL to 0.9 ng/mL. Fetal breathing was absent in patient ofthe infected group and in 11 study patients ofthe group without infection, while fetal movement was present in all patients (Table 3). Reactivity was absent in one patient of the infected group and in four patients of the group without infection. The time from obtaining amniotic fluid and bio- physical profile was greater than 2 days in the ma- jority of patients; therefore, we independently ana- lyzed the group ofpatients who delivered within 48 hours of sampling (Table 4). In the PPROM group, two patients had evidence of infection, with one patient culture-positive and another with LPS present. Cytokines were present in both patients with evidence ofinfection (mean level IL- [3 5.6 ng/mL; IL-6 23.7 ng/mL; and IL-8 148.2 ng/mL). In only one patient was fetal movement absent, and this pregnancy was in the infected group. Two fetuses had absent fetal breathing and movement, and both were in the noninfected group. One fetus was nonreactive (in the infected group). In the pregnancies delivered within 48 hours and complicated by preterm labor, four patients (23.5%) had infection (Table 4). The mean levels of cytokines in these four patients were: IL-I[ 4.8 ng/mL; IL-6 13.3 ng/mL; and IL-8 154.7 ng/mL. Fetal breathing was absent in three study patients, all from the noninfected group. Fetal movement was present in all patients in both groups. Nonreactivity was identified in one patient of the infected group and in two patients of the group without infection. DISCUSSION Current management strategies for preterm prema- ture rupture of fetal membranes employ evaluation of fetal behavioral state. A low biophysical profile score is correlated with clinical chorioamnionitis, 16 but the correlation with subclinical infection is less well studied. Roussis and colleagues in 199114 re- ported that absent fetal breathing movements and a nonreactive nonstress test should serve as indicators for further testing (i.e., amniocentesis) to exclude infection. Other investigators have also examined the relationship between infection and lack of fetal activity.

ell studied. Roussis and colleagues in 199114 re- ported that absent fetal breathing movements and a nonreactive nonstress test should serve as indicators for further testing (i.e., amniocentesis) to exclude infection. Other investigators have also examined the relationship between infection and lack of fetal activity. The conclusions from these studies sup- port the premise that decreased fetal activity is ac- curate in predicting neonatal sepsis and/or infec- tion. 11,13 Recently, a growing body of evidence suggests that inflammatory markers may be a more sensitive index of intraamniotic infection than bacteriologic studies. To document subclinical IAI, we quanti- tated several of the inflammatory markers (i.e., INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 79 INFECTION AND BIOPHYSICAL PROFILE COX ET AL. IL-113, IL-6, and IL-8) in AF. The results of our cytokine data are similar to the results obtained in Dr. Romero's laboratory. We both found high lev- els ofIL- [3 and IL-6 in AF from pregnancies with intraamniotic infection.4'5 With respect to IL-8, Dr. Romero observed mean AF levels of 120 ng/ml in preterm labor pregnancies complicated by infec- tion. The AF ofwomen with PTL and no evidence of infection contained higher levels of IL-8 if they did not respond to tocolysis and they delivered than those not in labor (7 ng/mL vs. undetectable). These levels ofIL-8 are very similar to the levels of IL-8 found in the present study (preterm delivery with infection 154.7 ng/mL; preterm delivery without infection 5.2 ng/mL; and PTL with no evidence of infection 1.9 ng/mL). In the present study subclinical IAI was not associated with a low biophysical profile score. We found no correlation between subclinical infection and a lack of reactivity, absent fetal breathing, or absent fetal movement. Thus alterations in the bio- physical profile may not be an accurate predictor of subclinical IAI. However, it is possible that with the small sample size (only 14 patients had biomo- lecular evidence of subclinical infection), our fail- ure to identify such a relationship may represent a type II error. If the prevalence of abnormal tests (absent fetal breathing, absent fetal tone, and non- reactivity) is 0.25 in the noninfected and 0.75 in the infected group, then inadequate power was present in this study to detect such differences (power 0.67, 0.18 for total and <48 hours in preterm labor group; power 0.41, 0.03 for total and <48 hours in preterm PROM group).

etal breathing, absent fetal tone, and non- reactivity) is 0.25 in the noninfected and 0.75 in the infected group, then inadequate power was present in this study to detect such differences (power 0.67, 0.18 for total and <48 hours in preterm labor group; power 0.41, 0.03 for total and <48 hours in preterm PROM group). Fur- ther, the validity of ascribing predictive value in the total sample when not stratifying for time to delivery is questionable. Developing infection cannot be determined from static lipopolysaccha- ride values and positive cultures obtained by am- niocentesis on admission if the infection is subse- quently initiated. Still, six of these pregnancies delivered within 48 hours of evaluation, and there was still no association with low biophysical param- eters (i.e., absent fetal breathing and movement). Likewise, of the 85 patients without IAI, none were culture-positive, none had LPS present, and none of the patients had elevated levels of cyto- kines, although the biophysical profile was altered in up to 20% ofthese patients (Table 3, fetal breath- ing absent). From these data we could not conclude that fetal biophysical parameters correlate with subclinical intraamniotic infection. Oligohydramnios limits the general usefulness of amniocentesis and subsequent analysis of biomolecular markers of inflammation. At the present time, there is no ideal test available to diagnose infection in PPROM and PTL. Obvi- ously, further studies are needed in this critical area. ACKNOWLEDGMENTS The authors are indebted to Mr. Kevin M. Knox and Ms. Pamela S. Conway for their technical assistance. The help of Mr. Sam Keeble and Ms. Carole Moore during isolation, preparation, and storage of the amniotic fluid samples is gratefully acknowledged. The authors are appreciative of the assistance of Ms. Darleen Chamberlain in the preparation of the manuscript. This work was sponsored by a grant from the NIH, 1-KO8- HDO0853. REFERENCES 1. Cox SM: Infection-induced preterm labor. In Glistrap LC, III, Faro S (eds): Infections in Pregnancy. pp 247- 253, 1990. 2. Cox SM, MacDonald PC, Casey ML: Assay of bacterial endotoxin lipopolysaccharide in human amniotic fluid: Potential usefulness in diagnosis and management of pre- term labor. Am j Obstet Gynecol 159:99-106, 1988. 3. Romero R, Rolanksy P, Oyarzun E, et al.: Labor and infection: Bacterial endotoxin in amniotic fluid and its relationship to the onset of preterm labor. Am J Obstet Gynecol 158:1044-1049, 1988. 4.

mniotic fluid: Potential usefulness in diagnosis and management of pre- term labor. Am j Obstet Gynecol 159:99-106, 1988. 3. Romero R, Rolanksy P, Oyarzun E, et al.: Labor and infection: Bacterial endotoxin in amniotic fluid and its relationship to the onset of preterm labor. Am J Obstet Gynecol 158:1044-1049, 1988. 4. Romero R, Brody DT, Oyarzun E, et al.: Infection and labor; Interleukin- A signal for the onset of parturition. Am J Obstet Gynecol 160:1117-1123, 1989. 5. Romero R, Avila C, Santhanam U, Sehgal PB: Amniotic fluid interleukin 6 in preterm labor. J Clin Invest 85: 1392-1400, 1990. 6. Romero R, Ceska M, Avila C, Mazor M, Behnke E, Lindley I: Neutrophil attactant/activating peptide-1/ interleukin-8 in term and preterm parturition. Am J Ob- stet Gynecol 165:813-820, 1991. 7. Casey ML, Cox SM, Beutler B, Milewich L, MacDon- ald PC: Cachectin/tumor necrosis factor-or formation in human decidua. J Clin Invest 83:430-436, 1989. 8. Romero R, Manogue KR, Mitchell MD, et al.: Infection and labor: Cachectin-tumor necrosis factor in the amni- otic fluid of women with intraamniotic infection and pre- term labor. Am J Obstet Gynecol 161:336-441, 1989. 9. Romero R, Emamian M, Wan M, et al.; Prostaglandin concentrations in amniotic fluid of women with intra- amniotic infection and preterm labor. Am J Obstet Gyne- col 157:1461-1467, 1987. 80 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY INFECTION AND BIOPHYSICAL PROFILE COX ET AL. 10. Casey ML, Cox SM, Word RA, MacDonald PC: Cytok- ines and infection-induced preterm labour. Reprod Fertil Dev 2:499-509, 1990. 11. Vintzileos AM, Campbell WA, Nochimson DJ, Wein- baum PJ: Fetal breathing as predictors of intraamniotic infection in premature rupture of membranes. Obstet Gynecol 57:813-817, 1986. 12. Goldstein J, Romero R, Merrill S, et al.: Fetal body and breathing movements as predictors ofintraamniotic infec- tion in preterm premature rupture of membranes. Am J Obstet Gynecol 159:363-368, 1988. 13. Vintzileos AM, Winston WA, Nochimson DJ, Connolly ME, Fuenfer MM, Hoehm GJ: The fetal biophysical profile in patients with premature rupture of the mem- branes: An early predictor of fetal infection. Am J Obstet Gynecol 152:510-516, 1985. 14. Roussis P, Rosemond RL, Glass C, Boehm FH: Preterm premature rupture of membranes: Detection of infection. AmJ Obstet Gynecol 165: 1099-1104, 1991. 15. Manning FA, Baskett TF, Morrison I, Lange I: Fetal biophysical profile scoring: A prospective study in 1184 high risk pregnancies.

Obstet Gynecol 152:510-516, 1985. 14. Roussis P, Rosemond RL, Glass C, Boehm FH: Preterm premature rupture of membranes: Detection of infection. AmJ Obstet Gynecol 165: 1099-1104, 1991. 15. Manning FA, Baskett TF, Morrison I, Lange I: Fetal biophysical profile scoring: A prospective study in 1184 high risk pregnancies. Am J Obstet Gynecol 140:289- 294, 1981. 16. Ohlsson A, Wang E: An analysis of antenatal tests to detect infection in preterm premature rupture ofthe mem- branes. Am J Obstet Gynecol 162:809-818, 1990. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:82-84 (1993) (C) 1993 Wiley-Liss, Inc. Effectiveness of Repeated Gonorrhea Cultures in the Third Trimester Juan G. Torres, T. Fleming Mattox, and Joseph G. Pastorek II Department ofObstetrics and Gynecology, Louisiana State University Medical Center, New Orleans, LA ABSTRACT Objective: With the high cost of health care today, the universal prophylactic measures recom- mended, and the availability of effective treatment should infection occur, the practice of routinely repeating the endocervical gonorrhea (GC) culture in the third trimester of pregnancy may be unwarranted. Methods: To test this hypothesis, we reviewed charts from patients who had received routine prenatal care during a 2-year period at the Lafayette and Opelousas parish health units. Those charts, which had documented results ofboth the initial and repeat GC cultures, were then used for retrospective review. The results ofthe initial GC culture were compared with that taken in the third trimester. Other data recorded included age, gravidity, race, and history of gonorrhea, syphilis, or multiple sexual partners. Results: Two hundred fifty charts were available for extraction; 130 of these had documentation ofboth GC cultures. Ofthe 130 cultures obtained during the initial prenatal visit, only 6 (4.6%) were positive. Of the repeat cultures taken during the third trimester, none were positive. Thirteen patients (10.0%) had a documented history of GC infection; none of them had positive cultures during the study period. Conclusions: Screening for GC during pregnancy is important and appropriate. This is commonly accomplished by taking a GC culture during the initial prenatal visit. Based upon the present study, we found that repeating this culture in the third trimester, even in a relatively high-risk population, seems unnecessary, whether the initial culture is negative or not. (C) 1993 Wiley-Liss, Inc. KEY WORS Prenatal care, STD screening, Neisseria gonorrhoeae, cervical cultures, maternal infection he prevalence of gonorrhea (GC) during preg- nancy varies from 1.0 to 7.0%, 1-3 depending on the patient population studied.

n, seems unnecessary, whether the initial culture is negative or not. (C) 1993 Wiley-Liss, Inc. KEY WORS Prenatal care, STD screening, Neisseria gonorrhoeae, cervical cultures, maternal infection he prevalence of gonorrhea (GC) during preg- nancy varies from 1.0 to 7.0%, 1-3 depending on the patient population studied. Higher rates are found among populations with risk factors such as single status, adolescence, multiple sexual partners, substance abuse, lack of prenatal care, and the pres- ence of other sexually transmitted diseases.2 Dis- seminated gonococcal infection has been noted to be more frequent during pregnancy, with up to 40% ofthe cases occurring in pregnant women.4 A preg- nant woman with gonococcal infection is more likely to experience preterm delivery, premature rupture of membranes, chorioamnionitis, and postpartum infection. 1'3 Therefore, a cervical screening cul- ture is recommended at the time ofthe first prenatal visit. A repeat culture in the third trimester (after 28 weeks) is also recommended by the Centers for Disease Control (CDC) in high-risk populations.5 Though the first recommendation of taking an ini- tial culture is almost universally followed, the latter one is not. This study questions the validity of repeating the GC culture in the third trimester. Address correspondence/reprint requests to Dr. Joseph G. Pastorek II, Section of Infectious Disease, Department ofObstet- rics and Gynecology, Louisiana State University Medical Center, 1542 Tulane Avenue, New Orleans, LA 70112-2822. Clinical Study Received February 15, 1993 Accepted May 17, 1993 GONORRHEA CULTURES IN PREGNANCY TORRES ET AL. MATERIALS AND METHODS Two hundred fifty medical charts from patients receiving routine prenatal care from the Lafayette and Opelousas parish health units were obtained for review during the years 1989 and 1990. The charts included those available for review by two of the investigators (J.G.T., T.F.M.) when they were on site at the respective parish health units. As such, the charts represented a relatively, but unstrin- gently, randomized sample of patients attending these clinics. Information recorded included age, gravidity, parity, race, gestational ages at the time of the initial and repeat culture, and results of these cul- tures. Also recorded were the presence of risk fac- tors such as a history of GC, syphilis, and multiple sexual partners. Patient charts were automatically excluded if the result of a third-trimester repeat culture was not recorded.

stational ages at the time of the initial and repeat culture, and results of these cul- tures. Also recorded were the presence of risk fac- tors such as a history of GC, syphilis, and multiple sexual partners. Patient charts were automatically excluded if the result of a third-trimester repeat culture was not recorded. Most of these excluded cases involved patients who began their initial pre- natal care at the health units but then switched to a private medical office once they obtained their med- ical assistance certification. RESULTS One hundred thirty charts met the criteria for in- clusion in the study. Forty-three patients (33%) presented for their initial culture during the first trimester (< 12 weeks). Another 74 patients (57%) presented during the second trimester (13-26 weeks), and 13 patients (10%) had their initial culture taken in the third trimester (27-40 weeks). Eighty-five of the study patients (65.4%) were black, 37 patients (28.5%) were Caucasian, 2 pa- tients (1.5%)were Hispanic, and 6 patients (4.6%) were classified as other races (Asian, Indian, Mid- dle Eastern). One hundred eleven patients (86%) had had three or fewer pregnancies, 47% of which were primigravidas. Ninety-six patients (74%) were between the ages of 15 and 25. Of the 130 initial cultures taken, 6 (4.6%) were positive. From the 130 repeat cultures taken in the third trimester, none were positive. Since the pre- cise number of patients seen during this time is not recorded, no absolute prevalence of GC in this population could be calculated. Thirteen patients (10%) had a history of GC infection, 7 patients (5.4%) had a history of syphi- litic infection, and 18 patients (!3.8%) reported having had multiple sexual partners in the past. All of these patients had negative initial and repeat cultures. Forty-three patients did not indicate whether they had multiple sexual partners; three of them had a positive initial GC culture. Five ofthe six patients with positive initial cultures had normal, spontane- ous vaginal deliveries at term. All of their babies had uncomplicated courses in the nursery. The de- livery records of the sixth patient with a positive initial culture could not be located for review. Though it was not a primary aim of the study, information was retroactively sought concerning the incidence of gonococcal ophthalmitis among neonates in this population.

had uncomplicated courses in the nursery. The de- livery records of the sixth patient with a positive initial culture could not be located for review. Though it was not a primary aim of the study, information was retroactively sought concerning the incidence of gonococcal ophthalmitis among neonates in this population. According to inter- views with the attending pediatricians in these health units, no case ofgonococcal eye infection was noted during the study period. Indeed, neonatal gonococ- cal infection is historically extremely rare in this population. DISCUSSION Screening for GC during pregnancy is important and appropriate, especially since the majority of female patients with gonococcal colonization are asymptomatic.3 Screening is best done by obtaining a GC culture during the initial prenatal visit, con- sidering the usual low cost of a Thayer-Martin plate and the standard recovery techniques in use today. Still, the cost of a GC culture is not negligi- ble. If it is to be repeated, there should be compel- ling rationale. The incidence of GC in this study is 4.6%, which is consistent with the reported incidence of between and 7.5%. 1-3 Prenatal screening for GC is important because it helps prevent many serious maternal and neonatal gonococcal complications, e.g., preterm delivery, premature rupture of the membranes, chorioamnionitis, and intrauterine growth retardation. 1'3 It has also been recom- mended that a repeat GC culture be obtained in the third trimester ofpregnancy, especially among those patients considered high risk. ,3 This report indi- cates that a repeat GC culture in the third trimester is unnecessary, at least in this population of "high- risk" public assistance obstetric patients. Of the 130 patients in the study, none had a positive repeat culture. Even the presence of risk factors, such as history of GC, syphilis, or multiple sexual part- ners, did not increase the likelihood that the repeat culture would be positive. Though the size of this study population is small, the practice of repeating INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY GONORRHEA CULTURES IN PREGNANCY TORRES ET AL. the GC culture in the third trimester appears to be in need of reevaluation, particularly during these times of budgetary constraints. Screening for GC during pregnancy is an ac- cepted part of prenatal care in this country. How- ever, how necessary is it to repeat the GC culture in the third trimester?

Y TORRES ET AL. the GC culture in the third trimester appears to be in need of reevaluation, particularly during these times of budgetary constraints. Screening for GC during pregnancy is an ac- cepted part of prenatal care in this country. How- ever, how necessary is it to repeat the GC culture in the third trimester? Are the additional time, effort, and expense worth the benefits derived from re- peating the culture? This study suggests that repeat- ing the GC culture in the third trimester is not useful as an additional precaution as surveillance for GC during pregnancy. No additional carriers of GC were found in 130 repeat cultures, even though the patient population was primarily of low socioeconomic standing and adolescent, and had other risk factors such as a history of gonorrhea, syphilis, or multiple sexual partners. During these times of limited health care budgets, for indigent care in particular, tests or procedures that are not cost effective should be discontinued. The routine (repeat) performance ofthe GC culture in the third trimester may be such a test. REFERENCES 1. Wendel GD, Cunningham FG: Sexually transmitted dis- eases in pregnancy. Williams Obstetrics, Suppl 13, 1991. 2. Edwards LE, Barraha MI, Hamann AA, et al.: Gonor- rhea in pregnancy. Am J Obstet Gynecol 132:637, 1978. 3. McNeeley SG: Gonococcal infections in women. Obstet Gynecol Clin North Am 16:467-478, 1989. 4. A1-Suleiman SA, Grimes EM, Jonas HS: Disseminated gonococcal infections. Obstet Gynecol 61:48, 1983. 5. Centers for Disease Control: 1989 Sexually transmitted diseases treatment guidelines. MMWR 38(Suppl 8): 1-43, 1989. 84 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:85-90 (1993) (C) 1993 Wiley-Liss, Inc. Presence of Chlamydia, Mycoplasma, Ureaplasma, and Other Bacteria in the Upper and Lower Genital Tracts of Fertile and Infertile Populations Mark G. Martens, Ronald L. Young, Marco Uribe, V.C. Buttram, Jr., and Sebastian Faro Department ofObstetrics and Gynecology, University of Texas Medical Branch, Galveston (M.G.M.), and Department ofObstetrics and Gynecology, Baylor College ofMedicine, Houston (R.L.Y., M.U., V.C.B.), TX; Department ofGynecology and Obstetrics, University ofKansas School ofMedicine, Kansas City (S.F.), KS ABSTRACT Objective: The genital mycoplasmas (Mycoplasma hominis and Ureaplasma urealyticum) and Chlamy. dia trachomatis have been implicated as possible etiologic factors in infertility. Their role in patients with infertility needs to be further defined. Methods: Seventy-nine infertile patients underwent laparoscopy with cultures obtained for aero- bic and anaerobic bacteria, Chlamydia, Mycoplasma, and Ureaplasma from the peritoneal fluid, fallopian tube, endometrium, and endocervix. Cultures for similar organisms were taken from the endocervix of80 fertile women in their first trimester. Culture results were also compared according to ovulatory status and laparoscopic findings in the infertile group. Results: There were no differences in the recovery of Ureaplasma (29% vs. 28%) or Chlamydia (4% vs. 0%) positive cervical cultures in the fertile and infertile groups, respectively. However, a significantly higher number of Mycoplasma positive cervical cultures (14% vs. 5%, P 0.05) were found in the fertile group. Only two upper genital tract cultures were found to be positive (Urea- plasma). Conclusions: Therefore, if these organisms play a role in infertility, they are present and eradi- cated prior to infertility work-up and thus do not support the use of a routine trial ofantibiotics prior to laparoscopy. (C) 1993 Wiley-Liss, Inc. KEY WORDS Chlamydia, Mycoplasma, Ureaplasma, infertility ver the past several decades, remarkable progress has been made in the treatment of infertility.

present and eradi- cated prior to infertility work-up and thus do not support the use of a routine trial ofantibiotics prior to laparoscopy. (C) 1993 Wiley-Liss, Inc. KEY WORDS Chlamydia, Mycoplasma, Ureaplasma, infertility ver the past several decades, remarkable progress has been made in the treatment of infertility. There still, however, continues to be a subset ofcouples with reproductive failure in whom there is no demonstrable etiology. An association of the genital mycoplasmas (Mycoplasma hominis and Ureaplasma urealyticum), as well as Chlamydia tra- chomatis, with infertility has long been suspected. 1-3 However, neither epidemiologic evaluation nor treatment data from females in infertile partner- ships have offered consistent reproducible results to implicate any ofthese organisms as etiologic agents. Since all 3 of these organisms are sensitive to cur- rently available antibiotics, routine empiric treat- ment of these organisms often occurs, despite the lack of substantial proof of its efficacy. Overall, considerable controversy exists cover- ing the role of all 3 of these organisms in human infertility. The major problem with previous stud- ies is that infertility is a syndrome, not a specific Address correspondence/reprint requests to Dr. Mark G. Martens, Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, TX 77555-0587. Received March 30, 1993 Clinical Study Accepted June 14, 1993 GENITAL TRACT CULTURES AND INFERTILITY MARTENS ET AL. entity, and that these organisms are likely to be related only to certain clinicopathological subsets. Much of the data conflict, and the clinician or infertility specialist is left with several questions: 1) Do the genital mycoplasmas play a pathogenic role in infertility? 2) Are cultures for Mycoplasma and Ureaplasma a necessary part of the infertility work- up? 3) Does cervical colonization correlate with upper genital tract involvement? 4) Will treatment result in increased fertility rates? Most of the studies include patients with "idio- pathic infertility," of whom many have had inade- quate diagnostic evaluations. IfMycoplasma, Urea- plasma, and Chlamydia are factors in infertility, they may only be contributing factors rather than primary ones.

t? 4) Will treatment result in increased fertility rates? Most of the studies include patients with "idio- pathic infertility," of whom many have had inade- quate diagnostic evaluations. IfMycoplasma, Urea- plasma, and Chlamydia are factors in infertility, they may only be contributing factors rather than primary ones. Because effective therapy may require treatment for several interrelated factors or organisms, it seems reasonable to first identify which organisms place patients at risk before attempting trial of anti- biotics which may not be specific for these organ- isms. Our particular study was designed to deter- mine the incidence of the genital mycoplasmas and chlamydia colonization in a group of fertile and infertile patients and to possibly identify a clinical subset of patients at a higher risk of colonization. SUBJECTS AND METHODS This study consisted of a group of 80 private prac- tice, infertile couples and a control group of 80 consecutive private practice couples with proven fertility in their first trimester ofpregnancy. Infor- mation obtained for the investigation included: duration of infertility (at least year); significant past medical history and social history; age and race of each partner; oral contraceptive (OC) or intrauterine device (IUD) use; history of pelvic inflammatory disease (PID) or sexually transmitted disease (STD); evidence of ovulation; hysterosalpingogram (HSG); laparoscopic findings; semen analysis and other male factors; results ofcultures for C. trachomatis, Mycoplasma, Ureaplasma, Neisseria gonorrhoeae, aerobes, and anaerobes from the endocervix, endometrium, and peritoneal fluid obtained at time of laparos- copy, as previously described. 1-z Information taken from the control (fertile) group included: age and race of each partner; OC or IUD use; history of STD or PID; history of previous infertility work-up. Similar cultures were collected from the endo- cervix for the previously mentioned organisms at time of initial examination for this group. Endo- metrial cultures utilized biopsy specimens obtained with Novak curette divided into 2 portions, for routine histological studies and another for culture. At time of laparoscopy, a collection of peritoneal fluid was obtained. If fluid in the cavity was inade- quate, lavage with normal saline and aspiration were performed. The fluid was centrifuged to pel- let cellular material and then transferred to tissue culture.

r routine histological studies and another for culture. At time of laparoscopy, a collection of peritoneal fluid was obtained. If fluid in the cavity was inade- quate, lavage with normal saline and aspiration were performed. The fluid was centrifuged to pel- let cellular material and then transferred to tissue culture. Direct specimens using swabs of the right oviduct were obtained in 16 patients. Specimens collected for M. hominis and U. urealyticum were done in a similar fashion and placed in a Mycotrans Mycoplasma Transport System (Irvine Scientific, Irvine, CA). These were refrigerated for 25 h, inoculated into a biphasic Mycotrim (Irvine Scien- tific) GU system, incubated, and then interpreted. N. gonorrhoeae, aerobic, and anaerobic cultures were obtained and evaluated by routine bacteriolog- ical techniques.4 Chlamydia specimens were pro- cessed by tissue culture according to standard meth- odology. 5 For statistical analysis, Fisher's exact test or chi-square analysis was utilized. P < 0.05 was determined to be significant. RESULTS The infertile and control groups were comparable with respect to the demographic characteristics listed in Table (P > 0.05). None of the patients in either group related a history of PID and none of the patients in the fertile group related a history of infertility evaluation. The demographic data and laparoscopic results are listed in Table 2. At the time oflaparoscopy, endometriosis was found in 33 (41%) of the patients. Adhesions were identified in 47 (70%) of all patients, but were present in only 31 (39%) of the patients without any evidence of endometriosis. Tubal occlusion, defined as distal occlusion as evidenced by hydrosalpinx, phimosis, 86 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY GENITAL TRACT CULTURES AND INFERTILITY MARTENS ET AL. TABLE I. Demographic data of infertile and pregnant control groups Infertile Control Population group group No. 80 80 Age Mean (years) 30.9 29. I* Range (years) 20-40 18-39 Race Caucasian 63 55 Black 8 12 Hispanic 5 9 Others 4 4 History of PID 0 0 Duration of unexplained primary 36.3 infertility (months) Primary infertility 43 (54%) Secondary infertility 37 (46%) *P > 0.05. or severe adhesions occurring in at least tube, was noted in 17 (21%) of the patients. Cultures were obtained properly from 79 of the patients in the infertile group (Table 3). These included positive cervical cultures in 5 (6%) of the patients for M. homin# and 22 (28%) patients for U. urealyticum.

37 (46%) *P > 0.05. or severe adhesions occurring in at least tube, was noted in 17 (21%) of the patients. Cultures were obtained properly from 79 of the patients in the infertile group (Table 3). These included positive cervical cultures in 5 (6%) of the patients for M. homin# and 22 (28%) patients for U. urealyticum. Of the 76 endometrial cultures obtained, 2 (3%) had M. hominis and 4 (5%) grew U. urealyticum. Two of these 4 patients were found to have evidence of pelvic adhesions and histologic evidence ofendometritis. Thirteen endometrial cul- tures were positive for other bacteria, with pa- tient also having a concomitant associated positive Ureaplasma culture. Bacteria isolated from the en- docervix and endometrium consisted ofaerobic and anaerobic species consistent with normal vaginal flora. Of the 70 spccimcns o peritoneal fluid recov- ered, (1%)grew Urel. Ths patient, who had had a previous unilateral salpngcctomy or an cctopic pregnancy, was ound to have extensive pelvic adhesions. Othe 20 tubal cultures obtained, all wcrc ncgafivc. There wcrc no positive cultures for C. trachomatis from any specimen, nor were there positive cultures for N. gonorrhoeae. Comparing cervical cultures in the overall study group (Table 4) in patients with or without evi- dence oftubal occlusion as defined earlier, we found no statistical difference in the incidence of either of the 3 organisms found. In addition, when patients with tubal factor and unexplained infertility were TABLE 2. Demographic data and the laparoscopic findings according to ovulation status epidemiology (N 67) Ovulation Ovulation positive negative [N 53 (79%)] [N 14 (21%)] Age Mean (years) 31.8 20.2 Range (years) 21-40 25-35 Race Caucasian 41 14 Hispanic 3 0 Black 5 0 Others. 4 0 Infertility Primary 22 10 Secondary 31 4 Average UPI 38.2 28.2 (months) Miscellaneous Term infants 16 0 Preterm infants 2 0 Miscarriage/abortions 18 0 Live child 16 3 Prior OC users 36 7 Prior OC non-users 17 4 Prior IUD users 7 0 Dysmenorrhea 33 8 Amenorrhea 2 Laparoscopic findings Endometriosis 25 (47%) 8 (57%) Pelvic adhesions 40 (75%) 7 (50%) Tubal occlusion 13 (25%) 4 (29%) Post-salpingectomy 5 (9%) 0 (0%) Polycystic ovary 3 (6%) 2 (I 5%) disease Fibroids 5 (9%) (7%) Hydrosalpinx (2%) (7%) Dysfunction uterine 0 (7%) bleeding Cervical stenosis 0 (7%) Endopolyps 0 (7%) aUnexplained primary infertility. combined, no statistical differences in their culture results were noted.

3 (25%) 4 (29%) Post-salpingectomy 5 (9%) 0 (0%) Polycystic ovary 3 (6%) 2 (I 5%) disease Fibroids 5 (9%) (7%) Hydrosalpinx (2%) (7%) Dysfunction uterine 0 (7%) bleeding Cervical stenosis 0 (7%) Endopolyps 0 (7%) aUnexplained primary infertility. combined, no statistical differences in their culture results were noted. Thirteen of 17 patients with occlusion had histo- ries of previous pelvic surgery. Of the 4 patients who had had no previous pelvic surgery, only had a positive culture (Ureaplasma). In the subpopula- tion of 53 ovulating patients (i.e., a group of pa- tients in which anovulation cannot be considered to be a contributing factor to the etiology of their infertility), the demographic characteristics were similar to the control group and infertile groups. Patients in the ovulatory group with laparoscopic INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY I]7 GENITAL TRACT CULTURES AND INFERTILITY MARTENS ET AL. TABLE 3. Culture results in the infertile group Ovulation Ovulation Positive cultures positive negative Uncertain or inconsistent intermediate ovulation Cervix 52 14 13 (n 79) Bacteria 52 14 13 GC 0 0 0 Chlarnydia 0 0 0 Mycoplasrna 3 Ureaplasrna 14 2 6 Endometrium 51 13 12 (n 76) Bacteria 10 2 GC 0 0 0 Chlarnydia 0 0 0 Mycoplasrna 0 0 2 Ureaplasrna 0 0 Peritoneal fluid 48 II II (n 70) Bacteria 0 0 0 GC 0 0 0 Chlarnydia 0 0 0 Mycoplasrna 0 0 0 Ureaplasrna 0 0 Fallopian tube 16 4 0 (n 20) Bacteria 0 0 0 GC 0 0 0 Chlamydia 0 0 0 Mycoplasrna 0 0 0 Ureaplasrna 0 0 0 an number of patients in group. bAerobic or anaerobic bacteria. cGC N. gonorrhoeae. evidence of endometriosis were compared to those with pelvic adhesions (without endometriosis). The findings did not demonstrate a statistical difference in cervical culture results for M. hominis or C. trachomatis, but a statistically higher incidence of ureaplasma colonization (P 0.04) was found in patients with endometriosis when compared to those with pelvic adhesions (36% vs. 12%). Sixteen of25 patients with documented endometriosis in the ovu- latory group also had concomitant adhesions. Fur- thermore, 18 of 23 patients with pelvic adhesions, but without endometriosis, had histories of previ- ous pelvic surgery. Of the 5 patients in the pelvic adhesion group ofovulating patients without previ- ous surgery, all had positive cervical cultures for Mycoplasma or Ureaplasma. The outcome of pregnancy in the 80 patients in the fertile group was recorded.

dhesions, but without endometriosis, had histories of previ- ous pelvic surgery. Of the 5 patients in the pelvic adhesion group ofovulating patients without previ- ous surgery, all had positive cervical cultures for Mycoplasma or Ureaplasma. The outcome of pregnancy in the 80 patients in the fertile group was recorded. Sixty-seven (84%) ofthe patients carried their infants to term; 8 (10%) ofthe patients delivered prematurely; 4 (5%)ofthe patients had spontaneous abortions; and of the patients had an ecotpic pregnancy. Cervical culture results in these patients revealed no statistical dif- ference in colonization in any of these groups (Ta- ble 5). DISCUSSION Female genital tract infections have long been im- plicated as a major cause of infertility. Gnarpe and Friberg first suggested an etiologic role of Myco- plasma in infertility when they demonstrated a high frequency of positive cultures recovered from the cervices ofwomen with unexplained infertility com- pared with those of the fertile pregnant control subjects. The unexplained infertility groups had normal basal body temperature charts, HSGs, and semen analyses. Others, however, have found no statistical difference in the incidence ofMycoplasma or Ureaplasma between fertile and infertile cou- ples.6'7 deLouvois et al.7 studied 120 patients with infertility of various etiologies and found a 52% incidence ofcervical ureaplasma. They also found a 55% incidence in 92 pregnant patients. Matthews et al. and Nagata et al. 9 found similar results. Gump et al. 10 studied 20 patients with infertility for longer than year and obtained cultures from the cervix and endometrium for Mycoplasma and Ureaplasma. They found no difference in the inci- dence of colonization with these organisms in pa- tients with laparoscopic evidence of previous PID. They also found genital mycoplasma colonization in only 10 of 203 endometrial biopsy (EMB) spec- imens, and of these was associated with endome- trial inflammation. During the same time, several investigators have attempted to demonstrate that treatment of these organisms would result in an increased pregnancy rate.Z, 11 Most regimens involve attempted eradica- tion of the organisms by treatment of both partners with either doxycycline or tetracycline, usually in- 12,13 volving a 28-day course. The role of C. trachomatis in infertility has long been suggested.

eatment of these organisms would result in an increased pregnancy rate.Z, 11 Most regimens involve attempted eradica- tion of the organisms by treatment of both partners with either doxycycline or tetracycline, usually in- 12,13 volving a 28-day course. The role of C. trachomatis in infertility has long been suggested. 14-18 Several investigators have demonstrated an association between serum anti- chlamydial antibodies and infertility in patients with tubal factors as the source of their infertility. 14-16 Despite these implications, few studies have been 88 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY GENITAL TRACT CULTURES AND INFERTILITY MARTENS ET AL. TABLE 4. Cervical culture results for diagnostic group No. patients Mycoplasma (%) Ureaplasma (%) Chlamydia (%) Control group (fertile) 80 II (14%)* 23 (29) 3 (4) Infertile group 79 4 (5) 22 (28) 0 Ovulation positive 53 (2) 14 (26) 0 Pelvic adhesions without endometriosis 25 (3) 3 (I 2)** 0 Endometriosis 25 0 (0) 9 (36) 0 Tubal occlusion 17 2 (12) 4 (24) 0 At least one patent tube 63 2 (3) 17 (27) 0 *The control group and the infertile groups were statistically different with respect to mycoplasma isolation (P 0.05). **The pelvic adhesion and the endometriosis groups were statistically different with respect to ureaplasma isolation (P 0.04). TABLE 5. Cervical culture results in the control pregnant group (N 80) Outcome No. Mycoplasma positive (%) Ureaplasma (%) Chlamydia (%) Term 67 Preterm 8 Spontaneous abortion 4 Ectopic pregnancy Total 80 0 ( s) 9 (zs) z (3) (3) 3 (38) 0 0 (5) (s) 0 0 0 II 23 3 successful in isolating C. trachomatis from the endocervix or endometrium of infertile patients. Cleary and Jones19 studied 19 patients with pos- itive serum anti-chlamydia antibodies greater than 1:32 and found C. trachomat# in 32% of their cervical cultures and 26% of their endometrial cul- tures. However, controls of seropositive fertile or seronegative infertile women were not done. In 1984, Kane et al.2 found that 22% of 164 infertile women were seropositive for anti-chlamydial anti- bodies, while a control group had an 11% rate. However, they were unable to isolate Chlamydia from the cervices or fimbriae in any of the patients studied. However, in this investigation no statisti- cal increase was found in the incidence of cervical colonization with U. urealyticum or C. trachomatis in the infertile groups. There was, however, a sta- tistically significant increased incidence ofM.

e Chlamydia from the cervices or fimbriae in any of the patients studied. However, in this investigation no statisti- cal increase was found in the incidence of cervical colonization with U. urealyticum or C. trachomatis in the infertile groups. There was, however, a sta- tistically significant increased incidence ofM. hom- inis isolation in the pregnant group compared to the infertile group (P 0.05). The lower incidence in the infertile group may have been the result of the previous use ofantibiotics in earlier infertility work- ups or for other non-gynecologic indications. Addi- tionally, seminal fluid cultures from the partners of our study group may have been helpful. Both Mat- thews et al. 8 and deLouvois et al.7 found fewer than 5% of their couples studied had male positive cul- tures, with all female cultures being negative. Two studies in which couples were treated for Urea- plasma on the basis of positive semen cultures pro- duced contradictory results. The first study by Re- hewy et al.21 failed to achieve a higher pregnancy rate, while Toth et al.z2 demonstrated that 60% of partners treated for Ureaplasma resulted in preg- nancy compared to only 5% in cases where the post-therapy cultures remained positive. As stated earlier, confounding variables include the possibil- ity of previous treatment of unrecognized infec- tions. This study did not document a higher incidence ofactive colonization with any ofthese 3 organisms in patients with infertility, tubal occlusion, or pel- vic adhesions when compared to pregnant patients. However, one cannot exclude the possibility that these patients were infected in the past, at which time fertility damage might have occurred, and/or the possibility that subsequent treatment with anti- biotics for any number of reasons resulted in a lower incidence ofpositive cultures. There does not appear to be a high rate of isolation of the genital mycoplasmas or Chlamydia at the time of infertility work-up. Either C. trachomatis, M. hominis, and U. urealyticum do not have a role in infertility or they are factors which manifest themselves prior to the formal work-up of the individual for infertil- ity. Therefore, routine antibiotic treatment without INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 89 GENITAL TRACT CULTURES AND INFERTILITY MARTENS ET AL. culture confirmation at the time of infertility work-up in a similar patient population is not indi- cated.

to the formal work-up of the individual for infertil- ity. Therefore, routine antibiotic treatment without INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 89 GENITAL TRACT CULTURES AND INFERTILITY MARTENS ET AL. culture confirmation at the time of infertility work-up in a similar patient population is not indi- cated. It may also be stated that in this, and perhaps similar infertility settings, culture of the endocer- vix for these organisms is unwarranted. However, work-ups performed by a physician at an earlier stage or in a population with a higher rate of PID may necessitate the consideration of obtaining cul- tures. If previous infections are proven at some point to be a factor in infertility, diagnosis and treatment of infection need to occur prior to the point at which patients seek infertility care. This challenge then falls on the patient's earlier obstetric and gynecologic care providers, perhaps even in the patient's teenage and early adulthood years be- fore conception is often considered. REFERENCES 1. Gnarpe H, FribergJ: Mycoplasma and human reproduc- tive failure. The occurrence of different mycoplasmas in couples with reproductive failure. Am J Obstet Gynecol 114:727, 1972. 2. Kundsin RB: Mycoplasma in genitourinary tract infec- tions and reproductive failure. In Sturgis SH, Taymore ML (eds): Professions in Gynecology. Vol 5. New York: Grune & Stratton, p 275, 197 0. 3. FribergJ, Gnarpe H: Mycoplasma and human reproduc- tive failure. Pregnancies in "infertile" couples treated with doxycycline for T-mycoplasmas. AmJ Obstet Gynecol 116: 23, 1973. 4. Berenson AB, Hamill HA, Martens MG, Faro S: Bacte- riologic findings of post-cesarean endometritis in adoles- cents. Obstet Gynecol 75:627, 1990. 5. Ripa K, Mardh PA: Cultivation ofChlamydia trachomatis in cycloheximide treated McCoy cells. J Clin Microbiol 6:328, 1977. 6. Taylor-Robinson D: Evaluation ofthe role of Ureaplasma urealyticum in infertility. Pediatr Infect Dis 5:262, 1986. 7. deLouvis J, Blades M, Harrison RF, Hurley R, Stanley VC: Frequency of mycoplasma in fertile and infertile couples. Lancet 1073, 1974. 8. Matthews CD, Elmslie RG, Clapp KH, Svigos JM: The frequency of genital mycoplasma infection in human in- fertility. Fertil Steril 26:988, 1975. 9. Nagata Y, Iwaska T, Wada T: Mycoplasma infection and infertility. Fertil Steril 31:392, 1979. 10. Gump DE, Gibson M, Ashikaga T: Lack of association between genital mycoplasmas and infertility. N Engl J Med 310:937, 1984. 11.

equency of genital mycoplasma infection in human in- fertility. Fertil Steril 26:988, 1975. 9. Nagata Y, Iwaska T, Wada T: Mycoplasma infection and infertility. Fertil Steril 31:392, 1979. 10. Gump DE, Gibson M, Ashikaga T: Lack of association between genital mycoplasmas and infertility. N Engl J Med 310:937, 1984. 11. Horne HW, Hertig AT, Kundsin RB: Subclinical endo- metrial inflammation and T-mycoplasmas: A possible cause ofhuman reproductive failure. IntJ Fertil 18:266, 1973. 12. Harrison RF, Blades M, deLouvisJ, Hurley R: Doxycy- cline treatment and human infertility. Lancet 1:605, 1975. 13. Hinton RA, Egdell LM, Andrews BE, Clark SK, Rich- mond SJ: A double-blind crossover study of the effect of doxycycline on mycoplasma infection and infertility. Br J Obstet Gynaecol 86:379, 1979. 14. Jones RB, Ardery BR, Hui SL, Cleary RE: Correlation between serum antibodies and tubal factor as a cause of infertility. Fertil Steril 38:553, 1982. 15. Moore DE, Foy HM, Daling JR, Grayston JT, Spadoni LR, Wang S, Kuo C, Eschenbach DA: Increased fre- quency of serum antibodies to Chlamydia trachomatis in infertility due to distal tubal disease. Lancet 2:574, 1982. 16. Sellors JW, Mohony JB, Chernesky MA, Rath DJ: Tu- bal factor infertility: An association with prior chlamydial infection and asymptomatic salpingitis. Fertil Steril 49: 451, 1988. 17. Anestad G, Lunde O, Moen M, Dalaker K: Infertility and chlamydial infection. Fertil Steri148:787, 1987. 18. Henry-Suchet J, Utzmann C, De-Brux J, Ardoin P, Catalan F: Microbiological study of chronic inflamma- tion associated with tubal factor infertility: Role of Chlamydia trachomatis. Fertil Steril 47:274, 1987. 19. Cleary RE, Jones RB: Recovery of Chlamydia trachomatis from the endometrium in infertile women with serum anti-chlamydial antibodies. Fertil Steril 44:233, 1985. 20. Kane JL, Woodland RM, Forsey T, Darougar S, Elder MG: Evidence of chlamydia infection in infertile women with and without fallopian tube obstruction. Fertil Steril 42:843, 1984. 21. Rehewy MSE, Thomas AJ, Hafez ESE, Brown WJ, Moghissi KS, Jaszczak S: Ureaplasma urealyticum (T- mycoplasma) in seminal plasma and spermatozoa from infertile and fertile volunteers. Eur J Obstet Gynecol Reprod Biol 8:247, 1978. 22. Toth A, Lesser ML, Brooks A, Labriola D: Subsequent pregnancies among 161 couples treated for T-mycoplasma genital-tract infection. N Engl J Med 308:505, 1983. 90 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:91-93 (1993) (C) 1993 Wiley-Liss, Inc. Epidemiology and Hospital Course of Patients With Acute Salpingitis and Coincident HIV Iris Ayala-Rodriguez and Joseph Apuzzio Department of Obstetrics and Gynecology, New Jersey Medical School, Newark, NJ ABSTRACT Objective: To compare the epidemiology and hospital course of patients with acute salpingitis with and without coincident human immunodeficiency virus (HIV) seropositivity. Methods: Patients admitted to the UMDNJ-University Hospital in Newark, New Jersey from January 1, 1991, to December 31, 1991, with acute salpingitis were studied. Results: Eight percent of all hospitalized patients with acute salpingitis were HIV-positive. The mean age ofthe HIV-negative group was 25.4 compared with 29.6 years in the HIV-positive group. Gonorrhea and chlamydia were present in 49% and 22%, respectively, in HIV-negatives and in 40% and 20% of HIV-positives. Two of 5 (40%) HIV-positive patients had tuboovarian abscesses com- pared with 12 of 59 (20%) HIV-negative patients. Three of 5 (60%) HIV-positive patients had admission WBC counts fewer than 10,000/mm3 compared to 6 of 59 (12%) of HIV-negatives (P 0.024). The hospital stay was 5.4 days for HIV-positives and 5.8 days for HIV-negatives. Conclusions: Eight percent of hospitalized patients with acute salpingitis were HIV-seropositive. Neisseria gonorrhoeae and chlamydia were commonly found organisms in both groups. The initial WBC count was lower for HIV-positive patients. The hospital course ofboth groups was similar. (C) 1993 Wiley-Liss, Inc. Kvx woRs HIV seropositivity, sexually transmitted diseases cute salpingitis is a major cause of both acute and chronic morbidity among young women with the potential for long-term reproductive con- sequences. Women at risk of acquiring pelvic in- flammatory disease (PID) tend to be young women of low socioeconomic background with multiple sexual partners. Human immunodeficiency virus (HIV) infectivity appears to be increasing among similar groups. Sexual risk behaviors are associated with the acquisition of both PID and HIV.

omen at risk of acquiring pelvic in- flammatory disease (PID) tend to be young women of low socioeconomic background with multiple sexual partners. Human immunodeficiency virus (HIV) infectivity appears to be increasing among similar groups. Sexual risk behaviors are associated with the acquisition of both PID and HIV. Coinci- dent infection with HIV may alter the clinical course of acute salpingitis. The objectives of the study were to determine the seroprevalence and epidemiology of HIV in hospitalized women with acute salpingitis and to compare the hospital course ofpatients who have acute salpingitis with and with- out coincident HIV. MATERIALS AND METHODS Charts were reviewed of all women admitted to UMDNJ-University Hospital in Newark, New Jersey, between January 1, 1991, and December 31, 1991, who fulfilled the clinical criteria for the diagnosis of PID. Only those patients with known HIV status were entered in the study. On the patient's admission, cervical cultures were obtained for Neisseria gonorrhoeae and a Chlamydi- azyme(R) (Abbott Laboratories, North Chicago, IL) was performed for chlamydia. Blood was drawn for CBC, SMA18, and venereal disease research labo- Address correspondence/reprint requests to Dr. Joseph Apuzzio, Department of Obstetrics and Gynecology, New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103-2714. Clinical Study Received April 6, 1993 Accepted June 2, 1993 AYALA-RODRIGUEZ AND APUZZIO HIV AND ACUTE SALPINGITIS ratory (VDRL). Fluorescent treponemal antibody test (FTA) was performed if the VDRL was posi- tive. HIV testing was offered after appropriate counseling was given and consent obtained. HIV serologic testing was performed at the University Hospital serology laboratory. Samples positive by enzyme-linked immunosorbent assay (ELISA) were confirmed by Western blot. All patients had pelvic sonograms to detect tuboovarian disease. Antibiot- ics were prescribed by protocol: Either the combi- nation of cefoxitin, 2 g IV q 6 h, and doxycycline, 100 mg IV q 12 h, or clindamycin, 900 mg IV q 8 h, gentamicin 1.5 mg/kg IV q 8 h, and ampicillin, 2 g IV q 6 h, at the discretion of the admitting physician. Peak and trough serum levels of gen- tamicin were obtained to adjust the dose ofgentam- icin. Antibiotics were continued for at least 4 days and at least 48 hours after the patient's deferves- cence. Upon discharge from the hospital, patients were prescribed oral doxycycline, 100 mg twice a day, for 7 days.

physician. Peak and trough serum levels of gen- tamicin were obtained to adjust the dose ofgentam- icin. Antibiotics were continued for at least 4 days and at least 48 hours after the patient's deferves- cence. Upon discharge from the hospital, patients were prescribed oral doxycycline, 100 mg twice a day, for 7 days. Initial antibiotic therapy was considered a failure if, after 48-72 hours oftreatment, the WBC count increased and clinical symptomatology worsened from the initial examination. Statistical analysis was performed by computer using the Fisher's exact test. RESULTS During the 12 month period fromJanuary 1, 1991, to December 30, 1991, 77 patients with acute sal- pingitis were admitted to University Hospital; 74 charts were available for review. Ten patients were excluded because of unknow.n HIV status. Of the 64 patients with known I--IIV status, 5 (8%) were found to be seropositive; no patient had AIDS. The average age ofpatients who were HIV-negative was 25.4 5.7 years, while those patients who were HIV-positive were all multiparous and older, with a mean age of 29.6 + 6.3 years. The youngest patient was 18 years old. The demographics are listed in Table 1. The incidence of positive cervical cultures for gonorrhea and the reactive Chlamydiazyme(R) as well as VDRL results are listed in Table 2. All patients with acute salpingitis had ultrasound examinations to document tuboovarian abscesses (TOA). TOA was noted in 2 of 5 (40%) HIV- seropositive patients and in 12 of 59 (20%) HIV- negative patients. TABLE I. Demographics of patients with acute salpingitis Age range (yrs) HIV (-)(n 59) HIV (+)(n 5)(8%) (mean age, (mean age, 25.4-5.7 yrs) 29.6 6.3 yrs) [no. (%)] 15-20 13 (22.0) 21-25 18 (30.5) 0 26-30 18 (20.3) 31-35 7 (11.8) 2 36-40 2 (3.3) 41-45 1(I.6) 0 TABLE 2. STDs among patients with acute salpingitis HIV status Positive Negative (n 5) (n- 59) N. gonorrhoeae 2 (40%) 29 (49. I%) Reactive Chlamydiazyme 13 (22.0%) Positive VDRL and FTA 0 5 (8.5%) Seven patients with TOA initially received ce- foxitin and doxycycline. Two ofthese patients failed initial antibiotic therapy but responded to a change in antibiotics to clindamycin, gentamicin, and ampi- cillin. One of these patients was HIV-positive. Seven other patients with TOA initially received clindamycin, gentamicin, and ampicillin. Two of these failed initial therapy. One responded when imipenem-cilastatin was prescribed.

tic therapy but responded to a change in antibiotics to clindamycin, gentamicin, and ampi- cillin. One of these patients was HIV-positive. Seven other patients with TOA initially received clindamycin, gentamicin, and ampicillin. Two of these failed initial therapy. One responded when imipenem-cilastatin was prescribed. The other pa- tient required surgical intervention for TOA and experienced a refractory clinical course. She was HIV-negative. DISCUSSION Because of its increasing incidence among women in inner cities, HIV will become more prevalent in the gynecologic setting. Several studies on coinci- dent infection of HIV in patients with acute salpin- gitis have been published, revealing a 6-17% inci- dence of HIV positivity among women treated for PID. Hoegsberg et al.2 in New York found that 14% of inner-city women hospitalized with PID were also HIV-positive. Safrin and co-workers3 in San Francisco found that 6.7% ofwomen with PID were HIV-positive, and Sperling et al.4 found 5 of 30 (16.7%) in their population to be HIV-positive. 92 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY AYALA-RODRIGUEZ AND APUZZIO HIV AND ACUTE SALPINGITIS TABLE 3. Hospital course of patients with acute salpingitis HIV status Positive (n 5) Negative (n 59) [no. (%)1 [no. (%)] Admission temp. 5 (100) 46 (78) > 38C (I00.4F) Admission WBC 2 (40) 52 (88. I)* > O,O00/mm3 Duration of hospital 5.4 -+ 1.02 5.8 -+ 2,48 stay (days) Tuboovarian abscess 2 (40) 12 (20.3) Surgery 0 (0) (I.6) Failed initial (20) 3 (5. I) antibiotic therapy *P 0.024 Among our study population of 64 patients ad- mitted with acute salpingitis and known HIV sta- tus, 5 of 64 (8%) tested positive for HIV. Perhaps attributable to the small study group, HIV seropos- itivity did not appear to be associated with a higher frequency of STDs, longer hospital stay, a more refractory clinical course, or an increased rate of surgical intervention (Table 3). The only statisti- cally significant finding was that 3 of 5 (60%) HIV-positive patients had initial WBC counts fewer than 10,000/mm,3 compared to 7 of 59 (12%) HIV-negatives (P 0.024). The lower initial WBC may be related to a mild immunosuppression of HIV-positive patients. A larger study group is needed to verify this trend in morbidity and the clinical course in HIV-infected women with acute salpingitis compared with women with acute sal- pingitis who are HIV-negative. The majority of cases with TOA (13 of 14) responded to IV antibiotics.

mild immunosuppression of HIV-positive patients. A larger study group is needed to verify this trend in morbidity and the clinical course in HIV-infected women with acute salpingitis compared with women with acute sal- pingitis who are HIV-negative. The majority of cases with TOA (13 of 14) responded to IV antibiotics. Seven of the patients with TOA received cefoxitin and doxycycline, with two patients requiring a change of antibiotics after 48 hours of therapy. Seven other patients received triple antibiotics ofclindamycin, gentamicin, ampi- cillin, with two failing initial therapy. One ofthese patients required surgical intervention for cure. Because the rate ofheterosexual transmission and HIV infectivity among women is increasing, HIV testing should be offered to all patients treated or suspected of having any STD. More importantly, counseling and patient education about STDs and sexual behaviors should assume a greater role in health care practice. REFERENCES 1. Hager WD, Eschenbach DA, Spence MR, Sweet RL: Criteria for diagnosis and grading of salpingitis. Obstet Gynecol 61 113-114, 1983. 2. Hoegsberg B, Abulafia O, Sedlis A, Feldman J, DesJalais D, Landesman S, Minkoff H: Sexually transmitted dis- eases and human immunodeficiency virus infection among women with pelvic inflammatory disease. Am J Obstet Gynecol 163:1135-1139, 1990. 3. Safrin S, Dattel BJ, Hauer L, Sweet RL: Seroprevalence and epidemiologic correlates of human immunodeficiency virus infection in women with acute pelvic inflammatory disease. Obstet Gyneco175:666-670, 1990. 4. Sperling R, Friedman F Jr, Joyner M, Brodman M, Dottino P: Seroprevalence of human immunodeficiency virus in women admitted to the hospital with pelvic inflam- matory disease. J Reprod Med 36:122-124, 1991. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 93

Infectious Diseases in Obstetrics and Gynecology 1:94-97 (1993) (C) 1993 Wiley-Liss, Inc. Vaginal Flora in Postmenopausal Women: The Effect of Estrogen Replacement Paul D. Ginkel, David E. Soper, Richard C. Bump, and Harry P. Dalton Departments of Obstetrics and Gynecology (P.D.G., D.E.S., R.C.B.) and Pathology (H.P.D.), Medical College ofVirginia, Virginia Commonwealth University, Richmond, VA ABSTRACT Objective: To determine the effect of estrogen replacement therapy (ERT) on the vaginal flora of postmenopausal women. Methods: Vaginal cultures were obtained from 15 postmenopausal women whose hormonal statuses were documented by serum follicle-stimulating hormone (FSH) and serum estrogen levels. After 8 weeks of ERT, consisting of 0.1 mg of estradiol delivered daily by dermal patch, the vaginal cultures were repeated, as were measurements ofthe vaginal pH, serum FSH, and serum estrogen levels. Results: Vaginal cultures revealed no significant change in the incidence of lactobacilli or of all aerobes. However, the incidence ofanaerobic species fell after treatmentfrom 47% to 13% (P 0.05), and the incidence of anaerobic gram-negative rods declined after treatment from 40% prior to ERT to 7% (P 0.035). Prior to ERT, the difference in mean vaginal pH between lactobacilli-positive and lactobacilli-negative subjects was not significant, but, following the administration ofexogenous estrogen, the lactobacilli-positive subjects exhibited a significantly lower mean vaginal pH (4.4 _+ 0.4) relative to the lactobacilli-negative population (5.2 + 0.3) (P 0.02). Conclusions: We conclude that women on ERT are less likely to have vaginal colonization with anaerobic bacteria when compared with women not using replacement therapy. Estrogen replace- ment may potentiate the effect oflactobacilli on vaginal pH. (C) 1993 Wiley-Liss, Inc. KE'r WORDS Estrogen, vaginal flora, postmenopausal n order to examine the effect of hormonal status on the vaginal flora, previous investigators have compared bacterial isolates of premenopausal and postmenopausal women. Tashijian et al.

may potentiate the effect oflactobacilli on vaginal pH. (C) 1993 Wiley-Liss, Inc. KE'r WORDS Estrogen, vaginal flora, postmenopausal n order to examine the effect of hormonal status on the vaginal flora, previous investigators have compared bacterial isolates of premenopausal and postmenopausal women. Tashijian et al. noted a higher incidence of aerobic gram-negative rods in postmenopausal women; however, the hormonal sta- tus of the subjects was never documented. Al- though a subsequent study revealed no significant difference between the bacterial isolates obtained from premenopausal and postmenopausal popula- tions, some of the subjects in the postmenopausal population were receiving estrogen replacement therapy (ERT) and, again, the hormonal status of the subjects was not documented by any objective parameter. 2 In contrast, a more recent study by Larsen et al. used vaginal cytology to document menopausal status and compared vaginal bacterial isolates obtained from postmenopausal women re- ceiving ERT with the isolates obtained from a sep- arate postmenopausal population not receiving ex- ogenous estrogen. 3 This study found no significant difference in the incidence of aerobic organisms, although anaerobic species were more frequently isolated in the untreated population. The purpose of the present investigation was to compare the vaginal microbial flora in postmeno- Address correspondence/reprint requests to Dr. David E. Soper, Box 34, MCV Station, Richmond, VA 23298. Received April 28, 1993 Clinical Study Accepted July 20, 1993 EFFECT OF ESTROGEN ON VAGINAL FLORA GINKEL ET AL. pausal women prior to (the control group) and 8 weeks after beginning ERT (the study group). MATERIALS AND METHODS The subjects used in the study were 15 postmeno- pausal outpatients of Medical College of Virginia hospitals who were eligible for ERT and whose postmenopausal status was confirmed by serum es- trogen and follicle-stimulating hormone (FSH) lev- els. At the time of the patient's initial visit, a com- plete gynecological history was obtained, detailing the menstrual, reproductive, contraceptive, and sex- ual history of the subject as well as accounting for recent antibiotic exposure and significant underly- ing disease. Thereafter, a complete gynecological physical examination was performed including a test ofvaginal pH using ColorpHast pH test strips (EM Science, Cherry Hill, NJ).

reproductive, contraceptive, and sex- ual history of the subject as well as accounting for recent antibiotic exposure and significant underly- ing disease. Thereafter, a complete gynecological physical examination was performed including a test ofvaginal pH using ColorpHast pH test strips (EM Science, Cherry Hill, NJ). A vaginal wash- ing was obtained using 3 ml of sterile, nonbacteri- ostatic saline that was instilled into the vagina, agi- tated with a sterile swab, and aspirated into a syringe. After excess air was expelled, the syringe was capped and transported immediately to the lab- oratory. ERT consisted of estrogen delivery by means of a patch providing 0.1 mg of estradiol per day. Following 8 weeks ofERT, the assessment detailed above was repeated including a repeat sampling of serum estradiol and FSH levels. Anaerobic cultures of the vaginal washings were incubated in an anaerobic chamber for 7 days using reduced brucella-base blood agar with 10 Ig/ml of menadione and 0.5 Ig/ml ofheroin (BMB), BMB with 75 Ig/ml of kanamycin and 7.5 Ig/ml of vancomycin, and BMB containing 100 Ixg/ml of neomycin sulfate. Media for the isolation of aer- obes and facultative anaerobes included 5% sheep's blood agar (BAP), MacConkey agar, and Pfizer Selective Enterococcus agar (PSE). These were in- cubated in either air (BAP) or in 5% carbon diox- ide (MacConkey agar and PSE). All organisms were identified by the standard methods previously outlined.4 Differences in the number of positive cultures obtained before and after ERT were tested for sig- nificance using a Chi square or Fisher's exact test. Quantitative data including vaginal pI-I, serum FSH, and serum estradiol were tested using a two- tailed test. All P values were based on two-tailed tests with a P < 0.05 considered significant. TABLE I. Population characteristics before and after estrogen replacement therapy (ERT) Variable Pre-ERT Post-ERT P value Serum FSH (mU/ml) 144.3 _+ 58.6 67.9 +- 28.0 Serum estradiol (mU/ml) 18.4 -+ 2.0 40.9 -19.6 Superficial cells (%) 4.0 -+ 9. 34. 19. Vaginal pH (units) 5.7 -+ 1.3 4.7 -+ 0.6 0.0002 0.0006 0.0002 0.0283 ayalues are mean SD. TABLE 2. Vaginal cultures Pre-ERT Post-ERT Microorganism (No. [%]) (No. [%]) P value Lactobacilli 7 [47] 9 [60] NS Any aerobe 14 [93] 13 [87] NS Any anaerobe 7 [47] 2 [I 3] 0.05 Anaerobic gram-negative 6 [40] [7] 0.035 rods aNS, not significant. RESULTS The mean age ofthe subjects was 53.9 -+- 8.6 years.

s are mean SD. TABLE 2. Vaginal cultures Pre-ERT Post-ERT Microorganism (No. [%]) (No. [%]) P value Lactobacilli 7 [47] 9 [60] NS Any aerobe 14 [93] 13 [87] NS Any anaerobe 7 [47] 2 [I 3] 0.05 Anaerobic gram-negative 6 [40] [7] 0.035 rods aNS, not significant. RESULTS The mean age ofthe subjects was 53.9 -+- 8.6 years. Nine (60%) were sexually active, and 5 (33%) gave a history of douching. Throughout the course of the study, the subjects maintained a relatively con- stant level ofsexual activity. No subject douched or had sexual intercourse within 3 days of having the vaginal cultures taken. No vaginal medications were used during the study period. None of the subjects were exposed to any antimicrobial agents. Seven of the 15 subjects had undergone hysterectomy. Prior to ERT, all of the subjects exhibited se- rum FSH and estradiol levels consistent with ova- rian failure. After 8 weeks of estrogen replace- ment, the sample mean serum estradiol levels were significantly increased, while serum FSH levels were suppressed. After the subjects received ERT, an increased superficial cell count was noted, re- flecting the enhanced epithelialization ofthe vagina under the influence of estrogen. Finally, the mean vaginal pH was noted to be decreased significantly following ERT. These results are summarized in Table 1. Detailed in Table 2 are the results of the vaginal cultures obtained before and after ERT. Although lactobacilli were among the most frequently iso- lated species both before and after estrogen replace- ment, the proportion of isolates did not signifi- cantly change following treatment. The non- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 95 EFFECT OF ESTROGEN ON VAGINAL FLORA GINKEL ET AL. enterococcal group D streptococci (5), Staph- ylococcus epidermidis (4), and gram-negative enter- ics (4) were also commonly isolated. No change was noted in the frequency with which the subjects tested positive for any aerobe. However, the prevalence of anaerobic isolates decreased considerably after ERT. The difference was most pronounced for the anaerobic gram-negative rods, which were isolated from 43% of the participants prior to treatment compared with only 7% after estrogen replacement. The majority [5/6 (83%)] of the anaerobic gram- negative rods disappearing on ERT were bile-re- sistant Bacteroides spp. [B. capillosus, B. fragilis ov t s (two)].

for the anaerobic gram-negative rods, which were isolated from 43% of the participants prior to treatment compared with only 7% after estrogen replacement. The majority [5/6 (83%)] of the anaerobic gram- negative rods disappearing on ERT were bile-re- sistant Bacteroides spp. [B. capillosus, B. fragilis ov t s (two)]. Finally, the mean vaginal pH after ERT in lactobacilli-positive subjects was 4.4 -+ 0.4 com- pared with 5.2 -+ 0.3 in lactobacilli-negative sub- jects (P 0.02). There was no difference between vaginal pH before ERT in lactobacilli-positive sub- jects (5.5 -+ 1.3) compared with lactobacilli-nega- tive subjects (5.9 -+ 1.4) (P 0.65). Although the subjects colonized with lactobacilli exhibited a consistently lower mean vaginal pH, this differ- ence was only significant following estrogen re- placement. DISCUSSION Unlike previous investigations detailed in the liter- ature, the present study utilized a single postmeno- pausal population, obtaining vaginal bacterial iso- lates before and after ERT and documenting the hormonal status at each step by serum FSH and estradiol levels and by serial examination ofvaginal cytology. Consequently, each patient served as her own control, allowing a more direct appraisal ofthe effects of estrogen on the vaginal pI-i and microbi- ology. These data confirm previous reports that a change in hormonal status is associated with changes in vaginal bacterial flora. In accord with the find- ings of"Larsen et al., the serial cultures employed in this study revealed a decreased prevalence oanaer- obic species, especially anaerobic gram-negative rods, following the administration of" exogenous estrogen,a The number of'aerobic isolates remained relatively constant. The incidence of'lactobacilli did not significantly change following estrogen replacement despite a documented increase in serum estrogen to levels sufficient to suppress FSH, supporting the theory that hormonal status alone plays an important inde- pendent role in the maintenance of a low vaginal pH. These findings are consistent with an estro- gen-induced increase in epithelial cell metabolism ofglycogen, resulting in a lower vaginal pH. How- ever, following ERT, the mean vaginal pH was significantly lower in lactobacilli-positive subjects. This finding suggests that lactobacilli contribute to a decrease in vaginal pH and raises the possibility that estrogen may in some way potentiate the effect of lactobacilli on vaginal pH.

ing in a lower vaginal pH. How- ever, following ERT, the mean vaginal pH was significantly lower in lactobacilli-positive subjects. This finding suggests that lactobacilli contribute to a decrease in vaginal pH and raises the possibility that estrogen may in some way potentiate the effect of lactobacilli on vaginal pH. By increasing the carbohydrates available for metabolism to lactic acid by lactobacilli, ERT may help decrease vaginal pH without producing a commensurate change in the incidence of lactobacilli. The lower pH associated with ERT yields an environment less suitable to anaerobes. 6'7 In addition, estrogen may enhance the ability of lactobacilli to produce hydrogen per- oxide, which may control the quantity of catalase- negative anaerobic microorganisms. Further inves- tigation is needed in these areas. This investigation is limited by the small num- ber of subjects studied and by the brief time over which the subjects were followed. A decrease in vaginal pH should select for acidiphilic species; however, any increase in the isolation ofacidophilic species first requires new colonization, a process that may take more time than the 8 weeks allowed in this study. In the small population utilized here, only three subjects had negative cultures for lacto- bacilli both before and after ERT. Given a larger number of such subjects, we might have been able to test for a significant change in mean vaginal pH in this subpopulation, thereby better demonstrating the effect ofestrogen on vaginal pH independent of its effect on lactobacilli. In summary, vaginal cultures obtained from postmenopausal women prior to the initiation of ERT were more likely to grow anaerobic bacteria when compared with vaginal cultures obtained from these same women after the initiation ofERT. ERT may potentiate the effect of lactobacilli on vaginal pH. REFERENCES 1. Tashjian JH, Coulan CB, Washington JA: Vaginal flora in asymptomatic women. Mayo Clin Proc 51:557-561, 1976. 2. Osborne NG, Wright RC, Grulin L: Genital bacteriology: A comparative study of premenopausal women with post- menopausal women. Am J Obstet Gynecol 135:195-198, 1979. 96 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY EFFECT OF ESTROGEN ON VAGINAL FLORA GINKEL ET AL. 3. Larsen B, Goplerud CP, Petzold CR, Ohm-Smith MJ, Galask RP: Effect ofestrogen treatment on the genital tract flora of postmenopausal women. Obstet Gynecol 60:20- 24, 1982. 4. Lennette EH, Balows A, Hausler WJ, Shadomy HJ: Manual of Clinical Microbiology. 4th ed.

LOGY EFFECT OF ESTROGEN ON VAGINAL FLORA GINKEL ET AL. 3. Larsen B, Goplerud CP, Petzold CR, Ohm-Smith MJ, Galask RP: Effect ofestrogen treatment on the genital tract flora of postmenopausal women. Obstet Gynecol 60:20- 24, 1982. 4. Lennette EH, Balows A, Hausler WJ, Shadomy HJ: Manual of Clinical Microbiology. 4th ed. Washington, DC: American Society for Microbiology, 1985. 5. Kienlin H: Die reaktion des vaginakelkrebs neugeborener. Zentralbl Gynak 50:644, 1926. 6. Brown WJ: Microbial ecology of the normal vagina. In Haafex ESE, Evans N (eds): The Human Vagina. New York, Elsevier/North Holland Biomedical Press, pp 407- 422, 1978. 7. Paavonen J: Physiology and ecology ofthe vagina. Scand J Infect Dis Supp140:31-35, 1983. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:98-103 (1993) (C) 1993 Wiley-Liss, Inc. Effect of Isoflurane on Neutrophil Phagocytic Function During Pregnancy Penny Clark, A. Joseph Layon, and Patrick Duff Division ofMaternal-Fetal Medicine (P.C., P.D.) and Departments ofAnesthesiology and Medicine (A.J.L.), University ofFlorida College ofMedicine, Gainesville, FL ABSTRACT Objective: General anesthesia has been considered an independent risk factor for postcesarean infection, but the mechanism for this association has not been delineated. The purpose of this prospective investigation was to determine if phagocytic response of neutrophils was impaired by in vitro exposure to isoflurane, a commonly used anesthetic. Methods: Twelve milliliter venous blood samples were withdrawn from 18 term patients during labor. Neutrophils were separated by Ficoll gradient centrifugation. Aliquots of 2 x 106 neutrophils/ml were exposed to anesthesia using an airtight modular incubator chamber through which a 0.5% isoflurane:50% N20 + 50% 02 mixture flowed at a rate of 4 l/min for 90 min at 37C. Neutrophils were assayed for phagocytosis by incubation with Escherichia coli conjugated with fluorescein isothiocyanate for 30 min at 37C. Phagocytosis was assessed by flow cytometry. Neu- trophils from the same patient that were not exposed to anesthesia served as controls. Results: The mean percentage of phagocytizing neutrophils in the isoflurane-treated group was 82.8 +_ 24 compared to 83.5 +- 22 in the control group. The difference between the two groups was not significant. Conclusions: In vitro exposure to the general anesthetic isoflurane for 90 min does not signifi- cantly alter the phagocytic capacity ofneutrophils. (C) 1993 Wiley-Liss, Inc. KEY WORDS Host defenses, phagocytosis, leukocyte function, anesthesia "r'here is a high frequency of postoperative infec- 2 |tion following cesarean delivery. Ofthe clin- ical risk factors for infection, general anesthesia was identified by Green and Sarubbi3 to be the most statistically significant variable.

iss, Inc. KEY WORDS Host defenses, phagocytosis, leukocyte function, anesthesia "r'here is a high frequency of postoperative infec- 2 |tion following cesarean delivery. Ofthe clin- ical risk factors for infection, general anesthesia was identified by Green and Sarubbi3 to be the most statistically significant variable. However, ac- cording to Gibbs,2 general anesthesia was not a consistent determinant for increased rate of postop- erative infection. Clearly, the role of anesthesia as a predisposing risk factor for infection following ob- stetric surgery is not yet well established. Inhalational anesthetic agents have been shown to suppress the immune system.4'5 Some agents cause impairment of phagocytic function, an im- portant aspect of nonspecific host resistance. 6-9 In- creased susceptibility to bacterial infections has been closely associated with defective neutrophil func- tion. The host depends initially on neutrophils to eliminate invading bacterial pathogens. In obstetric patients, the stresses of surgery and anesthesia dur- ing cesarean delivery may diminish the activity of phagocytes, thus predisposing the patient to post- partum infection. The purpose of the present prospective study was to determine the effect of isoflurane on the phagocytic function of neutrophils collected from laboring patients. Isoflurane is an inhalation anes- thetic commonly used in clinical obstetrics in the United States. The technique of flow cytometry Address correspondence/reprint requests to Dr. Patrick Duff, P.O. Box 100294, University of Florida College of Medicine, Gainesville, FL 32610-0294. Clinical Study Received April 29, 1993 Accepted June 7, 1993 ANESTHESIA AND NEUTROPHIL FUNCTION IN PREGNANCY CLARK ET AL. was used to evaluate phagocytosis of fluorescein- conjugated Escherichia coli by purified neutrophils after exposure in vitro to isoflurane. Flow cytome- try allowed single cell analysis of phagocytic activ- ity. 10--12 MATERIALS AND METHODS The study population consisted of 18 normal term patients between the ages of 18 and 40 years who were in labor when admitted to Shands Hospital, University of Florida, from September to Decem- ber, 1991. Written informed consent was obtained from each patient in accordance with guidelines established by the Institutional Review Board. Twelve-milliliter samples of peripheral venous blood were withdrawn into heparinized vacutainer tubes from each of the 18 patients.

versity of Florida, from September to Decem- ber, 1991. Written informed consent was obtained from each patient in accordance with guidelines established by the Institutional Review Board. Twelve-milliliter samples of peripheral venous blood were withdrawn into heparinized vacutainer tubes from each of the 18 patients. Granulocytes from whole blood were isolated using a modifica- tion of Boyum's Ficoll-I--Iypaque density gradient centrifugation. 13 Briefly, 3 ml ofHistopaque-1119 (Sigma Chemical Co., St. Louis, MO) was trans- ferred into a 15 ml conical centrifuge tube, then layered with 3 ml of Histopaque-1077 (Sigma Chemical Co.). Six milliliters of whole blood was carefully overlayed onto the upper gradient and centrifuged in a swinging bucket rotor at room temperature for 30 min at 700g. At the end of centrifugation, a distinct layer of lymphocytes, platelets, and other mononuclear cells settled at the plasma/1077 interphase while a layer of predomi- nantly granulocytes settled at the 1077/1119 inter- phase. Erythrocytes gravitated to the bottom. Each layer was aspirated; the granulocyte layer was saved and washed 3 times with Hank's balanced salt solu- tion (HBSS; Sigma Chemical Co.) by centrifuga- tion at 200g for 10 min. The washed cells were resuspended in RPMI 1640 with 25 mM I-IEPES and sodium bicarbonate (Sigma Chemical Co.) and counted in a Coulter Counter (Coulter Electronics, Inc., Hialeah, FL). The final suspension contained approximately 2 106 cells/ml. Viability of the granulocytes was determined to be over 95% by trypan blue exclusion. E. coli was labeled with fluorescein 5-isothiocy- anate (Sigma Chemical Co.) by the method de- scribed by Gelfand. 14 An overnight culture of E. coli on trypticase soybroth was heat-killed at 60C for 30 min, washed 3 times with normal saline, then resuspended in 0.5 M carbonate/bicarbonate buffer, pI-I 9.5. Fluorescein 5-isothiocyanate dis- solved in the same buffer was added to the bacterial suspension at a final concentration of mg/ml and incubated in the dark for 2 h at room temperature. The fluorescein-conjugated bacteria were washed several times and resuspended in HBSS to a final concentration of 109 cells/ml. One milliliter aliquots were stored at -80C and thawed to room temperature immediately before use. One milliliter suspensions of purified neutro- phils were distributed into 35 10 mm sterile polystyrene disposable suspension culture dishes (Corning Glass Works, Corning, NY).

SS to a final concentration of 109 cells/ml. One milliliter aliquots were stored at -80C and thawed to room temperature immediately before use. One milliliter suspensions of purified neutro- phils were distributed into 35 10 mm sterile polystyrene disposable suspension culture dishes (Corning Glass Works, Corning, NY). Neutro- phils that were exposed to anesthesia were placed inside an airtight Modular Incubator Chamber (Billups-Rothenberg, Inc., Del Mar, CA) through which a 0.5% isoflurane (Forane USP, Anaquest, Madison, WI):50% N20 + 50% O2 mixture flowed at a rate of 4 1/min for 90 min at 37C. The isoflurane vaporizer was calibrated using a Perkin- Elmer mass spectrometer (Perkin-Elmer, Nor- walk, CT). Gas samples were taken from the cir- cuit limb containing exhaust gas after chamber equilibration was achieved. For controls, cell sus- pensions from each patient were incubated at 37C but not exposed to the anesthetic. The 90 min expo- sure period was selected because it should be near the maximum duration ofexposure to general anes- thesia that a patient would encounter during cesar- ean delivery. Phagocytosis of fluorescein-conjugated E. coli was measured in a reaction mixture containing 0.5 ml cell suspension, 0.1 ml of fluorescent E. coli, and 0.4 ml diluted pooled sera. The ratio ofneutro- phils to bacteria was routinely 1:25 to 1:60. The mixture was allowed to incubate with an end-over- end rotation for 30 min at 37C. The reaction was terminated by the addition of 3 ml cold 3 mM EDTA in phosphate-buffered saline, then analyzed by flow cytometry. Flow cytometric analyses were carried out in a FACSCAN (Becton-Dickinson Immunocytometry Systems, Mountain View, CA) equipped with a 15 mW argon laser. The excitation wavelength was set at 488 nm, and green fluorescence was selected by a 530 30 nm band pass filter. Various cell types were discriminated by means of their combined forward and 90 light scatter. The mean channel number of green fluorescence intensity was simul- taneously measured. Ten thousand blood cells from each reaction tube were routinely analyzed. Free INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY ANESTHESIA AND NEUTROPHIL FUNCTION IN PREGNANCY CLARK ET AL. extracellular bacteria were excluded by their small size. The neutrophils were identified and this pop- ulation was gated. Consort 30 software (Becton- Dickinson Immunocytometry Systems) was used for data collection and analyses.

IN OBSTETRICS AND GYNECOLOGY ANESTHESIA AND NEUTROPHIL FUNCTION IN PREGNANCY CLARK ET AL. extracellular bacteria were excluded by their small size. The neutrophils were identified and this pop- ulation was gated. Consort 30 software (Becton- Dickinson Immunocytometry Systems) was used for data collection and analyses. The number of neutrophils, the percent fluorescent neutrophils, as well as the mean fluorescence per neutrophil were calculated within the gated region. The percent fluorescent neutrophils represented those cells that phagocytized fluorescent E. coli. For each patient, the percent phagocytizing neutrophils after in vitro exposure to isoflurane was compared to control cells that were not exposed to isoflurane. Flow cytomet- ric observations were confirmed by fluorescent mi- croscopy (Nikon Optiphot, Nikon, Inc., Garden City, NY). To ensure that the measured fluorescence was due to actual phagocytosis, we determined the effect of sodium azide, a known inhibitor of phagocyto- sis. Sodium azide in 100 mM concentration was incorporated into the phagocytosis assay mix- ture. is To further distinguish fluorescence due to internalization of bacteria vs. fluorescence result- ing from simple adherence of bacteria to the cell surface of the phagocytes, fluorescence-quenching experiments were performed with trypan blue at low pH. 16 After termination of the phagocytosis reaction, the cells were suspended in cold phos- phate-buffered saline with 0.25 mg/ml trypan blue at pH 4.5, then analyzed by flow cytometry. Diluted pooled sera in the phagocytosis reaction mixture were used for opsonization to improve the efficiency of phagocytosis. Serum was collected from normal pregnant women and frozen in ali- quots. Immediately prior to use, an aliquot was thawed and diluted 1:4 with RPMI 1640. Statistical analysis was performed using the two- tailed paired t-test. P < 0.05 was considered sig- nificant. RESULTS Different cell types were discriminated by their combined forward light scatter which measured cell size and side scatter which measured granular- ity. Figure is a representative computer-gener- ated dot plot of the distribution of 10,000 blood cells after neutrophil purification, showing the gated neutrophil population which comprised ap- proximately 71 ---21% of the total cells enumer- ated. The percentage of fluorescent neutrophils de- II191B001 Fig. I. Representative distribution of 10,000 blood cells in a sample of purified neutrophils from a pregnant patient.

after neutrophil purification, showing the gated neutrophil population which comprised ap- proximately 71 ---21% of the total cells enumer- ated. The percentage of fluorescent neutrophils de- II191B001 Fig. I. Representative distribution of 10,000 blood cells in a sample of purified neutrophils from a pregnant patient. Measurement of forward (FSC) and 90 (SSC) angle light scatter discriminated (A) lymphocytes, (B) platelets and erythrocytes, and (RI) neutrophils. The percent fluorescent neutrophils were calculated within the gated region. termined within the gated region was considered the percent phagocytizing neutrophils. The optimal ratio of phagocytes to bacteria and length of incubation for routine phagocytosis assay were first determined. Figure 2 demonstrates the rate of increase in percentage of phagocytizing neu- trophils with length of incubation, as well as with increasing neutrophil to E. coli ratios. Maximal phagocytosis was achieved after 30 min incubation, where approximately 90% of neutrophils were flu- orescent. Nonsignificant increases in phagocytosis occurred with longer incubation periods ofup to 60 min. The rate of ingestion increased with bacterial concentration and reached saturation at a neutrophil to E. coli ratio of 1:25. There was no further in- crease in the rate of uptake when the number of bacteria was 50, 100, and 150 times the number of neutrophils. Based on these findings, a neutrophil to E. coli ratio of 1:25 to 1:60 with 30 min incubation time was routinely used for the phagocytosis assay. Quenching with trypan blue at pH 4.5 did not alter the percent fluorescent neutrophils after 30 min of phagocytosis, indicating that at the end of this period fluorescent bacteria had been internal- ized. Sodium azide at 100 mM concentration de- creased phagocytosis to 5-7% in both the control and isoflurane-exposed groups. Panels A in Figure 3 represent two-parameter dot displays of the gated neutrophil population of control cells and isoflurane-treated cells at times 0 100 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY ANESTHESIA AND NEUTROPHIL FUNCTION IN PREGNANCY CLARK ET AL. 100 /., uJ 80 / 40 5 10 15 20 25 30 tt 45 50 55 60 TIME (minutes) Fig. 2. Rate of phagocytosis and neutrophil to E. coli ratio. Neutrophil to E. coli (r, 1:4; o, I:10; o, 1:25; , 1:50, I:100, 1:150). and 30 min incubation with fluorescent E. coli. The units represent channel numbers or arbitrary values proportional to the intensity of forward and side light scatter.

60 TIME (minutes) Fig. 2. Rate of phagocytosis and neutrophil to E. coli ratio. Neutrophil to E. coli (r, 1:4; o, I:10; o, 1:25; , 1:50, I:100, 1:150). and 30 min incubation with fluorescent E. coli. The units represent channel numbers or arbitrary values proportional to the intensity of forward and side light scatter. Panels B in Figure 3 represent the corresponding histograms ofgreen fluorescence intensity of neutrophils within the gated region. The mean percent fluorescent neutrophils in this region after in vitro exposure to isoflurane was 82.8 24% compared to 83.5 +-- 22% in the unexposed control (NS). DISCUSSION The response of neutrophils to bacterial invasion includes chemotaxis, phagocytosis, oxidative and hydrolytic intracellular killing, and release of ly- sosomal components. Several studies have demon- strated that inhalation anesthetics depress certain phases ofthe phagocytic response. Significant inhi- bition of microbicidal capacity of human neutro- phils has been observed with the volatile anesthetic halothane.6'7 Nakagawara and colleagues8 studied the effects of halothane, isoflurane, and enflurane on phagocytosis, superoxide production, and intra- cellular calcium mobilization on neutrophils ob- tained from healthy adult volunteers. Their results showed that these volatile agents caused a decrease in superoxide production that appeared to be due, at least in part, to inhibition of intracellular cal- cium mobilization. Welch9 reported that enflurane depressed bacte- rial killing and chemiluminescence only in neutro- phils that were stressed by high bacterial challenge, but this inhibition was reversed by exposure to air for 30 min. Earlier investigations demonstrated that halothane, trichloroethylene, diethyl ether, and methoxyflurane inhibited the migration of human neutrophils toward a chemoattractant, casein. 17 Neutrophil chemotaxis was similarly depressed by nitrous oxide and enflurane, but not by enflurane's chemical isomer, isoflurane. 18 In fact, isoflurane stimulated chemotaxis ofelicited rabbit neutrophils in vitro. 19 Therefore, different anesthetic agents appear to have different mechanisms by which they affect neutrophil function. In our experiment, the profound decrease in percentage of fluorescent neutrophils in the pres- ence of an inhibitor of phagocytosis such as sodium azide confirms that the amount of fluorescence in both the control cells and those exposed to isoflu- rane was due to actual engulfment ofthe bacteria by phagocytosis.

tion. In our experiment, the profound decrease in percentage of fluorescent neutrophils in the pres- ence of an inhibitor of phagocytosis such as sodium azide confirms that the amount of fluorescence in both the control cells and those exposed to isoflu- rane was due to actual engulfment ofthe bacteria by phagocytosis. Additional proof of actual internal- ization of fluorescent E. coli was the fact that no further fluorescence quenching was seen with addi- tion of trypan blue in acid pH after 30 min of phagocytosis. The dye would quench fluorescence from bacteria that were attached externally to the cell surface but not those inside the cell. 16 Our study demonstrates that the ability to phago- cytize bacteria remains active in neutrophils of nor- mal pregnant women after exposure to isoflurane. Although this investigation did not examine micro- bial killing by phagocytes, Welch9 previously re- ported that the ability of neutrophils of normal healthy adults to kill E. coli, Klebsiella pneumoniae, or Staphylococcus aureus was not significantly altered when exposed to 1-3% isoflurane for h. Further- more, no inhibition of microbicidal activity of hu- man neutrophils was observed with 70% nitrous oxide and 30% oxygen, alone, or in combination with isoflurane. INFECTIOUS DISEASES 1N OBSTETRICS AND GYNECOLOGY [01 ANESTHESIA AND NEUTROPHIL FUNCTION IN PREGNANCY CLARK ET AL. I12191B085 CONTROL 112191BOO5\FL1\ B . FLUORESCENCE 1I "'i' i'6 i' i'" 112191B007 112191B006 CONTROL 30 MIN ';::" 112191BO06\FL1\ FLUORESCENCE .ISOFLURANE 0 MIN ".'." ..:..'. 112191B00?\FL1\ B FLUORESCENCE 110 i i i) 112191B008 ISOFLURANE 30 MIN Fig. 3. Representative light scatter diagrams (A) showing forward (FSC) and 90 (SSC) side scatter and corresponding histograms offluorescence intensity (B), at 0 and 30 min of phagocytosis assay for control (top 4 panels) and isoflurane-treated (bottom 4 panels) neutrophils. Therefore, bacterial infections occurring imme- diately after cesarean delivery do not appear to be the direct result of use of a general anesthetic such as isoflurane. The increased risk of infection is more likely due to the complex emergencies that create the need for general anesthesia for delivery. 102 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY ANESTHESIA AND NEUTROPHIL FUNCTION IN PREGNANCY CLARK ET AL. REFERENCES 1. Sweet RL, Ledger WJ: Puerperal infectious morbidity. Am J Obstet Gynecol 117:1093-1100, 1973. 2. Gibbs RS: Clinical risk factors for puerperal infections.

for general anesthesia for delivery. 102 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY ANESTHESIA AND NEUTROPHIL FUNCTION IN PREGNANCY CLARK ET AL. REFERENCES 1. Sweet RL, Ledger WJ: Puerperal infectious morbidity. Am J Obstet Gynecol 117:1093-1100, 1973. 2. Gibbs RS: Clinical risk factors for puerperal infections. Obstet Gynecol 55(Suppl): 178S-184S, 1980. 3. Green SL, Sarubbi FA: Risk factors associated with post cesarean section febrile morbidity. Obstet Gynecol 49: 686-690, 1977. 4. Thomson DA: Anesthesia and the immune system. J Burn Care Rehabil 8:483-487, 1987. 5. Lippa S, De Sole P, Meucci E, Littaru GP, De Francisci G, Magalini SI: Effect of general anesthetics on human granulocyte chemiluminescence. Experientia 39:1386- 1388, 1983. 6. Welch WD: Halothane reversibly inhibits human neu- trophil bacterial killing. Anesthesiology 55:650-654, 1981. 7. Welch WD, Zaccari J: Effect of halothane and N20 on the oxidative activity of human neutrophils. Anesthesiol- ogy 57:172-176, 1982. 8. Nakagawara M, Takeshige K, Takamatsu J, Takahashi S, Yoshitake J, Minakami S: Inhibition of superoxide production and Ca + mobilization in human neutrophils by halothane, enflurane, and isoflurane. Anesthesiology 64:4-12, 1986. 9. Welch WD: Effect of en(lurane, isoflurane, and nitrous oxide on the microbicidal activity of human polymorpho- nuclear leukocytes. Anesthesiology 61:188-192, 1984. 10. Bassoe CF, Solberg CO: Phagocytosis of Staphylococcus aureaus by human leucocytes: Quantitation by a (low cyto- metric and microbiological method. Acta Pathol Micro- biol Immunol Scand Sect C 92:43-50, 1984. 11. Bjerknes R, Bassoe CF, Sjursen H, Laerum OD, Sol- berg CO: Flow cytometry for the study of phagocyte functions. Rev Infect Dis 11:16-33, 1989. 12. Steinkamp JA, Wilson JS, Saunders GC, Stewart CC: Phagocytosis: Flow cytometric quantitation with fluores- cent microspheres. Science 215:64-66, 1982. 13. Boyum A: Separation of leukocytes from blood and bone marrow. Scand J Clin Lab Invest 21(Suppl 97):77-89, 1968. 14. Gelfand JA, Fauci A, Green I, Frank M: A simple method for the determination of complement receptor bearing mononuclear cells. J Immunol 116:595-599, 1976. 15. Oda T, Maeda H: A new simple fluorometric assay for phagocytosis. J Immunol Methods 88:175-183, 1986. 16. Hed J, Hallden G, Johansson SGO, Larsson P: The use offluorescence quenching in flow cytofluorometry to mea- sure attachment and ingestion phases in phagocytosis in peripheral blood without prior cell separation.

. Oda T, Maeda H: A new simple fluorometric assay for phagocytosis. J Immunol Methods 88:175-183, 1986. 16. Hed J, Hallden G, Johansson SGO, Larsson P: The use offluorescence quenching in flow cytofluorometry to mea- sure attachment and ingestion phases in phagocytosis in peripheral blood without prior cell separation. J Immunol Methods 101:119-125, 1987. 17. Moudgil GC, Allan RB, Russel RJ, Wilkinson PC: Inhi- bition by anesthetic agents, of human leucocytic locomo- tion towards chemical attractants. Br J Anaesth 49:97- 104, 1977. 18. Moudgil GC, Gordon J, Forrest JB: Comparative effects of volatile anesthetic agents and nitrous oxide on human leucocyte chemotaxis in vitro. Can Anaesth Soc J 31:631- 637, 1984. 19. Erskine R, James MFM: Isoflurane but not halothane stimulates neutrophil chemotaxis. Br J Anaesth 64:723- 727, 1990. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 103

Infectious Diseases in Obstetrics and Gynecology I:115-117 (1993) (C) 1993 Wiley-Liss, Inc. Editorial Practical Guidelines for Preventing Congenital Syphilis A recent case of congenital syphilis at our hospital made us aware of the many potential pitfalls in the prevention ofthis disease. The patient in question presented in labor at term, and the intern noted a negative VDRL at the first prenatal visit. The attending physician on call (one of the authors) noted the low birth weight; however, the newborn had good Apgar scores and a normal cord pH. Another resident rotating through our department did not check the VDRL drawn post partum before discharging the mother. The laboratory did not perform the VDRL until 3 days after the patient delivered because it was a long holiday weekend. When the laboratory notified the floor, the nursing staff said, "Don't worry about it" because the patient had already been discharged. The case was finally tracked down through our local public health department, and the mother had the classic palmar-plantar rash of secondary syphilis. Therefore, the neonate was readmitted for treatment of congenital syphilis. This editorial only emphasizes what many readers already know--cases of early syphilis among reproductive-age women have risen rapidly in the past few years, and subsequently the number of cases of congenital syphilis has reached record numbers (Fig. 1). Some of the news is good, with overall cases of syphilis declining over 1990 to 1992. In our area, the number of cases has remained high, with Kansas City being 6th and St. Louis 1st in per capita syphilis cases in 1992 (Table 1). The current epidemic has been characterized as urban, heterosexual, and related to illicit drug use. There is evidence that syphilis has diffused into rural areas as well. The Centers for Disease Control (CDC) reported six cases in a rural western Kansas county in 1992, the first cases reported since 1989.2 Two of the these cases were pregnant women. Unfortunately, physician error can play a role in cases ofcongenital syphilis.

vidence that syphilis has diffused into rural areas as well. The Centers for Disease Control (CDC) reported six cases in a rural western Kansas county in 1992, the first cases reported since 1989.2 Two of the these cases were pregnant women. Unfortunately, physician error can play a role in cases ofcongenital syphilis. In a recent case series from Detroit, errors in detection, treatment, or diagnosis were responsible for many of the 51 cases of congenital syphilis. In 18 cases, no serological test for syphilis was done on maternal or cord blood. In two-thirds of cases with positive serological tests, no treatment was initiated primarily because physicians were not aware of the positive results (similar to our case). Three cases were due to inadequate therapy. We wish to present these pragmatic suggestions for the prevention ofcongenital syphilis: 1. Adequate screening. The recently published 19th edition of Williams' Obstet- rics states that a nontreponemal test for syphilis should be done at the first prenatal visit and at delivery. The authors also state that in half of all cases of congenital syphilis the mothers receive adequate prenatal care but are not screened according to the above suggestions. In areas of high prevalence, an additional screen should be done early in the 3rd trimester. At our institution, we obtain a VDRL at the time of the glucose challenge, thereby detecting and treating several cases of syphilis. 2. Laboratory measures. A nontreponemal test for syphilis should be done in a timely manner (5 or 6 times a week regardless of holidays) on each maternal CONGENITAL SYPHILIS PREVENTION AULT AND FARO Thousands of cases Congenital r'-]Early Syphilis nCongenital in women 1987 1988 19 9 Thousands of cases Early Syphilis 1990 1991 1992 25 2O 15 10 Fig. I. Congenital syphilis, 1987-1992. Source: CDC. TABLE I. Early syphilis in selected cities in the United States, 1992 Cases/100,000 City Rank population St. Louis, MO Ist 153.3 Kansas City, MO 6th 64.8 Chicago, IL 7th 64. Detroit, MI 14th 5 I. Miami, FL 3 st 16.8 Source: CDC sample. Maternal samples should be given priority. Laboratory personnel should regard a reactive serological test as a "panic" value and contact the responsible physician immediately. 3. Penicillin. Penicillin is the treatment of choice for syphilis in pregnancy. Erythromycin does not cross the placenta and thus does not prevent congenital syphilis. Penicillin-allergic patients should undergo inpatient penicillin desensiti- zation. 4.

"panic" value and contact the responsible physician immediately. 3. Penicillin. Penicillin is the treatment of choice for syphilis in pregnancy. Erythromycin does not cross the placenta and thus does not prevent congenital syphilis. Penicillin-allergic patients should undergo inpatient penicillin desensiti- zation. 4. Education. All health care providers including nurses, residents, and com- munity physicians must be made aware that syphilis is on the rise. No mother should be released from the postpartum unit without a serological test result. As a result of the above case, we have devised a "worksheet" for the postpartum patient that must be completed prior to discharge. This worksheet contains maternal blood type, hemoglobin, rubella status, plans for contraception, and results ofhepatitis-B surface antigen test. Congenital syphilis is a preventable disease. Errors in patient care are a factor on the current epidemic, and implementing some form of the above guidelines along with increased awareness should help decrease the incidence of congenital syphilis. 116 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY CONGENITAL SYPHILIS PREVENTION AULT AND FARO REFERENCES 1. Berry M, Dajani A: Resurgence ofcongenital syphilis. Infect Dis Clin N Am 6:19-29, 1992. 2. CDC: SyphilismFord County, Kansas, 1992. MMWR 41:644-645, 1992. Kevin A. Ault and Sebastian Faro INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 17

Infectious Diseases in Obstetrics and Gynecology I:118-122 (1993) (C) 1993 Wiley-Liss, Inc. Evaluation of Infectious Disease Knowledge in Obstetrics and Gynecology and the Effects of Varying Durations of Training Mark G. Martens Division ofInfectious Diseases, Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, TX ABSTRACT Objective: The amount, origin, and resources ofinfectious disease knowledge in the field ofobstetrics and gynecology were investigated. If this knowledge is lacking, the exact length of the specific infectious disease training during residency should be defined to meet the ever-increasing knowledge required in training. Methods: A 50-question test was developed by one faculty member utilizing questions that incor- porated the basic sciences of microbiology and pharmacology and clinical knowledge of infectious diseases in the area of obstetrics and gynecology. Multiple choice and matching questions were structured so as to ascertain the source of the knowledge, including medical school curriculum, recent journal articles, and clinical experience. Results: The test was given yearly to all students and residents on the Obstetric and Gynecology Service over a period of 2 years. The questions were the same for each group, but were reshuffled each exam period. Three hundred and seven tests were properly administered and recorded. There was no statistical improvement in any successive year's scores unless specific infectious disease training occurred. Increasing improvement in scores was noted, with an increasing duration of infectious disease training specific for obstetrics and gynecology, beginning at 2 weeks (22% im- provement), 4 weeks (30% improvement), and 6 weeks (31% improvement) (P .05-.001). Basic science questions were most frequently answered correctly by medical students and early residents, while correctly answered clinical questions correlated with increasing clinical experience except in the area of ambulatory care. Conclusions: The infectious disease knowledge of residents in obstetrics and gynecology can be improved with 4 weeks ofintensive training.

correctly by medical students and early residents, while correctly answered clinical questions correlated with increasing clinical experience except in the area of ambulatory care. Conclusions: The infectious disease knowledge of residents in obstetrics and gynecology can be improved with 4 weeks ofintensive training. Re-exposure to basic science knowledge and improved training in ambulatory care in this resident group appear to be necessary. This test or similar tests can be helpful in defining areas ofdeficiencies and their possible remedies. (C) 1993 Wiley-Liss, Inc. KEY WORDS Ob-Gyn residency, infectious disease training, subspecialty evaluation nfectious disease training is usually accomplished formally in medical school for persons entering the field of obstetrics and gynecology or the surgi- cal specialties. Obstetrics and gynecology residents are expected to expand upon this knowledge base during their training through a variety of methods including faculty interaction, lectures, courses, con- ferences, and individual reading. The efficacy of each of these methods has not been assessed, nor have the specific areas of expertise or deficiency in residency been fully examined. If obstetrics and gynecology-specific infectious disease training is Address correspondence/reprint requests to Dr. Mark G. Martens, Division of Infectious Diseases, Department ofObstetrics and Gynecology, University ofTexas Medical Branch, Galveston, TX 77555-0587. Clinical Study Received March 31, 1993 Accepted September I, 1993 INFECTIOUS DISEASE EVALUATION MARTENS available, the proper duration and efficacy of such training are also not known. An understanding of the appropriate duration of subspecialty training is imperative, in light of the ever-increasing fund of knowledge and experience that a residency pro- gram must cover in 4 years. Redundant training is as much a waste of valuable time and effort as is ineffective training. The fund of knowledge of infectious diseases in obstetrics and gynecology was measured in medical students, residents, and faculty. In addition, the areas of adequate or deficient specialty-specific in- fectious disease knowledge were determined in stu- dents and obstetrics and gynecology residents be- fore and after varying durations of intensive training.

eases in obstetrics and gynecology was measured in medical students, residents, and faculty. In addition, the areas of adequate or deficient specialty-specific in- fectious disease knowledge were determined in stu- dents and obstetrics and gynecology residents be- fore and after varying durations of intensive training. MATERIALS AND METHODS A 50-question test was developed by the author that incorporated questions related to the basic sciences of microbiology and pharmacology as well as the clinical knowledge of infectious diseases in the area of obstetrics and gynecology. The test questions were multiple choice or matching. Twenty-one questions were related to the micro- biology of pelvic infections. Ten questions were related to the pharmacology of infections and their treatment. The remaining 19 questions were based upon clinical situations that were deemed to be reflective of the current clinical practice on the university service. The questions were also structured to ascertain the source of the infectious disease knowledge. Six- teen questions were derived from information that was believed to be acquired in medical school. Thir- teen questions required correlation with clinical ex- perience in order to determine the correct answer. The remaining 21 questions could only be answered correctly if the individual had read the recent liter- ature (of the past 2 years) or had specifically at- tended lectures or received training in a subspe- cialty. A representative sample of 5 questions is given in Table 1. The test was administered in a monitored setting at the University of Texas Medical Branch in Galveston to all 3rd-year medical students at the end of their obstetrics and gynecology rotation; to all 4th-year medical students before and after their 1-week rotation on the obstetrics and gynecology infectious disease service; to obstetrics and gynecol- TABLE I. Sample questions I. #2 The second most common organism found in cultures from women with urinary tract infection (UTI) is: a) E. coli b) Enterococci c) Staphylococcus saprophyticus d) Gardnerella vaginalis 2. #5 Cefazolin's trade names include all but: a) Ancef b) Ceftin c) Kefzol 3. #11 Antibiotics that cross the placenta adequately include all but: a) Mezlocillin b) Erythromycin c) Cefizoxime d) Acyclovir (Zovirax) 4. #21 Each of these antibiotics has activity versus chlamydia except: a) Tetracycline b) Erythromycin c) Cefoxitin d) Clindamycin 5.

s include all but: a) Ancef b) Ceftin c) Kefzol 3. #11 Antibiotics that cross the placenta adequately include all but: a) Mezlocillin b) Erythromycin c) Cefizoxime d) Acyclovir (Zovirax) 4. #21 Each of these antibiotics has activity versus chlamydia except: a) Tetracycline b) Erythromycin c) Cefoxitin d) Clindamycin 5. #40 A patient comes to your office after noticing a nontender ulcer on her labia majora. Upon examination, you note a 2 2 shallow nontender ulcer, along with ipsilateral nontender enlarged inguinal lymph nodes. The next course of action is: a) Obtain RPR and treat per results b) Arrange for darkfield examination c) Start tetracycline, 500 mg PO QID d) Observe the patient for week ogy residents at all four levels of training annually for 2 years; and to university faculty. The tests were reviewed by physician members of the Infectious Diseases Society for Obstetrics and Gynecology (IDSOG). After validation, the remaining ques- tions and answers were utilized, and the participat- ing IDSOG group of physicians served as the con- trol group. Halfofthe 3rd-year residents each year over the period of 2 years completed an obstetrics and gynecology infectious disease elective. The elec- tive was comprised of 4 weeks of intensive experi- ence including twice daily rounding for a mini- mum of 2 hours a day, reading requirements, consultations, and 2 hours of informal lectures a week. One-half of the 1990 2nd-year residents un- dertook a 6-week rounds-only infectious disease elective, while half of the current 3rd-year resi- dents completed 2 weeks of infectious disease rounds. Repeat tests were given each year with the order of the questions being reshuffled each testing period. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 19 INFECTIOUS DISEASE EVALUATION MARTENS TABLE 2. Obstetrics and gynecology infectious disease test scores Initial test Group No. % Correct Repeat test No. % Correct P value Medical students 3rd year 91 44 -- 4th year 72 42 60 53 <.005 Ob/Gyn residents st year 9 58 7 62 NS 2nd year 5 54 4 66 .05 4 70 <.001 3rd year 7 62 7 83 <.001 4th year 9 58 University Ob/Gyn faculty 9 56 Pharmaceutical representatives 8 52 IDSOG 15 72 Total 225 82 aTests not repeated. bFollowing 2 weeks of infectious disease service rounding twice daily. CFollowing 4 weeks of full-time infectious disease service. dFollowing 6 weeks of full-time infectious disease service rounding only. elnfectious Diseases Society for Obstetrics and Gynecology.

ives 8 52 IDSOG 15 72 Total 225 82 aTests not repeated. bFollowing 2 weeks of infectious disease service rounding twice daily. CFollowing 4 weeks of full-time infectious disease service. dFollowing 6 weeks of full-time infectious disease service rounding only. elnfectious Diseases Society for Obstetrics and Gynecology. Test scores were analyzed using the paired Stu- dent's t-test, with a P value < 0.05 achieving sta- tistical significance. RESULTS Three hundred seven tests were administered. Ta- ble 2 demonstrates the year-by-year results. Ninety- one tests were given to junior medical students following their 6-week rotation on obstetrics and gynecology. No specific infectious disease training was provided for these students except for one lec- ture on sexually transmitted diseases and one lecture on pelvic inflammatory disease. Their mean score at the end of their rotation was 44%. Fourth-year medical students took the test during their 4-week obstetrics and gynecology subspecialty rotation be- fore and after their 1-week infectious disease rota- tion. Seventy-two students took the test prior to the rotation and obtained a mean score of42%, demon- strating no statistical difference between the 3rd and 4th year of medical school. After the infectious disease rotation, 60 4th-year students had a mean score of 52% (P < .001). Tests were given to all obstetrics and gyneco- logic residents 6 months into the 1990 academic year. Nine st-year obstetrics and gynecology resi- dents scored a mean of 58%, five 2nd-year resi- dents scored a mean of 53%, seven 3rd-year resi- dents scored a mean of 62% and nine 4th-year residents scored a mean of 58%. An infectious dis- ease service in obstetrics and gynecology was begun in 1990. Therefore, all residents were exposed to unlimited consultations ofthe infectious disease ser- vice and to a series of eight formal and two to six informal noon lectures over the period of a year. Interns, with a mean score of 58%, did not signifi- cantly improve their scores after year of passive training (P .22). Second-year residents under- went one of two regimens of formal infectious disease training. The first group had 2 weeks of infectious disease service rounding only, while the second group had 4 weeks offull-time service duty. Each group's score improved significantly from a mean of 54% before training (P < .001, respec- tively).

residents under- went one of two regimens of formal infectious disease training. The first group had 2 weeks of infectious disease service rounding only, while the second group had 4 weeks offull-time service duty. Each group's score improved significantly from a mean of 54% before training (P < .001, respec- tively). However, there was no significant differ- ence between the mean scores of 66% and 70% following the 2- or 4-week training period, respec- tively. Seven 4th-year residents completed 6 weeks of infectious disease service rounds and significantly improved their test scores from 62% in 1990 to 82% in 1991 (P < .001). University obstetrics and gynecology faculty took the test and scored a mean of 56%; eight hospital pharmaceutical repre- sentatives took the test with a mean score of 52%, which was not statistically different from those of the initial tests from the resident group. Fifteen 120 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY INFECTIOUS DISEASE EVALUATION MARTENS IDSOG members scored a mean of 72%, and these scores served as the control. Analysis of the areas of knowledge of the junior medical students demonstrated that the most com- mon questions incorrectly answered dealt with the microbiology or antibiotic treatment that was spe- cific to obstetrics and gynecology, with fewer than 5% of these questions answered correctly. The four questions most frequently answered correctly were related to basic microbiology and pharmacology, with greater than 90% of the students answering these questions accurately. Senior medical students' correct and incorrect answers followed similar pat- terns. The residents as a group were more knowl- edgable in the clinical areas, with greater than 70% of these questions answered correctly. However, answers in the areas ofbasic microbiology and phar- macology were correct less than 30% of the time. Surprisingly, fewer than 50% of questions pertain- ing to outpatient or office management ofinfections were answered correctly. Answers requiring knowledge primarily ob- tained from reading the recent literature reflected less than 40% accuracy. IDSOG members had questions marked incorrect when subspecialty or regional differences or personal ongoing unpub- lished research affected their answers. Overall, ID- SOG members initially scored statistically higher than all other groups, as expected (P < 001).

g the recent literature reflected less than 40% accuracy. IDSOG members had questions marked incorrect when subspecialty or regional differences or personal ongoing unpub- lished research affected their answers. Overall, ID- SOG members initially scored statistically higher than all other groups, as expected (P < 001). DISCUSSION Infectious disease training in medical school and in obstetrics and gynecology residency programs is often assumed to be acquired by exposure to the various aspects of individual specialties (such as maternal-fetal medicine or operative gynecology) and to the literature. Due to the diverse nature of obstetrics and gynecology, an integration of the basic sciences, especially microbiology and phar- macology, with the variety of acute, operative, and outpatient clinical situations must be accomplished. This integration of basic and clinical sciences tradi- tionally occurs separately in medical school and residency. An ideal situation would be to combine both entities or at least reinforce them during the same training period. Through the examination of the specific areas of expertise and deficiencies, it appears that much ba- sic science knowledge is lost, as the average obstet- rics and gynecology clerkship lasts only 7 weeks. It is, however, replaced over the 4 years of resi- dency by knowledge obtained from clinical experi- ence. In this investigation, medical students from the 3rd to 4th year and obstetrics and gynecology residents from the 1st to the 4th year were not shown to acquire obstetrics and gynecology-specific infectious disease knowledge passively. Scores on the infectious disease test did not significantly im- prove for any year except when formal training occurred. Scores increased proportionally with longer durations of infectious disease training. From the change in which questions were scored correctly over time, it is evident that re-exposure to basic science education would be helpful in expand- ing the fund of infectious disease knowledge for obstetrics and gynecology residents. This need ap- pears early in the residency period and continues after residency. Despite the improvement in scores with training of infectious disease faculty members, specific trends in knowledge lost and gained were identifi- able from the test questions. Therefore, with direct attention, any institution should be able to correct these deficiencies.

residency period and continues after residency. Despite the improvement in scores with training of infectious disease faculty members, specific trends in knowledge lost and gained were identifi- able from the test questions. Therefore, with direct attention, any institution should be able to correct these deficiencies. The unimpressive scores of the university faculty can be seen in the light of indi- vidual scores versus the entire faculty complement- ing each other in the training of the residency group. Ifspecific departmental deficiencies are sus- pected, a method of evaluation through quality as- surance records as proposed by Silberman may be utilized.2 Maternal-fetal medicine faculty mem- bers were knowledgable in the infections of their subspecialty, whereas they performed less efficiently in gynecology areas. The converse was true for the gynecologist. Therefore, a concerted effort by the entire faculty should provide a complete spectrum of infectious disease knowledge for the resident in training. This should be accomplished with greater ease in the future as the number of medical school faculty continues to grow.3 However, specific areas such as outpatient medicine need to be included. Re-education in the basic sciences, specifically mi- crobiology and pharmacology, have already been identified as necessary, but will have to be specifi- cally addressed by an alteration in the teaching cur- ricula, as upper level residents and university fac- ulty apparently have similar deficiencies. While these deficits are serious, fortunately they do not INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 121 INFECTIOUS DISEASE EVALUATION MARTENS often result in poor patient care in a university setting, as there are usually several "layers" of care with supervision of each training level from stu- dent to resident to faculty. In addition graduated residents usually have partner-to-partner or other colleague contact to help in areas in which they are uncertain. Lastly, even if support systems fail, the isolated practicing physician is best trained in clin- ical matters, and weaknesses in basic science knowl- edge should not present a critical deficit. However, improving the education of medical students and residents is the important issue and concern. In summary, infectious disease training in ob- stetrics and gynecology does not appear to expand with passive learning, but is replaced with clinical knowledge over the years of training from medical school through residency.

improving the education of medical students and residents is the important issue and concern. In summary, infectious disease training in ob- stetrics and gynecology does not appear to expand with passive learning, but is replaced with clinical knowledge over the years of training from medical school through residency. Four to 6 weeks ofexpo- sure to a concentrated obstetrics and gynecology- specific infectious disease service appears to be the most effective method of training and achieves a knowledge base equivalent to that of recognized infectious disease experts. Alternative sources of training and education should directly address the basic sciences and outpatient medicine. However, each institution will have its own strengths and weaknesses that can be examined through testing, following which the deficiencies can be directly addressed. REFERENCES 1. Herbert WNP, Cummings RV, Droegemueller W: Pro- file of student clerkship administration in obstetrics and gynecology. Obstet Gynecol 76:153-155, 1990. 2. Silberman L: Quality assurance in obstetrics: A model. Obstet Gynecol 76:466-470, 1990. 3. Pearse WH, Graham KK: Trends in obstetric-gynecologic academic manpower and research. Obstet Gynecol 78:141- 143, 1991. 122 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology I" 123-129 (1993) (C) 1994 Wiley-Liss, Inc. Inpatient Treatment for Uncomplicated and Complicated Acute Pelvic Inflammatory Disease: Ampicillin/Sulbactam Vs. Cefoxitin David L. Hemsell, George D. Wendel, Jr., Patricia G. Hemsell, Molly L. Heard, and Brenda J. Nobles Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center at Dallas, and Parkland Memorial Hospital, Dallas, TX ABSTRACT Objective: Ampicillin plus sulbactam, an irreversible [3-1actamase inhibitor, was compared to cefox- itin in the treatment of women with acute pelvic inflammatory disease (PID) with and without inflammatory mass(es). Methods: Participation in an open, prospective, randomized clinical trial was offered to all women given the clinical diagnosis of acute PID who required inpatient therapy. Neisseria gonorrhoeae and Chlamydia trachomatis were sought in cervical and endometrial samples and aerobic and anaerobic species were sought in endometrial samples prior to treatment initiation. Treatment was given on at least 4 days and until women were afebrile for at least 48 h. Daily examinations were performed to assess response to therapy and safety. Only women in whom C. trachomatis was identified were discharged from the hospital on oral doxycycline to be taken for 10-14 days. Results: One hundred twenty-four women were evaluated for safety; 117 (94%) were evaluated for efficacy. Demographic characteristics were similar for women in each treatment group. N. gonorrhoeae was recovered from 59% and C. trachomatis was recovered from 42% of study subjects. Inflammatory masses were identified in 35/76 (46%) women given ampicillin/sulbactam and 17/41 (41%) women given cefoxitin. Ampicillin/sulbactam cured 75 of 76 women (98.7%) [95% confidence interval (CI) 92.9-100.0%] and cefoxitin cured 37 of41 women (90.2%) (95% C176.9-97.3% in that treatment regimen. Conclusions: Overall ampicillin/sulbactam was more effective (P 0.05) than cefoxitin, due to superior efficacy in infection complicated by inflammatory mass(es) 35/35 vs. 12/17 cured; P 0.007). (C) 1994 Wiley-Liss, Inc.

92.9-100.0%] and cefoxitin cured 37 of41 women (90.2%) (95% C176.9-97.3% in that treatment regimen. Conclusions: Overall ampicillin/sulbactam was more effective (P 0.05) than cefoxitin, due to superior efficacy in infection complicated by inflammatory mass(es) 35/35 vs. 12/17 cured; P 0.007). (C) 1994 Wiley-Liss, Inc. KEY WORDS Pelvic inflammatory disease, inflammatory mass(es), antibiotic therapy variety of broad-spectrum single-agent and combination regimens have been highly suc- cessful when given to women hospitalized for treat- ment of acute pelvic inflammatory disease (PID). The optimal antibiotic regimen for inpatient ther- apy of such women has not been established. The etiology of this acute pelvic infection is generally thought to involve Neisseria gonorrhoeae, Chlamy- dia trachomatis, and a wide variety ofanaerobic and aerobic bacteria. Treatment regimens must include broad-spectrum coverage ofthese likely pathogens. Additionally, therapy must be effective in the vari- ous forms and complications of PID (e.g., endo- metritis, salpingitis, and pelvic inflammatory mass/ abscess). Combination antimicrobial therapy has been recommended by the Centers for Disease Con- Address correspondence/reprint requests to Dr. David L. Hemsell, Department ofObstetrics and Gynecology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, G6.226, Dallas, TX 75235-9032. Received August 23, 1993 Clinical Study Accepted December 2, 1993 ACUTE SALPINGITIS THERAPY HEMSELL ET AL. trol and Prevention (CDC) since 1982.1 However, many single-agent therapeutic regimens, including cefoxitin, have been successful in the treatment of women with PID.2'3 Ampicillin alone was shown to be effective in one small clinical trial.4 Since many potentially significant pathogens of pelvic infection produce ]3-1actamase enzyme, the addi- tion ofa suicide-type [3-1actamase inhibitor to ampi- cillin should enchance and extend its antibacterial activity. A prospective, randomized, open clinical trial was designed in which women with clinical symp- toms and signs ofacute PID who required hospital- ization for therapy would be treated with intrave- nous (IV) ampicillin/sulbactam or cefoxitin. This report details results of early (inpatient) therapy only.

terial activity. A prospective, randomized, open clinical trial was designed in which women with clinical symp- toms and signs ofacute PID who required hospital- ization for therapy would be treated with intrave- nous (IV) ampicillin/sulbactam or cefoxitin. This report details results of early (inpatient) therapy only. SUBJECTS AND METHODS Women 18 years of age or older who presented to the emergency room ofParkland Memorial Hospi- tal complaining of lower abdominal and/or pelvic pain and who had abdominal, cervical motion, uter- ine, and adnexal tenderness were considered for inclusion in this clinical trial. They also had to have one or more of the following: temperature >38C; leukocyte count >10,000/mm3; Gram-negative diplococci in the endocervix; pelvic inflammatory mass(es); or leukocytes and bacteria from a perito- neal specimen. Pregnant or breastfeeding women were excluded as were those who were suspected to have a ruptured abscess; required concomitant anti- microbial therapy; had a history of known hyper- sensitivity reaction to cephalosporins, penicillin, or tetracyclines; had received successful antibiotic ther- apy within the 4 days preceding consideration for study entry; were receiving another investigational drug; had impaired immunological function and/or leukopenia (< 1,500/mm); and had clinically sig- nificant renal dysfunction. Women who met the inclusion and exclusion criteria and who signed an Institutional Review Board-approved consent form were sequentially enrolled in this clinical trial. Prior to antimicrobial administration, material recovered from the endocervix with a sterile swab was cultured for N. gonorrhoeae and tested for C. trachomatis by culture and/or for fluorescent antibody detection by MicroTrak(R) (Syva Com- pany, San Jose, CA). An endometrial sample also was obtained using a Pipelle(R) (Unimar, Wilton, CT) for identification of N. gonorrhoeae, C. tra- chomatis, and aerobic and anaerobic bacteria in the departmental research microbiology laboratory. N. gonorrhoeae specimens were plated directly onto Martin-Lewis Transgrow(R) media (Remel, Len- exa, KS) for identification using the quadFERM(R) system (Analytab Products, Plainview, NY). C. trachomatis specimens were placed into refrigerated Multi-Microbe(R) medium (MicroTest, Inc., Snell- ville, GA) for transport and cultured in McCoy(R) cells (Ortho Diagnostic, Ranton, NJ). Identifica- tion was made using the Syva(R) confirmation test (Syva Company).

FERM(R) system (Analytab Products, Plainview, NY). C. trachomatis specimens were placed into refrigerated Multi-Microbe(R) medium (MicroTest, Inc., Snell- ville, GA) for transport and cultured in McCoy(R) cells (Ortho Diagnostic, Ranton, NJ). Identifica- tion was made using the Syva(R) confirmation test (Syva Company). Gram-negative aerobic isolates were identified using the API 20-E(R) system (Analytab Products) and conventional methods were employed for identification of Gram- positive aerobic bacteria. Anaerobic isolates were processed in a Coy(R) anaerobic glove box (Coy Laboratory Products, Ann Arbor, MI) on stan- dard media and identified using the API 20-A(R) system (Analytab Products). Agar dilution suscep- tibility testing by minimal inhibitory concentration was performed using National Committee for Clin- ical Laboratory Standards (NCCLS) guidelines for aerobic (MT-T2) and anaerobic (M1 I-A) bacteria. Standard reference powder of each antibiotic was provided by the manufacturer. [3-Lactamase was detected using a nitrocephin disk (Becton-Dickin- son, Cockeyville, MD). The following clinical laboratory studies were performed prior to the initiation of therapy, at the completion of successful therapy, when therapy failed, or when patients left the hospital against medical advice (AMA): complete blood count (CBC) with differential; hepatic and renal function tests; direct Coombs test; prothrombin time (PT); partial thromboplastin time (PTT); and urinalysis. Women treated under this protocol were given consecutive study numbers as they were enrolled, and treatment was randomly assigned according to a 2:1 (ampicillin/sulbactam:cefoxitin) computer- generated randomization schedule. The regimen doses for ampicillin/sulbactam and cefoxitin were 3 and 2 g, respectively. Each therapeutic regimen was to be administered IV every 6 h on at least 4 consecutive days and until the patient was afebrile for at least 48 h. Sonography was performed on all study subjects to identify and document inflamma- tory pelvic mass(es). Results were interpreted by of 4 faculty sonographers using the criteria of a 124 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY ACUTE SALPINGITIS THERAPY HEMSELL ET AL. complex enlargement that was predominantly so- nolucent and septated with irregular borders in the region of an adnexa without an ovary detected sepa- rately. Doppler flow imaging was not performed. Women with C.

criteria of a 124 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY ACUTE SALPINGITIS THERAPY HEMSELL ET AL. complex enlargement that was predominantly so- nolucent and septated with irregular borders in the region of an adnexa without an ovary detected sepa- rately. Doppler flow imaging was not performed. Women with C. trachomatis at study entry were given 100-mg doxycycline tablets to be taken orally twice daily for 10-14 days when discharged from the hospital. Subjects negative for C. trachomatis were discharged on no oral antibiotic. The study subjects were evaluated clinically twice daily during hospitalization. Clinical cure was de- fined as resolution of abnormal tenderness, disap- pearance of temperature elevation for at least 48 h, normalization of the leukocyte count, and no re- quirement for other antimicrobial or surgical ther- apy for the original infection. It was necessary to receive a regimen for at least 48 h to be evaluable for efficacy. Clinical failure was defined as persis- tent or increasing symptoms, physical findings, and temperature after at least 48 h of" the original regi- men necessitating alternative antimicrobial or sur- gical therapy. A power analysis indicated that 120 study sub- jects (2:1 :ampicillin/sulbactam: cefoxitin) would be required to have an 80% chance of detecting a difference in efficacy of 20%, assuming an efficacy of 95% for ampicillin with sulbactam. Data were analyzed utilizing two-tailed Fisher's exact, log like- lihood X2, Welch's approximation, and Student's t-tests. Exact confidence intervals (CI) were calcu- lated. Clinical significance was defined as P < 0.05. RESULTS One hundred twenty-four women were entered into this clinical trial over 37 months; 117 (94%) were evaluable for clinical efficacy determination. Seven women, given cefoxitin and 6 given ampicillin/ sulbactam, were exluded from clinical evaluation. The woman given cefoxitin was treated for suba- cute bacterial endocarditis. Three women given ampicillin/sulbactam left the hospital AMA and 2 others were given additional antimicrobial treat- ment for syphilis and Trichomonas vaginalis vagini- tis prior to completion of protocol therapy. One other study subject experienced recurrent pruritus which began on her third day of ampicillin/ sulbactam therapy; she was clinically improving and was treated for 2 more days with clindamycin TABLE I.

timicrobial treat- ment for syphilis and Trichomonas vaginalis vagini- tis prior to completion of protocol therapy. One other study subject experienced recurrent pruritus which began on her third day of ampicillin/ sulbactam therapy; she was clinically improving and was treated for 2 more days with clindamycin TABLE I. Demographic and contraception characteristics of II 7 hospitalized women with acute PIDa Treatment regimen Ampicillin/sulbactam Cefoxitin Characteristic (N 76) (N 41) Age (years) 24.0 -+ 4.7 24.5 +- 5.9 Height (in.) 64.0 +- 2.7 64.0 +- 3.2 Weight (Ib) 139. -+ 41.9 142.7 30. Gravidity 1.2 +-1.4 I.I- 1.8 Parity 0.8 -+ I.I 0.9 -1.8 Race Black 58 (76) 31 (76) Caucasian 10 (I 3) 7 (I 7) Hispanic 8 (I I) 3 (7) Contraception None 51 (67) 32 (78) Oral contraceptives (14) 6 (I 5) Barrier 7 (9) (2) Tubal ligation 5 (7) (2) Intrauterine device 2 (3) (2) Previous PID 14 (18) 9 (22) Previous N. gonorrhoeae 25 (33) 10 (24) Data are presented as mean standard deviation or number with per- cent in parentheses. and gentamicin. Study numbers for these subjects were 5, 41, 44, 65, 86, 112, and 123. Demographic, contraceptive, and prior infec- tion variables for the evaluable women were statis- ticall similar and are presented by therapeutic reg- imen in Table 1. Infection-related variables (Table 2) were also similar, except for a higher mean temperature reading at study entry in women given ampicillin/sulbactam. Of the 52 women with in- flammatory mass(es), 24 (46%) denied a history of PID or positive culture for N. gonorrhoeae. Excluding N. gonorrhoeae and C. trachomatb, 489 aerobic and anaerobic bacteria were recovered from endometrial specimens obtained from these 117 evaluable women (Table 3). The predominant aerobic endometrial isolates were Gram-positive or- ganisms (86%); peptostreptococci accounted for 44% of anaerobic isolates. Prevotella bivius was the most frequently isolated Gram-negative anaerobe, accounting for 23% of all identified anaerobic iso- lates. The median number of isolates per patient was 5 organisms. One hundred fifteen isolates (23%) produced [3-1actamase. [3-Lactamase produc- tion was observed most frequently in enterobacteri- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 125 ACUTE SALPINGITIS THERAPY HEMSELL ET AL. TABLE 2. Entry infection-related variables for II 7 women treated for acute PIDa Ampicillin/sulbactam Clinical variable (N 76) Treatment regimen Cefoxitin ( 4) Duration of pain (days) 3.9 _+ 4.7 4.0 -+ 4.

tly in enterobacteri- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 125 ACUTE SALPINGITIS THERAPY HEMSELL ET AL. TABLE 2. Entry infection-related variables for II 7 women treated for acute PIDa Ampicillin/sulbactam Clinical variable (N 76) Treatment regimen Cefoxitin ( 4) Duration of pain (days) 3.9 _+ 4.7 4.0 -+ 4. Interval to last menstrual period (days) 12.5 10. 16. -+ 18.7 Leukocyte count (x 103/ram3) 16.4 +_ 6.0 17.1 +_ 5.4 Hemoglobin (g %) 12.6 -+ 1.6 12.7 _+ 1.5 Temperature (C)* 38.5 _+ 0.7 38.2 -+ 0.8 Inflammatory mass(es) 35 (46) 17 (41) <6 cm size 15 (43) 8 (47) 6-10 cm size 17 (49) 7 (41) >10 cm size 3 (8) 2 (12) Data are presented as mean -+ standard deviation or number with percent in parentheses. *P 0.04. TABLE 3. Endometrial isolates recovered prior to therapy of women with acute PID No. of 13-Lactamase Ampicillin/ Bacterial species/group isolates positive isolates sulbactam MIC 90b Cefoxitin Neisseria gonorrhoeae 69 3 (4) Chlamydia trachomatis 49 Staphylococcus epidermidis 78 13 (17) Staphylococcus aureus 10 3 (30) Streptococcus species 46 <0.25 Streptococcus agalactiae 27 <0.25 Enterococcus faecalis IS Escherichia coli 21 9 (43) 32 Proteus species 2 0.5 Pseudomonas aeruginosa 2 <0.25 Klebsiella pneumoniae (100) 4 Citrobacter diversus <0.25 Acinetobacter calcoaceticus var. (100) 0.5 anitratus Peptostreptococcus species 126 (I) C,Iostridium species 4 0.5 Propionibacterium species II (9) Prevotella/Bacteroides species 66 34 (52) Prevotella bivius 51 33 (65) 2 Bacteroides fragilis fragilis 6 6 (I00) 4 distasonis 16 thetaiotaomicron 2 2 (100) vulgatus 3 3 (100) 4 Prevotella disiens 7 3 (43) <0.25 Fusobacterium species 8 2 (25) 0.5 2 4 2 2 >128 4 2 2 0.5 64 4 4 8 4 2 8 32 32 8 0.5 Data are presented as number or number with percent in parentheses. bAntibiotic concentration in ig/ml inhibiting 90% of bacterial species. Patients with recovery at endocervical and/or endometrial sites. aceae and Prevotella or Bacteroides species. Only 3 gonococcal strains (4%) produced [3-1actamase. The recovery of N. gonorrhoeae and C. Tracho- matis from the endocervix and endometrium was similar in both treatment regimens (data not shown). Of the 117 evaluable women, 40 (34%) had N. gonorrhoeae only, 20 (17%) had C. tracho- mat# only, 29 women (25%) had both microorgan- isms, and 28 women (24%) had neither of these species. Of the 69 women (59%) with N. gonor- 126 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY ACUTE SALPINGITIS THERAPY HEMSELL ET AL. TABLE 4.

117 evaluable women, 40 (34%) had N. gonorrhoeae only, 20 (17%) had C. tracho- mat# only, 29 women (25%) had both microorgan- isms, and 28 women (24%) had neither of these species. Of the 69 women (59%) with N. gonor- 126 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY ACUTE SALPINGITIS THERAPY HEMSELL ET AL. TABLE 4. Clinical outcome of 117 women treated for acute PID Treatment regimen Outcome Ampicillin/sulbactam Cefoxitin Success without mass 40/41 (97.6) 24/24 (100) Days of therapy 4.4 0.9 4.3 -+ 0.6 Success with mass* 35/35 (100) 13/17 (76.5) Days of therapy** 4.9 -+ I. 6.0 -+ 2. Overall success** 75/76 (98.7) 37/41 (90.2) 95% CI 92.9-100.0 76.9-97.3 Days of therapy 4.6 -+ 1.0 5.0 -+ 1.6 Data are presented as mean --+ standard deviation or number of patients cured/number of patients treated with percent in parentheses. P 0.007. **P- 0.05. Cefoxitin failures were diagnosed after a mean du- ration of therapy of 5 days (range 4-7 days) with a mean temperature of 3 8.3C (range 3 8.1-38.5C) at therapy alteration. The altered therapy (clin- damycin, gentamicin, and ampicillin) was admin- istered for a mean of4 days (range 3-6 days) before discharge from the hospital.. Clinical and laboratory adverse events were not clinically serious and were comparable for women in each antibiotic treatment regimen (Table 5). Only the previously mentioned women with an allergic reaction to ampicillin/sulbactam had an ad- verse reaction dictating removal from the study protocol. rhoeae, 61 (88%) had isolates recovered from both cervical and endometrial sites, 6 (9%) had isolates only in the cervix, and 2 (3%) had isolates recov- ered only from endometrial specimens. The same recovery rates by site for the 49 women (42%) with C. trachomatis were 33 (68%), 9 (18%), and 7 (14%), respectively. N. gonorrhoeae was found in both the cervix and endometrium ofinfected women more frequently (_P 0.015) than was C. tracho- mat#. The absence of N. gonorrhoeae and/or C. trachomatis was associated with failure of the origi- nal regimen (P 0.011). Clinical outcome data for the study subjects are presented in Table 4. The overall rate of cure was higher for women treated with ampicillin/ sulbactam, due primarily to its efficacy in the treat- ment of PID complicated by inflammatory mass(es). All 5 women who failed initial therapy required alteration in their therapy, which they received for a mean duration of 9 days.

nted in Table 4. The overall rate of cure was higher for women treated with ampicillin/ sulbactam, due primarily to its efficacy in the treat- ment of PID complicated by inflammatory mass(es). All 5 women who failed initial therapy required alteration in their therapy, which they received for a mean duration of 9 days. All 4 of the cefoxitin failures occurred in women with inflam- matory masses of 6 cm or larger, and of the 4 women required abscess drainage by colpotomy. Only of the 5 treatment failures had either N. gonorrhoeae or C. trachomatis; she had both patho- gens, an inflammatory mass larger than 6 cm, and was in the cefoxitin group. The woman who ulti- mately failed therapy with ampicillin/sulbactam had therapy altered on day 6 of therapy. Her tempera- ture was 3 8.3C, and she had moderate tenderness. Her therapy was altered to clindamycin and gen- tamicin, and she was discharged 3 days later. Her admission leukocyte count was 38.3 103/mm3, and it was 20.5 103/mm3 at therapy alteration. DISCUSSION According to data from the CDC through 1991,5 the reported number of women from whom N. gonorrhoeae was recovered has been steadily decreas- ing over the past 10 years in the United States. The number ofwomen hospitalized for therapy of acute PID has decreased even more markedly over the same time period.6 The explanation for these trends in the United States is unclear, especially since rates ofsyphilis have increased during the same period.7 N. gonorrhoeae continues to be the sexually trans- mitted disease species most frequently recovered from women admitted to our hospital for parenteral therapy. Although the percentage ofwomen admit- ted for inpatient therapy who had N. gonorrhoeae has remained relatively constant at between 50 and 60% over the past 7 years (data not shown), the number of women admitted has decreased almost 70% from 226 in 1985 to only 60 in 1992. We found that the broad-spectrum antimicro- bial regimen of ampicillin/sulbactam given 3g IV every 6 h was clinically superior to cefoxitin 2 g IV every 6 h in the treatment of women with acute PID diagnosed in an emergency room. On sub- group analyses, the efficacy of the 2 regimens was similar for uncomplicated PID. However, in women with infection complicated by pelvic in- flammatory mass(es) detected by physical and ultra- sound examinations, ampicillin/sulbactam had sig- nificantly greater efficacy.

cute PID diagnosed in an emergency room. On sub- group analyses, the efficacy of the 2 regimens was similar for uncomplicated PID. However, in women with infection complicated by pelvic in- flammatory mass(es) detected by physical and ultra- sound examinations, ampicillin/sulbactam had sig- nificantly greater efficacy. Furthermore, in this latter group the mean number of days of therapy for women treated with ampicillin/sulbactam was less than that of women treated with cefoxitin, sug- gesting a more rapid therapeutic response. Not unexpectedly, the treatment failures were INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 127 ACUTE SALPINGITIS THERAPY HEMSELL ET AL. TABLE S. Adverse events in 124 women treated as inpatients for acute PIDa Ampicillin/Sulbactam Adverse event (N 82) Treatment regimen Cefoxitin ( 4) Clinical Diarrhea 3 (4) 4 (10) Pruritus 2 (2) 2 (5) IV site pain/edema 2 (2) 0 (0) IV site phlebitis (I) (2) Rash 0 (0) () Laboratory test Aspartate aminotransferase 3 (4) 3 (7) Alkaline phosphatase 3 (4) (2) PTT () (2) Data are presented as number with percent in parentheses. Elevation out of normal range at end oftherapy with normal value prior to study entry. generally associated with non-gonococcal, non- chlamydial infection and large (>6 cm) pelvic in- flammatory masses at enrollment. Almost half of these women denied previous pelvic infection, in- dicating that inflammatory mass formation can com- plicate an initial infection. All of the cefoxitin- treated failures were women with pelvic masses. In this protocol, we followed our usual practice of clinically following all women after treatment for PID, especially to detect resolution or persistence of inflammatory mass(es). In a previous clinical trial, 1 only 12% of women had a persistent ad- nexal mass that required surgical diagnosis/therapy. A sterile hydrosalpinx was found in each instance. Neither of the regimens presented here is rec- ommended for adequate treatment ofC. trachomatis infection. Previous investigators have confirmed that 13-1actam antimicrobial agents have clinical ef- ficacy in acute PID, but may fail to eradicate C. trachomatis. 8'9 Clinical success of initial regimens not active against chlamydia has been observed in patients treated in our hospital under prospective protocols. 1-13 If the addition of doxycycline to PID regimens is only to eradicate C. trachomat#, it seems appropriate to identify those women and pro- vide specific therapy.

s. 8'9 Clinical success of initial regimens not active against chlamydia has been observed in patients treated in our hospital under prospective protocols. 1-13 If the addition of doxycycline to PID regimens is only to eradicate C. trachomat#, it seems appropriate to identify those women and pro- vide specific therapy. If testing is not done, all women should be treated. Administration of doxy- cycline for acute PID did not prevent adhesion formation or tubal occlusion in a report by Teisala and coworkers. 14 In that report, women with C. trachomatis prior to treatment with penicillin and metronidazole did not have it when retested at sec- ond-look laparoscopy. According to Westrom and coinvestigators, is tetracycline treatment did not re- sult in a significantly lower incidence of post-infec- tion tubal occlusion in an earlier report on a large number of Scandinavian women treated for PID. All women in both treatment regimens were given oral doxycycline at hospital discharge if they had chlamydia at enrollment into the clinical trial. Ex- perimental polymerase chain reaction (PCR)-based testing appears to be more sensitive and specific than currently available techniques, including cul- ture. When developed and available, it may pro- vide the most exact means of identifying C. tra- chomatis in clinical settings. There are several limitations to this study. All diagnoses were clinical and not laparoscopic. In 1969 Jacobson and Westrom16 reported that an accurate clinical diagnosis of acute PID was made in only 65% of women undergoing laparoscopy. Hadgu and coworkers17 reported that, if women were young, unmarried, had vaginal discharge, fever, tender adnexal swelling, an elevated erythro- cyte sedimentation rate, and N. gonorrhoeae in the cervix, the probability of the accuracy of the clini- cal diagnosis was 97%. Cumming et al. 18 sug- gested that normal laparoscopic findings did not exclude significant endosalpingeal infection, and Sellors and colleagues19 reported that laparoscopic visualization of the fallopian tube was 50% sensi- tive with 80% specificity for the diagnosis of sal- pingitis when fimbrial histopathology was utilized to establish the diagnosis. In our opinion, data do not support routinely performing laparoscopy in all women given the clinical diagnosis of PID using 128 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY ACUTE SALPINGITIS THERAPY HEMSELL ET AL. criteria proposed by Hager and coworkers,2 which were utilized in this clinical trial.

the diagnosis. In our opinion, data do not support routinely performing laparoscopy in all women given the clinical diagnosis of PID using 128 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY ACUTE SALPINGITIS THERAPY HEMSELL ET AL. criteria proposed by Hager and coworkers,2 which were utilized in this clinical trial. Follow-up endocervical and endometrial cultures were only performed for clinical treatmentfailures. Thus, the relation between bacteriologic and clini- cal cure was not examined. This report does not address long-term clinical response and fertility, both important components in long-term efficacy analyses. The potential for biased selection for en- try did exist since this was an open clinical trial. All women given the diagnosis of acute PID who re- quired inpatient therapy and met inclusion/ exclusion criteria were approached for participation and consent. The 2 study groups were similar in terms ofdemography, clinical presentation, micro- biologic spectrum of pelvic pathogens, and the fre- quency and relative sizes of inflammatory mass(es) were similar in both sets of patients. Thus, there appeared to be no selection biases in the hospital- ized treatment groups. In summary, we conclude that ampicillin/ sulbactam was significantly more effective than cefoxitin for the inpatient treatment of" acute PID, especially when complicated by pelvic inflamma- tory mass(es). ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid from Roerig Pharmaceuticals. REFERENCES 1. Centers for Disease Control: Sexually transmitted diseases treatment guidelines 1982. Morbidity and Mortality Weekly Report 3 l(Suppl 2):33S-60S, 1982. 2. Peterson HB, Walker CK, Kahn JG, Washington AE, Eschenbach DA, Faro S: Pelvic inflammatory disease. Key treatment issues and options. JAMA 266:2605-2611, 1991. 3. Peterson HB, Galaid EI, Zenilman JM: Pelvic inflam- matory disease: Review of treatment options. Rev Infect Dis 12(Suppl 6): $656-$664, 1990. 4. Spence MR, Genadry R, Raffel L: Randomized prospec- tive comparison ofampicillin and doxycycline in the treat- ment of acute pelvic inflammatory disease in hospitalized patients. Sex Transm Dis 8(Suppl 2):164-166, 1981. 5. Centers for Disease Control: Summary of notifiable dis- eases, United States, 1991. Morbidity and Mortality Weekly Report 40:24, 1991. 6. Centers for Disease Control: Pelvic inflammatory disease: Guidelines for prevention and management. Morbidity and Mortality Weekly Report 40:4, 1991. 7.

8(Suppl 2):164-166, 1981. 5. Centers for Disease Control: Summary of notifiable dis- eases, United States, 1991. Morbidity and Mortality Weekly Report 40:24, 1991. 6. Centers for Disease Control: Pelvic inflammatory disease: Guidelines for prevention and management. Morbidity and Mortality Weekly Report 40:4, 1991. 7. Gersham KA, Rolls RT: Diverging gonorrhea and syph- ilis trends in the 1980's: Are they real? Am J Public Health 81:1263-1266, 1991. 8. Sweet RL, Schachter J, Robbie MO: Failure of 13-1actam antibiotics to eradicate Chlamydia trachomatis in the en- dometrium despite apparent clinical cure of acute salpin- gitis. JAMA 250:2641-2645, 1983. 9. Kousseim M, Ronald A, Plummer FA, D'Costa L, Brun- ham RC: Treatment of acute pelvic inflammatory disease in the ambulatory setting: Trial of cefoxitin and doxycy- cline versus ampicillin-sulbactam. Antimicrob Agents Chemother 35:1651-1656, 1991. 10. Hemsell DL, Santos-Ramos R, Cunningham FG, No- bles BJ, Hemsell PG: Cefotaxime treatment for women with community acquired pelvic abscesses. Am J Obstet Gynecol 151:771-777, 1985. 11. Hemsell DL, Hemsell PG, Heard ML, Nobles BJ: Pip- eracillin and a combination of clindamycin and gentami- cin for hospital and community acquired acute pelvic infections including pelvic abscess. Surg Gynecol Obstet 165:223-229, 1987. 12. Hemsell DL, Nobles BJ, Heard MC, Hemsell PG: Up- per and lower reproductive tract bacteria in 126 women with acute pelvic inflammatory disease: Microbial suscep- tibility and clinical response to four therapeutic regimens. J Reprod Med 33:799-805, 1988. 13. Hemsell DL, Wendel GD, Gall SA, Newton ER, Gibbs RS, Knuppel RA, Lane TW, Sweet RL: Multicenter comparison of cefotetan and cefoxitin in the treatment of acute obstetric and gynecologic infections. Am J Obstet Gynecol 158:722-728, 1988. 14. Teisala K, Heinonen PK, Aine R, Punnonen R, Paa- vonen S: Second look laparoscopy after treatment of acute pelvic inflammatory disease. Obstet Gyneco169:343-346, 1987. 15. Westrom L, Iosif S, Svensson L, Mirdh P-A: Infertility after acute salpingitis: Results of treatment with different antibiotics. Curr Ther Res 26:752-759, 1979. 16. Jacobson L, Westrom L: Objectivized diagnosis of acute pelvic inflammatory disease. Diagnostic and prognostic values of routine laparoscopy. Am J Obstet Gynecol 105: 1088-1098, 1969. 17. Hadgu A, Westrom L, Brooks CA, Reynolds GH, Thompson SE: Predicting acute pelvic inflammatory dis- ease: A multivariate analysis.

16. Jacobson L, Westrom L: Objectivized diagnosis of acute pelvic inflammatory disease. Diagnostic and prognostic values of routine laparoscopy. Am J Obstet Gynecol 105: 1088-1098, 1969. 17. Hadgu A, Westrom L, Brooks CA, Reynolds GH, Thompson SE: Predicting acute pelvic inflammatory dis- ease: A multivariate analysis. Am J Obstet Gynecol 155: 954-960, 1986. 18. Cumming DC, Honor6 LH, Scott JZ, Williams KE: Microscopic evidence of silent inflammation in grossly normal fallopian tubes with ectopic pregnancy. Int J Fer- til 33:324-328, 1988. 19. Sellors J, Mahony J, Goldsmith C, et al.: The accuracy of clinical findings and laparoscopy in pelvic inflam- matory disease. Am J Obstet Gynecol 164:113-120, 1991. 20. Hager WD, Eschenbach DA, Spence MR, Sweet RL: Criteria for diagnosis and grading of salpingitis. Obstet Gynecol 61 113-114, 1983. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 129

Infectious Diseases in Obstetrics and Gynecology 1:130-133 (1993) (C) 1993 Wiley-Liss, Inc. Management of Intrauterine Device-Associated Actinomycosis Ashwin Chatwani and Soheil Amin-Hanjani Department ofObstetrics, Gynecology, and Reproductive Sciences, Temple University School of Medicine, Philadelphia, PA ABSTRACT Objective: To assess various methods ofmanagement ofactinomyces-like organisms associated with intrauterine devices. Methods: A retrospective chart review of 173 patients with intrauterine device-associated actino- myces-like organisms detected on Pap smear was performed. The patients were managed by IUD removal with or without antibiotic therapy, antibiotic therapy alone, or no treatment at all. Results: The success rate as reflected in negative follow-up smear was 100% for IUD removal combined with antibiotics, 97.4% for IUD removal alone, and 36.8% for antibiotics therapy alone. Conclusions: The best way to manage intrauterine device-associated actinomyces-like organisms is removal ofthe device with or without antibiotics. (C) 1993 Wiley-Liss, Inc. KEY WORS IUD, actinomyces, Pap smear uman actinomycosis was first described by Is- rael. The first report ofgenital actinomycosis infection was made in 1926 in a woman who was using an intrauterine pessary.2 Henderson's3 re- port in 1973 brought the association of pelvic acti- nomycosis and intrauterine contraceptive devices (IUDs) to the attention ofgynecologists. Since then, actinomyces colonization has been described in oth- erwise healthy women without IUDs by Persson and Holmberg.4 Gupta and associatess-7 first described the histo- morphologic identification of the carriage of acti- nomyces-like organisms (ALOs) and their associa- tion with IUDs on Pap smears. Gupta7 has demonstrated that Pap-stained cervicovaginal smear diagnosis ofALOs has both a high specificity (98%) and sensitivity (94%) when correlated with fluores- cence antibody staining. The management ofIUD-associated ALOs iden- tified on a cervicovaginal Pap smear, especially in an asymptomatic woman, is difficult, controver- sial, and without any uniform consensus.

al smear diagnosis ofALOs has both a high specificity (98%) and sensitivity (94%) when correlated with fluores- cence antibody staining. The management ofIUD-associated ALOs iden- tified on a cervicovaginal Pap smear, especially in an asymptomatic woman, is difficult, controver- sial, and without any uniform consensus. It ranges from leaving the IUD in situ to removal, with or without reinsertion of a new IUD or antibiotic treatment. 8-11 Our study assesses some of the ways to manage ALOs diagnosed by cervicovaginal Pap smears in asymptomatic IUD users. MATERIALS AND METHODS Data were compiled from records of patients at- tending the Temple University Hospital Family Planning Clinic from 1975 to 1985. We accumu- lated a list of 1,745 patients utilizing the IUD as a means of contraception. These patients' Pap smear results were reviewed for the presence of ALOs. One hundred seventy-three patients were found to have ALOs on their Pap smears. Pap smears were the only method used for diagnosis of ALOs. The patients' charts were then reviewed to determine the Address correspondence/reprint requests to Dr. Ashwin Chatwani, Temple University Hospital, Department ofOB/GYN & RS, 3401 North Broad Street, 7-OPD, Philadelphia, PA 19140. Clinical Study Received June 8, 1993 Accepted September 15, 1993 IUD-ASSOCIATED ACTINOMYCOSIS CHATWANI AND AMIN-HANJANI date of IUD insertion and removal and if antibiot- ics were taken while the IUD was in situ with presence ofALOs on their Pap smears. Criteria for inclusion in this study were: 1) Pap smear without ALOs prior to the insertion of the IUD; 2) Pap smear positive for ALOs after insertion ofthe IUD; 3) follow-up Pap smear within 6 months after treat- ment; and 4) asymptomatic status at the time of initial positive Pap smear. The patients were divided into 4 groups accord- ing to their management: Group 1: Removal of IUD alone Group 2: Antibiotics alone Group 3: Removal of IUD and antibiotics Group 4: No treatment It should be noted that removal ofthe IUD with or without antibiotics was often carried out for reasons other than treatment of cervical actinomy- ces: the presence of abnormal uterine bleeding (n 27), abnormal uterine bleeding with concom- itant unrelated infections (n 27), a change to other forms of contraception (n 31), or the de- sire for future fertility (n 29). Patients in group 2 received antibiotics for concomitant infections and not ALOs.

vical actinomy- ces: the presence of abnormal uterine bleeding (n 27), abnormal uterine bleeding with concom- itant unrelated infections (n 27), a change to other forms of contraception (n 31), or the de- sire for future fertility (n 29). Patients in group 2 received antibiotics for concomitant infections and not ALOs. Patients with negative follow-up Pap smears af- ter treatment were classified as successes, and patients with persistent ALOs after treatment were deemed failures. A small number of patients in group 4 with ALOs were eventually lost to follow-up. Antibiotics received by patients in groups 2 and 3 were divided into four groups: A) ampicillin, 500 mg 4 times a day for 7-10 days/penicillin, 250 mg 4 times a day for 7-10 days; B) doxycycline, 100 mg twice a day for 7 days/tetracycline, 500 mg 4 times a day for 7 days; C) metronidazole, 250 mg 3 times a day for 7 days; and D) multiple antibiotics (any combination of the aforementioned antibiotics). Statistical analyses were performed using Epi Info (Centers for Disease Control, Atlanta, GA). The Mantel-Haenszel chi square formula was used. When a cell value of less than 5 was encountered, a 2-tailed P value was obtained with the Fisher's exact test. RESULTS There was a total of 173 patients with IUD-associ- ated ALOs on cervicovaginal Pap smear. Table TABLE I. Management of IUD-associated ALOs Follow-up Type of No. of Negative Positive Group treatment patients [no. (%)] [no. (%)] Removal of IUD 76 74 (97.4) 2 (2.6) 2 Antibiotics 38 14 (36.8) 24 (63.2) 3 Antibiotics and 38 38 (100) 0 removal of IUD 4 No treatment 21 3 (14.3) 18 (85.7) details the results of the management in each of the four groups described. Group consisted of 7 6 patients who had ALOs on the Pap smear. Removal of the IUD alone gave a success rate of 97.4%. Two patients with persis- tent ALOs on their post-treatment smears were lost to follow-up. In group 2 all of the patients who failed initial antibiotic therapy had their IUDs re- moved subsequently and had a negative Pap smear within 2 weeks to 6 months ofIUD removal. Eigh- teen patients in group 4 who continued to have positive smears were eventually lost to follow-up. All patients in this group had a minimum fol- low-up of 6 months. The results ofmanagement with removal ofIUD alone or removal of IUD with antibiotics were found to be significantly better than management with antibiotics alone (P 0 and P 0).

up 4 who continued to have positive smears were eventually lost to follow-up. All patients in this group had a minimum fol- low-up of 6 months. The results ofmanagement with removal ofIUD alone or removal of IUD with antibiotics were found to be significantly better than management with antibiotics alone (P 0 and P 0). There was no difference when removal of IUD alone was compared with IUD removal with antibiotics (P 0.31). There were 27 patients who had their IUDs removed because of abnormal uterine bleeding. Seventeen ofthese patients had IUD removal alone, while ten patients had IUD removal and antibiot- ics. All 27 patients had subsequent negative Pap smears. The types of antibiotics used in group 2 patients are presented in Table 2. The ampicillin/penicillin group was not found to be different from the doxycycline/tetracycline and metronidazole groups (P 0.30 and P 0.23). However, patients who received multiple antibiotics had a better chance of cure when compared with patients who received doxycycline/tetracycline (P 0.04). DISCUSSION Our retrospective analysis indicates that the best form of treatment for IUD-associated ALOs is INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 31 IUD-ASSOCIATED ACTINOMYCOSIS CHATWANI AND AMIN-HANJANI TABLE 2. Antibiotic therapy only Follow-up No. of Negative Positive Group patients [no. (%)] [no. (%)] Ampicillin/penicillin 8 3 (37.5) 5 (62.5) Doxycycline/tetracycline 12 2 (16.5) 10 (83.3) Metronidazole 10 4 (40) 6 (60) Multiple antibiotics 8 5 (62.5) 3 (37.5) Total 38 14 (36.8) 24 (63.2) removal of the IUD. Addition of antibiotics at the time of removal of the IUD does not increase the overall success rate. Seventy-four of 77 patients who had only removal of the IUD had subsequent negative Pap smears, and all 38 patients who had IUD removal and antibiotics had negative fol- low-up Pap smears. This difference was not statis- tically significant (P 0.31). Treatment with antibiotics without removal of the IUD gave an overall success rate ofonly 36.8%, which was significantly less than IUD removal or IUD removal and antibiotics (P 0). As the pres- ence of the IUD acts as the focus for this foreign- body-induced infection, its removal seems essential for successful therapy. None ofour patients with IUD-associated ALOs developed serious upper genital tract infection.

, which was significantly less than IUD removal or IUD removal and antibiotics (P 0). As the pres- ence of the IUD acts as the focus for this foreign- body-induced infection, its removal seems essential for successful therapy. None ofour patients with IUD-associated ALOs developed serious upper genital tract infection. This finding has also been reported by other authors.4'8'9 However, women with IUD-associated ALOs on Pap smears ofthe cervix have been shown to have a 3.6-fold greater incidence ofhospitalization for pel- vic inflammatory disease (PID). 12 Tubo-ovarian abscesses have also been reported to be more com- mon in women with IUDs and colonization of ALOs when compared with women with IUDs but not colonized with ALOs. 12 In our study, there were 27 patients who had their IUDs removed because of abnormal uterine bleeding. It is proba- ble that this symptom was secondary to endometri- tis, but none of these patients had endometrial bi- opsy for confirmation. Singh et al.9 suggested expectant management in asymptomatic women if IUD-associated ALOs are noted on Pap smears. This recommendation is based on their findings of only 4.8% with positive cul- tures for ALOs in the endometrial biopsies, imply- ing to them that there is only superficial coloniza- tion of this organism of the cervix and IUD. This policy of leaving the device in situ needs further evaluation. Mao and Guillebaud have suggested removal and reinsertion ofanother IUD as satisfac- tory management of IUD-associated ALOs. In their study, seven patients who had !UDs removed for ALOs and a new IUD inserted at the same time had no evidence of ALOs on subsequent Pap smears. Spontaneous resolution of IUD-associated ALOs has also been reported. This occurred in three of our patients. Expectant management, as some investigators have advised, is often an attractive option. Even though spontaneous resolution does occasionally oc- cur, it is recommended that patients with IUD- associated ALOs be managed with removal of the device. The addition of antibiotics does not signifi- cantly alter the success rate. These patients should be followed with repeat Pap smears any time after 2 weeks of treatment. Once a negative smear is ob- tained, consideration may be given to reinsertion of a new IUD. REFERENCES 1. Israel J: Neue Beobachtungen auf dem Gebiete der My- kosen des Menschen. Arch Pathol Anat 74:15, 1878. 2. Draper JW, Studdiford WE: Report of a case of actino- myces of the tubes and ovaries.

2 weeks of treatment. Once a negative smear is ob- tained, consideration may be given to reinsertion of a new IUD. REFERENCES 1. Israel J: Neue Beobachtungen auf dem Gebiete der My- kosen des Menschen. Arch Pathol Anat 74:15, 1878. 2. Draper JW, Studdiford WE: Report of a case of actino- myces of the tubes and ovaries. Am J Obstet Gynecol 11:603-608, 1926. 3. Henderson SR: Pelvic actinomycosis associated with an intrauterine device. Obstet Gynecol 41:726-732, 1973. 4. Persson E, Holmberg K: A longitudinal study of actino- mycosis Israelii in the female genital tract. Acta Obstet Gynecol Scand 63:207-217, 1984. 5. Gupta PK, Hollander DH, Frost JK: Actinomycetins in cervicovaginal smears: An association with IUD usage. Acta Cytol 20:295-297, 1976. 6. Gupta PK, Erozan YS, Frost JK: Actinomycetes and the IUD: An update. Acta Cytol 22:281-282, 1978. 7. Gupta PK: Intrauterine contraceptive devices: Vaginal cytology, pathologic changes and clinical implications. Acta Cytol 26:571-613, 1982. 8. Mao K, Guillebaud J: Influence of removal of intrauter- ine contraceptive devices on colonization of the cervix by actinomyces-like organisms. Contraception 30:535-544, 1984. 9. Singh MM, Ingham HR, Wadehra V, Morris K: En- dometrial culture in IUD users with actinomycosis-like organisms (ALOs) on cervical smears. Br J Family Plan- ning 15:3-6, 1989. 10. Hager WD, Majmudar B: Pelvic actinomycosis in women 132 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY IUD-ASSOCIATED ACTINOMYCOSIS CHATWANI AND AMIN-HANJANI using intrauterine contraceptive devices. Am J Obstet Gynecol 133:60-63, 1979. 11. Bhagavan BS, Gupta PK: Genital actinomycosis and in- trauterine devices: Cytopathologic diagnosis and clinical significance. Hum Pathol 9:567-578, 1978. 12. Burkman R, Schlesselman S, McCaffrey L, Gupta PK, Spence M: The relationship ofgenital tract actinomycoses and the development of pelvic inflammatory disease. Am J Obstet Gynecol 143:585-589, 1982. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

irect microscopic detection and latex agglutination methods.4,s Isolation of a yeast (with growth) in culture from a vaginal specimen is con- sidered to be definitive proofofits presence and the "gold standard" in clinical microbiology and re- search. However, the true sensitivity ofculture has not yet been determined. Few studies have been published concerning the sensitivity ofculture tech- niques in establishing the presence of yeast from clinical specimens. Furthermore, no studies have been published with regard to the quantitation of yeasts from clinical specimens or in media to establish precise concentrations of yeast cells for study. The classical method of determining the concen- tration of a yeast cell suspension is counting in a Neubauer's chamber or similar method (Burke's chamber, hemocytometer, etc.). However, its use is time-consuming and not practical for evaluating a large number of suspensions. The aim of" the present study was to compare the counts obtained with a Neubauer's chamber to the values obtained by turbidimetry (spectrophotome- try) and by nephelometry. Address correspondence/reprint requests to Dr. Acicio Rodrigues, Department of Microbiology, Porto School of Medicine, Alameda Prof. Hernani Monteiro, 4200 Porto, Portugal. Received April 2, 1993 Basic Science Article Accepted August 25, 1993 CANDIDA ALBICANS SUSPENSION RODRIGUES ET AL. TABLE I. Results of the three different methods of assessing the concentration of yeast suspensions Absorbance/ Volt/ Cells/ml/chamber spectrophotometry nephelometry 1,008 0. 116 15.5 540 0.058 8.9 387 0.037 5.78 284 0.029 4.74 252 0.022 3.76 234 0.018 3.2 208 0.014 3 162 0.012 2.52 120 0.01 2.17 70 0.004 1.24 MATERIALS AND METHODS Clinical isolates of C. albicans were grown on Sa- bouraud's agar and identified using auxanography (API32C, BioMrieux, France). A dense yeast sus- pension was prepared in 0.9% NaC1 solution at room temperature from a 24-hour-old culture grown on Sabouraud's agar medium at 37C. The suspension of yeast cells was homogenized to break up aggregates of yeast cells into individual cells in suspension. Then, serial dilutions were performed 11 times (each one with a 10-fold dilution from the preceding one). One-milliliter aliquots were re- moved from each dilution and analyzed spectro- photometrically (Spectronic 2001) at 540 nm.

as homogenized to break up aggregates of yeast cells into individual cells in suspension. Then, serial dilutions were performed 11 times (each one with a 10-fold dilution from the preceding one). One-milliliter aliquots were re- moved from each dilution and analyzed spectro- photometrically (Spectronic 2001) at 540 nm. A similar sample was analyzed by nephelometry (Bhering laser automatic nephelometer); 0.001 ml ofthe suspension was placed in a Neubauer's cham- ber and counted under 400 magnification. Quan- titative determinations were made in duplicate in a double-blind fashion by two independent ob- servers. RESULTS The results obtained by each of the three proce- dures are shown in Table 1. The results obtained from Neubauer's chamber were expressed as the mean ofthe two determinations. These results were compared to the values obtained from both the spectrophotometric (Fig. 1) and the nephelometric determinations (Fig. 2). The results obtained uti- lizing spectrophotometry and nephelometry were converted to quantitative values utilizing the for- mulas presented in Table 2. These formulas allow a conversion to the number of cells per milliliter of medium. This method provides a simple and quick method to determine the number of yeast cells per milliliter in a suspension. DISCUSSION Inocula of known concentrations as well as suspen- sions of precise concentrations require a method of measurement that is fast, accurate, and reliable. Determination of the number of cells per milliliter using a counting chamber is commonly considered to be the "gold standard" or reference method. However, it is a tedious and inconvenient method when preparing a large number of suspensions. The use of the visual McFarland scale or Browne tubes does not seem to be accurate. 6 Turbidimetry is based upon a measurement ofthe reduction ofthe intensity of a light passing through a suspension by a photometer or spectrophotometer.7 Nephelome- 1200 =, 1000 800 600 = 400 E .=: 200 i1 0 0,02 0,04 0,06 0,08 0,1 0,12 Spectrofotometry (Ab$) Comparison of the results of spectrophotometry with counts in a counting chamber. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 135 CANDIDA ALBICANS SUSPENSION RODRIGUES ET AL. 1200 800 F... 1000 600 E 400 0 200 0 5 10 15 20 Nephelometry (Volt.) Fig. 2. Comparison of the results of nephelometry with counts in a counting chamber. TABLE 2.

lts of spectrophotometry with counts in a counting chamber. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 135 CANDIDA ALBICANS SUSPENSION RODRIGUES ET AL. 1200 800 F... 1000 600 E 400 0 200 0 5 10 15 20 Nephelometry (Volt.) Fig. 2. Comparison of the results of nephelometry with counts in a counting chamber. TABLE 2. Formulas for conversion Chamber (x 103/ml) 64.3 + 8,206 x spectrophotometry (absorbance) Chamber (x 103/ml) -0.2 + 64 nephelometry (volt) try measures the intensity of a reflected light beam, by the particles in the suspension, at a 90 angle to the incident beam.7 The present study shows a direct and reliable correlation between the three methods employed. Any one of these methods can be used indepen- dently to evaluate the concentration of yeast cells in suspension or to prepare a suspension of a definite concentration. The use ofthe spectrophotometer or the nephelometer facilitates these calculations as well as makes it simpler and faster than utilizing a cell counting chamber. The yeast cell suspensions used in the present study were easily converted from readings obtained with the spectrophotometer or nephelometer into a quantitative number of yeast cells per milliliter of medium. These two methods were found to be as accurate as counting individual yeast cells with a counting chamber such as Neu- bauer's chamber. REFERENCES 1. Wilkinson JS: Some remarks upon the development of epiphytes with the description of new vegetable formation found in connection with the human uterus. Lancet 2:448, 1849. 2. Hillier SL: Laboratory diagnoses of yeast vaginitis. In Horowitz BJ, Mrdh PA (eds): Vaginitis and Vaginosis. New York: Wiley-Liss, pp 121-124, 1991. 3. Odds FC: Candidosis of the genitalia. In Odds FC (ed): Candida and Candidosis. London: Balliere Tindall, pp 124-135, 1988. 4. Odds FC: Isolation, identification and other laboratory aspects of Candida. In Odds FC (ed): Candida and Candi- dosis. London: Balliere Tindall, pp 60-67, 1988. 5. Martinez-de-Oliveira J, Fonseca AF: Diagnosing vaginal candidosis by a slide latex agglutination test. In Guaschino S (ed): Infectious Diseases in Obstetrics and Gynecology. Bologna: Monduzi Editore, pp 171-172, 1990. 6. Raphael SS, Spencer F, Culling CFA: Microbiology-- Applied microbiology: Some basic concepts, techniques, and methods. In Raphael SS (ed): Lynch's Medical Labo- ratory Technology. Vol 1. Toronto: W.B. Saunders, pp 532-573, 1976. 7. Hyde TA, Mellor LD, Raphael SS: Chemistry: Analytical systems and applications.

1990. 6. Raphael SS, Spencer F, Culling CFA: Microbiology-- Applied microbiology: Some basic concepts, techniques, and methods. In Raphael SS (ed): Lynch's Medical Labo- ratory Technology. Vol 1. Toronto: W.B. Saunders, pp 532-573, 1976. 7. Hyde TA, Mellor LD, Raphael SS: Chemistry: Analytical systems and applications. In Raphael SS (ed): Lynch's Med- ical Laboratory Technology. Vol 1. Toronto: W.B. Saun- ders, pp 73-126, 1976. 136 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology I:137-143 (1993) (C) 1994 Wiley-Liss, Inc. Placental Transport of Zidovudine in the Rhesus Monkey Louis E. Ridgway III, Thomas S. King, George I. Henderson, Steven Schenker, and Robert S. Schenken Departments of Obstetrics and Gynecology, Medicine, and Pharmacology, University ofTexas Health Science Center at San Antonio, San Antonio, TX ABSTRACT Objective: This study was undertaken to characterize the pharmacokinetics of zidovudine (ZDV) and ZDV-glucuronide (ZDVG) in the material and fetal circulations ofthe rhesus monkey. Methods: Cannulas were placed in the maternal external jugular and the fetal internal jugular and carotid artery in 8 pregnant monkeys at 120-130 days gestation. ZDV (3.5 mg/kg) was adminis- tered to 5 monkeys and ZDVG (3.5 mg/kg) to 3 monkeys as single intravenous bolus infusions through the maternal catheter. Maternal and fetal blood samples were collected every 20 min for the first 2 h and then every hourfor the next 4 h. Maternal and fetal concentrations ofZDV and ZDVG were determined using high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. Results: In monkeys who received ZDV, the terminal half-life (T1/2) for ZDV was 37 -+ 15 and 33 -+ 13 min in the maternal and fetal compartments, respectively. The apparent T1/2 for maternal ZDVG was 124 +_ 44 and 142 +- 50 min in the maternal and fetal compartments, respectively. Peak levels ofZDV and ZDVG in the fetal compartment were reached 40 min after injection. The mean fetal/maternal concentration ratios for ZDV and ZDVG ranged from 0.20 -+ 0.20 at 20 min to a maximum of 0.74 + 1.0 at 120 min and from 0.28 + 0.08 at 20 min to 1.4 _+ 1.3 at 180 min, respec- tively. In monkeys who received ZDVG, the T1/2 for ZDVG in the maternal and fetal compart- ments was 47 _+ 26 and 119 _+ 164 min, respectively. ZDVG reached its peak in the fetal compart- ment at 60 min post-injection. The fetal/maternal ratio ranged from 0.08-+ 0.11 at 20 min to 4.2 -+ 4.2 at 180 min post-injection.

- tively. In monkeys who received ZDVG, the T1/2 for ZDVG in the maternal and fetal compart- ments was 47 _+ 26 and 119 _+ 164 min, respectively. ZDVG reached its peak in the fetal compart- ment at 60 min post-injection. The fetal/maternal ratio ranged from 0.08-+ 0.11 at 20 min to 4.2 -+ 4.2 at 180 min post-injection. Conclusions: These data demonstrate that 1) ZDV and ZDVG rapidly cross the placenta to the fetal compartment, 2) ZDV crosses more rapidly than ZDVG, and 3) some metabolism of ZDV to ZDVG occurs in the fetal compartment. (C) 1994 Wiley-Liss, Inc. KEY WOlmS Zidovudine, zidovudine-glucuronide, Macacca m#latta, pharmacokinetics n the United States there are an estimated 6,000 pregnancies annually in women who are human immunodeficiency virus (HIV) positive. Treat- ment with zidovudine (ZDV) is recommended for non-pregnant HIV-infected individuals whose CD4 cell counts drop below 500/mm3.2 In the pregnant patient, treatment with ZDV is not specifically rec- ommended until the CD4 cell count drops below 200/mm3.3 Some feel ZDV treatment should be offered during pregnancy when CD4 counts drop below 500/mm3.3 Regardless of when treatment is initiated, it is apparent that an increasing number of pregnant women and their fetuses are being ex- posed to ZDV.4 While there is considerable information on the pharmacology and metabolism of ZDV in men, Address correspondence/reprint requests to Dr. Louis E. Ridgway III, Department ofGynecology and Obstetrics, University ofKansas School ofMedicine, 3901 Rainbow Boulevard, Kansas City, KS 66160.7316. Basic Science Article Received August 3, 1993 Accepted December 6, 1993 PLACENTAL TRANSPORT OF ZDV RIDGWAY ET AL. there are limited data on the pharmacokinetics of ZDV in the pregnant mother and her fetus. Hankins et al. 5 examined maternal, fetal, and am- niotic fluid levels of ZDV and ZDV-glucuronide (ZDVG) after multiple oral doses in near-term pregnant baboons. In their study, maternal and fetal levels of ZDV and ZDVG were not signifi- cantly different at sampling. Ho.wever, amniotic fluid levels of ZDV and ZDVG were significantly greater than maternal levels. The authors do not give sampling time after oral dose and no fetal or maternal pharmacokinetic information can be de- rived from their study. A recent phase I study of ZDV in women in the third trimester of preg- nancy6 demonstrated the pharmacokinetic parame- ters to be similar to non-pregnant adults.

maternal levels. The authors do not give sampling time after oral dose and no fetal or maternal pharmacokinetic information can be de- rived from their study. A recent phase I study of ZDV in women in the third trimester of preg- nancy6 demonstrated the pharmacokinetic parame- ters to be similar to non-pregnant adults. Unfortu- nately, no information on fetal pharmacokinetics can be obtained in human pregnancies. In the present study, we utilized a chronically cannulated rhesus monkey model to assess the phar- macokinetics of ZDV and ZDVG in both the ma- ternal and fetal circulations after maternal injection of ZDV or ZDVG. SUBJECTS AND METHODS Primate Husbandry Rhesus monkeys (Macacca mulatta; n 8) of 120- 130 days gestation (term 167 days) were stud- ied. Weights ranged from 6.4 to 11.8 kg and all were drug naive. The animals were individually caged in climate-controlled rooms and provided with water ad libitum and food twice daily. The study was approved by the Institutional Animal Care and Use Committee. Catheterization After the monkey was sedated with ketamine hy- drochloride (15 mg/kg, i.m.), a cuffed endotra- cheal tube was placed and anesthesia maintained using a combination of halothane, nitrous oxide, and oxygen. Maternal stability was assured by con- tinuous monitoring of heart rate and blood pres- sure. The monkey's neck was prepared and draped for surgery. The skin was incised over the right sternocleidomastoid, and the maternal external jug- ular vein was exposed and cannulated with a polyvi- nyl catheter, which was advanced to the level of the superior vena cava. The catheter was then tunneled subcutaneously over the right scapula and exterior- ized at a point midway between the scapulae. The fetal position and placental location were determined using real-time ultrasonography. A midline incision was made through the abdominal wall after the maternal abdomen was sterilely pre- pared and draped. A 3-4 cm transverse uterine incision was made over the fetal head. The chorion and amnion were incised.after removal ofthe amni- otic fluid, which was stored at 37C. The fetal skin overlying the sternocleidomastoid muscle was ele- vated into the uterine incision. A 2 cm incision was made and a self-retaining retractor placed. The fetal internal jugular vein and carotid artery were exposed and catheterized using polyvinyl catheters. The fetal neck and uterus were closed in layers and the amniotic fluid was replaced.

ocleidomastoid muscle was ele- vated into the uterine incision. A 2 cm incision was made and a self-retaining retractor placed. The fetal internal jugular vein and carotid artery were exposed and catheterized using polyvinyl catheters. The fetal neck and uterus were closed in layers and the amniotic fluid was replaced. The fetal catheters were then tunneled subcutaneously and exteriorized at the same point as the maternal catheter. The abdomen was closed in layers and the monkey placed in a vest with a mobile tether to allow blood collec- tion without anesthesia. Drug Injection and Blood Collection ZDV (3.5 mg/kg; 3'-azido-3'-deoxythymidine; Sigma Chemical Co., St. Louis, MO) was given as a bolus injection through the maternal catheter in 5 animals after they had fully recovered from gener- alized anesthesia. ZDVG (3.5 mg/kg; Sigma Chem- ical Co.) was given to 3 other animals as a bolus injection. Each ZDVG animal received 2 doses of ZDVG 24 h apart. Maternal and fetal samples were collected every 20 min for the first 2 h and then every hour for the next 4 h. ZDV and ZDVG Assay Serum concentrations ofZDV and its major metab- olite ZDVG were determined by solid-phase (car- tridge C18 columns: Bond Elut, Analytichem In- ternational, Harbor City, CA) extraction of each serum sample and subsequent analysis by high- performance liquid chromatography (HPLC) with ultraviolet (UV) detection (modified from Good et al.7). Briefly, a 50 I1 aliquot of each extracted sample was injected onto a Supelcosil LC-18-S col- umn [250 4.6 mm; 5 Im particle size (Supelco, Bellefonte, PA)]. The mobile phase consisted of 0.5 M KHzPO4 (pH 2.7) and CH3CN (88:12, v/v) at a low rate of 1.0 ml/min. The UV detector was set at a sensitivity of 0.02 AUFS and 254 mm wavelength. Retention times for the internal stan- dard (3'-azido-3'-deoxyuridine, Sigma Chemical :}8 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY PLACENTAL TRANSPORT OF ZDV RIDGWAY ET AL. nglml 1,000 100 I0 0,1 0 100 200 300 400 minutes mean _+ sem Fig. I. Concentrations (mean -SEM) of ZDV in maternal and fetal circulations following maternal administration of ZD (3.5 mg/kg) at time 0; n 5. Co.), ZDV, and ZDVG were approximately 4.0, 7.3, and 10.9 min, respectively. Concentrations were determined by comparison of peak areas with those of standards using a programmed integrator interfaced with the UV detector. Detection limits for ZDV and ZDVG were 0.008 and 0.01 pg/ml, respectively.

mg/kg) at time 0; n 5. Co.), ZDV, and ZDVG were approximately 4.0, 7.3, and 10.9 min, respectively. Concentrations were determined by comparison of peak areas with those of standards using a programmed integrator interfaced with the UV detector. Detection limits for ZDV and ZDVG were 0.008 and 0.01 pg/ml, respectively. Values were linear through 10 g/ml of authentic ZDV and ZDVG standards. Intra-as- say variation was 5.1% for ZDV and 5.9% for ZDVG. Statistics Pharmacokinetic parameters were estimated using a non-compartmental model and the computer-mod- eling program MK MODEL. The terminal half- life (T1/2) was estimated using the log-linear model with the least-squares regression of the terminal portion of the plasma concentration curve utilizing the formula T1/2 0.693/ke, where ke is the elim- ination rate constant. Using the iterated data, we estimated the clearance (C1) and volume of distri- bution (Vd) by dividing the dose ofthe drug by the area under the curve and substituting this value into the formula T1/2 0.693"Vd/CI. Wilcoxon sign-rank and rank-sum tests were used where ap- propriate. P < 0.05 was considered to be signifi- cant. RESULTS The concentration profiles for ZDV and ZDVG in the maternal and fetal circulations for animals re- ceiving ZDV are shown in Figures and 2. Figure 3 shows the maternal and fetal concentrations of ZDVG in animals who received ZDVG. Table 1 shows the fetal/maternal ratio over the sampling interval for the animals given ZDV and ZDVG. If the serum concentration in the numerator or de- nominator was zero, the time value was not used in the calculation ofthe ratios. Tables 2 and 3 summa- rize the pharmacokinetic parameter estimates for ZDV and ZDVG. In animals receiving ZDVG, complete data were not obtained in monkey 3 for maternal levels after the first dose and the fetal levels after the second dose. Other placental metab- olites of AZT have been reported8'9 and were not sought in this study. In the animals receiving ZDV, the maternal and fetal T1/2 for ZDVG were significantly longer than for ZDV (P < 0.016). Peak fetal levels of ZDV and ZDVG were both reached at 40 min post- injection. In animals receiving ZDVG, peak levels of ZDVG in the fetal compartment were reached at 60 min. The fetal/maternal ZDVG ratio was con- sistently higher than that of ZDV.

fetal T1/2 for ZDVG were significantly longer than for ZDV (P < 0.016). Peak fetal levels of ZDV and ZDVG were both reached at 40 min post- injection. In animals receiving ZDVG, peak levels of ZDVG in the fetal compartment were reached at 60 min. The fetal/maternal ZDVG ratio was con- sistently higher than that of ZDV. DISCUSSION ZDV is being used with increasing frequency in the treatment of pregnant women with acquired immu- nodeficienc syndrome (AIDS).3'4 To date there is limited information on the in vivo placental trans- port of ZDV and its major glucuronidated metabo- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY PLACENTAL TRANSPORT OF ZDV RIDGWAY ET AL. ng/ml O,000 1,000 100 10 1 0 "Maternal ZDVG -Fetal ZDVG 100 200 300 400 minutes mean _+ sem Fig. 2. Concentrations (mean SEM) of ZDVG in maternal and fetal circulations following mater- nal administration of ZD (3.5 mg/kg) at time 0; n 5. ng/ml 100,000 10,000 1,000 0 100 200 300 minutes 400 mean _sem Fig. 3. Concentrations (mean +- SEM) of ZDVG in maternal and fetal circulations following mater- nal administration of ZDYG (3.5 mg/kg) at time 0; n 3. lite ZDVG in pregnancy. The rhesus monkey model, with a placentation very similar to the hu- man, provides a useful approach to the pharmaco- kinetic study of ZDV and ZDVG in pregnancy. The model provides useful quantitative informa- tion as to the elimination of the parent drug and its metabolite in the maternal compartment, as well as its transfer across the placenta and disposition in the fetal circulation. However, it is limited as to mech- anistic data on the nature of the placental transfer process. The latter is best investigated using the isolated perfused placental cotyledon system, as em- ployed by us8 and others9' 10 earlier. Using the in vivo model, we show clearly that 1) ZDV is removed rapidly from the maternal circula- tion and appears quickly in the fetal plasma, 2) 140 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY PLACENTAL TRANSPORT OF ZDV RIDGWAY ET AL. TABLE I. Fetal/maternal ratio Monkeys given Time Monkeys given ZDV ZDVG (min) ZD (n) ZDVG (n) ZDVG (n) 20 0.20 0.2 (5) 0.28 0.08 (5) 0.08 _+ 0. (4) 40 0.40 +_ 0.33 (5) 0.67 o.17 (5) 0.52 +_ 0.61 (4) 60 0.35 0.21 (5) 0.81 0.31 (5) 2.17 _+ 0.99 (4) 80 0.68 -+ 0.46 (5) I.

NECOLOGY PLACENTAL TRANSPORT OF ZDV RIDGWAY ET AL. TABLE I. Fetal/maternal ratio Monkeys given Time Monkeys given ZDV ZDVG (min) ZD (n) ZDVG (n) ZDVG (n) 20 0.20 0.2 (5) 0.28 0.08 (5) 0.08 _+ 0. (4) 40 0.40 +_ 0.33 (5) 0.67 o.17 (5) 0.52 +_ 0.61 (4) 60 0.35 0.21 (5) 0.81 0.31 (5) 2.17 _+ 0.99 (4) 80 0.68 -+ 0.46 (5) I. 19 +_ 0.76 (5) 2.65 -+ 1.7 (4) 100 0.65 0.69 (5) 1.12 -+ 0.92 (5) 2.47 +- 0.93 (4) 120 0.74 1.0 (5) 1.19 0.70 (5) 2.72 +_ 0.71 (4) 180 0.48 -+ 0.68 (5) 1.43 _+ 1.3 (5) 4.19 -+ 4.2 (3) 240 0.45 0.50 (3) 1.42 1.7 (5) 2.32 +- 1.9 (3) 300 0.86 (I) 0.70 _+ 0.32 (4) 1.45 +_ 0.64 (2) 360 0.92 (I) 0.76 0.73 (3) ZDVG is generated rapidly as a result of ZDV metabolism in the maternal compartment, and 3) ZDVG crosses the placenta into the fetal circula- tion, albeit apparently more slowly than ZDV. The T1/2 of ZDV in the maternal circulation is comparable to values reported earlier in non-preg- 6,12 nant11 and pregnant women, as well as in the pregnant monkey model. 13 Moreover, the clear- ance and volume of distribution of ZDV in our studies approximate the data of Lopez-Anaya et al. 13 in the pregnant monkey and O'Sullivan et al.6 in pregnant women. The plasma binding ofZDV is low; 1 hence, the low albumin concentration seen in pregnancy should have no effect on ZDV clear- ance. It appears, therefore, that the pharmacokinet- ics ofZDV in pregnancy are similar to those seen in non-pregnant patients. Moreover, in our model, there was rapid formation of ZDVG from injected ZDV, with peak maternal ZDVG already evident on initial sampling. The T1/2 of ZDVG in the maternal circulation after ZDV administration was much longer than the T1/2 of ZDV. This may be explained by ZDVG derivation from ZDV. This interpretation is consistent with lower ZDVG than ZDV concentrations in maternal plasma and by a shorter ZDVG T1/2 in maternal plasma when the agent is administered as ZDVG. In other studies, a rapid formation of ZDVG from ZDV was also noted after ZDV administration, although the half- life of both drugs was very similar. 1 Similarly, in our studies, clearances of administered ZDV and ZDVG in the maternal circulation were almost iden- tical. Both ZDV and ZDVG appeared rapidly in the fetal circulation after ZDV administration. The T1/2 of ZDV in the fetal compartment was not significantly different from that in maternal circu- lation.

r. 1 Similarly, in our studies, clearances of administered ZDV and ZDVG in the maternal circulation were almost iden- tical. Both ZDV and ZDVG appeared rapidly in the fetal circulation after ZDV administration. The T1/2 of ZDV in the fetal compartment was not significantly different from that in maternal circu- lation. In order to determine whether the ZDVG present in the fetal circulation is a result of placental transfer or fetal metabolism, we looked at the mon- keys given ZDVG alone. After ZDVG administra- tion to the mother, peak ZDVG in the fetal circula- tion was noted at 60 min. ZDVG disappeared more slowly than ZDV from the fetal circulation and the fetal/maternal ZDVG ratio was consistently greater than the ZDV ratio. The fetal/maternal ZDVG ra- tio after maternal ZDVG administration was also lower at 20 and 40 min than after ZDV administra- tion, but this was substantially reversed thereafter. The most likely explanations for these composite findings are that the transfer ofZDVG from mater- nal to fetal circulations is slower than ZDV passage across the placenta and some metabolism of ZDV to ZDVG occurs in the fetal compartment. This is notwithstanding the known relative immaturity of fetal hepatic enzymes from glucuronidation. 14-16 Moreover, the rapid clearance of ZDVG from the maternal circulation and the slower transfer of ZDVG across the placenta probably contribute to the retention of this metabolite in the fetus and the increased fetal/maternal ratio. Other investigators5 have reported high levels of ZDVG in fetal serum and amniotic fluid ofpregnant monkeys given mul- tiple oral doses of ZDV and have proposed that the amniotic fluid may serve as a reservoir for ZDVG. Verification of this overall concept will require in- fusions ofboth ZDV and ZDVG into thefetalcom- partment with monitoring of relative rates of trans- fer of each substance into the maternal circulation and into fetal bile and urine (amniotic fluid). Pre- liminary data in our unit, however, using the per- fused isolated placental cotyledon support a more rapid diffusion of ZDV than of ZDVG across the human placenta. This is consistent with the greater polarity ofZDVG8'9 and with the concept that these drugs cross the placenta passively. 8-10 While transfer of animal data to clinical medi- cine is difficult, our data suggest that ZDV dosing does not need to be modified in pregnancy as ZDV should not accumulate in the fetal compartment.

is consistent with the greater polarity ofZDVG8'9 and with the concept that these drugs cross the placenta passively. 8-10 While transfer of animal data to clinical medi- cine is difficult, our data suggest that ZDV dosing does not need to be modified in pregnancy as ZDV should not accumulate in the fetal compartment. While ZDVG may accumulate in this compart- ment, this should not be clinically important as long as the metabolite remains inactive. In conclusion, our data along with those of the previously cited investigators suggest that both ZDV INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY PLACENTAL TRANSPORT OF ZDV RIDGWAY ET AL. TABLE 2. Pharmacokinetic parameter estimates in monkeys given ZDV TV:z (min) Monkey No. Maternal ZDV Fetal ZD Maternal ZDYG Fetal ZDVG Maternal ZDV Vd (ml/kg) CI (ml/min/kg) 45 28 148 229 667 12 2 56 54 169 138 806 8 3 22 37 52 120 379 13 4 23 25 125 103 487 16 5 37 20 123 120 981 39 Mean 37 33 124 142 664 18 SD 15 13 44 50 242 12 TABLE 3. Pharmacokinetic parameter estimates in monkeys given ZDVG TV2 (min) Maternal ZDYG Monkey Maternal Fetal Vd CI No. ZDVG ZDVG (ml/kg) (ml/min/kg) 42 51 1,931 39 37 42 428 10 2 12 36 26 20 70 413 840 13 3 54 74 1,417 13 Mean 47 119 928 19 SD 26 164 761 12 and ZDVG rapidly appear in the fetal circulation following maternal ZDV injection. Placental trans- fer most likely occurs by passive diffusion with ZDV crossing more rapidly than ZDVG crosses. ZDVG may then accumulate in the fetal compart- ment with amniotic fluid serving as a reservoir. Fetal ZDVG accumulation appears to result from diffusion of maternal ZDVG and from fetal metab- olism of ZDV accompanied by slow back diffusion of fetal ZDVG and its decreased excretion by fetal organs. ACKNOWLEDGMENTS We gratefully acknowledge the technical support of Arturo Moreno and Diane Frasier and the assis- tance of Gretta Small and Patricia Littleton in the preparation of the manuscript. This work was sup- ported by NIH Career Development (RSDA) KO 2 AA00 121 and by NIAAA R01 HD 26707. REFERENCES 1. Centers for Disease Control: HIV prevalence estimates and AIDS case projections for the United States: Report based upon a workshop. Morbidity and Mortality Weekly Report 39(RR-16): 1-31, 1990. 2. Volberding PA, Lagakos SW, Koch MA, et al.: Zidovu- dine in asymptomatic human immunodeficiency virus in- fection: A controlled trial in persons with fewer than 500 CD4 positive cells per cubic millimeter. N Engl J Med 322:941-949, 1990. 3. HIV Infections.

Morbidity and Mortality Weekly Report 39(RR-16): 1-31, 1990. 2. Volberding PA, Lagakos SW, Koch MA, et al.: Zidovu- dine in asymptomatic human immunodeficiency virus in- fection: A controlled trial in persons with fewer than 500 CD4 positive cells per cubic millimeter. N Engl J Med 322:941-949, 1990. 3. HIV Infections. ACOG Technical Bulletin. Number 169, June 1992. The American College of Obstetricians and Gynecologists, Washington, DC. 4. Minkoff HL, Moreno JD: Drug prophylaxis for human immunodeficiency virus-infected pregnant women: Ethi- cal considerations. AmJ Obstet Gynecol 163:1111-1114, 1990. 5. Hankins ZDV, Lowery CL, Scott RT, et al.: Transpla- cental transfer of zidovudine in the near-term pregnant baboon. Am J Obstet Gynecol 163:728-732, 1990. 6. O'Sullivan MJ, Boyer PJ, Scott GB, et al.: The pharma- cokinetics and safety of zidovudine in the third trimester of pregnancy for women infected with human immuno- deficiency virus and their infants: Phase I Acquired Im- munodeficienc Syndrome Clinical Trials Group study (protocol 082). Am J Obstet Gynecol 168:1510-1516, 1993. 7. Good SS, Reynolds DJ, de Miranda P: Simultaneous quantification ofzidovudine and its glucuronide in serum by high performance liquid chromatography. J Chro- matogr 431:123-133, 1988. 8. Schenker S, Johnson RF, King TS, Schenken RS, Hend- erson GI: Azidothymidine (zidovudine) transport by the human placenta. Am J Med Sci 299(1):16-20, 1990. 9. Liebes L, Mendoza S, Wilson D, Dancis J: Transfer of zidovudine (AZT) by human placenta. J Infect Dis 161: 203-207, 1990. 10. Bawdon RE, Sobhi S, Dax J: The transfer of anti-human immunodeficiency virus nucleoside compounds by the term human placenta. Am J Obstet Gynecol 167:1570- 1574, 1992. 11. Kiecker RW Jr, Collins JM, Yarchoan R, et al.: Plasma and cerebrospinal fluid pharmacokinetics of 3'-azido-3'- deoxythymidine: A novel pyrimidine analog with poten- tial application for the treatment of patients with AIDS and related diseases. Clin Pharmacol Ther 41:407-412, 1987. 12. Watts DH, Brown ZA, Tartaglione T, et al.: Pharma- 142 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY PLACENTAL TRANSPORT OF ZDV RIDGWAY ET AL. cokinetic disposition of zidovudine during pregnancy. J Infect Dis 163:226-232, 1991. 13. Lopez-Anaya A, UnadkatJD, Schumann LA, Smith AL: Pharmacokinetics of zidovudine (azidothymidine). III. Effect ofpregnancy. J Acquired Immune Deficiency Synd 4:64-68, 1991. 14.

D GYNECOLOGY PLACENTAL TRANSPORT OF ZDV RIDGWAY ET AL. cokinetic disposition of zidovudine during pregnancy. J Infect Dis 163:226-232, 1991. 13. Lopez-Anaya A, UnadkatJD, Schumann LA, Smith AL: Pharmacokinetics of zidovudine (azidothymidine). III. Effect ofpregnancy. J Acquired Immune Deficiency Synd 4:64-68, 1991. 14. Dutton GJ: Developmental aspects of drug conjugation with special reference to glucuronidation. Annu Rev Phar- macol Toxicol 18:17-35, 1978. 15. Schenker S, Dawber NH, Schmid R: Bilirubin metabo- lism in the fetus. J Clin Invest 43:32, 1964. 16. Bashore RA, Smith F, Schenker S: Placental transfer and disposition of bilirubin in the pregnant monkey. Am J Obstet Gynecol 103:950, 1969. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 143

InfecUous Diseases in Obstetrics nd Gynecology I: 144-148 (1993) (C) 1993 VViley-Liss, Inc. CoccidJoidal Placentitis With Normal Umbilical Arter Velocimetr Stephen A. Nickisch, Luis Izquierdo, Maggie A. Viii, Luis Curet, and Gordon C. Wolf Divisions ofMaternal Fetal Medicine and Reproductive Endocrinology, Department ofObstetrics and Gynecology, University ofNew Mexico School ofMedicine, Albuquerque, NM ABSTRACT Background: Disseminated coccidiomycosis during pregnancy can lead to both maternal and neona- tal mortality. Placentitis is an uncommon sequelae and its effect on placental function remains speculative. The present report describes our management of such a case and describes serial umbilical artery velocimetry of an affected placenta. Case: A pregnant woman with coccidioidal placentitis confirmed histologically was treated with systemic and intrathecal amphotericin B starting at 28 weeks gestation. Serial umbilical artery velocimetry revealed that all systolic/diastolic ratios remained normal, and a normal infant was delivered at term. Conclusion: Coccidioidal placentitis was successfully treated with amphotericin B; serial umbili- cal artery velocimetry monitoring exhibited no abnormalities and, along with other reassuring fetal parameters, allowed continuation ofthe pregnancy to term. (C) 1993 Wiley-Liss, Inc. KEY WORDS Coccidiomycosis, Doppler, S/D ratios, amphotericin, meningitis occidiomycosis affects in every 1,000 preg- nancies in endemic areas of the southwestern United States; disseminated disease with meningi- tis and placentitis is much less common, with fewer than 60 cases having been reported. The use of amphotericin B and modern neonatal care have sig- nificantly reduced the previously high maternal and neonatal mortality associated with this disease, but a perinatal mortality of 4-14% remains for dissemi- nated disease in pregnant patients.2'3 Placental ab- normalities associated with coccidioidal placental invasion have been postulated as the pathologic mechanisms explaining the increased perinatal mor- bidity and mortality.

mortality associated with this disease, but a perinatal mortality of 4-14% remains for dissemi- nated disease in pregnant patients.2'3 Placental ab- normalities associated with coccidioidal placental invasion have been postulated as the pathologic mechanisms explaining the increased perinatal mor- bidity and mortality. We hypothesized that these lesions might lead to measurable placental resis- tance values inasmuch as the characteristic coccidio- idal changes (infarction, necrosis, and fibrin depo- sition) are common to the spectrum of lesions and lead to reduced placental parenchymal perfusion and increased umbilical artery resistance.4 We re- port umbilical artery velocimetry as an indicator of placental resistance in a patient with coccidioidal meningitis and histologically confirmed coccidio- idal placentitis. CASE REPORT A 27-year-old Hispanic female, G2PoAb, pre- sented at 24 weeks gestation with an upper respira- tory illness. She lived in central New Mexico though she had visited southern Arizona 6 weeks previously. Over a period of 2 weeks, the patient developed nausea, vomiting, headaches, blurred vision, fever, and weight loss. She had developed skin lesions on her upper lip and on her nose. A Address correspondence/reprint requests to Dr. G.C. Wolf, Department of Obstetrics and Gynecology, University of South Carolina, Two Medical Park, Columbia, SC 29203. Obstetrics Case Report Received August 12, 1993 Accepted October I, 1993 COCCIDIOIDAL PLACENTITIS NICKISCH ET AL. Fig. I. Skin biopsy revealing spherule engulfed by multinucleated giant cells. 7 2O GESTATIoNAL AGE (weeks) Umbilical artery S/D ratios in the 10th to 25th percentile range.4 75 50 25 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 145 COCCIDIOIDAL PLACENTITIS NICKISCH ET AL. Fig. 3. Placental biopsy confirming infarction necrosis and coccidioidal placentitis. biopsy confirmed Coccidioides immitis infection (Fig. 1). At 28 weeks, the coccidioidal complement fixation titer was found to be 1:8 in the cerebrospi- nal fluid and 1:64 in serum. An ultrasound examination confirmed a 28-week male fetus, appropriate for gestational age. The umbilical artery systolic/diastolic (S/D) ratio deter- mined at this time was in the 10th to 25th percentile at 2.5; ratios were subsequently determined at 3- to 7-day intervals (Fig. 2). Serial ultrasound exami- nations and nonstress tests likewise remained reas- suring.

ale fetus, appropriate for gestational age. The umbilical artery systolic/diastolic (S/D) ratio deter- mined at this time was in the 10th to 25th percentile at 2.5; ratios were subsequently determined at 3- to 7-day intervals (Fig. 2). Serial ultrasound exami- nations and nonstress tests likewise remained reas- suring. Treatment was initiated with systemic ampho- tericin B (to a maximum of 64 mg/day) in addition to intrathecal medication (0.3 mg on alternate days) until delivery at 38 weeks when the patient under- went spontaneous rupture of the fetal membranes. Oxytocin augmentation resulted in a normal spon- taneous vaginal delivery. The infant weighed 3,130 g with APGAR scores of 9 and 10 at and 5 minutes. The placenta appeared grossly normal ex- cept for three areas of infarction (confirmed micro- scopically), each smaller than 2 cm in diameter. Further microscopic examination revealed moder- ate amounts of intervillous fibrin deposition, nu- merous fungal spherules containing endospores and foreign.body giant cells, and an acute inflammatory reaction (Figs. 3, 4). The patient's symptoms gradually resolved after the initiation of therapy, and the skin lesions di- minished in size. Her total dose of amphotericin B at time of delivery had reached 4 g, and the regi- men was changed to fluconazole, 400 mg daily, along with continuation of intracisternal ampho- tericin B, 0.5 mg weekly. The postpartum course was uneventful; she and the infant were discharged on the third day following delivery. DISCUSSION The use ofDoppler velocimetry has been described in the evaluation of many conditions in human pregnancy. Diabetes mellitus, systemic lupus erythematosus, hypertensive disorders of preg- nancy, and sickle cell diSease have all illustrated the utility of uterine-umbilical artery velocimetry in conditions associated with placental vascular dis- ease. s-8 However, Doppler investigation of pla- 146 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY COCCIDIOIDAL PLACENTITIS NICKISCH ET AL. Fig. 4. Higher magnification of placenta with endospore-filled spherules. centitis secondary to a specific infectious agent has not been reported. A component of the pathophysiologic process in coccidiomycosis infection is that the hyphal outer wall of the fungal arthrocomidia of C. immitis re- sists phagocytosis, possibly as a result ofthe deposi- tion of fibrinous material that inhibits polymorpho- nuclear cell access to the infectious agent.

not been reported. A component of the pathophysiologic process in coccidiomycosis infection is that the hyphal outer wall of the fungal arthrocomidia of C. immitis re- sists phagocytosis, possibly as a result ofthe deposi- tion of fibrinous material that inhibits polymorpho- nuclear cell access to the infectious agent. The presence of placentitis with the concomitant fibrin- ous deposition and other pathologic features might lead to increased placental resistance and subse- quent fetal compromise. Umbilical artery veloci- metry serially throughout the third trimester, how- ever, provided no evidence of functional placental compromise. Despite the documentation of placental infection with the presence ofmultinucleated giant cells, fun- gal spherules and endospores, moderate intervil- lous fibrin deposition, and focal infarction with necrosis (Fig. 3), the umbilical artery measure- ments from 28 weeks to term showed no evidence of altered placental vascular resistance. The S/D ratio of the umbilical artery is plotted along the group of percentiles from Arabin's normative data (Fig. 4).4 The lack of correlation between pathologic find- ings and umbilical artery velocimetric data suggests that the functional effect of the placentitis in this case did not contribute to increased placental resis- tance and subsequent fetal compromise. It is possi- ble that even a significant infectious process might not be reflected in fetal compromise unless there is a significant reduction in the number of terminal arteries in the tertiary villi. 9 In summary, we report the birth ofan appropri- ate-for-gestational-age infant at term without objec- tive evidence of placental insufficiency despite dis- seminated maternal disease and significant coccidioidal placentitis. The lack of abnormal ve- locimetric measurements coupled with other reas- suring fetal parameters may allow continued obser- vation of preterm pregnancies affected by this and other infectious agents. REFERENCES 1. Peterson CM: Coccidiomycosis and pregnancy. Obstet Gy- necol Surv 48:149-158, 1993. 2. Peterson CM, Johnson SL, Kelly JV, Kelly PC: Coccidio- idal meningitis and pregnancy: A case report. Obstet Gyne- col 23:835-836, 1989. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 47 COCCIDIOIDAL PLACENTITIS NICKISCH ET AL. 3. Walker MPR, Brody CZ, Resnik, R: Reactivation of coc- cidiomycosis in pregnancy. Obstet Gynecol 79:815-817, 1992. 4. Arabin B: Doppler Blood Flow Measurement in Uteropla- cental and Fetal Vessels.

Gyne- col 23:835-836, 1989. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 47 COCCIDIOIDAL PLACENTITIS NICKISCH ET AL. 3. Walker MPR, Brody CZ, Resnik, R: Reactivation of coc- cidiomycosis in pregnancy. Obstet Gynecol 79:815-817, 1992. 4. Arabin B: Doppler Blood Flow Measurement in Uteropla- cental and Fetal Vessels. New York: Springer-Verlag, pp 46, 61-75, 1990. 5. Bracero L, Schulman H, Fleischer A, Farmakides G, Rochelson B: Umbilical artery velocimetry in diabetes and pregnancy. Obstet Gynecol 68:654-657, 1986. 6. DucyJ, Schulman H, Farmakides G, Rochelson B, Bracero L, Fleischer A, Guzman E, Winter D, Penny B: A classi- fication of hypertension in pregnancy based on Doppler velocimetry. Am J Obstet Gynecol 157:680-684, 1987. 7. Anyaegbunam A, Langer O, Brustman L, Damus K, Halpert R, Merkatz IR: The application of uterine and umbilical artery velocimetry to the antenatal supervision of pregnancies complicated by maternal sickle hemoglobinop- athies. Am J Obstet Gynecol 159:544-547, 1988. 8. Guzman E: Uterine-umbilical artery Doppler velocimetry in pregnant women with systemic lupus erythematosus. J Ultrasound Med 11:275-281, 1982. 9. Trudinger BJ, Giles WB, Cook CM, Bombardieri J, Col- lins L: Fetal umbilical artery flow velocity waveforms and placental resistance: Clinical significance. Br J Obstet Gy- naecol 92:23-30, 1985. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology I: 149-152 (1993) (C) 1993 Wiley-Liss, Inc. Candida Esophagitis in an Immunocompetent Pregnant Woman Jeffrey S. Greenspoon and Seth Kivnick Division ofMaternal-Fetal Medicine, Department of Obstetrics-Gynecology, Cedars-Sinai Medical Center (J.S.G.), and Department ofObstetrics-Gynecology, Kaiser Foundation Hospital-West Los Angeles (S.K.), Los Angeles, CA ABSTRACT Background: Nausea and vomiting are common during the first half of pregnancy and usually require only supportive measures. When symptoms are progressive and weight loss occurs, treatable causes should be sought by means of upper gastrointestinal endoscopy. We report a case of an immunocompetent gravida with invasive Candida albicans esophagitis. Case: The immunocompetent primigravida developed progressive nausea, vomiting, epigastric pain, and a 4.1 kg weight loss during the second trimester ofpregnancy. Treatmentwith metoclopra- mide and cimetidine for presumed gastroesophageal reflux was not effective. The patient had normal T-cell CD4 and CD8 subsets and was human immunodeficiency virus (HIV) antibody negative. Upper gastrointestinal endoscopy revealed C. albicans esophagitis which was treated with oral nystatin. The esophagitis had resolved completely when reassessed postpartum. The use of hista- mine2 blockers is associated with an increased risk for fungal esophagitis and may have been a contributing cause in this case. Conclusion: Pregnant patients with persistent nausea, vomiting, and weight loss should be evalu- ated by endoscopy for fungal esophagitis. (C) 1993 Wiley-Liss, Inc. KEY WORDS Candida albicas esophagitis, hyperemesis gravidarum, pregnancy complications, gastrointestinal e report a case of Candida albicans esophagi- tis in an immunocompetent gravida. We are unaware of other reports of this infection compli- cating pregnancy. We suggest that Canadida esoph- agitis be included in the differential diagnosis of refractory nausea, vomiting, and weight loss dur- ing pregnancy. CASE REPORT A 37-year-old primigravida woman with a history of leiomyomata presented for prenatal care at 8 weeks gestation with a uterus that was 20 weeks size.

regnancy. We suggest that Canadida esoph- agitis be included in the differential diagnosis of refractory nausea, vomiting, and weight loss dur- ing pregnancy. CASE REPORT A 37-year-old primigravida woman with a history of leiomyomata presented for prenatal care at 8 weeks gestation with a uterus that was 20 weeks size. At the age of 31 years, she had a positive tuberculin skin test and a normal chest radiograph. She refused isoniazid prophylaxis. Physical examination at that time showed a healthy, thin, black woman with a weight of 58.6 kg and a height of 167.5 cm. Pelvic ultrasonogra- phy showed a single, viable, 9.5-week fetus in a gestational sac near the cervix and a large fundal leiomyoma with dimensions of 17 10 13.5 cm. All routine prenatal laboratory values were normal. The patient declined human immunodefi- ciency virus (HIV) antibody screening. An amnio- centesis done at 15 weeks showed a normal karyo- type, 46 XX, and a normal alpha feto-protein. In the second trimester, she developed increas- ing uterine pain, tenderness, and loss of appetite. By 17 weeks gestation, her weight had decreased 1.8 kg to 56.8 kg. Address correspondence/reprint requests to Dr. Jeffrey S. Greenspoon, Room 1738, OB/GYN, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048. Gynecological Case Report Received June 8, 1993 Accepted October 19, 1993 CANDIDA ESOPHAGITIS IN PREGNANCY GREENSPOON AND KIVNICK Fig. I. Histologic section of esophagus demonstrating invasive Candida infection. Acute inflamma- tion and necrotic material are present in the lamina propria. Hematoxylin and eosin stain. 40. Despite enteral supplementation, her weight de- creased to 54.5 kg by 22 weeks gestation. She had nausea, vomiting, and a sensation of obstruction in her throat, but denied odynophagia. The uterus was markedly tender and was 30 cm at 22 weeks gestation. Degeneration of the leiomyomata was diagnosed. Hemoglobin was 10.7 g/dl, white blood cell (WBC) count 8,800/mm3 with a normal differen- tial, platelets 613,000/mm3, total protein 5.6 g/dl, and albumin 2.6 g/dl. All blood chemistries and liver function tests were normal. Ultrasound showed no hepatobiliary abnormalities and con- firmed appropriate fetal growth. Peripheral hyperalimentation was administered. Gastroesophageal reflux was diagnosed and cimeti- dine, 400 mg b.i.d., and metoclopramide, 10 mg 30-45 min before meals and at bedtime, were pre- scribed. Her symptoms resolved temporarily; she gained 2 kg.

atobiliary abnormalities and con- firmed appropriate fetal growth. Peripheral hyperalimentation was administered. Gastroesophageal reflux was diagnosed and cimeti- dine, 400 mg b.i.d., and metoclopramide, 10 mg 30-45 min before meals and at bedtime, were pre- scribed. Her symptoms resolved temporarily; she gained 2 kg. She refused further parenteral nutrition. At 27 weeks gestation, nausea, vomiting, and weight loss recurred. She had no oral thrush or vaginal candidiasis. She denied having odynopha- gia, retrosternal pain, hematemesis, or fever. The uterine fundal height had increased to 3 8 cm. The 1-hr post-50 g glucola blood glucose was 151 mg/ dl. A 3-hr glucose tolerance test met criteria for the diagnosis of gestational diabetes (fasting 77 mg/dl, 1-hr 194, 2-hr 167, and 3-hr 90). In order to provide adequate calories, we placed no restrictions and continued the Ensure (Ross Laboratories, Co- lumbus, .OH). Subsequent fasting and postpran- dial serum glucose concentrations remained nor- mal. A 10 French-feeding tube was inserted for enteral hyperalimentation. The tube became kinked and stopped functioning after week. The patient refused reinsertion ofthe tube until 32 weeks gesta- tion. Upper gastrointestinal endoscopy (for place- ment of a second feeding tube) revealed the classic patchy white exudates of Candida esophagitis. No feeding tube was placed. Fungal culture and histo- logic section confirmed invasive C. albicans infec- tion (Figs. 1, 2). Treatment was initiated with nystatin suspension, 500,000 units swish and swal- low q.i.d. Metoclopramide and cimetidine were discontinued. 150 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY CANDIDA ESOPHAGITIS IN PREGNANCY GREENSPOON AND KIVNICK Fig. 2. High-power view of Figure I. Histologic section of esophagus demonstrating invasive Can- dida infection. Pseudohyphae are present within necrotic debris. Silver methenamine stain. 450. The patient's and her spouse's serum HIV anti- body enzyme-linked immunosorbent assay (ELISA) tests were negative. The complete blood count (CBC), T-cell panel, and serum protein elec- trophoresis were normal. The WBC count was 10,600/mm3; lymphocytes were 18% of total (nor- mal 15-40%); T-helper cells (CD4) were 48% (normal 32-56%); and T-suppressor cells (CDS) were 24% (normal 17-40%). Skin testing for mumps showed 30 15 mm of erythema at 48 hr after injection; coccidioidin caused no reaction. Chest radiograph was normal.

The WBC count was 10,600/mm3; lymphocytes were 18% of total (nor- mal 15-40%); T-helper cells (CD4) were 48% (normal 32-56%); and T-suppressor cells (CDS) were 24% (normal 17-40%). Skin testing for mumps showed 30 15 mm of erythema at 48 hr after injection; coccidioidin caused no reaction. Chest radiograph was normal. During the course of antifungal therapy, the patient's dysphagia de- creased, but her oral intake remained poor and abdominal pain increased. She remained euglyce- mic. After fetal maturity was confirmed, a cesarean delivery was performed at 36.5 weeks for breech presentation and increasing abdominal pain. The female infant weighed 2,848 g and had Apgar scores of 9 and 9 at and 5 min, respec- tively. There was a single fundal leiomyoma which extended to the left hemidiaphragm. There was no evidence of Candida esophagitis when upper gas- trointestinal endoscopy was performed 3 days post- partum. Five months postpartum, a myomectomy was performed. The leiomyoma weighed 852 g and measured 15 15 x 7 cm. Pathologic examina- tion revealed a hyalinized and infarcted leiomy- oma. A preoperative HIV antibody test remained negative. DISCUSSION We were unable to find other reports of Candida esophagitis during pregnancy in immunocompe- tent or immunocompromised patients. This diag- nosis should be considered in patients with refrac- tory nausea, vomiting, and weight loss during pregnancy. This patient did not have the common symptom of odynophagia or the presence of oral thrush. The nystatin therapy improved the dys- phagia, and a repeat endoscopic examination con- firmed resolution of the esophagitis. The patient's dysphagia and the histologically proven Candida INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 151 CANDIDA ESOPHAGITIS IN PREGNANCY GREENSPOON AND KIVNICK esophagitis cannot be attributed to the mass effect of the gravid uterus and leiomyoma, although these may have caused some ofher epigastric discomfort. Histamine2 blockers are widely used and are usually well tolerated.2 A significant association exists between treatment with histamine2 blockers and the presence of fungal esophagitis.3 The prev- alence of fungal esophageal infection was 12 of 72 (16.7%) among patients exposed to histamine2 blockers, whereas only 3.5% of patients unexposed to the drug had fungal esophageal infection. The association is somewhat unexpected because cimeti- dine has been shown to enhance cell-mediated im- munity in humans.

e prev- alence of fungal esophageal infection was 12 of 72 (16.7%) among patients exposed to histamine2 blockers, whereas only 3.5% of patients unexposed to the drug had fungal esophageal infection. The association is somewhat unexpected because cimeti- dine has been shown to enhance cell-mediated im- munity in humans. Cimetidine augmented delayed hypersensitivity responses to skin tests including the Candida antigen in humans with duodenal-ul- cer disease.4 In 4 adult patients with chronic muco- cutaneous candidiasis, cimetidine stimulated the im- mune response to Candida antigen and the pro- duction of leukocyte migration inhibitory factor, although lymphocyte transformation was not af- fected, s Cimetidine and ranitidine are FDA cate- gory B drugs; their use during pregnancy should be limited to instances in which the benefit justifies the risk. Type I diabetes mellitus has been associated with Candida esophagitis. Although this patient met cri- teria for gestational diabetes mellitus, because she remained euglycemic, we do not think that her glucose intolerance was severe enough to be a cause for the fungal esophagitis. Perhaps the combina- tion of pregnancy, diabetes mellitus, and treatment with a histamine2 blocker may increase the risk for Candida esophagitis. Immunocompetent pregnant patients who have not responded to routine therapy (or gastroesoph- ageal reflux should undergo upper gastrointestinal endoscopy to diagnose Candida esophagitis. If they have been treated with histamine2 blockers, they are at increased risk for esophageal fungal infection. In immunocompromised pregnant patients, up- per gastrointestinal endoscopy is particularly im- portant to identify opportunistic esophageal infec- tions. As HIV infection becomes more common in obstetrical patients, Candida esophagitis and other opportunistic infections will be encountered more frequently. At this time, Candida esophagitis is the acquired immunodeficiency syndrome (AIDS)-in- dicator disease for 15% of HIV-infected adoles- cents and adults. Although this patient responded to oral nystatin therapy, immunocompromised patients frequently need prolonged systemic therapy with fluconazole, ketoconazole, or amphotericin B.6 The safety of these systemic drugs during pregnancy has not been established. ACKNOWLeDGmeNTS We thank Samuel H. Pepkowitz, M.D., for assis- tance in preparing the photomicrographs. REFERENCES 1. Haulk AA, Sugar AM: Candida esophagitis. In Stoller- man GH (ed): Advances in Internal Medicine.

nazole, or amphotericin B.6 The safety of these systemic drugs during pregnancy has not been established. ACKNOWLeDGmeNTS We thank Samuel H. Pepkowitz, M.D., for assis- tance in preparing the photomicrographs. REFERENCES 1. Haulk AA, Sugar AM: Candida esophagitis. In Stoller- man GH (ed): Advances in Internal Medicine. Vol. 36. St. Louis: Mosby Year Book, pp 307-318, 1991. 2. Lispy RJ, Fennerty B, Fagan TC: Clinical review of hista- mine receptor antagonists. Arch Intern Med 150:745- 751, 1990. 3. Vermeersch B, Rysselaere M, Dekeyser K, Rasquin K, De Vos M, Elewaut A, Barbier F: Fungal colonization of the esophagus. Am J Gastroenterol 84:1079-1083, 1989. 4. Avella J, Madsen JE, Binder HJ, Askenase PW: Effect of histamine I-I2-receptor antagonists on delayed hypersensi- tivity. Lancet 1(8065):624-626, 1978. 5. Jorizzo JL, Sams WM Jr, Jegasothy BV, Olansky AJ: Cimetidine as an immunomodulator: Chronic mucocutane- ous candidiasis as a model. Ann Intern Med 92:192-195, 1980. 6. Terrell CL, Hughes CE: Antifungal agents used for deep-seated mycotic infections. Mayo Clin Proc 67:69-91, I992. 152 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:153-161 (1993) (C) 1993 Wiley-Liss, Inc. Review of Vaginitis Sebastian Faro Department ofGynecology and Obstetrics, University ofKansas School ofMedicine, Kansas City, KS ABSTRACT A disruption ofthe dynamic equilibrium ofthe healthy vagina may have significant sequelae, leading to chronic or serious conditions. Therefore, all cases ofvaginitis should be accurately diagnosed and appropriately treated. (C) 1993 Wiley-Liss, Inc. KEY WORDS Vaginal microflora, vaginal discharge, vaginal pathogens aginitis is probably the most common reason for patients seeking a physician's care for prob- lems related to the reproductive tract. Vaginitis is estimated to be responsible for 10% of such office visits. Thomason et al.2 reported that bacterial vaginosis accounted for 31.1% of the cases seen in their ambulatory practice. In addition, they found that 23.9% of these cases were due to yeast, 12.9% due to human papillomavrius (HPV), 8.9% to un- known causes, 6.1% to bacterial vaginitis, and 2.7% to atrophic vaginitis. Although vaginitis is sometimes considered to be nothing more than a nuisance, it has been impli- cated in significant sequelae in perinatal, postpar- tum, and postgynecologic surgery patients. There is some disagreement on a direct cause-and-effect relationship between the presence of vaginitis and subsequent morbidity in the obstetric and gyneco- logic patient, but it stands to reason that a vaginal microflora dominated by pathogenic bacteria is po- tentially a threat to the patient's well being. An abnormal microflora becomes of even greater con- cern if conditions are present that increase the pa- tient's vulnerability. Vaginitis may be due to infectious or noninfec- tious causes. Infectious causes present the physican with a spectrum ofetiologic agents including bacte- ria, fungi, protozoa, and parasites. At times identi- lying the etiologic agent is simple, while at other times it is very difficult. Nevertheless, the most important factor leading to successful treatment is establishing the correct diagnosis.

es present the physican with a spectrum ofetiologic agents including bacte- ria, fungi, protozoa, and parasites. At times identi- lying the etiologic agent is simple, while at other times it is very difficult. Nevertheless, the most important factor leading to successful treatment is establishing the correct diagnosis. MICROBIOLOGY Understanding the various types of microbial vaginitis requires an understanding ofthe microbi- ology of the healthy vagina. The endogenous mi- croflora constituting a major part of the vaginal ecosystem consists predominantly of commensal bacteria. Potentially pathogenic bacteria, although in lower concentrations, are also present. The com- mensal organisms exist in a synergistic state with one another, but are antagonistic toward the poten- tially pathogenic bacteria. When environmental conditions are appropriate, the commensal bacteria suppress the growth of pathogens. The dominant bacteria in the healthy vagina are Lactobacillus, Corynebacterium, and diptheroids. The pathogenic bacteria consist of gram-positive and gram-negative aerobes and facultative as well as obligate anaerobic bacteria. There are many spe- cies of lactobacilli: L. acidophilus, L. fermentum, L. brevis, L. casei, L. leichmani, L. salivarious, L. lactis, and L. cellobiosis.3 Lactobacilli, especially L. acidophilus, play a central role in maintaining the proper environment by producing lactic acid and Address correspondence/reprint requests to Dr. Sebastian Faro, GYN/OB University of Kansas School of Medicine, 3901 Rainbow Boulevard, Kansas City, KS 66160-7316. Received October 5, 1993 Review Article Accepted October 19, 1993 REVIEW OF VAGINITIS FARO hydrogen perioxide (H202). The pH range of the healthy vagina is 3.8-4.5, and the proper hydro- gen ion concentration is maintained through the production of lactic acid. The pathogenic bacteria, especially anaerobic bacteria, tend not to grow well at the healthy pH range. Hydrogen peroxide is toxic to anaerobic bacteria because they do not pro- duce catalase and thus cannot convert hydrogen peroxide to water and oxygen. Hydrogen peroxide is a bacterial antagonist that functions directly on the bacterial cell or a peroxidase mediate enzymatic system.4'5 Eschenbach et al. 6 demonstrated that 96% of women with a healthy vaginal flora have hydrogen peroxide-producing lactobacilli.

thus cannot convert hydrogen peroxide to water and oxygen. Hydrogen peroxide is a bacterial antagonist that functions directly on the bacterial cell or a peroxidase mediate enzymatic system.4'5 Eschenbach et al. 6 demonstrated that 96% of women with a healthy vaginal flora have hydrogen peroxide-producing lactobacilli. On the other hand, they found that only 35% of women with a diagnosis of bacterial vaginosis had lactoba- cilli, ofwhich only 11% produced hydrogen perox- ide. Lactobacilli also produce lactocins which are pro- tein-inhibiting bacteriocins.7 These inhibitors have a narrow spectrum of activity that includes the strains from which they were produced. 8 These bacteriocins are an effective means of controlling bacterial growth. They also maintain lactobacilli counts within a range that prevents them from ex- erting an adverse effect on the host. They are lim- ited in their activity in that some are effective only against gram-positive bacteria and others only against gram-negative bacteria. 9 Data suggest that lactobacilli may inhibit bacterial adherence to epi- thelial cells, 0,1 which is a significant factor in view of the fact that the infectious potential of bac- teria depends upon their ability to adhere to epithe- lial cells. The maintenance of the vaginal microecosystem is dependent upon the dynamic interaction ofbacte- ria and host factors. An important aspect of this equilibrium concerns the synergism and antago- nism occurring in the endogenous microflora ofthe lower genital tract. Organisms introduced from the exogenous environment must overcome endogenous organisms in order to disrupt the equilibrium of this microecosystem. However, when Neisseria gonorrhoeae or Chlamydia trachomatis are introduced in the lower genital tract, the endogenous factors become significant because these organisms do not reside in the vagina and infect the more vulnerable columnar epithelium of the endocervix rather than squamous epithelial cells. PATIENT EVALUATION The initial step in the evaluation of the patient with a complaint of vaginitis is to obtain a detailed his- tory. This step is critical in the necessary consider- ation of all possible factors that may impinge upon the vaginal microecosystem and disrupt the equi- librium of the healthy state. This delicate equilib- rium can easily be disturbed by endogenous or exogenous factors.

aint of vaginitis is to obtain a detailed his- tory. This step is critical in the necessary consider- ation of all possible factors that may impinge upon the vaginal microecosystem and disrupt the equi- librium of the healthy state. This delicate equilib- rium can easily be disturbed by endogenous or exogenous factors. The physician should elicit information regard- ing the patient's hygienic practices, change in soaps, douching agents and frequency, and type, dosage, and frequency of any medications. Antibiotics have been shown to alter the endogenous flora. Cephalosporins, e.g., in a single dose, have been shown to cause an increase in colonization by en- 12 terococcl. Determining how sexually active the patient is is significant because an increase in prevalence ofbac- terial vaginosis has been associated with an increased frequency of sexual intercourse. Also important to determine is whether the patient's pattern of sexual behavior or practices provide an opportunity for vaginal colonization by oral or fecal flora. These bacteria may present a synergistic opportunity when placed in the same environment with endogenous pathogens, thereby becoming the dominant flora. The patient should be given the opportunity to describe her symptoms in detail, characterize the discharge, and specifically point out the anatomic site of her symptoms. Often times, if the patient does not specifically describe and locate her symp- toms, the physician and the patient mistakenly pre- sume that she has a vaginitis. She may believe that she has a vaginal infection yet identify the location of her discomfort (usually burning, itching, or pain) at the opening ofthe vagina or she may reveal that she has dyspareunia on insertion of the penis. Other important information is when the condi- tion first occurred and specific treatments that have been used. Knowing the duration ofthe disease and treatments already administered will help the phy- sician to explain the complexity of the problem to the patient. At the same time, the characteristics of a healthy vagina should be explained to the patient (Table 1). Typically, the patient with a healthy vagina has a slate-gray to white discharge with no odor. The estrogenized vagina normally is pink-white with 154 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY REVIEW OF VAGINITIS FARO TABLE I.

the characteristics of a healthy vagina should be explained to the patient (Table 1). Typically, the patient with a healthy vagina has a slate-gray to white discharge with no odor. The estrogenized vagina normally is pink-white with 154 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY REVIEW OF VAGINITIS FARO TABLE I. Characteristics of a healthy vagina pH Range 3.8-4.5 Discharge Liquid to creamy-pasty Color Slate-gray to white Odor None Background Free-floating bacteria, mostly bacilli WBCs Rare rugae. The presence of petechial hemorrhages on the vaginal epithelium indicates an abnormal vagi- nal microecosystem. The presence of an odor ema- nating from the pelvic area may not originate in the vagina but may indicate poor hygiene, hidradeni- tis, or fecal soiling. The vaginal discharge should be examined mi- croscopically by swabbing the lateral vaginal wall with a cotton-tipped swab. The swab is then in- serted into a tube containing approximately 2 ml of saline and swirled to dilute the vaginal discharge. A drop or two of the diluted discharge is placed on a glass slide and a coverslip is placed over the speci- men. The specimen should be examined at x40 magnification. The healthy vaginal discharge will contain squamous epithelial cells whose cytoplasmic and nuclear membranes are easily seen. Bacteria will be individually free floating in the vaginal fluid and will be largely bacillary morphotypes of equal size (lactobacilli). There may be a rare white blood cell (WBC). The pelvic examination should begin with a close inspection ofthe vulva to determine the presence of lesions, e.g., pyodermas, folliculitis, carbuncles, hidradenitis, raised white pyriform growths, raised umbilicate lesions, ulcers, erythema, or excoria- tions. Ifthere is any suggestion that the patient may have HPV, then later, after the vagina has been evaluated, the vulva should be painted with 5% acetic acid and examined under magnification. The Bartholin's and Skene's glands should be palpated and an attempt made to express the contents ofthese glands. If a purulent discharge is present, it should be Gram stained and cultures should be taken for aerobic, facultative, and obligate anaerobes as well as C. trachomatis. A vaginal speculum should be inserted with care to avoid traumatizing the cervix, which is likely to result in bleeding if the cervix is inflamed. The pH of the vagina should be ascer- tained by applying pH paper to the lateral vaginal wall.

en for aerobic, facultative, and obligate anaerobes as well as C. trachomatis. A vaginal speculum should be inserted with care to avoid traumatizing the cervix, which is likely to result in bleeding if the cervix is inflamed. The pH of the vagina should be ascer- tained by applying pH paper to the lateral vaginal wall. Both semen and spermicides will cause the pH to be more alkaline, so it is prudent to deter- mine ifthe patient has had sexual intercourse in the preceding 24 hr and if a spermicidal gel has been used. By performing a microscopic examination of a diluted specimen of the .vaginal discharge, the physician should be able to determine whether the vaginal microecosystem is healthy or not (Table 2). After performing the pelvic examination, the physician should be in a position to construct a differential diagnosis (Table 3). Following the mi- croscopic examination of the vaginal discharge, a definitive diagnosis will be possible in most cases. On occasion, the examination will not reveal the etiology of the patient's problem and specialized testing such as cultures for Candida or Trichomonas may be necessary. In 75-80% of the cases, T. vaginalis can be diagnosed by microscopic examination of the vagi- nal discharge. The organism is easily identified by the motile anterior flagella and characteristic move- ment on microscopic examination of an unstained preparation of diluted vaginal discharge. The vag- inal discharge in itself does not possess any specific characteristics that aid the physician in establishing a diagnosis without further analysis. A frothy, dirty- gray discharge can be seen with a trichomonas in- fection as well with bacterial vaginosis or Gardner- ella vaginalis. The classic "strawberry cervix" is seen in only approximately 25% of cases. Ifthere is strong suspicion of trichomoniasis but no identifi- able trichomonads, a portion of the vaginal dis- charge can be placed into a commercial system for trichomonas culture to allow the trichomonads to grow. This culture should be examined daily for the presence of trichomonads. Trichomonads can also be identified on a Pap smear. Typically, the bacterial flora will have shifted from lactobacillus- dominated flora to a polymicrobial one toward anaerobes. The presence of trichomonads should alert the physician that the patient is at risk for other sexually transmitted organisms and that further testing may be indicated. A patient with trichomoniasis may also have a concomitant bacterial vaginosis.

m lactobacillus- dominated flora to a polymicrobial one toward anaerobes. The presence of trichomonads should alert the physician that the patient is at risk for other sexually transmitted organisms and that further testing may be indicated. A patient with trichomoniasis may also have a concomitant bacterial vaginosis. In such a patient, treatment with metronidazole will often be effective in correcting both infections. Occa- sionally, a patient may have a concomitant candidi- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 155 REVIEW OF VAGINITIS FARO TABLE 2. Characteristics of vaginal discharge Healthy Bacterial Gardnerella Trichomonas Candida Atrophic Characteristic vagina vaginosis vaginalis vaginalis vaginitis vaginitis pH 3.8-4.5 >4.5 >4.5 >4.5 <5 >3.8 Color Gray-white Gray Gray Gray White White-yellow Odor None Fishy Fishy Foul None None Whiff test Negative Positive Positive Positive Negative Negative Clue cells Negative Positive Positive Negative Negative Negative Background Bacilli Mixed Clumps Mixed Bacilli Rare WBCs Rare + Rare Yes Yes Yes Bacteria Bacilli Mixed Bacilli Mixed Bacilli Rare TABLE 3. Differential diagnosis for vulvovaginitis due to infection Vulva Vagina Candida Bacterial vaginosis Folliculitis Gardnerella vaginalis Carbuncles Streptococcus agalactiae Hidradenitis Herpes simplex Herpes simplex Human papillomavirus Human papillomavirus Trichomonas vaginalis asis that can usually be detected on a wet-mount preparation. If difficulty is encountered in identi- fying pseudohyphae, yeast, or budding yeast forms, a drop or two ofthe vaginal discharge can be mixed with a drop or two of 10% potassium hydroxide (KOH). The chitinous cell walls of the fungi, which are resistant to strong alkaline solutions, will not dissolve as the squamous epithelial and bacterial cells will. A microscopic examination of the patient's vagi- nal discharge may not reveal the presence of trich- omonads. However, ifit contains a significant num- ber of WBCs and numerous bacteria of various morphotypes, but no clue cells or pathogens, a culture of the discharge specimen should be taken for T. vaginalis. Consideration should also be given to the possibility of an I-IPV infection. Some debate concerns the issue ofwhether bacte- rial vaginosis is different from G. vaginalis vagini- tis or whether the latter is just a marker organism for bacterial vaginosis. Additional so-called marker organisms are Mycoplasma and Mobiluncus.

ideration should also be given to the possibility of an I-IPV infection. Some debate concerns the issue ofwhether bacte- rial vaginosis is different from G. vaginalis vagini- tis or whether the latter is just a marker organism for bacterial vaginosis. Additional so-called marker organisms are Mycoplasma and Mobiluncus. A re- view ofthe original works ofGardner and Dukes13 makes it apparent that these two conditions may not be the same or may represent a spectrum of the same condition. These authors described clue cells as the presence of gram-negative bacteria (G. vagi- nalis) adhering to squamous epithelial cells. Their photomicrographs show that the bacteria in the vag- inal fluid not attached to squamous epithelial cells are not individually free floating but clumped or in aggregates with a characteristic lack of WBCs. G. vaginalis is a constant factor in the bacteriological assessment of bacterial vaginosis, as it is usually present in significant numbers. Perhaps the two conditions should be viewed as a spectrum of bacte- rial vaginitis. In the initial phase when the environ- ment is in the stage of being disrupted, a decline in lactobacilli occurs resulting in a shift in hydrogen ion concentration and allowing the growth of G. vaginalis. The continued growth of G. vaginalis results in further decline in the hydrogen ion con- centration and growth of the anaerobes. This con- tinual decline in hydrogen ion concentration favors the growth of anaerobes, eventually leading to the typical microbiological flora of bacterial vaginosis. This hypothesis has some support in the clinical setting by observing patients who have been repeat- edly treated with metronidazole but have failed to be cured. Microbiological analysis of the vaginal flora of these patients makes it possible to divide them into 2 groups: 1) those with nondescript flora dominated by Staphylococcus epidermidis, Enterococ- cus faecalis, or Escherichia coli and 2) those with flora dominated by G. vaginalis and a conspicuous absence ofanaerobes. 14,15 Therefore, it would seem logical that patients in the early development of a derangement of the vaginal microflora dominated by G. vaginalis, which is typically resistant to met- ronidazole, would not be cured by this drug. Individuals presenting with a clinical picture of bacterial vaginosis or G. vaginalis should not have their vaginal specimens sent for culture if the labo- ratory is not-equipped to perform a thorough workup.

ra dominated by G. vaginalis, which is typically resistant to met- ronidazole, would not be cured by this drug. Individuals presenting with a clinical picture of bacterial vaginosis or G. vaginalis should not have their vaginal specimens sent for culture if the labo- ratory is not-equipped to perform a thorough workup. The treatment options are limited and 156 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY REVIEW OF VAGINITIS FARO chosen empirically. Patients with repeated or chronic vaginitis should be thoroughly evaluated with specimens obtained for the culture of fungi, trichomonads, Streptococcus agalactiae, and, iflabo- ratory capabilities exist, aerobic, facultative, and obligate anaerobes. Again, if the laboratory is not equipped to carry out these studies, obtaining these specimens will be a waste of money. If vaginal specimens are sent to the laboratory, they must be transported in anaerobic vessels. Sending only aer- obic specimens will usually result in reports of a single organism, e.g., E. coli. This manner of handling specimens usually leads to treatment with an inappropriate antibiotic and failure ofthe patient to respond. It is extremely important that, when a patient complains of recurrent vaginitis, the physi- cian take time to examine the genital area thor- oughly and specifically avoid administering anti- microbial agents that may have an adverse effect on the vaginal flora and result in continuation of the patient's symptoms. Yeast cells are commonly seen in the patient's vaginal fluid, being isolated from approximately 40% of asymptomatic patients with a healthy vagi- nal microflora. 16 There has been much discussion regarding the development of more specific and sensitive tests for the identification ofyeast forms in patients with complaints of vaginitis. Approxi- mately 75% of cases can be diagnosed by micros- copy and KOH. The rapid tests that have been developed are only about 81% effective. Typically, the patient with a yeast vaginitis will present with complaints of vulvovaginal itching. Inspection of the vulva may or may not reveal the presence of erythema and excoriation. The vaginal epithelium as well as the cervical epithelium are erythematous. The discharge is classically thick and pasty, often referred to as "cottage cheese-like," but it may be homogenous and liquid. Usually, no odor is present, but when present it typically has the aro- matic odor of yeast.

rythema and excoriation. The vaginal epithelium as well as the cervical epithelium are erythematous. The discharge is classically thick and pasty, often referred to as "cottage cheese-like," but it may be homogenous and liquid. Usually, no odor is present, but when present it typically has the aro- matic odor of yeast. Microscopic examination of a diluted portion of the vaginal discharge may reveal the presence ofelliptical yeast cells that refract light and tend to have a greenish tinge to the cytoplasm. The cells may be budding or have short germina- tion tubes, which represent the beginning of the production of the characteristic pseudohyphae. When the pseudohyphae are quite long and branch- ing, they can usually be found among clumps of squamous epithelial cells. In the event that these structures cannot be identified, an aliquot of vagi- nal discharge can be placed on a glass slide and or 2 drops of 10% KOH can be added to it. Fungal and yeast cell walls are made of chitin and resistant to strong alkaline solutions. Therefore, all non- chitinous structures, e.g., bacteria and squamous cells, will dissolve in the alkaline solution. The remaining fungal cells are easy to identify. In re- current cases or in cases in which the organisms are not visually identified, a culture is recommended. Such a culture can easily be accomplished by plac- ing a specimen of the vaginal discharge on Sab- ouraud's or Nickerson's medium. In recalcitrant cases, the organism should be identified according to species. Antifungal sensitive studies may need to be performed in patients having tried without suc- cess all vaginal and oral preparations commonly used. TREATMENT The first error in the management of vaginitis oc- curs when an antimicrobial agent, even if nothing more than a sulfa-based cream, is administered to the patient presenting with vaginitis. Ifthe etiology ofthe vulvovaginitis has not been found, it is more appropriate to try to correct the pH if it is not within the acceptable range (3.8-4.5) than to pre- scribe an antimicrobial agent. Perhaps the patient should do no more than douche with vinegar water. Again, it is critical to establish the precise location of the symptoms. Patients whose complaints are truly localized to the inferior aspect of the vestibule, specifically the posterior fourchette, should be first managed by attempting to establish whether the problem is chronic inflammation, vestibulitis, or HPV, or a combination of these conditions.

the precise location of the symptoms. Patients whose complaints are truly localized to the inferior aspect of the vestibule, specifically the posterior fourchette, should be first managed by attempting to establish whether the problem is chronic inflammation, vestibulitis, or HPV, or a combination of these conditions. The diagnosis can be established by performing colposcopy and bi- opsy of the affected area. Patients with chronic inflammation are best managed by the local appli- cation ofhydrocortisone, 1% or 2.5% lotion, cream, or ointment. Although this therapy can be insti- tuted prior to performing a colposcopically directed biopsy, it can cause inconvenience and unnecessary cost to the patient if a return visit is necessary to continue the evaluation. A patient with vestibulitis requires histologic confirmation of her condition. The tissue typically reveals replacement ofthe gland acini (vestibular glands) with stratified squamous epithelium and inflammatory cells. Treatment in- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 157 REVIEW OF VAGINITIS FARO volves surgically excising the vulvar tissue contain- ing the vestibular gland. 17-21 In some instances, the patient may have either of these diagnoses asso- ciated with the presence of an HPV infection, in which case laser ablation may be of value. Prior to instituting any form of treatment, however, the presence of other possible causes of vulvar discom- fort such as herpes or lichen planus must be ruled out. The treatment of T. vaginalis is best accom- plished with oral metronidazole. Intravaginal met- ronidazole should not be used alone as it is not effective in eradicating trichomonads found, in the bladder, upper genital tract, or Skene's glands. Therefore, oral preparations should be used. The only drug approved in the United States is metron- idazole. Trinidazole, which can be obtained in other countries, has a longer half-life. A single oral 2-g dose of metronidazole has been shown to be 90% effective. Ifthe sexual partner is treated at the same time, the effectiveness is 95%.z2'z3 This drug can also be administered in dosages of 250 mg 3 times daily or 500 mg twice daily for 7 days. The most common side effects are metallic taste, nausea, and vomiting. Patients should be strongly advised not to consume alcoholic-containing liquids while tak- ing this medication and should refrain from them for 24 hr after the final dose.

nistered in dosages of 250 mg 3 times daily or 500 mg twice daily for 7 days. The most common side effects are metallic taste, nausea, and vomiting. Patients should be strongly advised not to consume alcoholic-containing liquids while tak- ing this medication and should refrain from them for 24 hr after the final dose. The combination of metronidazole and alcohol produces a disulfiram- like reaction with nausea and abdominal cramp- ing.24 There is some concern over the carcinogenic potential of metronidazole, which is related to the detection of mutagens in the urine of patients tak- ing this drug.25 These weak carcinogens have been reported to cause alterations in DNA. Thus far, no large studies have addressed this concern, but two small studies have shown no documented relation- ship between metronidazole use and cancer. 26'27 In cigarette smokers who have used metronidazole, a 10-fold increase in lung cancer has been reported.26 Metronidazole is relatively contraindicated in the pregnant patient. The role of trichomoniasis in adverse pregnancy is undergoing intensive investi- gation. This antibiotic does not appear to have ter- atogenic effects. However, in two relatively small studies, congenital anomalies were found in infants born to women who ingested metronidazole in the first trimester.26'28 Because of its possible teratoge- nic and carcinogenic effects, metronidazole should not be given in the first trimester and should be used cautiously in the second and third trimes- ters. 28 Treatment should be reserved for only those individuals who are truly symptomatic. An alterna- tive to metronidazole is clotrimazole. Clotrimazole can inhibit the growth of.T. vaginalis,29 but it will not eradicate the trichomoniasis and the infection will recur. Women who are breast feeding must be advised that metronidazole achieves detectable levels in breast milk. 3 For this reason, breast-feeding women who must be treated for symptomatic infec- tion should pump their breasts and discard the milk for at least the first 24 hr after discontinuing met- ronidazole. An appropriate course of management for the breast-feeding patient being treated for tri- chomoniasis is to collect an adequate supply ofbreast milk before instituting metronidazole therapy; dis- continue breast feeding but pump the breasts; take a single 2-g dose; and resume breast feeding 24 hr after completion of therapy.

ropriate course of management for the breast-feeding patient being treated for tri- chomoniasis is to collect an adequate supply ofbreast milk before instituting metronidazole therapy; dis- continue breast feeding but pump the breasts; take a single 2-g dose; and resume breast feeding 24 hr after completion of therapy. Any patient who fails routine treatment regi- mens should be questioned to determine the degree of her compliance in taking the medication as di- rected. In addition, any possibility of reinfection should be pursued. Reinfection can occur if her partner did not take the medication or she acquired a new partner. Although T. vaginalis has remained relatively sensitive to metronidazole, resistant strains have been reported.31 Sensitivity testing to metronidazole is complicated by the possibility that the test was not conducted in a truly anaerobic environment, as resistance has not been demon- strated in a truly anaerobic environment.31,32 Ox- ygen has been shown to affect the efficacy of met- ronidazole.33 Therefore, since the vaginal ecosystem is complex, the oxygen reduction poten- tial of the vaginal ecosystem will undoubtedly have a significant effect on the effectiveness of the drug in the treatment of trichomoniasis. Resistant or chronic infection can be treated by increasing the dose of metronidazole. One regimen is the admin- istration of 2-3 g orally daily for 14 days with the concomitant use of 500 or 1,000 mg vaginal sup- positories daily or intravaginal metronidazole 2-3 times a day for 14 days. Utilization ofhigh doses of metronidazole will, in all likelihood, cause signifi- cant nausea. One investigator reported that strains INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY REVIEW OF VAGINITIS FARO of T. vaginalis with an aerobic MIC >400 mg required a total of 40 g of metronidazole over a 14-day treatment course. 32'33 Some patients have also been treated with intravenously administered metronidazole, 3-4 g/day.34 However, it must be pointed out that the administration of metronida- zole at or above 4 g involves significant potential for the induction of seizures and disabling periph- eral neuropathy. Treatment of yeast vaginitis can be carried out with a number of antimycotic agents that can be placed in the vagina either in suppository form or as a cream. These agents are primarily azole deriv- atives: butoconazole, clotrimazole, miconazole, and terconazole. The duration oftherapy ranges from 3 to 7 days.

ropathy. Treatment of yeast vaginitis can be carried out with a number of antimycotic agents that can be placed in the vagina either in suppository form or as a cream. These agents are primarily azole deriv- atives: butoconazole, clotrimazole, miconazole, and terconazole. The duration oftherapy ranges from 3 to 7 days. Candida albicans is the most common cause of yeast vaginitis and remains sensitive to the azoles. However, C. glabrata and C. tropicalis both tend to be resistant to most azoles, with terconazole having good activity against these species.35'36 An- other effective treatment is nystatin (polyene), but it should be considered a second-line agent because it has not produced the success that the azoles have achieved. Another polyene commonly used in sys- temic fungal infections is amphotericin B, which is also supplied as a 3% cream or ointment. It is typically used for cutaneous or mucocutaneous in- fection and has not been approved for vaginitis. Although amphotericin B can be utilized for chronic or recurrent infection, it should be used with cau- tion as no data are currently available on its sys- temic absorption. Other forms of therapy that have been used with success are boric acid suppositories, 600 mg, placed intravaginally twice daily for 10-14 days. Boron has not been detected in the bloodstream following intravaginal usage, but it should not be used in pregnancy because no studies have been conducted on pregnant patients.37 Gentian violet used as tab- lets, paint, or tampons has proven to be effective, but it is somewhat difficult to use because it stains the clothing and skin. Oral therapy has been shown to be effective with ketoconazole, 400 mg/day for 14 days, followed by a maintenance dose of 100 mg/day for 6-12 months. The patient's liver function must be monitored be- cause liver toxicity in patients using ketoconazole for long periods is possible. Fluconazole can also be given orally, but there is no indication of it in the treatment of vaginal candidiasis. The dosage has not been established, but I have had success with an initial dose of200 mg followed by 100 mg daily for 7 days. Patients with recurrent or chronic infection should be evaluated for any underlying systemic illness such as diabetes or immunodeficiency. Again, a thorough history is important, especially with regard to any antimicrobial therapy that the patient may be on. The patient's hygiene and sexual behavioral patterns should be reviewed.

th recurrent or chronic infection should be evaluated for any underlying systemic illness such as diabetes or immunodeficiency. Again, a thorough history is important, especially with regard to any antimicrobial therapy that the patient may be on. The patient's hygiene and sexual behavioral patterns should be reviewed. In addi- tion, the possibility of gastrointestinal colonization should be considered as significant colonization of the gastrointestinal tract and oral cavity has been reported in patients with recurrent or chronic in- fection.37 Thus far, the available data do not indi- cate that oral nystatin is beneficial in these patients over a long period. The data do indicate a benefit over the short term. 38 The possibility that the or- ganism may colonize the sexual partner who, in turn, reintroduces the organism to the vagina ofthe patient with each subsequent intercourse should also be considered. Studies have demonstrated that ap- proximately 20% of the male sexual partners of women with candidiasis are colonized.39'4 For this reason, it is probably beneficial to treat the male partner with an agent such as fluconazole while treating the patient. I prefer an oral regimen over a topical one because the urethra may be colonized. Treatment of bacterial vaginosis has rested mainly with the use of metronidazole similar to the regimens used for the treatment of trichomoniasis. However, I believe that this agent should be used only for the first episode. If the patient fails to respond, consideration should be given to the pos- sibility that the infection may be in the early stages of development with the flora dominated by G. vaginalis, as discussed earlier. Alternative therapy consists of the use of metronidazole gel or clin- damycin cream. Oral clindamycin, 3 00 mg twice a day for 7-10 days, has been shown to be effec- tive.41 Other agents that can be used are amoxicillin/ clavulanic acid, 500 mg given orally 3 times daily for 7-10 days. Ampicillin and amoxicillin have been shown to be only 30-45% effective in the treatment ofbacterial vaginosis.49 This observation is compatible with the hypothesis that this disease may well be a spectrum of microbiological change that begins with a decrease in lactobacilli, a rise in INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY REVIEW OF VAGINITIS FARO pH, and an increase in G. vaginalis growth. Treat- ment at this stage would most likely be effective since G. vaginalis is sensitive to these agents but resistant to metronidazole.

cal change that begins with a decrease in lactobacilli, a rise in INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY REVIEW OF VAGINITIS FARO pH, and an increase in G. vaginalis growth. Treat- ment at this stage would most likely be effective since G. vaginalis is sensitive to these agents but resistant to metronidazole. Once the condition has progressed and the anaerobes have become domi- nant, the narrow-spectrum penicillins become inef- fective. SUMMARY Vaginitis due to "infection" can be defined as a disturbance in the microecosystem in which the endogenous microflora is prevented from main- taining its equilibrium. Commensal organisms ex- ist in a synergistic state with one another and exert an antagonistic effect on potential pathogens. This disruption of the dynamic equilibrium of the healthy vagina may have significant sequelae. These patients may develop significant symptoms that pre- vent them from leading normal lives, often pre- cluding them from having sexual relations because of odor or pain, and strain their relationships as a result. In some cases, these patients become "pelvic cripples." Another concern is the potential for de- veloping serious conditions such as premature rup- ture of amniotic membranes, premature labor and delivery, or low birth weight when vaginitis is associated with pregnancy. Finally, vaginitis may predispose a patient undergoing pelvic or vaginal surgery to significant postoperative infection. For these reasons, the patient with vaginitis must always be taken seriously, the condition must never be written offas a mild insignificant condition, and an accurate diagnosis must always be established. REFERENCES 1. PaavonenJ, Stamm WE: Lower genital tract infections in women. In Hansfield HH (ed): Sexually Transmitted Diseases. Infectious Disease Clinics of North America. Philadelphia: W.B. Saunders, pp 179-198, 1987. 2. Thomason JI, Gelbart SM, Broekhuizen FF: Office and clinical laboratory diagnosis of vulvovaginal infections: An overview. In Horowitz BJ, Mardh P-A (eds): Vagini- tis and Vaginosis. New York: Wiley-Liss, pp 93-108, 1991. 3. Mardh P-A: Vaginal microbial ecology. In Horowitz BJ, Mardh P-A (eds): Vaginitis and Vaginosis. New York: Wiley-Liss, pp 1-9, 1991. 4. Dahiya RS, Speck ML: Hydrogen peroxide formation by lactobacilli and its effect on Staphylococcus aureus. J Dairy Sci 51:1568-1572, 1968. 5. KlebanoffJJ, Smith DC: Peroxidase-mediated antimicro- bial activity of rat uterine fluid. Gynecol Invest 1:21-30, 1979. 6.

is and Vaginosis. New York: Wiley-Liss, pp 1-9, 1991. 4. Dahiya RS, Speck ML: Hydrogen peroxide formation by lactobacilli and its effect on Staphylococcus aureus. J Dairy Sci 51:1568-1572, 1968. 5. KlebanoffJJ, Smith DC: Peroxidase-mediated antimicro- bial activity of rat uterine fluid. Gynecol Invest 1:21-30, 1979. 6. Eschenbach DA, Davick PR, Williams BC: Prevalence of hydrogen peroxide producing Lactobacillus species in normal women and women with bacterial vaginosis. J Clin Microbiol 27:251-256, 1989. 7. Upreti GC, Hinsdill RD: Isolation and characterization of a bacteriocin from a homofermative lactobacillus. An- timicrob Agents Chemother 4:484-487, 1973. 8. Andersson RE, Daeschel MA, Hassan HN: Antibacte- rial activity of plantaricin SIK-83, a bacteriocin produced by Lactobacillus plantarum. Biochemistry 70:381-390, 1988. 9. Barefoot SK, Klaenhammer TR: Detection and activity of lactacin B, a bacteriocin produced by Lactobacillus acidophilus. Appl Environ Microbiol 45:1808-1815, 1983. 10. Reid G, Cook RL, Bruce AW: Examination of strains of lactobacilli for properties that may influence bacterial in- terference in the urinary tract. J Urol 138:330-335, 1987. 11. Sobel JD, Myers P, Levison ME, Kaye D: C. albicans adherence to vaginal epithelial cells. J Infect Dis 143: 767-782, 1981. 12. Cox SM, Phillips LE, Mercer LJ, Stager CE, Waller S, Faro S: Lactobacillemia of amniotic fluid origin. Obstet Gynecol 68:134-135, 1986. 13. Gardner HL, Dukes CD: Haemophilus vaginalis vagini- tis. Am J Obstet Gynecol 69:962-976, 1955. 14. Faro S, Phillips LE: Non-specific vaginitis or vaginitis of undetermined etiology. Int J Tissue Reactions IX:173- 177, 1987. 15. Faro S: Persistent vaginitis and vaginosis. In Horowitz BJ, Mardh P-A (eds): Vaginitis and Vaginosis. New York: Wiley-Liss, pp 237-245, 1991. 16. Sobel JD: Epidemiology and pathogenesis of recurrent vulvovaginal candidiasis. AmJ Obstet Gynecol 152:924- 935, 1985. 17. Friedrich EG Jr: Vulvar vestibulitis syndrome. J Reprod Med 32:110-114, 1987. 18. Pyka RE, Wilkinson EJ, Friedrich EG Jr, Croler BP: The histopathology of vulvar vestibulitis syndrome. Int J Gynecol Pathol 7:249-257, 1988. 19. Turner MLC, MarinoffSC: Association of human papil- loma virus with vulvodynia and the vulvar vestibular syndrome. J Reprod Med 33:533-537, 1988. 20. Friedrich EG Jr: Therapeutic studies on vulvar vestibuli- tis. J Reprod Med 33:514-518, 1988. 21. Woodruff D, Friedrich EG Jr: The vestibule. Clin Ob- stet Gynecol 28:134-141, 1985. 22.

inoffSC: Association of human papil- loma virus with vulvodynia and the vulvar vestibular syndrome. J Reprod Med 33:533-537, 1988. 20. Friedrich EG Jr: Therapeutic studies on vulvar vestibuli- tis. J Reprod Med 33:514-518, 1988. 21. Woodruff D, Friedrich EG Jr: The vestibule. Clin Ob- stet Gynecol 28:134-141, 1985. 22. Lossick JG: Treatment of Trichomonas vaginalis infec- tions. Rev Infect Dis 4:S-801-818, 1982. 23. Hager WD, Brown ST, Klaus SJ, et al.: Metronidazole in vaginal trichomoniasis. Seven day vs. single-dose re- gimes. JAMA 244:1219-1220, 1980. 24. O'Reilly RA: Stereospecific interaction of warfarin and metronidazole. Fed Proc 34:259, 1975. 160 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY REVIEW OF VAGINITIS FARO 25. Roe JFC: A critical appraisal ofthe toxicology of metron- idazole. In Phillips I, Collier J (eds): Metronidazole. London: Academic Press, pp 215-222, 1979. 26. Beard CM, Noller KL, O'Fallon WM, Kurkland LT, Dockerty MB: Lack of evidence for cancer due to use of metronidazole. N Engl J Med 301:519-522, 1979. 27. Friedman GD: Cancer after metronidazole (letter). N Engl J Med 302:519, 1980. 28. Peterson WF, Stauch JE, Ryder CO: Metronidazole in pregnancy. Am J Obstet Gynecol 94:343-349, 1966. 29. Erickson SH, Oppenheim GL, Smith GH: Metronida- zole in breast milk. Obstet Gynecol 57:48-50, 1981. 30. Schnell JD: The incidence of vaginal Candida and Trich- omonas infections and treatment of Trichomonas vaginalis with clotrimazole. Postgrad Med 50:79-81, 1974. 31. Pereyra AJ, Lansing JD: Urogenital trichomoniasis. Treatment with metronidazole in 2,002 incarcerated women. Obstet Gynecol 24:499-508, 1964. 32. Meingassner JG, Turner J: Strains of Trichomonas vagi- nalis resistant to metronidazole and other 5-nitro-imida- zoles. Antimicrob Agents Chemother 15:254-257, 1979. 33. Lossik JG, Muller M, Gorrell TE: In vitro drug suscep- tibility and doses of metronidazole required for cure in cases of refractory vaginal trichomoniasis. J Infect Dis 153:948-955, 1986. 34. Muller M, Lossick JG, Gorrell TE: In vitro susceptibil- ity of Trichomonas vaginalis to metronidazole and treat- ment outcome in vaginal trichomoniasis. Sex Transm Dis 15:17-24, 1988. 35. Frytak S, Mortel CG, Childs OS, Albers JW: Neuro- logic toxicity associated with high dose metronidazole. Ann Intern Med 88:361-362, 1978. 36. Redondo-Lopez V, Lynch M, Schmitt C: Torulopsis gla- brata vaginitis: Clinical aspects and susceptibility to anti- fungal agents. Obstet Gynecbl 76:651-655, 1990. 37.

1988. 35. Frytak S, Mortel CG, Childs OS, Albers JW: Neuro- logic toxicity associated with high dose metronidazole. Ann Intern Med 88:361-362, 1978. 36. Redondo-Lopez V, Lynch M, Schmitt C: Torulopsis gla- brata vaginitis: Clinical aspects and susceptibility to anti- fungal agents. Obstet Gynecbl 76:651-655, 1990. 37. Cook CW: Vulvovaginal candidiasis (letter). Obstet Gy- necol 48:631, 1976. 38. Miles MR, Olsen I, RogersA: Recurrent vaginal candid- iasis: Importance of an intestinal reservoir. JAMA 238: 1836-1837, 1977. 39. Nystatin Multicenter Study Group: Therapy of candidal vaginitis. The effect of eliminating intestinal Candida. AmJ Obstet Gynecol 155:651-655, 1986. 40. Horowitz BJ, Edelstein SW, Lippman L: Sexual trans- mission of Candida. Obstet Gynecol 69:883-886, 1987. 41. Greaves W, Chungafung J, Morris B, Haile A, Townsend JF: Clindamycin versus metronidazole in the treatment of bacterial vaginosis. Obstet Gynecol 72:799- 802, 1988. 42. Lee L, Schmale JD: Ampicillin therapy for Corynebacte- rium vaginalae (Haemophilus vaginalis) vaginitis. Am J Obstet Gynecol 115:786-788, 1973. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:163-165 (1994) (C) 1994 Wiley-Liss, Inc. Varicella in Pregnancy: Five Priorities for Clinicians Editorial Acute varicella (chicken pox) is one of the most highly contagious viral illnesses. The infection occurs commonly in childhood, and approximately 95% of individ- uals are immune by the time they reach childbearing age. Varicella occurs in fewer than 1:1,000 pregnancies each year. Although it is a relatively mild illness in children, varicella may cause severe complications in pregnant women and their fetuses. Accordingly, physicians and nurses who provide obstetric care must be aware of the following 5 priorities. First, at the time of the patient's first prenatal appointment, she should be questioned about a history of varicella. If she is certain she had chicken pox, she should be reassured that second infections are extremely unlikely and that she need not fear accidental exposure to a person with varicella. If the patient is uncertain about her history and a family member is unable to verify that infection occurred, a serologic test to determine immune status should be performed.2 This test is particularly important in women who have frequent occupational exposure to children with viral illnesses, e.g., school teachers, day care workers, and health care workers. The appropriate serologic test for determining immune status is measurement ofIgG antibody by an enzyme-linked immunosorbent assay (ELISA) or the fluorescent antibody test for membrane antigen (FAMA). Approximately 80% of patients with uncertain histories will, in fact, have evidence of immunity, and they can be reassured that they are not at risk during the present pregnancy. Susceptible women must be specifically counseled to avoid exposure to other individuals who may have varicella. The second priority is to promptly and correctly evaluate the obstetric patient who has been exposed to an individual with acute varicella and whose serologic status is either unknown or negative. In the former case a serologic test should immediately be performed. Laboratory personnel must be informed ofthe need for completion of the test within 24--48 h.

correctly evaluate the obstetric patient who has been exposed to an individual with acute varicella and whose serologic status is either unknown or negative. In the former case a serologic test should immediately be performed. Laboratory personnel must be informed ofthe need for completion of the test within 24--48 h. If the patient is seronegative or if the laboratory test cannot be completed promptly, the patient should be offered vari- cella-zoster immune globulin (VZIG). This preparation contains high titer of antibody to the varicella-zoster virus, and, if it is given within 4 days of exposure, is of benefit in preventing or attenuating subsequent illness. The appropriate dose of VZIG is vial (125 units) per 10 kg body weight, administered intramuscu- larly, up to a maximum of 5 vials (625 units).3 The approximate cost of VZIG is $4OO-$6OO. The third priority is to treat appropriately patients who present for care more than 4 days after exposure or who become infected despite receiving VZIG. These patients should be counseled about the major complications of varicella: dissemi- nated infection, which is extremely uncommon in immunocompetent patients; encephalitis, which occurs in <1% of patients; and pneumonia, which may occur in 20-50% of patients. In reports published prior to the availability of effective antiviral chemotherapy, the mortality in pregnant women with varicella pneumo- nia was as high as 40%.4 Patients should be instructed to report immediately if signs of severe systemic illness develop. If evaluation confirms the presence of a VARICELLA IN PREGNANCY DUFF serious complication, the patient should be hospitalized and treated with intrave- nous acyclovir, 10-15 mg/kg or 500 mg/m2 3 times a day for approximately 7 days.4 Consultation with specialists in infectious diseases, pulmonology, and criti- cal care may be indicated. The fourth priority is to evaluate exposed fetuses for possible congenital vari- cella infection. Recent reports have shown that the incidence ofanatomic anomalies related to maternal varicella infection ranges from 0 to 9%. 1,s Serologic evidence of congenital infection may be present in up to 25% of exposed neonates.

The fourth priority is to evaluate exposed fetuses for possible congenital vari- cella infection. Recent reports have shown that the incidence ofanatomic anomalies related to maternal varicella infection ranges from 0 to 9%. 1,s Serologic evidence of congenital infection may be present in up to 25% of exposed neonates. Several diagnostic testshave been proposed for identification of affected fetuses, including amniocentesis, chorionic villus sampling (CVS), cordocentesis, and ultrasound.6'7 Identification of virus, viral antigen, or antibody in amniotic fluid, placental tissue, or fetal blood confirms that infection has occurred but does not indicate that the fetus has been seriously injured. The best test for identifying the fetus with anomalies is ultrasound. Ultrasound findings suggestive of congenital varicella include, but are not limited to, polyhydramnios, hydrops, ventriculomegaly, microcephaly, cardiac anomalies, limb abnormalities, intrauterine growth retarda- tion, and dystrophic calcifications in multiple organs, especially the liver.7 Moth- ers with affected infants may be offered pregnancy termination if the gestational age is appropriate. The fifth priority is to provide appropriate care to the mother and neonate when the former is acutely infected at the time ofdelivery. When infection occurs during the period 5 days before to 2 days after delivery, the fetus may be exposed to an intense viremia without benefit oftransplacental transfer ofmaternal antibody. Up to 40% of these infants develop neonatal varicella, and the mortality in untreated infants approaches 20%. 8 Accordingly, the pediatrician who will be caring for the infant must be notified promptly that an acutely infected mother is about to deliver. Immediately following delivery, the infant should receive VZIG at the direction of the pediatrician. In addition, mother and infant should be separated at least until neonatal assessment has been completed and immunoprophylaxis has been admin- istered. In summary, varicella in pregnancy is a rare, but potentially fatal, disorder for both mother and fetus. The immune status of patients should be determined early in pregnancy. Susceptible patients are candidates for immunoprophylaxis if expo- sure occurs. Infected patients should be monitored for serious complications, and their fetuses should be assessed for congenital infection with ultrasound examina- tion. Infants at risk for neonatal varicella are also candidates for immunoprophy- laxis with VZIG.

patients are candidates for immunoprophylaxis if expo- sure occurs. Infected patients should be monitored for serious complications, and their fetuses should be assessed for congenital infection with ultrasound examina- tion. Infants at risk for neonatal varicella are also candidates for immunoprophy- laxis with VZIG. Patrick Duff, M.D. Division ofMaternal-Fetal Medicine University ofFlorida College ofMedicine Gainesville Florida REFERENCES 1. Balducci J, Rodis JF, Rosengren S, Vintzilleos AM, Spivey G, Vosseller C: Pregnancy outcome following first-trimester varicella infection. Obstet Gynecol 79:5-6, 1992. 2. McGregorJA, Mark S, Crawford GP, Levin MJ: Varicella zoster antibody testing in the care of pregnant women exposed to varicella. Am J Obstet Gynecol 157:281-284, 1987. 3. Varicella-zoster immune globulin for the prevention of chickenpox. Morbidity and Mortality Weekly Report 33:83-100, 1984. 4. Smego RA, Asperilla MO: Use of acyclovir for varicella pneumonia during pregnancy. Obstet Gynecol 78:1112-1116, 1991. 164 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY VARICELLA IN PREGNANCY DUFF 5. Paryani SG, Arvin AM: Intrauterine infection with varicella-zoster virus after maternal vari- celia. N Engl J Med 314:1542-1546, 1986. 6. Grose C, Itani O, Weiner CP: Prenatal diagnosis offetal infection: Advances from amniocentesis to cordocentesis--congenital toxoplasmosis, rubella, cytomegalovirus, varicella virus, parvovirus and human immunodeficiency virus. Pediatr Infect Dis 8:459-468, 1989. 7. Pretorius DH, Hayward I, Jones KL, Stamm E: Sonographic evaluation of pregnancies with maternal varicella infection. J Ultrasound Med 11:459-463, 1992. 8. Brunell PA: Varicella in pregnancy, the fetus, and the newborn: Problems in management. J Infect Dis 166(Suppl 1):$412-$417, 1992. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology I:166-172 (I 994) (C) 1994 Wiley-Liss, Inc. Amniotic Fluid Glucose Concentration: A Marker for Infection in Preterm Labor and Preterm Premature Rupture of Membranes Gary A. Dildy, Mark D. Pearlman, Leon G. Smith, Guillermo Tortolero-Luna, Sebastian Faro, and David B. Cotton Divisions ofMaternal-Fetal Medicine and Infectious Diseases, Department of Obstetrics and Gynecology, Baylor College ofMedicine, Houston, TX ABSTRACT Amniotic fluid Gram stain and culture have been utilized as laboratory tests ofmicrobial invasion of the amniotic cavity. The Gram stain ofamniotic fluid has a low sensitivity in the detection ofclinical infection or microbial invasion of the amniotic cavity, and amniotic fluid culture results are not immediately available for management decisions. Glucose concentration is used to diagnose infec- tion in other sites such as cerebrospinal fluid. Objective: The purpose of this study was to evaluate the usefulness of amniotic fluid glucose concentration in detecting microbial invasion of the amniotic cavity associated with preterm labor and preterm premature rupture ofmembranes. Methods: Amniocentesis was performed in 60 women with preterm labor and/or preterm prema- ture rupture of membranes. Gram stain and culture for Mycoplasma hominis, Ureaplasma urealyti- cum, aerobic, and anaerobic bacteria were performed. Subjects were studied prospectively for the development ofpositive amniotic fluid cultures and the development of clinical chorioamnionitis. Results: The diagnosis of clinical chorioamnionitis was made in 25% (15/60) of women entered into the study. Low amniotic fluid glucose concentration was considered < 15 mg/dl. The sensitivity, specificity, and positive predictive value of low amniotic, fluid glucose concentration to predict clinical chorioamnionitis were 73.3%, 88.1%, and 68.8% respectively, while positive amniotic fluid culture, had a sensitivity of 43.8%, specificity of 79.5%, and positive predictive value of43.8%. Conclusions: Amniotic fluid glucose concentration was more sensitive in predicting chorioam- nionitis than either Gram stain or culture.

ioamnionitis were 73.3%, 88.1%, and 68.8% respectively, while positive amniotic fluid culture, had a sensitivity of 43.8%, specificity of 79.5%, and positive predictive value of43.8%. Conclusions: Amniotic fluid glucose concentration was more sensitive in predicting chorioam- nionitis than either Gram stain or culture. Amniotic fluid glucose concentration was better in predicting clinical chorioamnionitis than predicting positive amniotic fluid culture results. Gesta- tional age-dependent normal ranges and pathologic conditions that may alter amniotic fluid glucose concentrations should be considered when interpreting amniotic fluid glucose values to diagnose microbial invasion ofthe amniotic cavity. (C) 1994 Wiley-Liss, Inc. KEY WORDS Chorioamnionitis, glucose, intra-amniotic infection nfection may be a significant etiologic factor in the development of preterm labor (PTL) and preterm premature rupture of" membranes (PPROM). Amniocentesis is sometimes employed to obtain amniotic fluid for analysis from patients presenting with PTL or PPROM in order to ex- clude infection as an underlying cause. 1-4 Amniotic fluid culture has been utilized for the diagnosis of microbial invasion ofthe amniotic cavity; however, the results of bacteriologic cultures may take in excess of 2 days. In addition, biologic properties of amniotic fluid such as bacteriolytic/static activity Address correspondence/reprint requests to Dr. Gary A. Dildy, Director, Perinatal Center, Utah Valley Regional Medical Center, 1034 North 500 West, Provo, UT 84604. Clinical Study Received September 21, 1993 Accepted December 31, 1993 AMNIOTIC FLUID GLUCOSE AND INFECTION DILDY ET AL. may potentially result in negative culture results, s The Gram stain is a rapid test that provides useful information for clinical management. However, at least 105 organisms/ml must be present to de- tect microbial invasion of the amniotic cavity by Gram stain, thus limiting the sensitivity of amni- otic fluid Gram stain to 45%.6 Low glucose concentration has been used as a marker for detecting infection in cerebrospinal fluid (CSF).7-1 Recently the use of amniotic fluid glu- cose concentration has been reported in the detec- tion of microbial invasion of the amniotic cavity in patients with PTL and PPROM.

f amni- otic fluid Gram stain to 45%.6 Low glucose concentration has been used as a marker for detecting infection in cerebrospinal fluid (CSF).7-1 Recently the use of amniotic fluid glu- cose concentration has been reported in the detec- tion of microbial invasion of the amniotic cavity in patients with PTL and PPROM. 11-4 The pur- pose ofthis study was to compare the value ofGram stain ofamniotic fluid and amniotic fluid glucose in the identification ofmicrobial invasion ofthe amni- otic cavity and the development of clinical chorio- amnionitis in patients presenting with PTL or PPROM. The relationship between presenting clinical diagnoses (PTL, PPROM, PTL with PPROM) and incidence of positive diagnostic tests was also evaluated. MATERIALS AND METHODS This study was conducted at Baylor College ofMed- icine's affiliated hospitals after approval by the Bay- lor College ofMedicine and Harris County Hospi- tal District institutional review boards. The period ofresearch was between February and December of 1990. Patients with the primary diagnosis of PTL or PPROM who were undergoing amniocentesis to rule out microbial invasion ofthe amniotic cavity were asked to enroll in the study. Patients were enrolled if chorioamnionitis was suspected as an underlying cause of PTL or PPROM, but not if a clinical diagnosis of chorioamnionitis had already been made. PTL was defined as regular painful uterine con- tractions with a frequency of at least three contrac- tions within 10 minutes or documented cervical change. PPROM was confirmed by speculum ex- amination with nitrazine and fern testing. The clin- ical diagnosis ofchorioamnionitis was made accord- ing to the criteria described by Gibbs and co- workerss as a syndrome of fever, with either maternal or fetal taehycardia, uterine tenderness, foul-smelling amniotic fluid, or peripheral blood leukocytosis. Patients with antecedent antimicrobial therapy within month ofpresentation were excluded from the study. Patients were enrolled in the study re- gardless of previous tocolytic therapy, which in- cluded terbutaline or magnesium sulfate. Patients with known glucose intolerance were excluded from the study. Amniocentesis was performed under local anes- thesia and with ultrasound guidance. Samples of amniotic fluid were submitted to the hospital labo- ratory for clinically indicated tests such as Gram stain, culture and sensitivity, and pulmonary matu- rity studies. The remainder of the amniotic fluid specimen was used for this study.

s was performed under local anes- thesia and with ultrasound guidance. Samples of amniotic fluid were submitted to the hospital labo- ratory for clinically indicated tests such as Gram stain, culture and sensitivity, and pulmonary matu- rity studies. The remainder of the amniotic fluid specimen was used for this study. Amniotic fluid was placed in a BBL Port-a-cul vial (Becton Dickinson and Company, Cockeysville, MD) and in separate ster- ile polypropylene tubes (Becton Dickinson Lab- ware, Lincoln Park, NJ) that were immediately refrigerated and transported for storage in a 70C freezer until glucose assay. Gram stain was performed with reagents (BBL Microbiology Systems, Cockeysville, MD) under standard conditions. Quantitation of both microor- ganisms and white blood cells (WBCs) were as follows: 0, none present; < -I-, to occasional/oil immersion field (OIF); +, 1/OIF; 2 +, 2-5/OIF; 3 +, 5-10/OIF; and 4+, > 10/OIF. Culture media were obtained from Regional Me- dia Laboratories, Inc. (REMEL, Lenexa, KS). Samples were placed on blood agar (Trypticase Soy Agar (TSA) with 5% sheep blood), chocolate agar, Thayer Martin agar (improved), and phenylethyl alcohol (PEA) agar with 5% sheep blood at 35C in a CO2 incubator. Amniotic fluid was plated on MacConkey's agar in a 35C non-CO2 incubator. Anaerobic cultures were plated on anaerobic KV blood agar [Centers for Disease Control (CDC) formulation] and anaerobic PEA blood agar (CDC formulation) and grown in an anaerobic chamber at 35C. A-7 medium was utilized for the isolation of Mycoplasma and Ureaplasma (Scott Lab) and incu- bated at 35C in CO2. Quantitative amniotic fluid cultures were performed with a Il/ml loop. Am- niotic fluid cultures were considered positive if Mycoplasma or Ureaplasma were isolated. Amniotic fluid glucose concentrations were de- termined in singlicate using the glucose oxidase method. Amniotic fluid glucose measurements were not used in the clinical management of patients. Subjects with positive amniotic fluid Gram stains were placed on antibiotics and then were delivered. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY AMNIOTIC FLUID GLUCOSE AND INFECTION DILDY ET AL. p<O.05 p=ns p=ns p=ns 80 70 60 Amniotic 50 Fluid Glucose 40 (mg/dl) 30 20 10 p<0.0001 0 Rare 1 /- 4/ (n=39) (n=11) (n=9) Neg. Pos. (n=52) (n=7) Neg. Pos. Neg. Pos.

tains were placed on antibiotics and then were delivered. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY AMNIOTIC FLUID GLUCOSE AND INFECTION DILDY ET AL. p<O.05 p=ns p=ns p=ns 80 70 60 Amniotic 50 Fluid Glucose 40 (mg/dl) 30 20 10 p<0.0001 0 Rare 1 /- 4/ (n=39) (n=11) (n=9) Neg. Pos. (n=52) (n=7) Neg. Pos. Neg. Pos. (n=44) (n=16) (n=47) (n=13) Amniotic Fluid Amniotic Fluid Amnitic Fluid Development of Gram Stain for Gram Stain Culture Clinical White Blood Cells for Organisms Chorioamnionitis Fig. I. Relationship of amniotic fluid glucose concentrations in patients with PTL and/or PPROM to amniotic fluid Gram stain, amniotic fluid culture, and development of clinical chorioamnionitis. Bars represent median values. 1+-4+ see text for explanation. Statistical analysis was performed using the Mann-Whitney test to compare non-parametric variables between the infected and non-infected groups. Fisher exact analysis (two-tailed test) was used to compare clinical outcomes between the groups with normal and low amniotic fluid glucose concentration. Diagnostic indices (sensitivity, spec- ificity, positive predictive value, and negative pre- dictive value) were used in the prediction of posi- tive amniotic fluid cultures and development of clinical chorioamnionitis. RESULTS Sixty women were enrolled in the study, 34 with PTL, 19 with PROM, and 7 with both PTL and PPROM. Median (range) maternal age was 25 years (15-41), and median (range) gravidity and parity were 2 (1-8) and (0-6), respectively. Subjects with positive amniotic fluid cultures had significantly lower (P < 0.05, Mann-Whit- ney) median amniotic fluid glucose concentrations (16.0 vs. 30.0 mg/dl), est.imated gestational age at delivery (30.6 vs. 33.4 weeks), amniocentesis-to- delivery interval (9.8 vs. 174.0 hours), and birth- weight (1,514 vs. 2,020 g). There was no statisti- ca1 difference in serum WBC (12.9 vs. 11.6 103/mm3) or estimated gestational age at presentation (30.4 vs. 32.0 weeks). Subjects who developed clinical chorioamnioni- tis had significantly lower median amniotic fluid glucose concentrations (9.0 vs. 33.0 mg/dl), esti- mated gestational age at presentation (29.7 vs. 32.0 weeks), estimated age at delivery (29.7 vs. 33.7 weeks), amniocentesis-to-delivery interval (8.5 vs. 203.0 hours), and birthweight (1,280 vs. 2,145 grams). There was no significant difference in se- rum WBC (14.8 vs. 11.3 103/mm3) at presen- tation.

3.0 mg/dl), esti- mated gestational age at presentation (29.7 vs. 32.0 weeks), estimated age at delivery (29.7 vs. 33.7 weeks), amniocentesis-to-delivery interval (8.5 vs. 203.0 hours), and birthweight (1,280 vs. 2,145 grams). There was no significant difference in se- rum WBC (14.8 vs. 11.3 103/mm3) at presen- tation. Figure depicts the relationship of amniotic fluid glucose concentration to amniotic fluid Gram stain for WBCs, amniotic fluid Gram stain for microorganisms, amniotic fluid culture, and the development ofclinical chorioamnionitis. A signif- icant difference (P < 0.05) was seen between pa- tients with negative Gram stain for WBCs versus those with + to 4+ WBCs. The median amniotic fluid glucose concentration for those patients with positive Gram stain for organisms was 17 mg/dl, while that of those with a negative Gram stain for organisms was 30 mg/dl. 168 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY AMNIOTIC FLUID GLUCOSE AND INFECTION DILDY ET AL. TABLE I. Laboratory tests to predict positive amniotic fluid culturesa Sensitivity + Amniotic fluid culture Specificity PPV NPV Amniotic fluid Gram stain for organisms Amniotic fluid Gram stain for WBCs Amniotic fluid glucose concentration < 15 mg/dl 43.8 100 100 82.7 68.8 79.1 55.0 87.2 43.8 79.5 43.8 79.S aPPV, positive predictive value; NPV, negative predictive value. TABLE 2. Laboratory tests to predict clinical chorioamnionitis Sensitivity + Clinical chorioamnionitis Specificity PPV NPV Amniotic fluid Gram stain for organisms Amniotic fluid Gram stain for WBCs Amniotic fluid culture Amniotic fluid glucose concentration < 15 mg/dl 21.4 95.2 60.0 78.4 64.3 81.0 52.9 87.2 60.0 88. 64.3 86.0 73.3 88. 68.8 90.2 aPPV, positive predictive value; NPV, negative predictive value. The sensitivity, specificity, and positive predic- tive value (PPV) ofamniotic fluid glucose to detect positive amniotic fluid culture were 43.8, 79.6, and 43.8%, respectively, at < 15 mg/dl; and 50.0, 79.6, and 47.1%, respectively, at < 16 mg/dl. The sensitivity, specificity, and PPV of an amniotic fluid glucose < 15 mg/dl to detect clinical chorio- amnionitis were 73.3, 88.1, and 68.8%, respec- tively. Raising the cutoff point to < 16 mg/dl changed the sensitivity, specificity, and PPV to 76.9, 85.1, and 58.8%, respectively. A cutoffvalue for low amniotic fluid glucose concentration was chosen as < 15 mg/dl for comparison with other laboratory techniques.

chorio- amnionitis were 73.3, 88.1, and 68.8%, respec- tively. Raising the cutoff point to < 16 mg/dl changed the sensitivity, specificity, and PPV to 76.9, 85.1, and 58.8%, respectively. A cutoffvalue for low amniotic fluid glucose concentration was chosen as < 15 mg/dl for comparison with other laboratory techniques. When cultures positive only for Mycoplasma and Ureaplasma were considered negative, the sensitivity and specificity of low am- niotic fluid glucose at a cutoff of 15 mg/dl to detect positive cultures were 37.5% and 75%, respec- tively. This was not appreciably different from values obtained by considering the isolation ofMy- coplasma and Ureaplasma as positive amniotic fluid cultures. Validity analysis of amniotic fluid glucose and other laboratory tests for the prediction of positive amniotic fluid cultures and clinical chorioamnioni- tis are depicted in Tables and 2. Amniotic fluid glucose appears to be more sensitive in detecting clinical chorioamnionitis than Gram stain for mi- croorganisms and Gram stain for WBCs and amni- otic fluid culture (Table 2). Low amniotic fluid glucose concentration appears to be a better predic- tor ofthe subsequent development ofclinical chori- oamnionitis (Table 2, sensitivity 73.3%) than the development of positive amniotic fluid culture (Table 1, sensitivity 43.8%). Of the 60 study subjects, 44 had normal amni- otic fluid glucose concentration and 16 had low amniotic fluid glucose concentration. There was a significantly increased incidence of clinical chorio- amnionitis (68.8 vs. 9.1%, P < 0.001, Fisher ex- act test) and delivery within 72 hours (75.0 vs. 38.6%, P < 0.004) in the group with low amni- otic fluid glucose concentration. The increased in- cidence ofpositive amniotic fluid cultures (43.8 vs. 20.5%) in the low amniotic fluid glucose group did not reach statistical significance. Among the 16 patients who were found to have positive amniotic fluid cultures, 8 cultures were positive only for Mycoplasma spp. (Table 3). Thus, at least one-half of patients with positive cultures would be expected to have negative Gram stains for organisms. There were nine false negative and no false positive Gram stains for microorganisms. Nine of the 16 patients with positive cultures did not develop clinical chorioamnionitis. Of these nine patients, eight demonstrated normal amniotic fluid glucose concentration.

d be expected to have negative Gram stains for organisms. There were nine false negative and no false positive Gram stains for microorganisms. Nine of the 16 patients with positive cultures did not develop clinical chorioamnionitis. Of these nine patients, eight demonstrated normal amniotic fluid glucose concentration. There was no statistical dif- ference between amniotic fluid glucose concentra- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 69 AMNIOTIC FLUID GLUCOSE AND INFECTION DILDY ET AL. TABLE 3. Results of 16 positive amniotic fluid cultures among 60 women with PTL and/or PPROM undergoing amniocentesis to rule out intra-amniotic infectiona Pt At amniocentesis Amniotic fluid Gram stain no. Diagnosis EGA (wks) Organisms WBC AF glucose Amniotic fluid culture mg/dl Clinical chorio. 3 PTL 29. NOS 2+ Ureaplasrna 4 No 5 PTL 32.4 NOS O+ Ureaplasrna 64 No 7 PROM 31. NOS O+ Ureaplasma 33 No 10 PROM 33.9 NOS O+ Ureaplasma 37 No II PROM 31.7 NOS O+ Ureaplasma, N. gonorrhoeae 6 Yes 12 PTL 17.6 NOS 2+ Ureaplasrna 9 Yes 14 PROM 30.3 < I+ yeast 0+ > l0 C,. albicans 18 No 21 PTL 30.4 NOS 4+ 1.8 X 104 t. ochracea 8 Yes 23 PROM, PTL 23.4 I+ GPC, 2+ GNR 4+ > l0 G. vaginalis 3 Yes 37 PROM 30.6 NOS < + Ureaplasrna 12 Yes 38 PROM 29.0 4+ GNDC 3+ > x l0 N. gonorrhoeae 17 Yes 41 PROM 33.3 + GNR < + 7.5 X 104 H. influenzae 15 No 45 PTL, PROM 26.3 4+ GPC I+ > l0 S. agalactiae 17 No 55 PROM, PTL 29.7 NOS 4+ Ureaplasrna Yes 56 PTL 29.4 3+ yeast, 4+ GPR 4+ Ureaplasrna, Mycoplasrna 40 No 58 PTL 3 I. < + GPR > x l0 Lactobacillus spp., 27 No < I+ yeast I+ 3 103 C. albicans aNOS, no organisms seen; GPC, Gram positive cocci; GNR, Gram negative rods; GNDC, Gram negative diplococci; GPR, Gram positive rods; EGA, estimated gestational age; AF, amniotic fluid. For organism and WBC counts, see text. tions in amniotic fluid cultures positive for Myco- plasma spp. versus bacterial species. No anaerobic organisms were isolated from culture. Among the 16 patients with positive amniotic fluid cultures, 7 had PPROM, 6 had PTL, and 3 had PPROM with PTL. There was no significant difference in amniotic fluid glucose concentration between patients with PPROM and PTL (19.7 vs. 25.3 mg/dl; P 0.58). Amniotic fluid WBC count was lower in associ- ation with PPROM than with PTL; however, this did not reach statistical significance (0.57 + vs. 2.08 + ;P 0.077). The relationships between primary clinical diagnoses (PTL, PPROM, or PTL with PPROM) and clinical tests or outcomes are shown in Figure 2.

25.3 mg/dl; P 0.58). Amniotic fluid WBC count was lower in associ- ation with PPROM than with PTL; however, this did not reach statistical significance (0.57 + vs. 2.08 + ;P 0.077). The relationships between primary clinical diagnoses (PTL, PPROM, or PTL with PPROM) and clinical tests or outcomes are shown in Figure 2. Among the 16 patients with low amniotic fluid glucose (<15 mg/dl), 6 had negative cultures and did not develop chorioamnionitis. One patient with Rh alloimmunization prematurely delivered 18 hours following amniocentesis. One patient deliv- ered in 6 hours with abruptio placentae, which was believed to be the underlying cause of PTL. An- other patient with PTL delivered in 91 hours with fetal distress and suspected pregnancy-induced hy- pertension. A patient with PPROM and oligohy- dramnios delivered after 232 hours with fetal dis- tress; the neonate was found to have congenital syphilis. Two patients with low amniotic fluid glu- cose and negative cultures had prolonged amnio- centesis-to-delivery times of 36 and 44 days. DISCUSSION The effects of leukocytes and bacteria on the glu- cose concentration of CSF in bacterial meningitis have been well established.7 Both activated leuko- cytes and bacteria are required to lower CSF glu- cose concentration. 8-10 Altered glucose transport across inflamed membranes has also been a pro- posed mechanism for hypoglycorrhachia in bacte- rial meningitis. 16 This understanding makes it plausible that the presence of bacteria andactivated leukocytes in amniotic fluid may result in decreased concentrations of glucose. Weiss and colleagues17 studied 2,295 samples of amniotic fluid obtained between the 14th and 42nd weeks of pregnancy. Mean amniotic fluid glucose concentration in normal pregnancy rose between the 14th and 17th weeks of pregnancy and then declined steadily toward term. They reported the tenth percentile of amniotic fluid glucose concen- tration for normal pregnancies at 28/29, 30/31, 32/33, 34/35, 36/37, and 38/39 weeks ofgestation to be 20, 18, 17, 16, 14, and 12 mg/dl, respec- tively. Amniotic fluid glucose concentration may be elevated in diabetic pregnancies and reduced in 170 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY AMNIOTIC FLUID GLUCOSE AND INFECTION DILDY ET AL.

ies at 28/29, 30/31, 32/33, 34/35, 36/37, and 38/39 weeks ofgestation to be 20, 18, 17, 16, 14, and 12 mg/dl, respec- tively. Amniotic fluid glucose concentration may be elevated in diabetic pregnancies and reduced in 170 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY AMNIOTIC FLUID GLUCOSE AND INFECTION DILDY ET AL. 60 50 40 o 30 2O 10 PTL n 341 Positive Positive Positive Low (<15mg/dl) Development Amniotic Fluid Amniotic Fluid Amniotic Amniotlc Fluid of Clinical Gram Stain for Gram Stain for Fluid Glucose Chorioamnionitis White Blood Cells Organisms Culture Concentration Fig. 2. Relationship of clinical diagnosis (PTL, PPROM, or PTL with PPROM) and incidence of positive clinical tests and development of clinical chorioamnionitis. The asterisk denotes P < 0.05, Fisher's exact test. pregnancies complicated by growth retardation or fetal malformation. 17-19 All papers published to date regarding amniotic fluid glucose concentration in detecting microbial invasion of the amniotic cavity have reported dif- ferent thresholds oflow amniotic fluid glucose. 11-14 Romero and colleagues11 studied 168 women with PTL and intact membranes. They found that a cutoff of <14 mg/dl resulted in a reasonable com- promise between the true positive and false positive rate. With this threshold, the sensitivity, specific- ity, and PPV were 86.9%, 91.7%, and 62.5%, respectively. Kirshon and associateslz studied 39 patients with PTL and/or PPROM. All nine pa- tients with microbial invasion of the amniotic cav- ity were noted to have amniotic fluid glucose con- centrations of < 10 mg/dl, and none with > 10 mg/dl developed chorioamnionitis. Kiltz and co- workers13 evaluated the amniotic fluid glucose con- centrations of 84 patients who were either in PTL, near term but not in labor, or at term in labor. They found that an amniotic fluid glucose concen- tration < 5 mg/dl had a PPV of 90% for positive culture and at > 20 mg/dl had a NPV of 98%. The PPV at < 15 mg/dl was 46%, and the NPV at > 15 mg/dl was 97%. The PPV for amniotic fluid glucose concentration in our study was 43.8% at < 15 mg/dl and 47.1% at < 16 mg/dl, similar to the findings of Kiltz and co-workers. In the clinical setting of PTL or PPROM, the ramifications of a false positive test include induc- tion of labor and premature delivery, which are highly undesirable.

V for amniotic fluid glucose concentration in our study was 43.8% at < 15 mg/dl and 47.1% at < 16 mg/dl, similar to the findings of Kiltz and co-workers. In the clinical setting of PTL or PPROM, the ramifications of a false positive test include induc- tion of labor and premature delivery, which are highly undesirable. Likewise, the results ofclinical action based upon a false negative test may include unwarranted tocolysis, delayed delivery and with- holding of antibiotic therapy, which may lead to adverse maternal and neonatal outcomes. We chose to use 15 mg/dl as a cutoff for normal amniotic fluid glucose concentration based upon the ability to predict positive cultures and the development of clinical chorioamnionitis. Analysis ofour data dem- onstrates that this threshold for amniotic fluid glu- cose concentration is comparable or superior to the other laboratory tests evaluated for detection ofclin- ical chorioamnionitis, but not for positive cultures. It should be recognized that immediate delivery for positive amniotic fluid Gram stain or culture may have a significant effect in reducing the number of subjects who would later develop clinical chorioam- nionitis. It appears that a cutofffor amniotic fluid glucose concentration at 15 mg/dl would be appropriate for general clinical use at this time. The use of a lower cutoff point would reduce the false positive rate at the expense ofsensitivity, especially at earlier gesta- tional ages. Studies examining the factor of gesta- tional age have not yet been conducted. Gestational age may play an important role in interpretation of INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY AMNIOTIC FLUID GLUCOSE AND INFECTION DILDY ET AL. these data, since an amniotic fluid glucose concen- tration of 14 mg/dl may actually be normal at 3 5 weeks but considered low at 32 weeks of gestation. This is demonstrated by our data in Table 3: four patients (numbers 14, 38, 41, and 45)were found to have normal (> 15 mg/dl) amniotic fluid glu- cose concentrations in the presence of both positive Gram stain for microorganisms and positive amni- otic fluid cultures. If gestational age were taken into consideration, these four patients would have been considered to have low amniotic fluid glucose. Further studies examining the effects of gestational age appear warranted.

ntrations in the presence of both positive Gram stain for microorganisms and positive amni- otic fluid cultures. If gestational age were taken into consideration, these four patients would have been considered to have low amniotic fluid glucose. Further studies examining the effects of gestational age appear warranted. Due to limitations in sensi- tivity and specificity, we recommend that amniotic fluid glucose concentration should not be used alone in making clinical decisions, but should be factored in with clinical signs, symptoms, and other labora- tory tests. REFERENCES 1. Garite TJ, Freeman RK: Chorioamnionitis in the pre- term gestation. Obstet Gynecol 59:539-545, 1982. 2. Miller JM, Hill GB, Welt SI, Pupkin MJ: Bacterial colonization of amniotic fluid in the presence of ruptured membranes. Am J Obstet Gynecol 137:451-458, 1980. 3. Bobitt JR, Hayslip CC, Damato JD: Amniotic fluid in- fection as determined by transabdominal amniocentesis in patients with intact membranes in premature labor. Am J Obstet Gynecol 140:947-952, 1981. 4. Zlatnik FJ, Cruikshank DP, Petzold CR, Galask RP: Amniocentesis in the identification ofinapparent infection in preterm patients with premature rupture of the mem- branes. J Reprod Med 9:656-660, 1984. 5. Bratlid D, Lindback T: Bacteriolytic activity of amniotic fluid. Obstet Gynecol 51:63-66, 1978. 6. Romero R, Emamian M, Quintero R, et al.: The value and limitations ofthe Gram stain examination in the diag- nosis of intraamniotic infection. Am J Obstet Gynecol 159:114--119, 1988. 7. Goldring S, Harford CG: Effect ofleukocytes and bacte- ria on glucose content ofthe cerebrospinal fluid in menin- gitis. Proc Soc Exp Biol Med 75:669-672, 1950. 8. Petersdorf RG, Garcia M, Swarner DR: Mechanism of hypoglycorrhachia in experimental pneumococcal menin- gitis. Proc Soc Exp Biol Med 102:669-672, 1959. 9. Petersdorf RG, Swarner DR, Garcia M: Synergistic ac- tion of pneumococci and leukocytes in lowering cere- brospinal fluid glucose. Proc Soc Exp Biol Med 103: 380-382, 1960. 10. Bretz G, Mauer AM: Glucose consumption by polymor- phonuclear leukocytes in the cerebrospinal fluid of pa- tients with bacterial meningitis. J Pediatr 70:767-771, 1967. 11. Romero R, Jimenez C, Lohda AK, et al.: Amniotic fluid glucose concentration: A rapid and simple method for the detection of intraamniotic infection in preterm labor. Am J Obstet Gynecol 163:968-974, 1990. 12. Kirshon B, Rosenfeld B, Mari G, Belfort M: Amniotic fluid glucose and intraamniotic infection.

7-771, 1967. 11. Romero R, Jimenez C, Lohda AK, et al.: Amniotic fluid glucose concentration: A rapid and simple method for the detection of intraamniotic infection in preterm labor. Am J Obstet Gynecol 163:968-974, 1990. 12. Kirshon B, Rosenfeld B, Mari G, Belfort M: Amniotic fluid glucose and intraamniotic infection. Am J Obstet Gynecol 164:818-820, 1991. 13. Kiltz RJ, Burke MS, Porreco RP: Amniotic fluid glucose concentration as a marker for intra-amniotic infection. Obstet Gynecol 78:619-622, 1991. 14. Gauthier DW, Meyer WJ, Bieniarz A: Correlation of amniotic fluid glucose concentration and intraamniotic infection in patients with preterm labor or premature rupture of membranes. Am J Obstet Gynecol 165:1105- 1110, 1991. 15. Gibbs RS, Castillo MS, Rodgers PJ: Management of acute chorioamnionitis. Am J Obstet Gynecol 136:709- 713, 1980. 16. Williams RDB: Alterations in the glucose transport mech- anism in patients with complications ofbacterial meningi- tis. Pediatrics 34:491-502, 1964. 17 Weiss PAM, Hofmann H, Winter R, Purstner P, Lich- tenegger W: Amniotic fluid glucose values in normal and abnormal pregnancies. Obstet Gynecol 65:333-339, 1985. 18. Spellacy WN, Buhi WC, Bradley B, Holsinger KK: Maternal, fetal and amniotic fluid levels ofglucose, insu- lin and growth hormone. Obstet Gynecol 41:323-331, 1973. 19. Drazancic A, Kuvacic I: Amniotic fluid glucose concen- tration. Am J Obstet Gynecol 120:40-48, 1974. 172 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology I:173-176 (I 994) (C) 1994 Wiley-Liss, Inc. Correlation Between the Clinical Diagnosis of Bacterial Vaginosis and the Results of a Proline Aminopeptidase Assay George H. Nelson and Janice L. Bacon Department ofObstetrics and Gynecology, University ofSouth Carolina School ofMedicine, Columbia, SC ABSTRACT Objective: The object of this study was to develop a simple and inexpensive test for detection of bacterial vaginosis (BV) in pregnant patients and to test its accuracy in a clinic population. Methods: We developed a modified proline aminopeptidase (PAMP) assay to detect BV and compared the results ofthe assay with the clinical diagnosis ofBV. Results: The results of the PAMP assay in 55 asymptomatic and 50 symptomatic subjects significantly correlated with a clinical diagnosis of BV. The prevalence of BV in the asymptomatic population was 42% (PAMP assay) and 38% (clinical diagnosis). In the symptomatic population, it was 50% (PAMP assay) and 54% (clinical diagnosis). The sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) of the PAMP assay were 86, 85, 86, 78, and 91%, respectively, in asymptomatic patients and 89, 96, 92, 96, and 88%, respectively, in symptomatic patients. Conclusions: The modified PAMP assay, which we describe, met our goals for simplicity, cost, and accuracy. We feel it could be best used as a screening test for BV in asymptomatic pregnant patients. (C) 1994 Wiley-Liss, Inc. KEY WORDS Enzyme method, pregnancy, vaginal inections ecause of growing evidence1-3 that bacterial vaginosis (BV) may be a contributing factor in preterm labor and delivery, a simple method for detection ofBV is highly desirable. Schoonmaker et al.4 recently described a new method for the diag- nosis of BV using a proline aminopeptidase (PAMP) assay. They compared the Gram stain with 2 PAMP assays using either L-proline B-naphthyamide or L-proline P-nitroanalide as sub- strate in the diagnosis ofBV. We have modified the proline nitroanalide assay and report here the re- suits ofthis modified assay in the diagnosis ofBV in asymptomatic and symptomatic pregnant patients.

compared the Gram stain with 2 PAMP assays using either L-proline B-naphthyamide or L-proline P-nitroanalide as sub- strate in the diagnosis ofBV. We have modified the proline nitroanalide assay and report here the re- suits ofthis modified assay in the diagnosis ofBV in asymptomatic and symptomatic pregnant patients. We chose the proline nitroanalide substrate because proline naphthyamide yields a carcinogenic end product. SUBJECTS AND METHODS Patients attended either the Teen or OB Clinic at Richland Memorial Hospital. All patients seen in these clinics were under the supervision of the au- thor (J.L.B.) who made the clinical diagnoses. A clinical diagnosis of BV, Trichomonas vaginitis (TV), a yeast vaginitis (YV), or no vaginitis (NV) was made using the following criteria: BV (3 or more of the following: presence of 20% or more Address correspondence/reprint requests to Dr. George H. Nelson, Two Richland Medical Park, Suite 208, Columbia, SC 29203. Clinical Study Received July 12, 1993 Accepted January 5, 1994 ENZYME ASSAY FOR BACTERIAL VAGINOSIS NELSON AND BACON clue cells, fishy odor with KOH, vaginal pH > 4.5, or thin gray-white discharge in patients who were diagnosed with BV alone; 2 or more of the following: presence of clue cells, fishy odor with KOH, or vaginal pH > 4.5 in patients with mixed infections); TV, presence of trichomonads on wet mount; YV, presence of hyphae on wet mount; and NV, absence of any diagnosis above. All study patients were tested for BV, TV, and YV. The PAMP assay was done in the following manner. Vaginal secretions were taken with a cot- ton-tipped applicator and placed in a 1.5 ml plastic centrifuge tube containing 1.0 ml ofnormal saline. The applicator tip was broken off and the tube was sealed with a plastic cap which comes attached to the tube. The tubes were placed in a refrigerator and were assayed within 3 days ofcollection. The appli- cator tip was removed from the tube with tweezers and the secretions were extracted into the saline using a gloved hand. The tubes were centrifuged for 5 min at 10,000g in a Fisher Scientific Microcentrifuge, Model 50-A (Fair Lawn, NJ). The supernatant was decanted and the following were added to the pellet in each tube: 50 txl solution (0.05 M Trizma, T-4003, pH 7.4) and 100 Il solution 2 (2 mg/ml ofL-pro- line p-nitroanalide, p-5267, made up in solution 1). A blank and a standard tube were run with each batch of sample tubes. The blank tube contained 100 Ixl each ofsolutions and 2.

the following were added to the pellet in each tube: 50 txl solution (0.05 M Trizma, T-4003, pH 7.4) and 100 Il solution 2 (2 mg/ml ofL-pro- line p-nitroanalide, p-5267, made up in solution 1). A blank and a standard tube were run with each batch of sample tubes. The blank tube contained 100 Ixl each ofsolutions and 2. The standard tube contained 100 1 solution (containing 20 mU of microsomal leucine aminopeptidase, L-5006, made up in solution 1) and 100 Ixl solution 2. All chem- icals were purchased from Sigma Chemical Com- pany (St. Louis, MO). The reason 100 I1 of solu- tion (blank tube) and 100 txl of enzyme (standard tube) are added instead of 50 I1 is because the volume of the pellet plus fluid remaining in the sample tube after centrifugation and decantation is about 50 I1. Therefore, all tubes contained approx- imately 200 Ixl total volume for color development. Leucine aminopeptidase was used as a standard be- cause PAMP is not available commercially and leucine aminopeptidase cleaves the proline from proline nitroanalide in a similar manner as PAMP. After the prepara.tion of the tubes (one or more samples, one blank, and one standard), they were incubated in a Dubnoff metabolic shaking incuba- tor (Fisher Scientific, Norcross, GA) at 37C for h. The samples were then removed, and the depth of the yellow color was compared visually to the standard and blank tubes. A positive test in a sam- ple tube is a depth of color > the color in the standard tube. This method differs from the original method described by Schoonmaker et al.4 in the following ways: 1. Sample collection, color development, and eval- uation take place in the same tube. The original method resuspends the cell pellet and transfers it to a microtiter plate well. 2. A water incubator is used instead of an air incu- bator. 3. Incubation time is reduced from 4 h to h. 4. A standard tube is used for comparison to the sample tube. The original article states: "A yel- low color indicated a diagnosis of bacterial vag- inosis; a clear color was scored as negative for bacterial vaginosis.''4 Statistical analyses were done using the X 2 test for independence. P < 0.05 was considered signif- icant. RESULTS All patients were pregnant at the time of the assay with gestational ages ranging from first trimester to 41 weeks gestation. No attempt was made to control for patient age or gestational age. Patients were asked ifthey had any complaints ofvaginal burning or itching or if they had an abnormal vaginal dis- charge.

ULTS All patients were pregnant at the time of the assay with gestational ages ranging from first trimester to 41 weeks gestation. No attempt was made to control for patient age or gestational age. Patients were asked ifthey had any complaints ofvaginal burning or itching or if they had an abnormal vaginal dis- charge. Those who responded negatively were listed as asymptomatic, while those who answered affir- matively were listed as symptomatic. A comparison of the results of the PAMP assay with the clinical diagnoses of 55 asymptomatic preg- nant patients is shown in Table 1. As can be seen, there was a highly significant correlation between the clinical diagnosis of BV and the PAMP assay. Likewise, Table 2 illustrates a highly significant correlation between the clinical diagnosis ofBV and the PAMP assay in symptomatic patients. Of the 105 subjects studied, 41 (39%) had BV alone, 6 (5.7%) had BV plus TV, (1%) had BV plus TV plus YV, 5 (4.8%) had TV alone, 9 (8.6%) had YV alone, and (1%) had TV plus YV. There- fore, in this population, 8 (7.6%) had mixed infec- tions. Interestingly, all mixed infections were found in symptomatic patients. 174 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY ENZYME ASSAY FOR BACTERIAL VAGINOSIS NELSON AND BACON TABLE I. Correlation between the clinical diagnosis of BV, NV, TV, or YV and the PAMP assay in asymptomatic pregnant patients Clinical diagnosis (no. of patients) PAMP assay BV positive* BV negative* Postive 18 5 Negative 3 29 BV alone NV TV YV Positive 18 4 0 Negative 3 25 2 2 *N 55; 2 26; P < 0.001. TABLE 2. Correlation between the clinical diagnosis of BV, NV, TV, or YV and the PAMP assay in symptomatic pregnant patients Clinical diagnosis (no. of patients) PAMP assay BV positive* BV negative* Positive 24 Negative 3 22 BV BV BV + TV TV alone +'IV +YV NV TV YV +YV Positive 18 Negative 2 5 0 0 0 0 13 7 *N 50; X 35; P < 0.001. TABLE 3. Sensitivity (Sen), specificity (Spec), accuracy (Acc), PP, and NP of the PAMP assay in asymptomatic and symptomatic pregnant patientsa Asymptomatic Symptomatic Sen (%) 86 89 Spec (%) 85 96 Acc (%) 85 92 PP (%) 78 96 NPV (%) 91 88 aSen true positive (TP) divided by TP + false negative (FN); Spec true negative (TN) divided by TN + false positive (FP); Acc= TP + TN divided by TP + TN + FP + FN; PPV TP divided by TP + FP; NPV TN divided by TN + FN. Table 3 illustrates the sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) of the PAMP assay.

negative (FN); Spec true negative (TN) divided by TN + false positive (FP); Acc= TP + TN divided by TP + TN + FP + FN; PPV TP divided by TP + FP; NPV TN divided by TN + FN. Table 3 illustrates the sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) of the PAMP assay. DISCUSSION When this study was established, our intention was to set up a PAMP assay that was easy, inexpensive, required no sophisticated equipment, demanded minimum technical expertise, and was reasonably accurate. The procedure described by Schoonmaker et al.4 seemed to serve as an excellent starting point. The modified procedure as described here required taking a specimen of vaginal fluid with a cotton swab, extracting the fluid into saline in a centrifuge tube, a 5 min centrifugation, decantation, making measurement each of 50 I1 and 100 Ixl, a h incubation in an ordinary water bath, and visually reading a positive or negative test comparing the sample to a standard. In our study, the sensitivity of the PAMP assay was 88% overall, which is comparable to that re- ported for this assay by Shoonmaker et al.4 (93%) and Thomason et al. s (81%). Shoonmaker et al.4 compared the PAMP assay with the Gram stain, while Thomason et al. 5 compared the assay with the clinical diagnosis of BV. The data reported in Table 3 assume that the clinical diagnosis ofBV is correct in every instance. Since the clinical diagnosis of BV is based on sub- jective evaluation, it is entirely possible that the false-positive and the false-negative measurements reported for the PAMP assay may not be truly false-positive and false-negative values. Therefore, the accuracy of the PAMP assay may actually be better than we report here. This would seem appropriate in light of the report by Eschenbach et al.6 They report that in 311 patients diagnosed with BV by Gram stain criteria, 29% had a homogeneous discharge, 43% had a fishy odor with KOH, 97% had a pH > 4.7, 81% had the presence of clue cells, and 78% had 20% or more clue cells. However, the PPV of the assay may be lower in a popula- tion of patients with a lower prevalence of BV. Perhaps it might be more appropriate not to use terms such as sensitivity, specificity, and PPV when conducting a study such as this. Neither of the techniques used (clinical diagnosis, PAMP as- say) provides a definitive diagnosis. Perhaps we should only conclude that the techniques appear to be evaluating the same process.

s it might be more appropriate not to use terms such as sensitivity, specificity, and PPV when conducting a study such as this. Neither of the techniques used (clinical diagnosis, PAMP as- say) provides a definitive diagnosis. Perhaps we should only conclude that the techniques appear to be evaluating the same process. The X 2 test for independence illustrates that the odds the 2 mea- surements (clinical diagnosis, PAMP assay)are totally independent ofone another are less than in 1,000. While the assay could be used in a variety of settings, we feel that a definite role would be its INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 175 ENZYME ASSAY FOR BACTERIAL VAGINOSIS NELSON AND BACON potential use as a screening test in asymptomatic pregnant clinic patients. This may be particularly applicable to public health settings. In our study in this population, 38% (by clinical diagnosis) and 42% (by positive PAMP assay) were found to be harboring the organisms associated with BV. While the assay described takes longer to com- plete than a clinical diagnosis by a physician, the assay can be performed by non-physician per- sonnel with minimal training in laboratory medi- cine. In addition, it appears that the assay will detect BV in the asymptomatic patient who would not normally be checked for BV in a clinical set- ting. This may be particularly pertinent in view of the recent data reported by Riduan et al.7 They showed that preterm delivery was more prevalent in patients with BV diagnosed at 16-20 weeks ges- tation but not in patients diagnosed with BV at 28-32 weeks gestation. Perhaps screening a large group of asymptomatic patients with the PAMP assay at 16-20 weeks gestation would yield promis- ing results. REFERENCES 1. Martius J, Krohn MA, Hillier SL, et al.: Relationships of vaginal Lactobacillus species, cervical Chlamydia trachoma- t/s, and bacterial vaginosis to preterm birth. Obstet Gyne- col 71:89-95, 1988. 2. Gravett MG, Nelson HP, DeRouen T, et al.: Independent associations of bacterial vaginosis and Chlamydia trachoma- t/s infection with adverse pregnancy outcome. JAMA 256: 1899-1903, 1986. 3. Gravett MG, Hummel D, Eschenbach DA, et al.: Pre- term labor associated with subclinical amniotic fluid infec- tion and with bacterial vaginosis. Obstet Gynecol 67:229- 237, 1986. 4. Schoonmaker JN, Lunt BC, Lawellin DW, et al.: A new proline aminopeptidase assay for diagnosis ofbacterial vag- inosis. Am J Obstet Gynecol 165:737-749, 1991. 5.

chenbach DA, et al.: Pre- term labor associated with subclinical amniotic fluid infec- tion and with bacterial vaginosis. Obstet Gynecol 67:229- 237, 1986. 4. Schoonmaker JN, Lunt BC, Lawellin DW, et al.: A new proline aminopeptidase assay for diagnosis ofbacterial vag- inosis. Am J Obstet Gynecol 165:737-749, 1991. 5. Thomason JL, Gelbart SM, Wilcoski LM, et al.: Proline aminopeptidase activity as a rapid diagnostic test to confirm bacterial vaginosis. Obstet Gynecol 71:607-611, 1988. 6. Eschenbach DA, Hillier S, Critchlow MS, et al.: Diagno- sis and clinical manifestations of bacterial vaginosis. Am J Obstet Gynecol 158:819-828, 1988. 7. Riduan JM, Hillier SL, Utomo B, et al.: Bacterial vagino- sis and prematurity in Indonesia: Association in early and late pregnancy. AmJ Obstet Gynecol 169:175-178, 1993. 176 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology I: 177-181 (I 994) (C) 1994 Wiley-Liss, Inc. Screening for Chlamydia trachomatis in Low-Risk Obstetric Patients Robert K. Gribble, Jean M. Ricci-Goodman, and Richard L. Berg Department ofObstetrics and Gynecology (R.K.G., J.M.R.-G.) and Department ofMedical Biostatistics ofthe Research Division (R.L.B.), Marshfield Clinic, Marshfield, WI ABSTRACT Objective: The purpose of this study was to evaluate the prevalence of Chlamydia trachomatis in our rural obstetric population and assess the appropriateness of selective vs. universal prenatal screen- ing. Methods: Between April 1, 1991 and May 1, 1993, 1,587 patients were screened at their first prenatal visit using a C. trachomatis antigen test. Patients who were unmarried, younger than 20 years of age, or had a history of a previous sexually transmitted disease (STD) were classified as being at high risk for C. trachomatis. All others were considered low risk for C. trachomatis. Results: The overall prevalence of C. trachomatis was 2.0%. There was a significant difference (P < 0.001) in the 1,128 patients considered low risk [0.5%, 95% confidence interval (CI) 0.2-1.2] compared to the 459 patients with one or more identifiable risk factors (5.7%, 95% CI 3.7-8.2). Conclusions:Routine prenatal screening for C. trachomatis in our population is not appropriate for low-risk patients. (C) 1994 Wiley-Liss, Inc. KEY WORDS Pregnancy, infection, prevalence hlamydia trachomatis is the most common bac- terial sexually transmitted disease (STD) in the United States. Many reports have described serious complications that may result from C. trachomatis infection during pregnancy. C. trachomatis has been implicated in premature rupture ofmembranes and premature labor and associated with low birth weight, stillbirth, and neonatal death. 1-4 In the postpartum period, C. trachomatis has been identi- fied as a cause of late postpartum endometritis, s It is important to note, however, that not all studies of C. trachomatis in pregnancy have supported the association of C. trachomatis with any or all ofthese complications.

, stillbirth, and neonatal death. 1-4 In the postpartum period, C. trachomatis has been identi- fied as a cause of late postpartum endometritis, s It is important to note, however, that not all studies of C. trachomatis in pregnancy have supported the association of C. trachomatis with any or all ofthese complications. Some have found no linkage, while others found an association only in the subset of C. trachomatis-infected patients who are also positive for C. trachomatis IgM antibodies. 6-1 Less con- troversial is the neonatal infectious morbidity asso- ciated with maternal C. trachomatis infection. Of neonates born to mothers with untreated C. tra- chomatis, it is estimated that 30% will develop C. trachomat# ophthalmia neonatorum and 35% will' develop Chlamydia pneumonia. 12 Although these are usually benign and easily treated illnesses, ap- proximately 25% of infants with pneumonia re- quire hospitalization and one report has described permanent respiratory sequelae in infants with pneumonia. 13--14 The fact that C. trachomatis screening of preg- nant patients and treatment of those infected can eliminate most or all ofthe complications described above has led to the recommendation for expanded C. trachomatis screening as a worthwhile preven- tive strategy. The Center for Disease Control (CDC), in a 1985 bulletin, recommended that pregnant women be screened for C. trachomatis if Address correspondence/reprint requests to Dr. Robert K. Gribble, Department of Obstetrics and Gynecology, Marshfield Clinic, 1000 North Oak Avenue, Marshfield, W154449. Clinical Study Received September 27, 1993 Accepted January 3, 1994 PRENATAL CHLAMYDIA SCREENING GRIBBLE ET AL. they were unmarried, younger than 20 years of age, had a history of STDs, or had multiple sexual partners. 15 In 1989, new recommendations were published calling for universal rather than selective prenatal C. trachomatis screening. 16 The Wiscon- sin Department of Health and Social Services and the Wisconsin Association for Perinatal Care have also recommended that all pregnant women be screened for C. trachomatis. Although the cost (and ease) of C. trachomatis screening has been reduced by the use oflaboratory techniques that do not require cell culture, it is still significant. Furthermore, it has been shown that the prevalence of C. trachomatis varies from one geographic area to the next and within geographic areas it varies according to patient risk fac- tors.

trachomatis screening has been reduced by the use oflaboratory techniques that do not require cell culture, it is still significant. Furthermore, it has been shown that the prevalence of C. trachomatis varies from one geographic area to the next and within geographic areas it varies according to patient risk fac- tors. 1,5,7,18 The purpose of this study was to de- termine whether it was appropriate, in our particu- lar patient population, to comply with the recommendation to screen all pregnant patients for C. trachomatis or if we should continue our policy of selective screening based on certain risk factors. SUBJECTS AND METHODS Between April 1, 1991 and May 1, 1993, all pa- tients who enrolled for routine prenatal care at a large multispecialty clinic in central Wisconsin were considered eligible for this study. Patients who had received C. trachomatis screening at other facilities were excluded. Each patient was interviewed at her first prena- tal visit regarding maternal age, marital status, and history ofSTDs. Patients were classified as being at high risk for C. trachomatis if they were younger than 20 years of age, unmarried (single, divorced, separated, or widowed), or had a history of a docu- mented STD. Classification as having had a previ- ous STD required culture-proven gonorrhea, chlamydia, or herpes, a positive serologic test for syphilis or human immunodeficiency virus, or gen- ital condylomata. Patients who had no identified risk factors were defined as being at low risk for C. trachomatis. All enrolled patients, regardless of identified risk factors, were tested for C. trachomat# at their first prenatal visit. The Syva MicroTrakTM Chlamy- dia Enzyme Immunoassay (EIA) Specimen Collec- tion Kit (Syra Company, San Jose, CA) was used to obtain and transport the specimen as per the manu- facturer's recommendations. Specimens were im- mediately transported to our in-house regional lab- oratory which performs Chlamydia EIA studies on a daily basis. Specimens are tested according to the Syva Company protocol and utilize the Syva Mi- croTrakTM II Chlamydia EIA kit along with a Den- ley Wellwash 4 autowasher (Denley Instruments, Sussex, England) and Syva autoreader. Results are reported as either positive or negative for Chlamy- dia antigen. Patients in the low-risk group who had positive antigen tests were offered confirmatory cell cul- tures.

Mi- croTrakTM II Chlamydia EIA kit along with a Den- ley Wellwash 4 autowasher (Denley Instruments, Sussex, England) and Syva autoreader. Results are reported as either positive or negative for Chlamy- dia antigen. Patients in the low-risk group who had positive antigen tests were offered confirmatory cell cul- tures. A specimen from the endocervix was ob- tained with a sterile swab, placed in a sucrose- phosphate broth, and transported at 4C to our in-house laboratory. Specimens were inoculated into McCoy cells and incubated for 48-72 h. Mono- clonal antibody for C. trachomatis was used for staining and the coverslips examined under fluores- cent microscopy. Estimates ofthe prevalence of C. trachomatis are presented by risk group along with exact binomial confidence limits. A comparison of the prevalence for high-risk vs. low-risk patients is based upon an exact chi-square test (CYTEL Software Corpora- tion: Software for Exact Nonparametric Inference User's Manual, Cambridge, MA, 1989). The as- sociation of 3 risk factors (marital status, age, and history of STD) with C. trachomatis infection was examined through a stepwise logistic regression analysis (SAS Institute, Inc.: SAS/STAT User's Guide, Version 6, Cary, NC, 1989). Results were deemed statistically significant with P < 0.05. RESULTS Of the 1,598 eligible prenatal patients, 11 patients did not have C. trachomatis studies performed due to patient refusal or the provider not obtaining a specimen. Patient history and C. trachomatis test results were available on the remaining 1,587 pa- tients. By history, 1,128 (71%) of these patients were determined to be at low risk for C. trachomatis and 459 (29%) at high risk. In the low-risk group, 6 had a positive C. trachomatis antigen study, a prevalence of 0.5% [95% confidence interval (CI) 0.2-1.2]. In the high-risk group, 26 had a posi- tive C. trachomatis antigen study, a prevalence of 5.7% (95% CI 3.7-8.2). The difference between these 2 groups was highly significant (P < 0.001). Of the 6 patients in the low-risk group who tested positive, 3 agreed to have confirmatory cell 178 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY PRENATAL CHLAMYDIA SCREENING GRIBBLE ET AL. TABLE I.

tudy, a prevalence of 5.7% (95% CI 3.7-8.2). The difference between these 2 groups was highly significant (P < 0.001). Of the 6 patients in the low-risk group who tested positive, 3 agreed to have confirmatory cell 178 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY PRENATAL CHLAMYDIA SCREENING GRIBBLE ET AL. TABLE I. Prenatal Chlamydia infection by risk factors with 95% confidence intervals History of any STD Currently Age No married group Frequency Percent 95% CI Yes Frequency Percent 95% CI Yes 20+ 6/I,128 0.5 0.1, 1.0 <20 0/23 0.0 0.0, 14.9 No 20+ 7/167 4.4 1.7, 8.5 <20 0/I 14 8.8 4.3, 15.6 1/93 I.I 0.0, 5.9 0/2 0.0 0.0, 84.2 7/52 13.5 5.6, 25.9 I/8 12.5 0.3, 52.7 TABLE 2. Prenatal Chlamydia screening: logistic regression analysis Risk factor Odds ratio (95% CI) P Unmarried 7. (2.7, 18.9) < 0.001 Age (years) 0.89 (0.82, 0.98) 0.016 History of STD 2.8 (I.2, 6.5) 0.017 cultures. Two ofthe 3 cultures were positive for C. trachomatis. Prevalence for each combination of the 3 risk factors is shown in Table 1. Small numbers in some cells were reflected by wide CIs. In particular, although none of the 23 patients whose only risk factor was maternal age younger than 20 years had a positive study, the small number of patients pre- cludes drawing conclusions about this group. A stepwise logistic regression model was used to fur- ther assess the relative importance of 3 risk factors. Stepwise logistic regression analysis with maternal age as a continuous variable showed all 3 risk fac- tors to be significant (Table 2). DISCUSSION Several studies have evaluated the question of the cost effectiveness of C. trachomatis screening dur- ing pregnancy. 12,19,20 Although different method- ologies were used, the prevalence of C. trachomatis at which screening ofa pregnant population became cost effective ranged from 5% to 6%. The major difficulty in determining cost effectiveness is the controversy and uncertainty regarding the impor- tance of C. trachomatis in the various pregnancy complications discussed previously. Screening costs and the cost of caring for patients with C. trachom- atis-related illnesses also vary. With all of this in mind, we believe it is reasonable to assume that C. trachomatis screening is not cost effective in a very low-prevalence population such as our low-risk pa- tients. Based on the results ofthis study, we propose that the vast majority ofthe benefit of C.

C. trachom- atis-related illnesses also vary. With all of this in mind, we believe it is reasonable to assume that C. trachomatis screening is not cost effective in a very low-prevalence population such as our low-risk pa- tients. Based on the results ofthis study, we propose that the vast majority ofthe benefit of C. trachoma- tis screening will be gained, at a much lower cost, by returning to selective rather than universal screening. This policy is also consistent with a re- cently published Canadian study. 18 One of the criticisms of selective screening for C. trachomatis is that providers would neglect to do an adequate assessment of risk factors and thus not screen all patients at risk. We decided to simplify risk factor assessment by using only 3 of the 4 risk factors described in the 1985 CDC recommenda- tions. We did not include multiple sexual partners as a risk factor because we were concerned about our ability to objectively assess the accuracy of this history. By limiting risk factors, for the purpose of determining the need to screen for C. trachomatis, the process becomes more straightforward and pro- video" compliance will likely improve. We have also subjected the 3 risk factors utilized for this study to logistic regression analysis which docu- mented the importance of each ofthe 3 risk (actors. Finally, we wish to emphasize that this risk factor assessment should not preclude the use of a provid- er's clinical judgment regarding C. trachomatis screening in individual patients. Another concern about C. trachomatis screening is the particular screening test being used. Cell culture remains the "gold standard" but is more costly and associated with greater transport difficul- ties than are non-culture techniques. Therefore, these non-culture assays have become the most com- mon methods ofC. trachomat# screening. Although they have excellent sensitivity and specificity, their specificity is still less than that of cell culture.21 This is not a significant problem in high-preva- lence populations, and it is not cost effective to do confirmatory cell cultures for patients in this group who have positive antigen tests. It does, however, INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 179 PRENATAL CHLAMYDIA SCREENING GRIBBLE ET AL. become more important in low-prevalence popula- tions. For example, an antigen test with a specific- ity of 98% and a sensitivity of 90% when applied to a population with a 2.5% disease prevalence has a 54% positive predictive value.

DISEASES IN OBSTETRICS AND GYNECOLOGY 179 PRENATAL CHLAMYDIA SCREENING GRIBBLE ET AL. become more important in low-prevalence popula- tions. For example, an antigen test with a specific- ity of 98% and a sensitivity of 90% when applied to a population with a 2.5% disease prevalence has a 54% positive predictive value. 19 This led to our decision to offer confirmatory cell cultures to low- risk patients with positive antigen tests. That of 3 of these confirmatory cultures was negative is con- sistent with the expected low positive predictive value. The newer DNA polymerase methods are reported to have the same specificity as cell culture and their future use may thus eliminate this con- cern. 22 Selective screening requires that one has first determined, as was done in this study, the preva- lence in the particular population that is presumed to be low risk. It should not be assumed that preva- lences in different populations are comparable. For example, even our high-risk patients had a lower prevalence of C. trachomatis than is usually re- ported for high-risk groups. This may be due to our particular patient demographics, including a rural, central Wisconsin population in which 98% of prenatal patients are Caucasian, 7 5% have pri- vate health insurance, and 8 5% have at least a high school education. In any case, it emphasizes the need to base screening policies on prevalence mea- surements from the practitioner's own patient group. Once it is determined that the population desig- nated as being at low risk does not have a preva- lence that justifies, on a cost-effectiveness basis, screening for C. trachomatis, the.next issue is how one would know if that prevalence changes signifi- cantly in the future. One option is to periodically screen low-risk patients until enough have been tested to indicate that the prevalence remains lower than the level at which screening is cost effective. A second option is to rely on statistics generated by the State Health Department (in those states where C. trachomatis is a reportable disease), although it is often difficult to draw conclusions about a particu- lar patient population from the .more general statis- tics in these data. We have chosen a third option which is to monitor the. prevalence of C. trachoma- tis in the high-risk pregnancy patients and assume that it mirrors the prevalence in low-risk patients.

h it is often difficult to draw conclusions about a particu- lar patient population from the .more general statis- tics in these data. We have chosen a third option which is to monitor the. prevalence of C. trachoma- tis in the high-risk pregnancy patients and assume that it mirrors the prevalence in low-risk patients. Therefore, if the prevalence increases in high-risk patients, we will need to reevaluate low-risk pa- tients to determine if they have also experienced a concurrent and significant increase. We also moni- tor the number of positive C. trachomatis studies in infants with conjunctivitis or pneumonia, and we would reevaluate the screening protocol if there were more than a minimal number of positive tests (there were none over the time span of this study). In summary, in an environment oflimited health care resources, we believe it is appropriate to use selective rather than universal prenatal screening for C. trachomatis. Prerequisites for this approach are a simple method for assessing risk, a knowledge of the C. trachomatis prevalence in the particular patient population determined to be low risk, and a mechanism to follow that population for any signif- icant change in prevalence. ACKNOWLEDGMENTS The authors acknowledge the significant assistance of Linda Stini in data collection and coordination. REFERENCES 1. Martin DH, Koutsky L, Eschenbach DA, Daling JR, Alexander ER, Benedetti JK, Holmes KK: Prematurity and perinatal mortality in pregnancies complicated by maternal Chlamydia trachomatis infections. JAMA 247: 1585-1588, 1982. 2. Gravett MG, Nelson HP, DeRouen T, Critchlow C, Eschenbach DA, Holmes KK: Independent associations ofbacterial vaginosis and Chlamydia trachomatis infection with adverse pregnancy outcome. JAMA 256:1899- 1903, 1986. 3. Alger LS, Lovchik JC, Hebel JR, Blackmon LR, Cren- shaw MC: The association of Chlamydia trachomatis, Neisseria gonorrhoeae, and group B streptococci with pre- term rupture of the membranes and pregnancy outcome. Am J Obstet Gynecol 159:397-404, 1988. 4. Johns Hopkins Study of Cervicitis and Adverse Preg- nancy Outcome: Association ofChlamydia trachomatis and Mycoplasma hominis with intrauterine growth retardation and preterm delivery. Am J Epidemiol 129:1247-1257, 1989. 5. Wager GP, Martin DH, Koutsky L, Eschenbach DA, Daling JR, Chiang WT, Alexander ER, Holmes KK: Puerperal infectious morbidity: Relationship to route of delivery and to antepartum Chlamydia trachomatis infec- tion.

a hominis with intrauterine growth retardation and preterm delivery. Am J Epidemiol 129:1247-1257, 1989. 5. Wager GP, Martin DH, Koutsky L, Eschenbach DA, Daling JR, Chiang WT, Alexander ER, Holmes KK: Puerperal infectious morbidity: Relationship to route of delivery and to antepartum Chlamydia trachomatis infec- tion. Am J Obstet Gynecol 138:1028-1033, 1980. 6. Heggie AD, Lumicao GG, Stuart LA, Gyves MT: Chlamydia trachomatis infection in mothers and infants. A prospective study. Am J Dis Child 135:507-511, 1981. 7. Hardy PH, Hardy JB, Nell EE, Graham DA, Spence MR, Rosenbaum RC: Prevalence of six sexually trans- mitted disease agents among pregnant inner-city ado- lescents and pregnancy outcome. Lancet 2:333-337, 1984. 8. FitzSimmons J, Callahan C, Shanahan B, Jungkind D: 180 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOIOGY PRENATAL CHLAMYDIA SCREENING GRIBBLE ET AL. Chlamydial infections in pregnancy. J Reprod Med 31: 19-22, 1986. 9. Harrison HR, Alexander ER, Weinstein L, Lewis M, Nash M, Sim DA: Cervical Chlamydia trachomatis and mycoplasmal infections in pregnancy. Epidemiology and outcomes. JAMA 250:1721-1727, 1983. 10. Berman SM, Harrison HR, Boyce WT, Haffner WJ, Lewis M, Arthur JB: Low birth weight, prematurity, and postpartum endometritis. Association with prenatal cervical Mycoplasma hominis and Chlamydia trachomatis infections. JAMA 257:1189-1194, 1987. 11. Sweet RL, Landers DV, Walker C, Schachter J: Chlamy- dia trachomatis infection and pregnancy outcome. Am J Obstet Gynecol 156:824--833, 1987. 12. Schachter J, Grossman M: Chlamydial infections. Annu Rev Med 32:45-61, 1981. 13. Schachter J, Sweet RL, Grossman M, Landers D, Rob- bie M, Bishop E: Experience with the routine use of erythromycin for chlamydial infections in pregnancy. N EnglJ Med 314:276-279, 1986. 14. Weiss SG, Newcomb RW, Beem MO: Pulmonary as- sessment of children after chlamydial pneumonia of in- fancy. J Pediatr 108:659-664, 1986. 15. Anon: Chlamydia trachomatis infections. Policy guidelines for prevention and control. MMWR Morb Mortal Wkly Rep 34:53S-75S, 1985. 16. Anon: 1989 sexually transmitted diseases treatment guide- lines (published erratum appears in MMWR Morb Mor- tal Wkly Rep 38:664, 1989). MMWR Morb Mortal Wkly Rep 38:1-43, 1989. 17. Glover DD, Gordon H, Moore G, Larsen B: Chlamydia trachomatis antigen prevalence among pregnant women in West Virginia. WV Med J 88:548-551, 1992. 18.

exually transmitted diseases treatment guide- lines (published erratum appears in MMWR Morb Mor- tal Wkly Rep 38:664, 1989). MMWR Morb Mortal Wkly Rep 38:1-43, 1989. 17. Glover DD, Gordon H, Moore G, Larsen B: Chlamydia trachomatis antigen prevalence among pregnant women in West Virginia. WV Med J 88:548-551, 1992. 18. Alary M, Joly JR, Moutquin J-M, Labrecque M: Strat- egy for screening pregnant women for chlamydial infec- tion in a low-prevalence area. Obstet Gynecol 82:399- 404, 1993. 19. Carroll JC: Chlamydia trachomatis during pregnancy. To screen or not to screen? Can Fam Physician 39:97-102, 1993. 20. Nettleman MD, Bell TA: Cost-effectiveness of prenatal testing for Chlamydia trachomatis. Am J Obstet Gynecol 164:1289-1294, 1991. 21. Baselski VS, McNeeley SG, Ryan G, Robison M: A comparison of nonculture-dependent methods for detec- tion of Chlamydia trachomatis infections in pregnant women. Obstet Gynecol 70:47-52, 1987. 22. Hosein IK, Kaunitz AM, Craft SJ: Detection of cervical Chlamydia trachomatis and Neisseria gonorrhoeae with deox- yribonucleic acid probe assays in obstetric patients. Am J Obstet Gynecol 167:588-591, 1992. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:182-187 (I 994) (C) 1994 Wiley-Liss, Inc. Rapid Polymerase Chain Reaction-Based Test for the Detection of Female Urogenital Chlamydial Infections Harold C. Wiesenfeld, Michael Uhrin, Bruce W. Dixon, and Richard L. Sweet Department ofObstetrics, Gynecology, and Reproductive Sciences, Magee-Womens Hospital (H.C.W., M.U., R.L.S.), and Department ofMedicine (B.W.D.), University ofPittsburgh School ofMedicine, Pittsburgh, PA ABSTRACT Objective: The purpose of this study was to evaluate the Amplicor Chlamydia trachomatis Test (Roche Molecular Systems, Branchburg, NJ), a polymerase chain reaction (PCR)-based technique, as a screening test for the detection offemale urogenital C. trachomatis infections, comparing it to an enzyme immunoassay method. Methods: Endocervical specimens for PCR and Chlamydiazyme (Abbott Laboratories, North Chicago, IL) analysis were obtained from 328 unselected patients at the outpatient Sexually Trans- mitted Diseases Clinic at the Allegheny County Health Department, Pittsburgh, PA. In addition, urethral swabs for PCR analysis were obtained from 256 ofthese patients. Results: By PCR analysis, the prevalence ofurogenital chlamydial infections was 15.6% and that ofcervical chlamydial infections was 10.7%. The sensitivity ofPCR in the detection ofendocervical chlamydial infections was 89.7% and the specificity was 100%. The positive and negative predictive values ofPCR were 100% and 99%, respectively. The sensitivity ofChlamydiazyme in the detection of cervical infections was 61.5% and the specificity was 99.7%, with a positive predictive value of 96.0%. Among all patients with urogenital chlamydial infections, concomitant infections in the urethra and cervix occurred in 52.5%, whereas the urethra or cervix was solely infected in 35.0% and 12.5%, respectively. Conclusions: This PCR-based technique is a rapid screening tool for the diagnosis of urogenital chlamydial infections and is more sensitive than Chlamydiazyme for endocervical infections in a sexually transmitted disease clinic population. (C) 1994 Wiley-Liss, Inc.

was solely infected in 35.0% and 12.5%, respectively. Conclusions: This PCR-based technique is a rapid screening tool for the diagnosis of urogenital chlamydial infections and is more sensitive than Chlamydiazyme for endocervical infections in a sexually transmitted disease clinic population. (C) 1994 Wiley-Liss, Inc. KEY WORDS Cervicitis, urethritis, PCR-based test, chlamydial infections hlamydia trachomatis, an obligatory intracellu- lar bacterium, is one of the most common sex- ually transmitted organisms in developed countries. The prevalence of chlamydial infections varies, de- pending on the population examined, from an esti- mated 4-5% of all sexually active women in the United States to as many as 33 % of patients attend- ing sexually transmitted disease (STD) clinics. In women, C. trachomatis is a major cause of urethri- tis, cervicitis, and pelvic inflammatory disease. Long-term sequelae of upper genital tract infec- tions include infertility, ectopic pregnancy, and chronic pelvic pain. Recent chlamydial infections may be associated with premature rupture of mem- branes, preterm labor, and late postpartum endo- myometritis.2-4 In men, C. trachomatis causes over 40% ofcases ofnon-gonococcal urethritis5 and is an important cause of epididymitis, prostatitis, and proctitis. Vertical transmission of C. trachomatis can lead to inclusion conjunctivitis in the newborn Address correspondence/reprint requests to Dr. Harold C. Wiesenfeld, Department ofObstetrics, Gynecology, and Reproduc- tive Sciences, Magee-Womens Hospital, 300 Halket Street, Pittsburgh, PA 15213. Received November 17, 1993 Basic Science Article Accepted January 8, 1994 PCR AND CHLAMYDIAL INFECTIONS WIESENFELD ET AL. and pneumonia in infants during the first months of life. In addition to the clinical manifestations mentioned, there is a high rate of asymptomatic infection in both males and females; up to 70% of infected females are asymptomatic.6 There are a variety of methods available for the detection of C. trachomatis. While the currently accepted gold standard diagnostic test is tissue cul- ture isolation of the organism, its sensitivity is esti- mated to be only 80%.7 Moreover, it requires up to 72 h for incubation and is costly. Antigen detection methods, based on fluorescent monoclonal antibody (MicroTrak, Syva, Palo Alto, CA) or enzyme- linked immunoassay (Chlamydiazyme, Abbott Lab- oratories, North Chicago, IL) methods are widely used in detecting chlamydial infections.

ly 80%.7 Moreover, it requires up to 72 h for incubation and is costly. Antigen detection methods, based on fluorescent monoclonal antibody (MicroTrak, Syva, Palo Alto, CA) or enzyme- linked immunoassay (Chlamydiazyme, Abbott Lab- oratories, North Chicago, IL) methods are widely used in detecting chlamydial infections. These tests are less expensive and can be performed more rap- idly than cell culture; however, they are less sensi- tive and specific. 8 The polymerase chain reaction (PCR) detects small quantities of deoxyribonucleic acid (DNA) by using a DNA polymerase to amplify a target DNA sequence. The PCR technique has been de- veloped to detect viral and bacterial infections. PCR detection of C. trachomatis infections has been eval- uated in several small studies. 9-11 This technique, however, is time consuming and labor intensive. A rapid PCR assay for the detection of C. trachomatis has been developed recently (Amplicor C. tracho- matis Test, Roche Molecular Systems, Branchburg, NJ). The Amplicor test has been demonstrated to be more sensitive than culture for the detection of endocervical infections in a high-prevalence obstet- ric population and more sensitive than Chlamydi- azyme in a low-prevalence population. 12 This tech- nique offers both excellent sensitivity and a total assay time of4.5 h, which are important character- istics for a diagnostic or screening tool. In this study, we evaluated the Amplicor test on lower genital tract specimens from female patients attend- ing an STD clinic and compared endocervical spec- imens with Chlamydiazyme, the primary diagnos- tic test for C. trachomatis used at our institution. SUBJECTS AND METHODS Our study population was comprised of patients attending the outpatient Sexually Transmitted Dis- eases Clinic at the Allegheny County Health De- partment, Pittsburgh, PA. Specimens were ob- tained from both symptomatic and asymptomatic women from September 1, 1992, to October 30, 1992. Patients were included in the study if one of the investigators (H.C.W.) or certain nurse clini- cians were present to collect the specimens. Cervi- cal swabs were obtained randomly from 328 unse- lected female patients attending the clinic, and urethral swabs were obtained concomitantly from 256 of these patients. Urethral specimens were obtained by passing a DacronM-tipped swab into the distal urethra.

se clini- cians were present to collect the specimens. Cervi- cal swabs were obtained randomly from 328 unse- lected female patients attending the clinic, and urethral swabs were obtained concomitantly from 256 of these patients. Urethral specimens were obtained by passing a DacronM-tipped swab into the distal urethra. A non-lubricated speculum was then placed into the vagina, excess cervical mucus was removed, and then endocervical samples were obtained for cul- ture ofNeisseria gonorrhoeae, Chlamydiazyme, and PCR (Amplicor C. trachomatis Test), in that order. Cervical swabs for Chlamydiazyme were obtained prior to those for the Amplicor test in order to obtain the greatest bacterial inoculum to facilitate antigen detection. The Chlamydiazyme specimens were processed as per the manufacturer's recom- mendation. The PCR DacronTM swabs were placed in ml of sodium dodecyl sulfate-based specimen transport media and agitated for 15 sec to displace the clinical sample into the media. The PCR speci- mens were sent at room temperature to a single research laboratory at Magee-Womens Hospital, Pittsburgh, PA, and these specimens were main- tained at 4C prior to processing. PCR amplification was performed on clinical specimens, negative controls, and positive plasmid DNA controls according to a procedure described elsewhere, lz Briefly, 50 I1 of each diluted speci- men was placed in a PCR reaction tube, to which was added a 50 I1 aliquot of master mix containing Taq polymerase, uracil-N-glycosylase, Tris buffer, KCI, glycerol, deoxynucleotide triphosphates, and biotinylated oligonucleotide primers specific for the cryptic plasmid of C. trachomatis. A 2-tempera- ture, 30-cycle amplification scheme was then con- ducted in a Perkin Elmer (Norwalk, CT) 9600 thermal cycler. Sodium hydroxide-based denaturation solution was added to the amplified specimens whereupon 25 Ixl of each specimen was removed and placed into a 96-well microtiter detection plate containing a hybridization buffer. This microtiter plate was coated with a complementary probe for the ampli- fied chlamydia cryptic plasmid. The amplified DNA was then permitted to hybridize, and the wells were then washed in order to remove unhy- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY PCR AND CHLAMYDIAL INFECTIONS WIESENFELD ET AL. TABLE I. Comparison of Chlamydiazyme to PCR for the detection of endocervical C.

li- fied chlamydia cryptic plasmid. The amplified DNA was then permitted to hybridize, and the wells were then washed in order to remove unhy- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY PCR AND CHLAMYDIAL INFECTIONS WIESENFELD ET AL. TABLE I. Comparison of Chlamydiazyme to PCR for the detection of endocervical C. trachomatis Chlamydiazyme PCR results results Positive Negative Total Reactive 20 5 25 (7.6%) Non-reactive 15 288 303 Total 35 (10.7%) 293 328 TABLE 2. Performance of PCR in the detection of endocervical C. trachomatis (after resolution of discrepant results) Resolved specimens PCR results Positive Negative Total Positive 35 0 35 Negative 4 289 293 Total 39 289 328 bridized excess amplification reagents. Avidin- horseradish peroxidase conjugate was added to each well, followed by a wash to remove unbound conju- gate. Horseradish peroxidase substrate, containing hydrogen peroxide and tetramethylbenzidine, was then added for 10 min. The optical densities were read at 450 nm in a Biotek ELISA plate reader (Winooski, VT). Following preestablished crite- ria, 12 we graded absorbencies >0,25 as positive. Specimens with initial absorbencies between 0,20 and 0,60 were deemed equivocal and retested, with the second amplification value used as the final result. Discrepant results were analyzed in the follow- ing manner. Specimens that were positive by PCR and negative by Chlamydiazyme were reamplified using primers directed against the major outer membrane protein (MOMP) gene of C. trachoma- t/s, as described elsewhere. 12 A negative result after reamplification indicated contamination by cryptic plasmid amplicons, whereas a positive result indi- cated a false negative Chlamydiazyme result. Spec- imens positive by Chlamydiazyme and negative by PCR were reamplified after phenol-chloroform, ethanol-precipitated extraction. A negative result indicated sampling error, infection with a plasmid- less strain of C. trachomat#, or a Chlamydiazyme false positive. A. positive result after extraction sig- nified the presence, of PCR inhibitors. RESULTS The results of Chlamydiazyme and Amplicor C. trachomatis Tests on cervical specimens are com- pared in Table 1. Twenty-five patients (7.6%) tested positive for C. trachomatis by Chlamydiaz- yme, and 35 patients (10.7%) were positive by PCR analysis. Compared to PCR, the sensitivity of Chlamydiazyme was only 57.1% and the specificity was 98.3%. The positive predictive value (PPV) of TABLE 3.

l specimens are com- pared in Table 1. Twenty-five patients (7.6%) tested positive for C. trachomatis by Chlamydiaz- yme, and 35 patients (10.7%) were positive by PCR analysis. Compared to PCR, the sensitivity of Chlamydiazyme was only 57.1% and the specificity was 98.3%. The positive predictive value (PPV) of TABLE 3. Performance of Chlamydiazyme in the detection of endocervical C. trachomatis (after resolution of discrepant results) Chlamydiazyme Resolved specimens results Positive Negative Total Positive 24 25 Negative S 288 303 Total 39 289 328 Chlamydiazyme was 80.0% and the negative pre- dictive value (NPV) was 95.0%. Discrepant results were noted in 20 specimens. Fifteen patients were positive by PCR and negative by Chlamydiazyme. All 15 specimens were con- firmed as positive after amplification with primers for the MOMP gene. Four of the 5 specimens positive by Chlamydiazyme and negative by PCR were resolved as positive. One specimen remained negative after phenol-chloroform extraction. The results of PCR and Chlamydiazyme after resolution of discrepant results are shown in Tables 2 and 3. The sensitivity of PCR in detecting endo- cervical C. trachomatis infections was 89.7% and the specificity was 100%. The PPV and NPV of PCR were 100% and 98.6%, respectively. Chlamy- diazyme demonstrated a sensitivity of 61.5% and a specificity of 99.7%. The PPV and NPV were 96.0% and 95.0%, respectively. The difference in sensitivities of PCR and Chlamydiazyme was sig- nificant (P < 0.01, 2 analysis). Table 4 displays the results of the Amplicor test from patients with both urethral and cervical spec- imens. These paired samples were separately run using the PCR technique. C. trachomatis was iden- tified in the cervix in 26 women (10.2%) and ure- thral chlamydia infections were detected in 35 pa- tients (13.7%). Forty patients (15.6%) were 184 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY PCR AND CHLAMYDIAL INFECTIONS WIESENFELD ET AL. TABLE 4. Comparison of the detection of cervical and urethral C. trachomatis by PCR Cervix Urethra Positive Negative Total Positive 21 14 35 (I 3.7%) Negative 5 216 221 Total 26 (10.2%) 230 256 positive for chlamydia in either the cervix or ure- thra. Twenty-one of 26 patients (80.8%) with cer- vical chlamydial infections had concomitant ure- thral chlamydia infections. Likewise, of35 patients with positive urethral PCR samples, 21 (60.0%) were positive for chlamydia in the cervix. Of all patients with urogenital chlamydial infections, C.

er the cervix or ure- thra. Twenty-one of 26 patients (80.8%) with cer- vical chlamydial infections had concomitant ure- thral chlamydia infections. Likewise, of35 patients with positive urethral PCR samples, 21 (60.0%) were positive for chlamydia in the cervix. Of all patients with urogenital chlamydial infections, C. trachomatis was identified in both sites in 21 (52.5%), while 14 (35.0%) were positive only in the urethra and only 5 (12.5%) were positive solely in the cervix. Forty-two women were positive for N. gonor- rhoeae in the endocervix. Cervical chlamydial in- fections were detected in 15 (35.7%) of these pa- tients using PCR, whereas 10 patients (23.8%) were positive by Chlamydiazyme. Among the 35 patients positive for C. trachomatis by PCR, cervi- cal coinfection with N. gonorrhoeae occurred in 15 (42.9%). DISCUSSION Our study demonstrates the superior sensitivity of PCR, using the Amplicor C. trachomatis Test, when compared to enzyme immunoassay (Chlamydiaz- yme) in the detection of cervical chlamydial infec- tions in women attending an STD clinic. The sen- sitivity of PCR in our study was 89.7%, whereas the sensitivity of Chlamydiazyme was 61.5% (P < 0.01). In our high-risk population, the PCR technique detected 15 additional patients who were negative by enzyme immunoassay. Over 35% of those patients infected were not identified by Chlamydiazyme. Chlamydiazyme detected 5 pa- tients not identified by PCR. Four of these speci- mens were resolved as positive, whereas remained negative after discrepant analysis. The 4 false neg- ative PCR specimens were among the first 15% of samples analyzed, and we speculate that this may represent inexperience in the early phases of the learning curve. Using PCR, the prevalences of chlamydial cer- vicitis and urethritis in our population are 10.7% and 13.7%, respectively, and the prevalence of urogenital chlamydial infections is 15.6%. Our data indicate that there is a high rate of concomitant urethral and cervical chlamydial infections and con- firm other screening studies in STD clinics that suggest that 50% ofwomen with urogenital chlamy- dial infections are infected in both the cervix and urethra. 13 In our population, C. trachomatis was detected from both sites in 52.5%, while 35.0% and 12.5% were infected solely in the urethra and cervix, respectively. Over one-third of urogenital chlamydial infections would be missed by failing to sample the urethra; therefore, this site should be evaluated when testing for C. trachomatis.

lation, C. trachomatis was detected from both sites in 52.5%, while 35.0% and 12.5% were infected solely in the urethra and cervix, respectively. Over one-third of urogenital chlamydial infections would be missed by failing to sample the urethra; therefore, this site should be evaluated when testing for C. trachomatis. Commonly used laboratory tests to confirm C. trachomatis infections include tissue culture and an- tigen detection techniques. While tissue culture us- ing cycloheximide-treated McCoy cells has long been considered the gold standard diagnostic test for C. trachomatis, it will not detect up to 20% ofall chlamydial infections.7 Its clinical use is limited, as it is costly, necessitates stringent transport condi- tions, including refrigeration, and requires at least 48-72 h ofincubation prior to interpretation. Rapid antigen detection tests have been developed using either direct immunofluorescence monoclonal anti- body staining or enzyme-linked immunoassay. The sensitivities ofthese rapid antigen detection tests are less than those ofculture. Compared to cell culture, the sensitivities ofdirect immunofluorescence stain- ing (MicroTrak) and enzyme immunoassay (Chlamydiazyme) range from 61 to 99% and 60 to 98%, respectively. 14 In addition, the low PPVs of the antigen detection tests in low-prevalence popu- lations are concerning. DNA probe assays are addi- tional diagnostic tests available for C. trachomatis. The PACE 2 assay (Gen-Probe, Inc., San Diego, CA) is rapid and convenient, but its sensitivity is less than cell culture. 15 We chose the Amplicor C. trachomatis Test, a PCR-based technique, as our gold standard for chlamydia detection, rather than cell culture. The efficacy of PCR in the detection of C. trachomatis infections has been documented.9-12 In a recent INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 185 PCR AND CHLAMYDIAL INFECTIONS WIESENFELD ET AL. study, the Amplicor C. trachomatis Test was dem- onstrated to have a sensitivity of97% and a specific- ity of 99.7% in the detection of C. trachomatis in cervical specimens, whereas the sensitivity of cul- ture was only 86%. 12 This PCR-based technique is highly sensitive in the detection ofcervical chlamy- dial infections, superior to culture or enzyme-linked immunoassays, and may be a better gold standard for the detection of C. trachomatis infections.

trachomatis in cervical specimens, whereas the sensitivity of cul- ture was only 86%. 12 This PCR-based technique is highly sensitive in the detection ofcervical chlamy- dial infections, superior to culture or enzyme-linked immunoassays, and may be a better gold standard for the detection of C. trachomatis infections. We chose to compare the Amplicor test to Chlamydiaz- yme, as Chlamydiazyme is the main test used at our STD clinic and is widely used as the sole diagnostic test for chlamydial infections. Detecting a greater proportion of people infected with C. trachomatis has major public health implications. Rapid testing may enable earlier treatment which, theoretically, may prevent the sequelae of chlamydial infections. Moreover, PCR may be shown, in the future, to be a better method to test for cure, differentiating suppression from eradication ofthe organism. This may be important in adequately treating pelvic in- flammatory disease where small inoculum of C. trachomatis can persist, causing ongoing tubal dam- age, perhaps through the immune response to chlamydial heat-shock proteins. 16 Unlike cell culture, clinical specimens for Am- plicor C. trachomatis Test analysis can be trans- ported at room temperature, and chlamydial DNA is stable for 6 months when refrigerated in trans- port medium. 12 The Amplicor system assay is rapid; laboratory processing is only 4.5 h, which compares favorably to the assay times ofthe antigen detection methods. When chlamydial organisms are present in small amounts, the rapid antigen detec- tion tests are less likely to detect the low levels of antigen present. The PCR method enables the de- tection of small quantities of specific DNA frag- ments and has the potential to identify DNA from organisms present in low concentrations. The mate- rial and labor costs ofthis kit depend on the volume of assays performed. In most centers, the total cost will be approximately $20, somewhat more than the cost of Chlamydiazyme but much less than that of cell culture. Our study demonstrates that the Amplicor C. trachomatis Test, a PCR-based process for the de- tection of C. trachomatis, is a rapid and simple diagnostic test for the detection offemale urogenital chlamydial infections and is superior to Chlamydi- azyme in detecting endocervical infections in an STD clinic population. ACKNOWLI:DGMENTS This study was partially supported by Roche Mo- lecular Systems, Branchburg, NJ. REFERENCES 1.

chomatis, is a rapid and simple diagnostic test for the detection offemale urogenital chlamydial infections and is superior to Chlamydi- azyme in detecting endocervical infections in an STD clinic population. ACKNOWLI:DGMENTS This study was partially supported by Roche Mo- lecular Systems, Branchburg, NJ. REFERENCES 1. Sweet RL, Gibbs RS: Infectious Diseases of the Female Genital Tract. 2nd Ed. Baltimore: Williams & Wilkins, p 49, 1990. 2. Sweet RL, Landers DV, Walker C, Schachter J: Chlamy- dia trachomatis infection and pregnancy outcome. Am J Obstet Gynecol 156:824-833, 1987. 3. Wager GP, Martin DH, Koutsky L, et al.: Puerperal infectious morbidity: Relationship to route of delivery and to antepartum Chlamydia trachomatis infection. Am J Obstet Gynecol 138:1028-1033, 1980. 4. Gravett MG, Nelson HP, Deroven T, Critchlow C, Eschenbach DA, Holmes KK: Independent associations ofbacterial vaginosis and Chlamydia trachomatis infection with adverse pregnancy outcome. JAMA 256:1899- 1903, 1986. 5. Holmes KK, Handsfield HH, Wang S-P, et al.: Etiol- ogy of nongonococcal urethritis. N Engl J Med 292: 1199-1206, 1975. 6. Mardh P-A, Paavonen J, Poulakkainen M: Chlamydia. New York: Plenum Press, p 151, 1989. 7. Mardh P-A, Paavonen J, Poulakkainen M: Chlamydia. New York: Plenum Press, p 79, 1989. 8. Barnes RC: Laboratory diagnosis of human chlamydial infections. Clin Microbiol Rev 2:119-136, 1989. 9. Griffais R, Thibon M: Detection of Chlamydia trachoma- tis by the polymerase chain reaction. Res Microbiol 140: 139-141, 1989. 10. Ostergaard L, Birkelund S, Christiansen G: Use of poly- merase chain reaction for detection of Chlamydia tracho- mat#. J Clin Microbiol 28:1254-1260, 1990. 11. Bobo L, Coutlee F, Yolken RH, Quinn T, Viscidi RP: Diagnosis of Chlamydia trachomatis cervical infection by detection of amplified DNA with an enzyme immunoas- say. J Clin Microbiol 28:1968-1973, 1990. 12. Loeffelholz MJ, Lewinski CA, Silver SR, et al.: Detec- tion of Chlamydia trachomatis in endocervical specimens by polymerase chain reaction. J Clin Microbio130:2847- 2851, 1992. 13. Stamm WE, Holmes KK: Chlamydia trachomatis infec- tions of the adult. In Holmes KK, Mardh PA, Sparling PF, et al. (eds): Sexually Transmitted Diseases. 2nd Ed. New York: McGraw-Hill, p 185, 1990. 14. Stamm WE: Diagnosis of Chlamydia trachomatis geni- tourinary infections. Ann Intern Med 108:710-717, 1988. 15.

1992. 13. Stamm WE, Holmes KK: Chlamydia trachomatis infec- tions of the adult. In Holmes KK, Mardh PA, Sparling PF, et al. (eds): Sexually Transmitted Diseases. 2nd Ed. New York: McGraw-Hill, p 185, 1990. 14. Stamm WE: Diagnosis of Chlamydia trachomatis geni- tourinary infections. Ann Intern Med 108:710-717, 1988. 15. Clarke LM, Sierra MF, Daidone BJ, Lopez N, Covino JM, McCormack WM: Comparison of the Syva Micro- trak enzyme immunoassay and Gen-Probe PACE 2 with 186 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY PCR AND CHLAMYDIAL INFECTIONS WIESENFELD ET AL. cell culture for diagnosis of cervical Chlamydia trachoma- t/s infection in a high-prevalence female population. J Clin Microbiol 31:968-971, 1993. 16. Morrison RP, Manning S, Caldwell HD: Immunology of Chlamydia trachomatis infections: Immunoprotective and Immunopathogenetic Responses. In Quinn TC (ed): Sexually Transmitted Diseases. New York: Raven Press, p 57, 1992. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 117

Infectious Diseases in Obstetrics and Gynecology I: 188-192 (I 994) (C) 1994 Wiley-Liss, Inc. Comparison of Five Methods for the Determination of Rubella Immunity Robert L. Sautter, Arthur E. Crist, Jr., Lynn M. Johnson, and William D. LeBar Department ofPathology, Harrisburg Hospital (R.L.S.), Departments ofPathology (A.E.C.) and Laboratories (L.M.J.), Polyclinic Medical Center, Harrisburg, PA, and Citation Clinical Laboratories, Providence Hospital (W.D.L.), Microbiology Laboratory, Southfield, MI ABSTRACT Objective: The purpose ofthis study was to compare the accuracy ofcommonly used methods for the detection ofrubella immunity, especially the fully automated IMx assay. Methods: A total of 190 sera (101 immune and 89 non-immune) submitted to Harrisburg Hospital or Polyclinic Medical Center for the determination of rubella immunity were tested by enzyme immunoassay (IMx and Rubazyme, Abbott Diagnostic Laboratories, North Chicago, IL), indirect immunofluorescence (FIAX, Whittaker Bioproducts, Walkersville, MD), and latex agglutination (Rubascan, Becton Dickinson Microbiology Systems, Cockeysville, MD, and Rubalex, Wellcome Diagnostics, Research Triangle Park, NC). Specimens were frozen at -30C until the study was initiated. Each ofthe assays was performed according to the manufacturers' specifications. Sensitiv- ity, specificity, accuracy, and positive and negative predictive values for each assay were calculated using a consensus result ofthe 5 methods tested. Results: The sensitivity, specificity, and accuracy, respectively, of the test systems were as fol- lows: IMx, 96%, 97%, and 96%; Rubazyme, 100%, 99%, and 99%; Rubascan, 100%, 98%, and 99%; Rubalex, 99%, 97%, and 98%; and FIAX 90%, 100%, and 95%. False negative reactions were seen with the FIAX system. Conclusions: The IMx system, a new "walk away" system from Abbott Diagnostic Laboratories and the Rubazyme systems performed well; however the latex agglutination tests proved to be the most rapid and convenient methods for screening sera for the presence ofrubella immunity. (C) 1994 Wiley-Liss, Inc.

h the FIAX system. Conclusions: The IMx system, a new "walk away" system from Abbott Diagnostic Laboratories and the Rubazyme systems performed well; however the latex agglutination tests proved to be the most rapid and convenient methods for screening sera for the presence ofrubella immunity. (C) 1994 Wiley-Liss, Inc. KEY WORDS IMx, enzyme immunoassay, latex agglutination, fluorescent immunoassay, comparison of viral immunological methods ublic health measures to reduce the transmission ofrubella are dependent upon the vaccination of children > 12 months of age, school-age children not previously immunized, and susceptible adults. The occurrence of rubella during pregnancy can result in severe congenital abnormalities ofthe new- born. Current guidelines suggest that women of childbearing age should be evaluated for immune status to rubella. If they are negative, they should be monitored throughout the pregnancy for sero- conversion. If these women remain negative fol- lowing delivery, then they should be vaccinated postpartum. In addition, health care personnel are monitored at most pre-employment physicals; if they are found to be non-immune, vaccination is recommended. The rubella antibody test is one of the most frequently ordered tests in the serology laboratory. Address correspondence/reprint requests to Dr. Robert L. Sautter, Director, Clinical Microbiology, Department of Pathol- ogy, Harrisburg Hospital, South Front Street, Harrisburg, PA 17101-2099. Received November 29, 1993 Basic Science Article Accepted January 28, 1994 COMPARISON OF RUBELLA IMMUNOASSAYS SAUTTER ET AL. In the past, the recommended method for deter- mining immune status to rubella was the hemagglu- tination inhibition (HI) test. For the most part, HI has been replaced with less cumbersome methods, including passive hemagglutination, 1-5 latex agglu- tination, 1-13 fluorescenceimmunoassay, 1--4,6,14,15 enzyme immunoassay, 1,2,4,5,10, 1,15--18 and radial hemolysis in gel.4'13 These methods have been shown to be as accurate as HI and in some cases more sensitive, specific, and reproducible,s'8'9'11 Recently, a fully automated IMx immunoassay analyzer was developed for the detection of IgG and IgM antibodies to rubella virus. 16,19 A fully automated system provides for a more objective result, decreases the technical time and cost re- quired to perform the assay, offers standardization of the test procedure, and lends itself to physician office testing.

say analyzer was developed for the detection of IgG and IgM antibodies to rubella virus. 16,19 A fully automated system provides for a more objective result, decreases the technical time and cost re- quired to perform the assay, offers standardization of the test procedure, and lends itself to physician office testing. The purpose of this study was to compare several commercially available methods for the determination of rubella immunity, includ- ing the fully automated IMx immunoassay ana- lyzer. MATERIALS AND METHODS Specimens A total of 190 serum specimens submitted to the clinical microbiology laboratory at Harrisburg Hospital and Polyclinic Medical Center were tested by 5 methods. The serum samples were obtained from specimens submitted for rubella serology. Sera were tested on receipt in the laboratory by fluores- cence immunoassay (FIAX, Whittaker Bioprod- ucts, Waldersville, MD) and latex agglutination (Rubascan, Becton Dickinson Microbiology Sys- tems, Cockeysville, MD) and then were stored at -30C until subsequent testing. The stored sera remained frozen for 6 months and then were tested using the remainder of the assays. IMx The IMx (Abbott Diagnostic Laboratories, North Chicago, IL) is an automated procedure based on microparticle enzyme immunoassay technology. Once the serum sample is placed into the reaction cell of the carousel, all additional steps are per- formed by the instrument. The principle and oper- ation of the IMx have been described previ- ously6,18 and are reviewed briefly here. Once controls and samples have been loaded onto the carousel and the instrument started, the probe/ electrode assembly delivers the sample and diluent buffer to the predilution well of the reaction cell. Next, the microparticles coated with rubella virus and an aliquot ofthe diluted sample are added to the incubation well. Ifantibodies to rubella are present in the patient's serum, they will bind to the antigen- coated microparticle forming an antigen-antibody complex. Diluent buffer is added to the reaction mixture, and an aliquot of the antigen-antibody complex is added to the glass fiber matrix. The microparticles bind irreversibly to the glass fiber matrix. The matrix is washed to remove unbound material, and an alkaline phosphatase-conjugated anti-human IgG is dispensed onto the matrix and binds to the antigen-antibody complex. The matrix is then washed again to remove unbound material.

e glass fiber matrix. The microparticles bind irreversibly to the glass fiber matrix. The matrix is washed to remove unbound material, and an alkaline phosphatase-conjugated anti-human IgG is dispensed onto the matrix and binds to the antigen-antibody complex. The matrix is then washed again to remove unbound material. The substrate 4-methylumbelliferyl phosphate is then added to the matrix, and the fluorescent prod- uct formed is measured by the optical assembly of the instrument. Those specimens that exhibited val- ues > 10 IU of IgG antibody to rubella virus were considered immune. At the beginning ofthe study, 6 calibrators were run to establish a calibration curve. Once established, this calibration curve has been shown to be stable for at least 2 weeks. Posi- tive and negative controls were included in each run. FlAX Fluorescence immunoassay was performed using Rubella G kits supplied by the manufacturer. The method was performed according to the manufac- turer's instructions and has been described previ- ously. s A result lower than 8 indicated susceptibil- ity to rubella and a result greater than 12 indicated immunity. An equivocal zone of 8-12 has been established by the manufacturer to avoid false posi- tive readings, and serum specimens in this range were retested. Latex Agglutination Latex agglutination was performed using the Rubascan and Rubalex (Wellcome Diagnostics, Re- search Triangle Park, NC). Both procedures were tested according to the manufacturers' instructions and have been described previously.2'6 Serum spec- imens tested with the Rubalex method were diluted 1:10 prior to testing. With the Rubascan method, serum samples were tested undiluted and diluted 1:10 as recommended by the manufacturer. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY JOe ) COMPARISON OF RUBELLA IMMUNOASSAYS SAUTTER ET AL. TABLE I. Performance characteristics (%) of the 5 methods for determining rubella immune status Method Sensitivity Specificity PP NP Accuracy IMx 96 (97/101) 97 (86/89) 96 (97/101) 96 (86/90) 96 (183/190) FlAX 90 (89/99)* 100 (89/89) 100 (89/89) 90 (89/99) 95 (I 78/188) Rubascan 100 (101/101) 98 (87/89) 98 (101/103) 100 (87/87) 99 (188/190) Rubalex 99 (100/101) 97 (86/89) 97 (100/103) 99 (86/87) 98 (186/190) Rubazyme 100 (101/101) 99 (88/89) 99 (101/102) 100 (88/88) 99 (189/190) aTwo specimens gave equivocal results using the FlAX method and were elminated from the comparison. *P 0.0008. TABLE 2. Samples with discrepant results Final No.

/87) 99 (188/190) Rubalex 99 (100/101) 97 (86/89) 97 (100/103) 99 (86/87) 98 (186/190) Rubazyme 100 (101/101) 99 (88/89) 99 (101/102) 100 (88/88) 99 (189/190) aTwo specimens gave equivocal results using the FlAX method and were elminated from the comparison. *P 0.0008. TABLE 2. Samples with discrepant results Final No. of IMx FlAX Rubascan Rubalex Rubazyme interpretation samples Negative Negative Positive Positive Negative Negative 2 Positive Negative Positive Positive Positive Positive 4 Positive Equivocal Positive Positive Positive Positive 2 Negative Negative Positive Positive Positive Positive 4 Positive Negative Negative Negative Negative Negative 2 Positive Positive Positive Negative Positive Positive Negative Negative Negative Positive Negative Negative Positive Negative Negative Negative Positive Negative Enzyme Immunoassay (EIA) EIA was performed using Rubazyme (Abbott Di- agnostic Laboratories) kits supplied by the manu- facturer. The method was performed and the results were interpreted according to the manu- facturer's instructions and have been descr'bed pre- viously, s Each serum sample was tested in dupli- cate. Comparison of Results and Statistical Analysis Specimens were considered positive or negative for rubella antibody when 3 or more methods were in agreement. Each assay method was compared to the "consensus" result in order to determine sensitivity, specificity, accuracy, and predictive values of the method. Statistical analysis and evaluation of the 5 methods were made using the chi-square test (StatView + Graphics, Abacus Concepts, Inc., Berkeley, CA). RESULTS Using a consensus of 3 or more methods, we found a total of 101 specimens to be positive for rubella antibody and 89 specimens to be negative. Perfor- mance characteristics of the methods for the detec- tion of rubella antibody can be seen in Table 1. The FIAX, with a sensitivity of 90%, was the least sensitive of the methods evaluated (P 0.0008). The other 4 methods were compa- rable in sensitivity, which ranged from 96% to 100%. The specificity, positive predictive value (PPV), negative predictive value (NPV), and accu- racy of the 5 methods were as follows: 97%, 96%, 96%, 96% for IMx; 100%, 100%, 90%, 95% for FIAX; 98%, 98%, 100%, 99% for Rubascan; 97%, 97%, 99%, 98% for Rubalex; and 99%, 99%, 100%, 99% for Rubazyme. There was no statisti- cally significant difference in specificity in the 5 methods (P 0.3158). Data on samples giving discrepant results are found in Table 2.

%, 96%, 96%, 96% for IMx; 100%, 100%, 90%, 95% for FIAX; 98%, 98%, 100%, 99% for Rubascan; 97%, 97%, 99%, 98% for Rubalex; and 99%, 99%, 100%, 99% for Rubazyme. There was no statisti- cally significant difference in specificity in the 5 methods (P 0.3158). Data on samples giving discrepant results are found in Table 2. There were 17 samples that gave discrepant results in assays. Four samples were neg- ative, and 2 were equivocal (even on repeat testing) by the FIAX method but positive by the other 4 methods. Four additional samples were negative by FIAX and IMx but positive by all other methods. The other 7 discrepancies were distributed among the other assays. DISCUSSION The Advisory Committee for Immunization Prac- tices of the Center for Disease Control, Atlanta, 190 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY COMPARISON OF RUBELLA IMMUNOASSAYS SAUTTER ET AL. GA, has recommended that a positive result by any test for rubella antibody be accepted as evidence of rubella immunity,z In support of this recommen- dation are several studies that have shown that low antibody titers, particularly those detected by latex agglutination, are sufficient to protect against in- fection with attenuated virus. 8'1z The latex aggluti- nation methods (Rubascan and Rubalex), as dem- onstrated in this study, are as sensitive, specific, and accurate as other methodologies. They also have the advantages of requiring no pretreatment of se- rum samples, small sample volume, rapid turn- around time (less than 10 min), and no purchase of capital equipment. The 3 remaining assays are either fully auto- mated (IMx) or semiautomated (FIAX and Ruba- zyme). Previous studies on the fully automated IMx rubella IgG assay have shown that the system is more sensitive than and as specific as conven- tional EIA technology. 16,18 Schaefer et al. 8 showed that the IMx had a sensitivity of 99.9% compared to a sensitivity of 96.5% for Rubazyme. Specificity was identical for both assays at 98.9%. Similar results were obtained by Abbott et al. 16 and Skurrie et al. 19 In these previous studies, the IMx was only compared to conventional EIA with discordant re- sults referred by passive hemagglutination. Other commercially available systems such as latex agglu- tination and fluorescent immunoassay were not eval- uated. In the present study, the IMx was found to be more sensitive and accurate than fluorescent im- munoassay but equivalent to the latex agglutination assays and conventional EIA.

y passive hemagglutination. Other commercially available systems such as latex agglu- tination and fluorescent immunoassay were not eval- uated. In the present study, the IMx was found to be more sensitive and accurate than fluorescent im- munoassay but equivalent to the latex agglutination assays and conventional EIA. The primary purpose of rubella screening is to identify non-immune women of childbearing age. Therefore, a false positive result would be an error with the most serious consequences since women who are truly non-immune would not be vaccinated and would be fully susceptible to rubella infection. Subsequent infection during pregnancy would pose a risk to the unborn fetus. On the other hand, a false negative result, although not as serious, could result in potential morbidity to the patient due to unnecessary vaccination. The FIAX method showed the lowest diagnostic accuracy (95%), primarily due to a 10% false negative rate. The other methods evaluated had diagnostic accuracies ranging from 96% to 99%, with few false positive or false nega- tive results. On the basis of the results presented here, all ofthe methods evaluated, with the possible exception ofthe FIAX assay, can be used reliably to determine rubella immune status. ACKNOWLEDGMENTS This work was funded in part by Carter Wallace, Inc., Cranbury, NJ. REFERENCES 1. Castellano GA, Madden DL, Hazzard GT, et al.: Eval- uation of commercially available diagnostic test kits for rubella. J Infect Dis 143:578-584, 1981. 2. Chernesky MA, DeLong DJ, MahonyJB, Castriciano S: Differences in antibody responses with rapid agglutina- tion tests for the detection of rubella antibodies. J Clin Microbiol 23:772-776, 1986. 3. Fayram SL, Akin S, Aarnaes SL, Peterson EM, de la Maza LM: Determination of immune status in patients with low antibody titers for rubella virus. J Clin Micro- biol 25:178-180, 1987. 4. Herrmann KL: Available rubella serologic tests. Rev Infect Dis 7(Suppl 1):108-112, 1985. 5. Kleeman KT, Kiefer DJ, Halbert SP: Rubella antibodies detected by several commercial immunoassays in hemag- glutination inhibition-negative sera. J Clin Microbiol 18: 1131-1137, 1983. 6. Fayram SL, Nakasone A, Aarnaes S, Zartarian M, Peter- son EM, de la Maza LM: Fluorescence immunoassay and passive latex agglutination as alternatives to hemag- glutination inhibition for determining rubella immune status. J Clin Microbiol 17:685-688, 1983. 7.

tion-negative sera. J Clin Microbiol 18: 1131-1137, 1983. 6. Fayram SL, Nakasone A, Aarnaes S, Zartarian M, Peter- son EM, de la Maza LM: Fluorescence immunoassay and passive latex agglutination as alternatives to hemag- glutination inhibition for determining rubella immune status. J Clin Microbiol 17:685-688, 1983. 7. Freeman S, Clark L, Dumas N: Evaluation of a latex agglutination test for detection of antibodies to rubella virus in selected sera. J Clin Microbiol 18:197-198, 1983. 8. Grangeot-Keros L, Badur S, Pons JC, Duthu S, Pillot J: Use of in vivo challenge to assess rubella immunity deter- mined by hemagglutination inhibition and latex aggluti- nation. Res Virol 140:437-442, 1989. 9. Leon P, deOry F, Domingo C, Lopez JA, Echevarria JM: Evaluation of a latex agglutination test for screening antibodies to rubella virus. Eur J Clin Microbiol Infect Dis 7:196-199, 1988. 10. Simor AE, Chua R, Low DE: Evaluation of a new latex test and a new enzyme immunoassay for determination of rubella immunity. J Clin Microbiol 26:1582-1583, 1988. 11. Steece RS, Talley MS, Skeels MR, Lanier GA: Compar- ison ofenzyme-linked immunosorbent assay, hemaggluti- nation inhibition, and passive latex agglutination for de- termination of rubella immune status. J Clin Microbiol 21:140-142, 1985. 12. Storch GA, Myers N: Latex-agglutination test for rubella antibody: Validity of positive results assessed by response to immunization and comparison with other tests. J Infect Dis 149:459-464, 1984. 13. Vaananen P, Haiva VM, Koskela P, Meurman O: Com- parison ofa simple latex agglutination test with hemolysis- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY COMPARISON OF RUBELLA IMMUNOASSAYS in-gel, hemagglutination inhibition, and radioimmunoas- say for detection of rubella virus antibodies. J Clin Microbiol 21:793-795, 1985. 14. Cremer NE, Hagens SJ, Cossen C: Comparison of the hemagglutination inhibition test and an indirect fluores- cent-antibody test for detection of antibody to rubella vi- rus in human sera. J Clin Microbiol 11:746-747, 1980. 15. Zartarian MV, Friedly G, Peterson EM, de la Maza LM: Detection of rubella antibodies by hemagglutination inhibition, indirect fluorescent-antibody test, and enzyme- linked immunosorbent assay. J Clin Microbiol 14:640- 645, 1981. 16. Abbott GG, Safford JW, MacDonald RG, Craine MC, Applegren RR: Development ofautomated immunoassays for immune status screening and serodiagnosis of rubella virus infection. J Virol Methods 27:227-240, 1990. SAUTTER ET AL. 17.

antibody test, and enzyme- linked immunosorbent assay. J Clin Microbiol 14:640- 645, 1981. 16. Abbott GG, Safford JW, MacDonald RG, Craine MC, Applegren RR: Development ofautomated immunoassays for immune status screening and serodiagnosis of rubella virus infection. J Virol Methods 27:227-240, 1990. SAUTTER ET AL. 17. Hossain A, Ramia S, Bakir TMF: Comparison of he- magglutination test, enzyme-linked immunosorbent assay and indirect immunofluorescence antibody test for deter- mination of rubella immune status. J Trop Med Hyg 91:216-221, 1988. 18. Schaefer LE, DykeJW, Meglio FD, Murray PR, Crafts W, Niles AC: Evaluation of microparticle enzyme im- munoassays for immunoglobulins G and M to rubella virus and Toxoplasma gondii on the Abbott IMx automated analyzer. J Clin Microbiol 27:2410-2413, 1989. 19. Skurrie IJ, Head IL, Garland SM: Detection of rubella- specific immunoglobulin G: Comparison of the enzyme- linked immunosorbent assay and an automated micropar- ticle enzyme immunoassay (IMx). J Clin Microbiol 29: 1752-1753, 1991. 20. Rubella prevention. MMWR 30:37-47, 1981. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:193-197 (1994) (C) 1994 Wiley-Liss, Inc. In Vitro Effect of Local Anesthetics on Candida albicans Germ Tube Formation Acficio Rodrigues, Cidfilia Pina Vaz, A. Freitas Fonseca, J. Martinez de Oliveira, and Henrique Barros Departments ofMicrobiology (A.R., C.P.V., A.F.F.), Gynecology (J.M.d.O.), and Hygiene and Epidemiology (H.B.), Porto Faculty ofMedicine, University ofPorto, Porto, Portugal ABSTRACT Objective: This study was planned to clarify the in vitro effect of lidocaine and bupivacaine on germ tube formation by Candida albicans isolates from cases ofclinical vaginal candidiasis. Methods: Fourteen C. albicans strains (clinical vaginal isolates) were grown on Sabouraud agar for 24 h at 37C and tested as follows: 100 1 of a yeast suspension [105 colony forming units (CFU)/ml of phosphate buffered saline (PBS)] was added to 500 1 of fresh human serum with lidocaine or bupivacaine (pure salts) in serial concentrations. The test was run in duplicate. Controls were prepared for each strain. After 4 h ofincubation at 37C, samples were taken from each vial and 200 yeasts were counted in a counting chamber. The pH ofeach suspension was measured. Results: The results are given as the mean ofthe 2 readings and are expressed as the percentage of blastoconidia with germ tubes/total blastoconidia. Conclusions: Our experiments show that both lidocaine and bupivacaine have a dose-dependent inhibitory effect, pH-independent, on germ tube formation by C. albicans and that both drugs seem to be promising in the treatment of genital candidiasis due to the combination of anesthetic and antifungal properties. (C) 1994 Wiley-Liss, Inc. KEY WORDS Local anesthetics, germ tube, antimicrobial activity, candidiasis n 1909 Jonnesco first described the antibacterial effect of local anesthetic drugs and in 1955 Mur- phy et al.2 reported the toxicity of tetracaine in relation to Pseudomonas. Kleinfeld and Ellis3'4 could demonstrate a toxic effect of tetracaine, benoxinate, and cocaine to Candida albicans. Erlichs showed that, while tetracaine at a 0.5% concentration in- hibited C.

l effect of local anesthetic drugs and in 1955 Mur- phy et al.2 reported the toxicity of tetracaine in relation to Pseudomonas. Kleinfeld and Ellis3'4 could demonstrate a toxic effect of tetracaine, benoxinate, and cocaine to Candida albicans. Erlichs showed that, while tetracaine at a 0.5% concentration in- hibited C. albicans as well as Staphylococcus aureus, greater concentrations oflidocaine were required to obtain a similar effect. In 1970 Schmidt and Rosen- kranz6 demonstrated the antimicrobial activity of lidocaine and procaine against several bacteria and yeasts. These authors did not study any genital spec- imens. Of 10 Cryptococcus neoformans and C. albi- cans studied (isolates from nongenital sources), all 5 Cryptococcus were inhibited by lidocaine and pro- caine in concentrations in excess of 1.0%. How- ever, none of the 5 Candida strains were inhibited by 2.0% lidocaine or procaine. Meanwhile, all 10 isolates were inhibited by 0.5-1.0 xg/ml of am- photericin B.6 Therefore, the results of Schmidt and Rosenkranz6 regarding C. albicans are in dis- agreement with those of previous authors. It is admitted that filamentation of Candida blas- toconidia is a phenotypical change related to patho- genesis.7'8 The presence of hyphae on the bedside "wet smear" examination is supposed to indicate invasiveness and an abnormal stage not accepted as colonization.9 Some years ago, Taschdjian et al. 10 used germ tube formation as a test for identification Address correspondence/reprint requests to Dr. Accio Rodrigues, Department of Microbiology, Porto Faculty of Medicine, Alameda Prof. Hernani Monteiro, 4200 Porto, Portugal. Basic Science Article Received August 12, 1993 Accepted January 5, 1994 LOCAL ANESTHETICS ON C. ALBICANS GERM TUBE RODRIGUES ET AL. 8O 2O Control 0,03% 0,07% a-b: p=O.O005 b-c- p not significant c-d-p=O.O005 Lidocaine C18 x C21 d = C24 O, 15% 0,25% 0,50% Lidocaine concentrations Fig. I. Effect of lidocaine. of C. albicans. In addition to antifungal properties, local anesthetics could help in relieving the very stressful symptoms commonly present in genital candidiasis. The present work was performed to clarify the in vitro effect of 2 local anesthetics, lidocaine, a common and well-known agent avail- able for topical use, and bupivacaine, a more recent and potent drug, on the germ tube formation by C. albicans. MATERIALS AND METHODS All 14 C.

commonly present in genital candidiasis. The present work was performed to clarify the in vitro effect of 2 local anesthetics, lidocaine, a common and well-known agent avail- able for topical use, and bupivacaine, a more recent and potent drug, on the germ tube formation by C. albicans. MATERIALS AND METHODS All 14 C. albicans strains used in this study were clinical isolates from microbiologically diagnosed cases of vaginal candidiasis, some of them from recalcitrant cases or cases resistant in vivo to several antifungal treatments. They were grown on Sab- ouraud agar (Difco, Detroit, MI) for 24 h at 37C and tested as follows: 100 I1 of a yeast suspension [105 colony forming units (CFU)/ml of phosphate buffered saline (PBS)] was added to 500 I1 of fresh human serum (pool) with lidocaine or bupi- vacaine in serial concentrations. Solutions of lidocaine and bupivacaine hydrochloride (pure salts, free of preservatives, purchased from Sigma, St. Louis, MO) were prepared in sterile PBS immedi- ately before experiments in order to guarantee good stability. For each concentration of both local anes- thetics, the test was run in duplicate. Duplicate controls, with plain serum, were prepared with each strain. After 4 h of incubation at 37C, the samples were taken from each vial and 200 yeasts were evaluated and counted using a Burke's cham- ber under a magnification of 400 on a blind reading. The pH value of each suspension was measured. For the statistical analysis of data, we used the Wilcoxon signed rank tests. RESULTS Results are given as the mean ofthe 2 readings, for each strain and concentration, and are expressed as the percentage of blastoconidia with germ tubes/ 194 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY LOCAL ANESTHETICS ON C. ALBICANS GERM TUBE RODRIGUES ET AL. 00 a b Bupivacalne Control 0,0175% 0,035% a-b: p not significant b-c: p=O.O01 a-c: p=O.O005 c-d: p=O.O005 C24 0,075% 0,15% 0,25% Bupivacaine concentrations Fig. 2. Effect of bupivacaine. total blastoconidia. No significant pI--I variation was found between controls and local anesthetic solu- tions. The effect of lidocaine is shown in Figure and the effect ofbupivacaine in Figure 2. The degree of germ tube inhibition of each local anesthetic at different concentrations is listed in Table 1. DISCUSSION The mechanisms involved in Candida pathogenesis are not yet well defined, even in C. albicans, which is commonly accepted as the most pathogenic spe- cies.

e and the effect ofbupivacaine in Figure 2. The degree of germ tube inhibition of each local anesthetic at different concentrations is listed in Table 1. DISCUSSION The mechanisms involved in Candida pathogenesis are not yet well defined, even in C. albicans, which is commonly accepted as the most pathogenic spe- cies. 12 In contrast to many bacteria, no single viru- lence factor can be identified as responsible for fungal infectivity. Yeast pathogenicity results from several different factors, the resultant being the overwhelming of the host defenses by the yeasts. Although not a crucial step, germ tube formation is believed to play an important pathogenic role in the initial phase oftissue invasion. In some studies, germ tubes were proved to be associated with better adhe- sion to epithelial cells in vitro, 13']4 and it has been repeatedly shown in vitro that C. albicans germ tubes grow from macrophages]5']6 as they do from neutro- phils (PMNs)17 after phagocytosis, in this way pre- venting the intracellular killing of the yeast. These and several other experimental facts helped to create the concept of a differential behavior of C. albicans germ tubes and pseudohyphae in relation to blasto- conidia. The reason why C. albicans is a common and persistent pathogen in the vaginas of a significant number ofwomen 8-20 still remains undetermined. In our study, we decided to use serum for the germ tube test due to the more impressive germ tube production in this substrate when compared to others. In a previous study (unpublished data), we evaluated the different sensitivities for germ tube formation of several media, namely Eagle's me- dium, RPMI medium, Sabouraud's dextrose broth, egg albumin, fetal calf serum, and human serum, INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY LOCAL ANESTHETICS ON C. ALBICANS GERM TUBE RODRIGUES ET AL. TABLE I. Percent of germ tube formation inhibition at serial concentrations of local anesthetic Concentration Lidocaine Bupivacaine (%) (%) (%) 0.0175 12.1-+ 12.0 0.035 24.0 13.8 27.9 -+ 18.4 0.075 33.0 20.0 91.8 -+ 5.3 0.15 91.6 -+ 5.5 I00 0.25 O0 I00 0.50 I00 I00 sis was found.6 Further testing is being carried out to clarify this effect. To our knowledge, this is the first report ofthe effect oflocal anesthetics, namely lidocaine and bupivacaine, on germ tube formation by C. albicans.

0 13.8 27.9 -+ 18.4 0.075 33.0 20.0 91.8 -+ 5.3 0.15 91.6 -+ 5.5 I00 0.25 O0 I00 0.50 I00 I00 sis was found.6 Further testing is being carried out to clarify this effect. To our knowledge, this is the first report ofthe effect oflocal anesthetics, namely lidocaine and bupivacaine, on germ tube formation by C. albicans. From a theoretical point of view, these drugs seem to be promising in the treatment of genital candidiasis due to the combination of anesthetic and antifungal properties. REFERENCES the latter being the most sensitive. That human serum (a pool, to avoid possible anticandidal factors present in a particular serum that might affect germ tubes) is a good substrate for germ tube production is also confirmed in this work, being the mean value ofgerm tube formation in our controls (plain human serum), 91.8 5.3%. Our results show that both lidocaine and bupi- vacaine have a potent dose-dependent inhibitory effect on germ tube formation by C. albicans. This effect is pH-independent, as there is no significant pH variation when compared to controls. From the values we found, bupivacaine seems to be more potent on that action than lidocaine. In fact, when the results are compared for the 0.07% concentra- tion (in which there is a significant effect of both drugs), bupivacaine seems to be 3 times more po- tent than lidocaine (91.8 5.3% vs. 33.0 +-- 20.0% of inhibition; P 7.3 10-6) in achiev- ing that effect. In very dilute concentrations such as 0.15% (compared to the ones used in clinical prac- tice for several purposes), we found 100% inhibi- tion of germ tube formation with bupivacaine and almost the same figure with lidocaine (91.6-+ 5.5%). When the 0.25% concentration is consid- ered, both lidocaine and bupivacaine produced an inhibition of 100%. Similarly, low concentration values may be easily attainable in the vaginal mi- lieu, in vivo, probably with similar effects. The mechanism by which local anesthetics exert their antimicrobial effects is still unclear to us. A plausi- ble hypothesis is the impairment of protein synthe- sis necessary to germ tube formation, namely, the cell wall or the cytoplasmic membrane. Biochemi- cal studies demonstrated that protein synthesis was more sensitive than RNA or DNA production to the inhibitory effect of lidocaine, although no dis- tinct selective inhibition of macromolecular synthe- 1. JonnescoT: Remarks on general spinal analgesia. Br Med J 2:1396-1401, 1909. 2.

the cytoplasmic membrane. Biochemi- cal studies demonstrated that protein synthesis was more sensitive than RNA or DNA production to the inhibitory effect of lidocaine, although no dis- tinct selective inhibition of macromolecular synthe- 1. JonnescoT: Remarks on general spinal analgesia. Br Med J 2:1396-1401, 1909. 2. Murphy JT, Allen HF, Mangiaracine AB: Preparation, sterilization, and preservation of ophthalmic solutions: Experimental studies and a practical method. Arch Oph- thalmol 53:63-78, 1955. 3. Kleinfeld J, Ellis PP: Effects of topical anesthetics on growth of microorganisms. Arch Ophthalmol 76:712- 725, 1966. 4. Kleinfeld J, Ellis PP: Inhibition of microorganisms by topical anesthetics. Appl Microbiol 15:1296-1298, 1967. 5. Erlich H: Bacteriologic studies and effects of anesthetic solutions on bronchial secretions during bronchoscopy. Am Rev Respir Dis 84:414-421, 1961. 6. Schmidt RM, Rosenkranz HS: Antimicrobial activity of local anesthetics: Lidocaine and procaine. J Infect Dis 121:587-607, 1970. 7. Sobel JD: Epidemiology and pathogenesis of recurrent vulvovaginal candidiasis. AmJ Obstet Gynecol 152:924- 935, 1985. 8. Odds FC: Morphogenesis in Candida, with special refer- ence to C. albicans. In Odds FC (ed): Candida and Candi- dosis. Baillire Tindall, London, pp 42-59, 1988. 9. Eschenbach DA. Clin Obst Gynecol 26:186-201, 1983. 10. Taschdjian CL, Burchall JJ, Kozinn PJ: Rapid identifica- tion of Candida albicans by filamentation on serum and serum substitutes. Am J Dis Child 99:212-215, 1960. 11. Hill B: The Wilcoxon signed rank test. In Hill B (ed): Principles of Medical Statistics, 12th Ed. London: Ed- ward Arnold, pp 126-127, 1991. 12. Hurley R: Pathogenicity of the genus Candida. In Win- ner HI, Hurley R (eds): Symposium on Candida Infec- tions. Edinburgh: Churchill Livingstone, pp 13-25, 1966. 13. Kimura LH, Pearsall NM: Relationship between germi- nation of Candida albicans and increased adherence to human buccal epithelial cells. Infect Immun 28:464-468, 1980. 14. Rotrosen D, Edwards JE, Gibson TR, et al.: Adherence of Candida to cultured vascular endothelial cells: Mecha- nisms of attachment and endothelial penetration. J Infect Dis 152:1264-1274, 1985. 15. Peterson EM, Calderone RA: Growth inhibition of Can- dida albicans by rabbit alveolar macrophages. Infect Im- mun 15:911-915, 1977. 196 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY LOCAL ANESTHETICS ON C. ALBICANS GERM TUBE RODRIGUES ET AL. 16.

ment and endothelial penetration. J Infect Dis 152:1264-1274, 1985. 15. Peterson EM, Calderone RA: Growth inhibition of Can- dida albicans by rabbit alveolar macrophages. Infect Im- mun 15:911-915, 1977. 196 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY LOCAL ANESTHETICS ON C. ALBICANS GERM TUBE RODRIGUES ET AL. 16. Evron R: In vitro phagocytosis of Candida albicans by peritoneal mouse macrophages. Infect Immun 28:963- 971, 1980. 17. Arai T, Mikami Y, Yokohama K: Phagocytosis of Can- dida albicans by rabbit alveolar macrophages and guinea pig neutrophils. Sabouraudia 15:171-177, 1977. 18. Sobel JD: Epidemiology and pathogenesis of recurrent vulvovaginal candidiasis. AmJ Obstet Gynecol 152:924- 935, 1985. 19. Sobel JD: Recurrent vulvovaginal candidiasis: What we know and what we don't. Ann Intern Med 101:390-392, 1984. 20. Martinez de Oliveira J, Silva Cruz A, Fonseca AF, Pina Vaz C, Rodrigues A: Prevalence of Candida albicans in the vaginal fluid of Portuguese women. J Reprod Med 38:41-42, 1993. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 197

Infectious Diseases in Obstetrics and Gynecology I" 198-201 (1994) (C) 1994 Wiley-Liss, Inc. Acute Obstructive Hydrocephalus Due to Cysticercosis During Pregnancy Ronald M. Ramus, Mark Girson, Diane M. Twickler, and George D. Wendel, Jr. Departments ofObstetrics and Gynecology (R.M.R., G.D.W.) and Radiology (M.G., D.M.T.), University ofTexas Southwestern Medical Center, Dallas, TX ABSTRACT Background: Cysticercosis, due to the parasite Taenia solium, can involve any organ. When central nervous system infection occurs, signs and symptoms depend on the location ofthe cerebral lesions. Most patients develop seizures, focal symptoms, or headaches with nausea and vomiting. Case: A case of extraparenchymal (intraventricular) cysticercosis was diagnosed in a patient at term who presented with acute alteration in mental status. Ventriculostomy was performed because of acute obstructive hydrocephalus. Labor ensued and was augmented with oxytocin. Intrapartum management included magnesium sulfate seizure prophylaxis and corticosteroids. Intracranial pres- sures ranged between 4 and 12 cm n20 peripartum with approximately 300 mL of cerebrospinal fluid drained over the first 24 hours. Postpartum management included craniotomy with resection of a larval cyst and oral praziquantel therapy. Conclusion: This case describes an uncommon presentation of neurocysticercosis that should be considered in gravidas with acute mental status changes. (C) 1994 Wiley-Liss, Inc. KEY WORS Neurocysticercosis, organic brain syndrome, Taenia solium Ysticercosis is a parasitic disease caused by Tae- nia solium, the pork tapeworm. Humans are the only known host of the adult cestode, which resides in the intestine. Infection is acquired by eating undercooked pork infested with cysticerci. In contrast, both humans and pigs can serve as intermediate hosts for the larval stage, which can develop in almost any tissue in the body, and is responsible for the clinical manifestations of this infection.

de, which resides in the intestine. Infection is acquired by eating undercooked pork infested with cysticerci. In contrast, both humans and pigs can serve as intermediate hosts for the larval stage, which can develop in almost any tissue in the body, and is responsible for the clinical manifestations of this infection. The routes by which a human may be- come an intermediate host include contact with food or water contaminated with human feces (the most common source), autoinfection from anus to mouth by a person with the adult tapeworm, or reverse peristalsis and internal infection. Cysticercosis is rare, but is the most common parasitic disease af- fecting the central nervous system, with a variety of clinical presentations. Most cases of neurocys- ticercosis have a relatively benign course and are due to cysts in the cerebral parenchyma. Extra- parenchymal disease (intraventricular, subarach- noid, and spinal) is due to cysts in the cerebrospinal fluid and carries a poorer prognosis. This report describes a case of"intraventricular cysticercosis that presented as acute obstructive hydrocephalus in a patient with a term gestation. Upon review of the literature, we found this case to be the second re- ported one of extraparenchymal neurocysticercosis associated with pregnancy. CASE REPORT The patient is a 28-year-old Hispanic female, G3P2, who initially presented for prenatal care at 19 weeks Address correspondence/reprint requests to Dr. Ronald M. Ramus, Department ofObstetrics and Gynecology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9032. Obstetrics Case Report Received September 28, 1993 Accepted December 17, 1994 OBSTRUCTIVE HYDROCEPHALUS DUE TO CYSTICERCOSIS RAMUS ET AL. gestation. I-Ier past medical history was unremark- able. She had delivered term infants in 1978 in Mexico and in 1987 in Dallas, after moving to the United States from Mexico in 1984. The patient's prenatal course was remarkable for acute right adnexal torsion at 25 weeks gestation necessitating exploratory laparotomy and right sal- pingo-oophorectomy. Her postoperative course was uncomplicated, and she was discharged on the fifth postoperative day. At 38.5 weeks gestation, the patient was brought to the emergency room by her family because of acute mental status changes. Two hours previously, the patient began mumbling inappropriate re- sponses to questions and became progressively less responsive.

mplicated, and she was discharged on the fifth postoperative day. At 38.5 weeks gestation, the patient was brought to the emergency room by her family because of acute mental status changes. Two hours previously, the patient began mumbling inappropriate re- sponses to questions and became progressively less responsive. There were no seizures, but the patient complained ofa severe headache several hours prior to her mental status changes. There was no history of nausea or vomiting. On examination, the patient was obtunded and unresponsive to stimuli. Her oral temperature was 38.4C, blood pressure 170/90 mmI-Ig, pulse 100, and respirations 24/rain. Her pupils were slug- gishly reactive, horizontal nystagmus was present, and her left eye was outwardly deviated. Mild papilledema was present. No other focal neurologic findings were present. The patient could move all extremities, and reflexes were normal. Fetal heart tones were 150 beats/min. I-Ier cervix was cm dilated and 25% effaced. Cephalic presentation was noted. There were no subcutaneous or intramuscu- lar nodules. The patient's white blood cell count was 11,300/ mm3, hemoglobin, 10.7 g/dL; hematocrit, 32.0%; and platelet count, 247,000/mm3. Electrolytes, liver function tests, urinalysis, and coagulation stud- ies were all normal. Arterial blood gas on room air was pI-I, 7.47; pCO2, 27 mmI-Ig; pO)., 132 mmI-Ig; with an oxygen saturation, 99%. The chest x-ray was normal. The patient was admitted to labor and delivery, and an emergency computed tomography (CT) scan of the head was obtained. It demonstrated hydro- cephalus with dilatation of the lateral and third ventricles. A soft tissue density was present within the posterior portion of the third ventricle, repre- senting the intraventricular cysticercus cyst (Fig. 1). Several parenchymal cysticercus cysts were present, manifest by low density areas with punc- tate high density scoleces. Fig. I. Axial CT scan on admission. There is hydroceph- alus, manifested by dilation of the third and lateral ventricles, with periventricular calcifications (arrowhead). The intra- ventricular cysticercus cyst (arrow) is seen as a soft-tissue density within the dilated third ventricle. The patient was started on magnesium sulfate for seizure prophylaxis and intravenous dexametha- sone. Magnesium sulfate was selected since it is the standard medication used for seizure prophylaxis in our labor and delivery unit.

ticercus cyst (arrow) is seen as a soft-tissue density within the dilated third ventricle. The patient was started on magnesium sulfate for seizure prophylaxis and intravenous dexametha- sone. Magnesium sulfate was selected since it is the standard medication used for seizure prophylaxis in our labor and delivery unit. 2 At that time, proto- cols for the administration of alternative anticon- vulsants had not yet been developed. Neurosurgical consultation was obtained, and a ventriculostomy shunt was placed in the right lateral ventricle to relieve the acute obstructive hydrocephalus. This was followed by a gradual return to normal neuro- logic function over 12-24 hours. Intracranial pres- sures were noted to vary between 4 and 12 cm H20 after ventriculostomy placement, with approxi- mately 300 mL of cerebrospinal fluid drained over the next 24 hours. Ventricular cerebrospinal fluid studies including a cell count, protein, glucose, and cultures were performed post-ventriculostomy and were normal. The patient's fever and hypertension present at admission spontaneously resolved. In addition, the patient began having regular contractions with cer- vical dilation to 2 cm. Oxytocin augmentation of labor resulted in the delivery of a 2,745-g male INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY OBSTRUCTIVE HYDROCEPHALUS DUE TO CYSTICERCOSIS RAMUS ET AL. discharged on the fifth postoperative day on diphe- nylhydantoin and a 14-day course of praziquantel. Fig. 2. MRI scan ofthe head following ventriculostomy. An axial T weighted image through the ventricles demonstrates the high signal intensity cysticercus cyst within the trigone region of the left lateral ventricle. There is disproportionate enlargement of the left lateral ventricle, but the right lateral and third ventricles are normal in size. infant over an intact perineum, with Apgar scores of 8 and 9 at and 5 minutes, respectively. Magnetic resonance imaging (MRI) scan of the head, performed 36 hours following ventriculos- tomy, showed the right lateral ventricle and third ventricle to be of normal size. The left lateral ven- tricle remained enlarged, and the cyst, previously seen within the third ventricle, was now apparent posteriorly in the left lateral ventricle. This sug- gested migration of the intraventricular cysticercus cyst from the third ventricle into the lateral ventri- cle. The cyst manifested high signal intensity on both T1 and T2 weighted sequences, with respect to cerebrospinal fluid and brain parenchyma (Fig.

ow apparent posteriorly in the left lateral ventricle. This sug- gested migration of the intraventricular cysticercus cyst from the third ventricle into the lateral ventri- cle. The cyst manifested high signal intensity on both T1 and T2 weighted sequences, with respect to cerebrospinal fluid and brain parenchyma (Fig. 2). The parenchymal cysticercus lesions, seen pre- viously on CT scan, were once again seen as cystic structures with the high signal intensity scoleces. The ventriculostomy was removed 6 days after placement, and the next day craniotomy was per- formed with resection of an intact larval cyst mea- suring 2.0 x 1.5 x 1.5 cm. The patient's postop- erative course was unremarkable. She was DISCUSSION There are a variety of clinical manifestations of central nervous system (CNS) cysticercosis. Sei- zures, focal neurologic deficits, decreased visual acuity, and altered mental status have all been de- scribed.3 The particular symptoms in any patient depend primarily upon the anatomic location of the cysts. In addition, the degree of associated inflam- mation and edema can influence the clinical pic- ture. The cysts usually remain viable for 3-5 years and then degenerate, which can induce a significant inflammatory response. The diagnosis of cerebral cysticercosis is usually made by CT or MRI of the CNS.4-6 Cerebrospi- nal fluid (CSF) studies can also help diagnose and classify CNS cysticercosis. Nonspecific findings, such as increased CSF protein, lymphocytosis, and eosinophilia can be indicators of the severity of an inflammatory response in the subarachnoid space. In addition, an immunologic response in blood or CSF to cysticercal antigens, as demonstrated in an enzyme-linked immunoelectrotransfer blot (EITB), is diagnostic. The EITB assay has a sensi- tivity of 95% and a specificity of 100%, and has been mostly utilized in areas of endemic infection where high resolution imaging techniques are im- possible and many asymptomatic individuals with 7 infection are present. The only other reported case of extraparenchy- real neurocysticercosis during pregnancy occurred in a Mexican visitor to Arizona in 1972. The patient presented at 12 weeks gestation with a 3-day history of nausea, vomiting, and headache. She became unresponsive and tachypneic and then res- pirations suddenly ceased. Upon arrival at the hos- pital, she was cyanotic with a weak pulse, no respi- rations, and pupils that were fixed and dilated.

zona in 1972. The patient presented at 12 weeks gestation with a 3-day history of nausea, vomiting, and headache. She became unresponsive and tachypneic and then res- pirations suddenly ceased. Upon arrival at the hos- pital, she was cyanotic with a weak pulse, no respi- rations, and pupils that were fixed and dilated. Aggressive supportive care was initiated with grad- ual deterioration of the patient's status and subse- quent demise 24 hours after admission. Autopsy revealed cysticercosis of the right lateral ventricle and obstructive hydrocephalus secondary to cys- ticerci. The optimal treatment of cysticercosis is preven- tion. Personal hygiene and sanitary health measures are critical to avoid human fecal contamination. In 200 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY OBSTRUCTIVE HYDROCEPHALUS DUE TO CYSTICERCOSIS RAMUS ET AL. addition, the larvae are destroyed by either freezing or thoroughly cooking pork. In patients with neu- rocysticercosis, treatment is primarily determined by the anatomic location of the disease. Algorithms of proposed treatment options have been devised.9 Inactive disease is usually characterized by granulo- mas or calcifications on imaging of the CNS, and only supportive measures are indicated. This would include anticonvulsants for those with recurrent seizures. Surgical removal of inactive neurocys- ticercosis or antihelminthic therapy is not indicated. Parenchymal disease from viable cysts can be treated medically with praziquantell or albendazole. 11 There is good evidence that medical therapy is effi- cacious, with success rates as good as surgical ther- apy in reducing the frequency of subsequent sei- zures in patients with parenchymal infection, lz Therapy is more complicated for extraparenchymal neurocysticercosis. Subarachnoid cysticercosis can also be treated medically after shunting for any associated hydrocephalus is accomplished. How- ever, these patients do not respond as well to medi- cal therapy and frequently require surgical removal of focal lesions.3 Intraventricular cysticercosis of- ten presents with hydrocephalus, requiring a ven- tricular shunt. Primary therapy of cysticerci is sur- gical, as anti-cysticercal drugs are not effective. 13 However, this concept has been recently challenged by a report of a patient with intraventricular cys- ticercosis successfully treated with praziquantel.

sis of- ten presents with hydrocephalus, requiring a ven- tricular shunt. Primary therapy of cysticerci is sur- gical, as anti-cysticercal drugs are not effective. 13 However, this concept has been recently challenged by a report of a patient with intraventricular cys- ticercosis successfully treated with praziquantel. 14 Spinal cysticercosis can be treated medically, with surgical resection by laminectomy reserved for per- sistent disease or cysts in a single location. Most patients require seizure prophylaxis with anticon- vulsants, and steroids may be utilized if associated cerebral edema is present. The possibility ofneurocysticercosis is most com- monly considered by the obstetrician in the differ- ential diagnosis of peripartum seizures, particu- larly in women from Mexico and South America. 15 However, as this patient illustrates, cysticercosis may also present with only acute mental status changes due to acute obstructive hydrocephalus. Aggressive medical and surgical intervention re- sulted in maternal survival and return of normal neurologic function. The diagnosis of neurocys- ticercosis should be considered in gravidas with signs and symptoms of an acute organic brain dys- function. REFERENCES 1. Sotelo J: Neurocysticercosis. In Kennedy PGE, Johnson RT (eds): Infections of the Nervous System. London: Butterworth, pp 145-152, 1987. 2. PritchardJA, Cunningham FG, Pritchard SA: The Park- land Memorial Hospital protocol for treatment of ec- lampsia: Evaluation of 245 cases. Am J Obstet Gynecol 148i951-960, 1984. 3. Del Brutto OH, Sotelo J: Neurocysticercosis: An update. J Infect Dis 10:1075-1087, 1988. 4. Suss RA, Maravilla KR, Thompson J: MR imaging of intracranial cysticercosis: Comparison with CT and anatomapathologic features. AJNR 7:235-242, 1986. 5. Ramos OM, Stiebel-Chin G, Altman N, Duchowny M: Diagnosis of neurocysticercosis by magnetic resonance imaging. Pediatr Infect Dis 5:470--473, 1986. 6. Barkovich AJ, Citrin CM, Klara P, Wippold FJ, Kattah J: Magnetic resonance imaging of cysticercosis. West J Med 145:687-690, 1986. 7. Garcia HH, Martinez M, Gilman R, et al.: Diagnosis of cysticercosis in endemic regions. Lancet 338:549-551, 1991. 8. Bazley WS: Maternal mortality due to cysticercus cere- bri: A case report. Obstet Gynecol 39:362-367, 1972. 9. Castero I: Tratado de Anatomia Patologica. Mexico City: Ed Atlante, pp 1485-1495, 1946. 10.

Garcia HH, Martinez M, Gilman R, et al.: Diagnosis of cysticercosis in endemic regions. Lancet 338:549-551, 1991. 8. Bazley WS: Maternal mortality due to cysticercus cere- bri: A case report. Obstet Gynecol 39:362-367, 1972. 9. Castero I: Tratado de Anatomia Patologica. Mexico City: Ed Atlante, pp 1485-1495, 1946. 10. Sotelo J, Escobedo F, Rodriquez-Carbajal J, Torres B, Rubio-Donnadieu F: Therapy of parenchymal brain cys- ticercosis with praziquantel. N Engl J Med 310:1001- 1007, 1984. 11. Escobedo F, Penagos P, Rodriques J, Sotelo J: Albenda- zole therapy for neurocysticercosis. Arch Intern Med 147: 738-741, 1987. 12. Vazquez V, Sotelo J: The course of seizures after treat- ment for cerebral cysticercosis. N Engl J Med 327:696- 701, 1992. 13. SoteloJ: Cysticercosis: InJohnson RT (ed): Current Ther- apy in Neurologic Disease. Vol 2. Philadelphia: BC Decker, pp 114-117, 1987. 14. Bandres JC, White AC, Samo T, Murphy EC, Harris RL: Extraparenchymal neurocysticercosis: Report of five cases and review ofmanagement. Clin Infect Dis 15:799- 811, 1992. 15. Brown CEL, Purdy P, Cunningham FG: Head com- puted tomographic scans in women with eclampsia. Am J Obstet Gynecol 159:915-920, 1988. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:202-208 (1994) (C) 1994 Wiley-Liss, Inc. Systemic vs. Topical Therapy for the Treatment of Vulvovaginal Candidiasis Sebastian Faro Department ofGynecology and Obstetrics, University ofKansas School ofMedicine, Kansas City, KS ABSTRACT It is estimated that 75% of all women will experience at least 1 episode of vulvovaginal candidiasis (VVC) during their lifetimes. Most patients with acute VVC can be treated with short-term regi- mens that optimize compliance. Since current topical and oral antifungals have shown comparably high efficacy rates, other issues should be considered in determining the most appropriate therapy. It is possible that the use of short-duration narrow-spectrum agents may increase selection of more resistant organisms which will result in an increase of recurrent VVC (RVVC). Women who are known or suspected to be pregnant and women of childbearing age who are not using a reliable means of contraception should receive topical therapy, as should those who are breast-feeding or receiving drugs that can interact with an oral azole and those who have previously experienced adverse effects during azole therapy. Because of the potential risks associated with systemic treat- ment, topical therapy with a broad-spectrum agent should be the method of choice for VVC, whereas systemic therapy should be reserved for either RVVC or cases where the benefits outweigh any possible adverse reactions. (C) 1994 Wiley-Liss, Inc. KEY WORDS Adverse effects, compliance, drug interactions, oral therapy, pregnancy, topical therapy ulvovaginal candidiasis (VVC) remains, after bacterial vaginosis, the most common vaginal infection in the United States. 1,2 Researchers esti- mate that 75% of all women will have VVC infection during their lifetimes, typically during their childbearing years. 1'3'4 Between 40% and 50% of women who have experienced episode of VVC are likely to have at least recurrence, and approximately 5% will have repeated episodes of the disorder.

es. 1,2 Researchers esti- mate that 75% of all women will have VVC infection during their lifetimes, typically during their childbearing years. 1'3'4 Between 40% and 50% of women who have experienced episode of VVC are likely to have at least recurrence, and approximately 5% will have repeated episodes of the disorder. Of particular concern to physicians and other health care professionals is the fact that the number of prescriptions written in the United States to treat yeast infections has almost doubled between 1980 and 1990, reaching 13 million. Statistical data derived from patients treated at genitourinary med- icine centers in the United Kingdom indicate a similarly sharp increase, from 118/100,000 to 200/ 100,000 patients during the last decade. More- over, although the causative organism in 85-90% of all cases of VVC is Candida albicans, some evi- dence suggests that the proportion of non-albicans Candida infections, including those caused by C. (Torulopsis) glabrata, C. tropicalis, and C. krusei, is rising substantially.2'5 Traditional imidazole treat- ment regimens have often shown less efficacy against those species than against most of the ap- proximately 200 known pathogenic strains of C. albicans. 1,6,7 Amid these causes for concern, however, posi- tive developments are the number of therapeutic agents and range of treatment options available for the management of VVC. Most women with acute VVC can be treated with short-term regimens that are likely to enhance compliance and thus minimize the risk of treatment failure.2'8'9 Newer triazole Address correspondence/reprint requests to Dr. Sebastian Faro, Department of Gynecology and Obstetrics, University of Kansas School ofMedicine, 3901 Rainbow Boulevard, Kansas City, KS 66160-7316. Review Article Received February 16, 1994 Accepted February 22, 1994 SYSTEMIC VS. TOPICAL CANDIDIASIS THERAPY FARO TABLE I.

Address correspondence/reprint requests to Dr. Sebastian Faro, Department of Gynecology and Obstetrics, University of Kansas School ofMedicine, 3901 Rainbow Boulevard, Kansas City, KS 66160-7316. Review Article Received February 16, 1994 Accepted February 22, 1994 SYSTEMIC VS. TOPICAL CANDIDIASIS THERAPY FARO TABLE I. Drug formulations and doses commonly used to treat VVCa Agent Formulation Dose Nystatin Clotrimazole Miconazole Tioconazole Econazole Terconazole 100,000 U vaginal tablet 100,000 U x 14 days 1% cream 5 g x 7-14 days 100 mg vaginal tablet 100 mg 7 days 100 mg vaginal tablet 200 mg 3 days 500 mg vaginal tablet 500 mg x dose 2% cream 5 g x 7 days 100 mg vaginal 100 mg 7 days suppository 200 mg vaginal 200 mg x 3 days suppository 1,200 mg vaginal 1,200 mg x dose suppository 2% cream 5 g x 3 days 6.5% cream 5 g dose 150 mg vaginal tablet 150 mg 3 days 0.4% cream 5 g x 7 days 0.8% cream 5 g x 3 days 80 mg vaginal 80 mg x 3 days suppository Ketoconazole 200 mg oral tablet Fluconazole 150 mg oral capsule Itraconazole 100 mg oral capsule 400 mg x 5 days3z 150 mg dose2s 200 mg x 3 days4 aAdapted in part from Sobel. formulations provide a broader spectrum of activ- ity against non-albicans species, thereby suggesting the possibility ofbetter cure rates and less recurrent disease. One decision for the physician, given this diversity of options, is whether to prescribe a topi- cal or an oral agent for the patient with VVC. This review discusses the potential benefits and liabilities of topical vs. systemic (oral) drug ther- apy, taking into account such factors as efficacy in acute and recurrent VVC (RVVC), potential for the development of resistance, pathogen selection or shift, VVC during pregnancy, potential for drug interactions, safety profiles, and patient acceptance. Table summarizes the formulations and dosages of those agents that are commonly used to treat VVC. TREATMENT OF TOPICAL INFECTIONS WITH TOPICAL PREPARATIONS As a rule, it is preferable to treat topical disorders with topical therapy. It is always advisable to in- volve the smallest possible number ofbody systems in the pharmacotherapeutic process. Even with the use of a systemic agent that has a good safety pro- file, concern about the development of potentially serious adverse effects remains.

rable to treat topical disorders with topical therapy. It is always advisable to in- volve the smallest possible number ofbody systems in the pharmacotherapeutic process. Even with the use of a systemic agent that has a good safety pro- file, concern about the development of potentially serious adverse effects remains. In an era of fiscal constraint, when the effective use of physician time and the reduction of patient costs are regarded as essential, the unnecessary monitoring of patients for untoward effects of systemic therapy and the possible need for additional physician visits are dif- ficult to defend. Concern about adverse.effects is compounded when therapy focuses on women of childbearing age whose acute or recurrent disorder may be an unrecognized sign of pregnancy. Whether or not preclinical investigations and existing data from human populations have suggested that a given sys- temic agent has the potential for teratogenesis, un- less the absence of pregnancy is confirmed, the prudent course is to treat the patient who could be pregnant with a topical agent that has a well-estab- lished reproductive health profile. Similarly, the risk of potentially serious drug- drug interactions always remains when a systemic drug is used in lieu of a topical agent. This risk is significantly increased when a systemic agent is to be used primarily in a population whose members are known to make extensive use of an interacting class ofdrugs, i.e., oral contraceptives. Even when the physician clearly delineates the implications of drug-drug interactions to a patient and explicitly questions her about current use of potentially inter- acting agents, full disclosure by the patient cannot be guaranteed. Many studies have shown that good compliance is unlikely to be achieved unless the patient regards the presenting problem as a severe disorder. De- spite VVC's potential seriousness, women tend to view it as a nuisance or an inconvenience rather than a major health problem. Thus, it cannot be assumed that patients who use a systemic agent will refrain from the concurrent use ofpotentially inter- acting drugs or modify their behaviors to minimize the risks of drug-drug interactions. EFFICACY IN ACUTE VVC In immunocompetent patients with acute VVC, the overall clinical and mycological cure rate is ex- 1,10 tremely high with both topical and oral agents. The forms of topical antimycotic drugs include creams, lotions, vaginal tablets, and suppositories.

o minimize the risks of drug-drug interactions. EFFICACY IN ACUTE VVC In immunocompetent patients with acute VVC, the overall clinical and mycological cure rate is ex- 1,10 tremely high with both topical and oral agents. The forms of topical antimycotic drugs include creams, lotions, vaginal tablets, and suppositories. There are no data that the formulation of a topical agent influences its clinical efficacy or that the ma- jority of women prefer a particular form of local administration. With nystatin, the average myco- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 203 SYSTEMIC VS. TOPICAL CANDIDIASIS THERAPY FARO TABLE 2. Risk factors for VVC'3 Predisposing conditions Pregnancy, especially in the 3rd trimester Diabetes mellitus that is poorly controlled Cushing's disease Addison's disease Hypo- or hyperthyroidism Debilitating disease, e.g., leukemia, severe vitamin deficiency AIDS or HIV infection Vaginal trauma Predisposing practices Use of high-estrogen oral contraceptives Use of antibiotics, especially tetracycline, ampicillin, and cephalosporins Use of therapies that compromise the immune system, including hormones, cytotoxic agents, immunosuppressive drugs, and radiotherapy Frequent, traumatic sexual intercourse Wearing of tight clothing or nylon underwear Use of intrauterine contraceptive devices Ingestion of a large amount of candy logical cure rate is generally considered to range from 75% to 80%, while it is estimated to be some- what higher, from 85% to 90%, with the azole derivatives. 1,3 Because of its broader spectrum of activity, the triazole terconazole has shown excel- lent efficacy against a wide range of pathogenic fungi.3'8 EFFICACY IN RVVC RVVC, the occurrence of at least 4 mycologically proven symptomatic episodes of VVC in year, occurs in approximately 5% ofpatients with VVC. 11 Although it occurs at extremely high rates among immunocompromised women, including approxi- mately 24% of human immunodeficiency virus (HIV)-infected patients, 12 it is also found in women who have none of the risk factors generally associ- ated with VVC (Table 2). ''The cause ofRVVC has not been determined, but several explanations have been proposed. RVVC has often been attributed to the existence of an intestinal reservoir of Candida that acts as a source of reinfection.2' However, while it is true that some patients with RVVC demonstrate rectal colonization by Candida species, such colonization has also been found in approximately 40% of as- ymptomatic women.

ed. RVVC has often been attributed to the existence of an intestinal reservoir of Candida that acts as a source of reinfection.2' However, while it is true that some patients with RVVC demonstrate rectal colonization by Candida species, such colonization has also been found in approximately 40% of as- ymptomatic women. Furthermore, biotyping of vaginal and rectal cultures simultaneously obtained from patients with RVVC has often shown a low concordance between cultures. A longitudinal study ofwomen with RVVC who were receiving ketocon- azole therapy showed that VVC sometimes recurred when rectal cultures were negative for Candida spe- cies. In addition, treatment with nystatin, which reduces intestinal yeast carriage, has failed to pre- vent symptomatic RVVC. Although the clinical rel- evance of existing Candida reservoirs remains open to investigation, systemic, oral therapy may be used as an effective means of destroying these reser- voirs. 10,4,15 Most patients with RVVC require long-term maintenance regimens with mycosuppressive pro- phylaxis. Extended therapy with topical clotrima- zole or miconazole or with oral ketoconazole has been shown to significantly reduce the frequency of RVVC for the duration of treatment, l Neverthe- less, approximately 50% of patients experience a relapse within several months after treatment ends. 'll Although some investigators have sug- gested that oral therapy is more likely than vaginal therapy to promote the necessary level of patient compliance during long-term treatment, '9 others have argued that the greater risk of systemic toxic- ity associated with oral agents points in favor of topical therapy for RVVC. 16 Oral therapy has the potential to increase the amount of physician time devoted to patient monitoring and to increase pa- tient expenses associated with physician visits and laboratory tests. RESISTANCE AND PATHOGEN SHIFT Some controversy exists as to whether the trend toward shorter courses of therapy for acute VVC and the use of long-term maintenance regimens in RVVC have led to the selection of more resistant strains of Candida species, such as C. tropicalis, C. krusei, and C. glabrata (formerly Torulopsis gla- brata). 1'2 It has been argued that, although C. albicans remains the most common cause of VVC, other, more resistant species are gradually assum- ing a more prominent role.

ave led to the selection of more resistant strains of Candida species, such as C. tropicalis, C. krusei, and C. glabrata (formerly Torulopsis gla- brata). 1'2 It has been argued that, although C. albicans remains the most common cause of VVC, other, more resistant species are gradually assum- ing a more prominent role. In examining the re- sults of 9 studies conducted in the 1970s, Horowitz et al.2'5 found that the prevalence of non-albicans species approximated 9.9%. By the 1980s, 7 stud- ies revealed a non-albicans prevalence of 21.3%. During that period, the incidence of C. glabrata was found to have increased from 4.6% to 6.7%, while that of C. tropicalis rose from 1.3 % to 8.2%. Although the phenomenon ofpathogen shift among yeasts has not been definitively proven, 11 most data seem to point in that direction. 204 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY SYSTEMIC VS. TOPICAL CANDIDIASIS THERAPY FARO Resistance to Topical Agents Both in vivo and in vitro studies have demonstrated the lack of development of resistance to topical antifungal agents. 8 One barrier to resistance may be the high drug concentration attained at the site of infection. This is especially true of the azole deriv- atives, which act on multiple targets on the plasma membrane and within the yeast cell. The only avail- able topical triazole, terconazole, has the added benefit ofa broad spectrum ofactivity against many non-albicans Candida species, which may prevent 3,8 selection of" more resistant yeasts. Resistance to Oral Agents Among the 3 oral azole antifungal agents, the fre- quency with which resistance has been described in a clinical setting differs considerably. 12,17 Flucon- azole has been implicated in emergent resistance more frequently than ketoconazole, and ketocona- zole more often than itraconazole. Although most reported cases of resistance have involved long- term administration to immunocompromised pa- tients with sexually transmitted diseases or neutro- penia, the development of resistance may also have some correlation in the treatment of VVC. For example, in patients with acquired immune defi- ciency syndrome (AIDS) and neutropenia and those receiving treatment in intensive care units, C. gla- brata and C. krusei are frequently cited as emergent colonizing flora during frequent or sustained flu- conazole therapy. Those species have also been as- sociated with treatment failures in patients with disseminated infections and mucosal Candida infec- tions.

hose receiving treatment in intensive care units, C. gla- brata and C. krusei are frequently cited as emergent colonizing flora during frequent or sustained flu- conazole therapy. Those species have also been as- sociated with treatment failures in patients with disseminated infections and mucosal Candida infec- tions. Odds17 observes that, although most ofthese treatment failures are probably related to the lim- ited susceptibility of C. glabrata and C. krusei to fluconazole, the development of secondary resis- tance by C. glabrata strains has also been described. Del Palacio18 has observed that, although empiric prophylactic use of fluconazole in neutropenic pa- tients has led to a decreased frequency of C. albicans and C. tropicalis infections, such therapy seems to encourage the emergence of less common strains with a native resistance to fluconazole, including C. krusei, C. glabrata, C. lambica, and C. lusitaniae. MANAGEMENT OF VVC DURING PREGNANCY During pregnancy, the vagina is particularly sus- ceptible to Candida infection because of a higher glycogen content, increased estrogen levels, and the presence of other reproductive hormones. Can- dida strains can be found in vaginal cultures of 10-20% of pregnant women, and the incidence of colonization has been reported to be even higher during the 3rd trimester of pregnancy. 10 The inci- dence of symptomatic VVC is twice as high as it is in the nonpregnant state. Treatment ofVVC during pregnancy is indicated not only to alleviate the wom- an's symptoms, but also to protect the fetus from potentially fatal Candida sepsis. 10 Although clinical response during pregnancy tends to be slower and recurrences are more fre- quent, most topical antifungal agents are effective, especially when administered for 1-2 weeks. Al- though little systemic absorption of topical antifun- gal drugs occurs, the risk to the fetus during the st trimester of pregnancy should be weighed against the benefit derived by the mother. Many practi- tioners consider treatment with topical agents to be relatively safe during the 2nd and 3rd trimesters. 9 Administration of oral ketoconazole, fluconazole, and itraconazole during pregnancy is not recom- mended. 19,20 Because the development ofVVC may be an early sign of pregnancy and since it is not always feasible to administer a pregnancy test to each woman of childbearing potential who presents to the physician with acute VVC, the safest course is the use of a topical agent for first-line therapy.

ncy is not recom- mended. 19,20 Because the development ofVVC may be an early sign of pregnancy and since it is not always feasible to administer a pregnancy test to each woman of childbearing potential who presents to the physician with acute VVC, the safest course is the use of a topical agent for first-line therapy. In addition, fluconazole, itraconazole, and prob- ably ketoconazole are excreted in breast milk. Therefore, the use of systemic agents should be limited to those women who choose not to breast- feed. Alternately, topical therapy can be used dur- ing this time. POTENTIAL FOR DRUG INTERACTIONS Although significant drug interactions have not been associated with the use of topical antifungal agents, the potential for such drug interactions ex- ists with oral therapy.21 Table 3 lists those agents that are associated with the greatest risk for signifi- cant interaction with ketoconazole, fluconazole, and itraconazole. Among the drug interactions associated with ad- ministration of the oral azoles, 2 are of particular concern because ofthe characteristics ofthe patients who typically present with VVC. It is ironic that, although VVC generally occurs in women of child- bearing age and is especially common among those INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY SYSTEMIC VS. TOPICAL CANDIDIASIS THERAPY FARO TABLE 3. Agents with the greatest potential for interaction with the oral azoles27-29 Antacids Isoniazid Anticholinergics Loratadine Anticoagulants Methylprednisolone Astemizole Oral contraceptives Cimetidine Phenytoin Cyclosporine Potassium Digoxin Rifampin Diuretics Sulfonylureas Hz antagonists Terfenadine Hypoglycemics who take estrogen-containing oral contraceptives, the oral azoles have the potential to interact with oral contraceptives.22 As noted previously, the oral azoles are not recommended for young women who are not using a reliable method of birth control. 10 Another area ofspecial concern is that ofdiabetic patients. Although women with diabetes are at heightened risk for the development of VVC, 1'13 the potential for interaction exists when oral azoles are concurrently taken with oral hypoglycemics.23 The fact that a diabetic woman has developed symp- tomatic VVC suggests that her blood glucose levels are poorly controlled, and treatment of VVC with an oral azole can further compromise that control.

t of VVC, 1'13 the potential for interaction exists when oral azoles are concurrently taken with oral hypoglycemics.23 The fact that a diabetic woman has developed symp- tomatic VVC suggests that her blood glucose levels are poorly controlled, and treatment of VVC with an oral azole can further compromise that control. In view of the increasing use of the nonsedating antihistamines terfenadine and astemizole, physi- cians must advise patients of" the severe cardiovas- cular adverse effects, including prolonged QT in- tervals, that may arise when oral azoles are coadministered with those agents, z3 Conversely, patients should be cautioned that concomitant use of He antagonists can impair the efficacy of the oral azoles. The extent to which drug interactions with oral azoles may occur in actual clinical practice was suggested by a cross-sectional observational study conducted over a 12-week period at an urban hospi- tal in Australia.23 Of 76 patients receiving flucon- azole, 3 5 had been prescribed potentially interact- ing drugs and 9 of those patients were judged to have experienced 10 possible adverse interactions. In patient who received concomitant cyclosporine and fluconazole, the cyclosporine plasma concen- tration rose from 7 53 ng/ml to 1,543 ng/ml after 6 days of combination therapy. Potential adverse re- actions also occurred in patients receiving flucona- zole in conjunction with the following drugs: war- farin (2 of 4 patients), theophylline (1 of 2), phenytoin (3 of 9), rifabutin (2 of4), and rifampin (1 orS). The risk of potentially serious drug interactions may place a significant burden on the physician who elects to prescribe one of the oral azoles as first-line therapy for VVC. Patients must be ques- tioned carefully about the medications they are cur- rently receiving, and the possibility of an inaccu- rate self-report always exists. The physician who consciously prescribes an oral azole for a woman who is also receiving a potentially interacting drug must expend significant effort in monitoring the patient. Similarly, the patient may be required to bear the economic burden of additional physician visits and laboratory tests to monitor plasma con- centrations or other relevant parameters. ADVERSE EFFECT PROFILES Overall, a low incidence ofadverse effects has been reported for both topical and oral antifungal agents used to treat VVC.

the patient may be required to bear the economic burden of additional physician visits and laboratory tests to monitor plasma con- centrations or other relevant parameters. ADVERSE EFFECT PROFILES Overall, a low incidence ofadverse effects has been reported for both topical and oral antifungal agents used to treat VVC. It is noteworthy that a similar incidence and range of adverse effects have some- times been reported by VVC patients receiving ac- tive drug and placebo. 14 Adverse Reactions and Topical Therapy Administration of topical azoles is associated with few local and systemic side effects. The most com- mon local effects, which are usually minor, are vaginal burning, stinging, itching, irritation, and pain; dyspareunia may also occur. 3'8'14'24'25 Over- all, approximately 7% of women using azole anti- fungal cream preparations have reported some de- gree of" treatment-related vaginal discomfort.9 Additional side effects reported with topical agents include increased frequency of urination, pelvic cramps, dysmenorrhea, paresthesia, rhinorrhea, headache, fever, and chills.3'8'15'22'24'25 Adverse Effects Associated With Ketoconazole The most common adverse effects reported with ketoconazole are mild gastrointestinal disorders, in- cluding nausea, vomiting, and abdominal pain; these have been reported in approximately 10% of patients receiving the drug. At doses greater than 400 mg/day, some cases of androgen biosynthesis blockade have been described, z A few cases of anaphylaxis have also been reported. Although he- patotoxicity was initially estimated to occur in 206 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY SYSTEMIC VS. TOPICAL CANDIDIASIS THERAPY FARO 1/10,000 patients treated, the reported incidence has more recently fallen to 1/70,000. Part of this decrease can probably be attributed to a tendency for physicians to prescribe shorter courses of ther- apy. The incidence of hepatic reactions appears to be higher in women; patients being treated for onychomycosis; patients with preexisting liver dis- ease or alcoholism; patients who receive more than 2 weeks oftherapy or who have previously received 16 griseofulvin; and those over the age of 50 years.

courses of ther- apy. The incidence of hepatic reactions appears to be higher in women; patients being treated for onychomycosis; patients with preexisting liver dis- ease or alcoholism; patients who receive more than 2 weeks oftherapy or who have previously received 16 griseofulvin; and those over the age of 50 years. Adverse Effects Associated With Fluconazole Mild gastrointestinal symptoms, including nausea, vomiting, abdominal pain, and diarrhea, have been reported in approximately 5% of patients receiving fluconazole for VVC.4'18'25'26 Headache, dizzi- ness, dry mouth, rash, and dry skin have also been reported with some regularity. Approximately 5.1% of patients treated with fluconazole for super- ficial infection show abnormal liver function test results, as opposed to 3.7% of patients receiving topical treatments with clotrimazole.2 Although severe toxic epidermal necrolysis has been reported in a small number of patients treated with fluconazole, those patients were typically AIDS patients who had been receiving concomitant drug therapy for tuberculosis. Thus, a clear association between fluconazole and epidermal necrolysis has not been established, z One case ofthrombocytope- nia has also been reported. A case of angioedema with buccal ulceration was reported in a 21-year- old woman who had taken the 6th oftwice-monthly prophylactic doses of 150 mg fluconazole that had been prescribed for RVVC of 3 years duration.27 The case of a healthy 22-year-old woman who experienced an anaphylactic reaction a few minutes after administration of 150 mg of oral fluconazole as treatment for VVC has been reported.28 Although the patient had had no previous contact with flucon- azole, she had been treated with ketoconazole 2.5 years before the reported reaction. She had also received vaginal metronidazole year before and oral metronidazole week before the event. Results of a prick test with fluconazole solution were posi- tive at a 1:10 dilution. It appears likely that the patient experienced an allergic reaction to flucona- zole that may have been caused by cross-sensitiza- tion to ketoconazole or metronidazole. Adverse Effects Associated With Itraconazole The overall incidence of minor adverse effects re- ported with itraconazole has been estimated to range from 7% to 10%.20 Most have consisted of mild gastrointestinal disturbances. 13 Malaise, headache, dizziness, transient faintness, psychiatric effects, fever, hypertension, and edema have also been re- ported.

nazole The overall incidence of minor adverse effects re- ported with itraconazole has been estimated to range from 7% to 10%.20 Most have consisted of mild gastrointestinal disturbances. 13 Malaise, headache, dizziness, transient faintness, psychiatric effects, fever, hypertension, and edema have also been re- ported. 14,16,22 Hypokalemia has been associated with high-dose therapy. Although a few cases of reversible peripheral neuropathy have been re- ported, a firm association with itraconazole has not been established, z The incidence of hepatic reac- tions, including hepatitis, is approximately 6 in almost 5 million treatments. A low frequency (0.9%) of reversible changes in liver function tests has also been reported. 13,14,20 PATIENT ACCEPTANCE Since acute VVC in immunocompetent patients usu- ally responds to antifungal treatment, it has often been argued that many apparent treatment failures are actually the results of poor patient compli- ance. 9'15'24 Because studies have repeatedly shown that key factors in achieving compliance are a brief, uncomplicated dosage regimen and the patient's sat- isfaction with the nature of the treatment, patient preferences are important considerations in evalu- ating treatment alternatives. Some studies have indicated that when efficacy is comparable, women tend to prefer oral to topical therapy.29' It has also been suggested, however, that application of a topical agent to the affected area may confer more rapid symptomatic relief for some patients. Whether this benefit is real or only perceptual, the physician may wish to consider concomitant ther- apy with a topical drug when using a systemic antifungal. CONCLUSIONS In light of the comparable levels of efficacy re- ported for topical and oral agents in the treatment ofVVC and RVVC, major criteria for determining whether to prescribe a local or systemic agent in- clude issues related to drug resistance and pathogen shift, safety in pregnancy, drug interactions, ad- verse effects, and patient acceptance. Patients who are pregnant or suspected to be pregnant and women of childbearing age who are not using a reliable method of contraception should receive topical therapy. Women who are breast- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 207 SYSTEMIC VS.

, drug interactions, ad- verse effects, and patient acceptance. Patients who are pregnant or suspected to be pregnant and women of childbearing age who are not using a reliable method of contraception should receive topical therapy. Women who are breast- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 207 SYSTEMIC VS. TOPICAL CANDIDIASIS THERAPY FARO feeding or receiving one or more drugs with the potential to interact with the oral azoles should also be treated topically, as should those with a history ofadverse reactions to the azoles or with significant liver impairment. Whichever form oftherapy is ultimately chosen, the shortest feasible course of treatment is advis- able. The potential for resistance and pathogen shift with oral drugs argues for the use of broad-spec- trum topical therapy. Because the potential risks and disadvantages asso- ciated with oral therapy tend to outweigh the benefits, treatment with topical agents should be regarded as the method ofchoice for patients with VVC, although oral agents may have a place in the treatment ofRVVC or in those patients in whom the benefits may out- weigh any possible adverse reactions. REFERENCES 1. Sobel JD: Candidal vulvovaginitis. Clin Obstet Gynecol 36:153-165, 1993. 2. Horowitz BJ, Giaquinta D, Ito S: Evolving pathogens in vulvovaginal candidiasis: Implications for patient care. J Clin Pharmacol 32:248-255, 1992. 3. Corson SL, Kapikian RR, Nehring R: Terconazole and miconazole cream for treating vulvovaginal candidiasis. A comparison. J Reprod Med 36:561-567, 1991. 4. Osser S, Haglund A, Westrom L: Treatment ofcandidal vaginitis. A prospective randomized investigator-blind multicenter study comparing topically applied econazole with oral fluconazole. Acta Obstet Gynaecol Scand 70: 73-78, 1991. 5. Horowitz BJ: Mycotic vulvovaginitis: A broad overview. Am J Obstet Gynecol 165:1188-1192, 1991. 6. Redondo-Lopez V, Lynch M, Schmitt C, Cook R, Sobel JD: Torulopsis glabrata vaginitis: Clinical aspects and sus- ceptibility to antifungal agents. Obstet Gynecol 76:651- 655, 1990. 7. Boag FC, Houang ET, Westrom R, McCormack SM, Lawrence AG: Comparison of vaginal flora after treat- ment with a clotrimazole 500 mg vaginal pessary or a fluconazole 150 mg capsule for vaginal candidosis. Geni- tourin Med 67:232-234, 1991. 8. Ernest JM: Topical antifungal agents. Obstet Gynecol Clin North Am 19:587-607, 1992. 9. Nixon SA: Vulvovaginitis: The role ofpatient compliance in treatment success. Am J Obstet Gynecol 165:1207- 1209, 1991.

mg vaginal pessary or a fluconazole 150 mg capsule for vaginal candidosis. Geni- tourin Med 67:232-234, 1991. 8. Ernest JM: Topical antifungal agents. Obstet Gynecol Clin North Am 19:587-607, 1992. 9. Nixon SA: Vulvovaginitis: The role ofpatient compliance in treatment success. Am J Obstet Gynecol 165:1207- 1209, 1991. 10. Merkus JMWM: Treatment ofvaginal candidiasis: Orally or vaginally?J Am Acad Dermato123:568-572, 1990. 11. Sobel JD: Pathogenesis and treatment of recurrent vul- vovagina! candidiasis. Clin Infect Dis 14(Suppl 1):S148- S153, 1992. 12. Ng TTC, Denning DW: Fluconazole resistance in Can- dida in patients with AIDS---A therapeutic approach. J Infect Dis 26:117-125, 1993. 13. Scudamore JA, Tooley PJ, Allcorn RJ: The treatment of acute and chronic vaginal candidosis. Br J Clin Pract 46:260-263, 1992. 14. Stein GE, Mummaw N: Placebo-controlled trial of itra- conazole for treatment of acute vaginal candidiasis. Anti- microb Agents Chemother 37:89-92, 1993. 15. Patel HS, Peters MD II, Smith CL: Is there a role for fluconazole in the treatment of vulvovaginal candidiasis? Ann Pharmacother 26:350-353, 1992. 16. Fong IW: The value of chronic suppressive therapy with itraconazole versus clotrimazole in women with recurrent vaginal candidiasis. Genitourin Med 68:374-377, 1992. 17. Odds FC: Resistance ofyeasts to azole-derivative antifun- gals. J Antimicrob Chemother 31:463-471, 1993. 18. Del Palacio A: Fungal skin and soft tissue infections. Curr Opin Infect Dis 5:687-694, 1992. 19. Timonen H: Shorter treatment for vaginal candidosis: Comparison between single-dose oral fluconazole and three-day treatment with local miconazole. Mycoses 35: 317-320, 1992. 20. Hay RJ: Risk/benefit ratio of modern antifungal therapy: Focus on hepatic reactions. J Am Acad Dermato129:$50- $54, 1993. 21. Baciewicz AM, Baciewicz FA Jr: Ketoconazole and flu- conazole drug interactions. Arch Intern Med 153:1970- 1976, 1993. 22. 1994 Physician's Desk Reference. Montvale, NJ: Medi- cal Economics Data Production Company, 1994. 23. Tett S, Carey D, Lee HS: Drug interactions with flucon- azole. Med J Aust 156:365, 1992. 24. Clark C, Cooper CL, Gordon SF, van Amerongen D, Smith FO, Upmalis DH: A multicenter comparison of one-dose ticonazole ointment with three-dose terconazole cream in vulvovaginal candidiasis. J Women's Health 2:189-196, 1993. 25. Stein GE, Christensen S, Mummaw N: Comparative study of fluconazole and clotrimazole in the treatment of vulvovaginal candidiasis.

rongen D, Smith FO, Upmalis DH: A multicenter comparison of one-dose ticonazole ointment with three-dose terconazole cream in vulvovaginal candidiasis. J Women's Health 2:189-196, 1993. 25. Stein GE, Christensen S, Mummaw N: Comparative study of fluconazole and clotrimazole in the treatment of vulvovaginal candidiasis. Drug Intelligence and Clinical Practice 25:582-585, 1991. 26. Brammer KW, Feczko JM: Single-dose oral fluconazole in the treatment of vaginal candidiasis. Ann NY Acad Sci 554:561-563, 1988. 27. Abbott M, Hughes DL, Patel R, Kinghorn GR: Angio- oedema after fluconazole. Lancet 338:633, 1991. 28. Neuhaus G, Pavcic N, Pletscher M: Anaphylactic reac- tion after oral fluconazole. Br Med J 302:1341, 1991. 29. Tobin JM, Loo P, Granger SE: Treatment of vaginal candidosis: A comparative study of the efficacy and ac- ceptability of itraconazole and clotrimazole. Genitourin Med 68:36-38, 1992. 30. Slavin MB, Benrubi GI, Parker R, Griffin CR, Magee MJ: Single dose oral fluconazole vs. intravaginal tercon- azole in treatment of candida vaginitis. Comparison and pilot study. J Fla Med Assoc 79:693-696, 1992. 208 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:209 (1994) (C) 1994 VViley-Liss, Inc. Immunizations During Pregnancy The time has come when physicians may be able to practice preventive medicine, which is particularly important in reference to those infectious diseases that can be prevented by immunizations. Even pregnant women should be candidates for certain immunizations. It is the scope of this editorial to cover just two of those immunizations that can be prescribed for pregnant women: the influenza vaccine and the hepatitis B vaccine. The influenza vaccine is changed on a yearly basis to immunize against the strains expected to cause disease that season. Although the influenza vaccine is available, it is often not prescribed or used in the pregnant patient. There are several reasons for not prescribing it during pregnancy, one of which is the physician's fear of medical/legal consequences. Another reason is that patients who have a bad outcome during pregnancy may have recall bias and blame the unfortu- nate outcome on a specific circumstance, drug, or immunization. Although this cause and effect is almost never the case, it is certainly difficult to convince the patient of this point. However, physicians should be proactive in offering the influenza vaccine to those patients who may suffer the highest complication rates, namely, patients who are severe asthmatics or those with heart disease. Therefore, we should remind ourselves, particularly in the northern hemisphere ofthe United States, that in the months of October and November before the winter flu season, we should offer the influenza vaccine to patients who have entered the second trimester or more of pregnancy without fear that the vaccine will cause a fetal problem. The hepatitis B vaccine, now offered in a recombinant form, should be made available to our female population, especially to individuals who either have a life style that may increase their risk of acquiring hepatitis B infection, such as health care workers, or live in a community where there is a high prevalence of hepatitis, such as in a large inner-city population.

nt form, should be made available to our female population, especially to individuals who either have a life style that may increase their risk of acquiring hepatitis B infection, such as health care workers, or live in a community where there is a high prevalence of hepatitis, such as in a large inner-city population. Unfortunately, the cost of the hepatitis B vaccine for an adult is approximately $50 per dose, with each patient requiring 3 doses over a course of 6 months. For this reason, the cost factor has limited the use of the hepatitis B vaccine. With the current active support of the Academy of Pediatrics to immunize newborns and children with the hepatitis B vaccine, many individuals may be immunized in the future. However, until that point is reached, we should offer immunization to women who are considered high risk, preferably before they get pregnant, and remember that pregnancy is not a contraindication for the hepatitis B vaccine. Joseph J. Apuzzio, M.D. Department ofOB/GYN New Jersey Medical School Newark, New Jersey Editorial

Infectious Diseases in Obstetrics and Gynecology 1:210-215 (I 994) (C) 1994 Wiley-Liss, Inc. Effect of Resident Physician Education Regarding Selective Chemoprophylaxis for the Prevention of Early Onset Group B Streptococcal Sepsis: An Outcome Study Jeffrey S. Greenspoon, Doron J.D. Rosen, and Anita P. Sumen Department of Obstetrics and Gynecology, Cedars-Sinai Medical Center, Los Angeles, CA ABSTRACT Objective: The aim of this study was to evaluate the effect of a voluntary protocol for selective intrapartum chemoprophylaxis on the incidence of early onset group B streptococcal sepsis (GBS EOS). Methods: Cases ofGBS EOS were defined as a positive GBS culture from a normally sterile fluid obtained during the first 7 days of life. All cases of GBS EOS at an urban, university-affiliated community hospital were reviewed during 2 time periods. The 2-year period before instituting a resident education program to promote selective chemoprophylaxis (1988-89) was retrospectively reviewed; the 2-year period after the education program was introduced (1990-91) was prospec- tively recorded. The outcome measure was the incidence ofGBS EOS. Results: The rate ofGBS EOS was 7/14,335 deliveries (0.05%) before and 9/13,999 (0.064%) after the introduction of the education program (observed difference between proportions 0.016%, 95% confidence interval [CI] for the difference between the proportions -0.071% to 0.04%, P not significant [NS]). The rate ofGBS EOS in preterm infants was 5/1,331 (0.376%) before and 3/1,297 (0.23%) afterward (observed difference between proportions 0.14%, 95% CI -0.28% to 0.56%, P NS). The incidence ofGBS EOS did not decrease during the latter period due to failure of antepar- tum cultures to predict intrapartum GBS colonization (2 cases); non-compliance with voluntary recommendations to administer chemoprophylaxis (2 cases); failure of chemoprophylaxis or ther- apy for intraamniotic infection to prevent neonatal infection (3 cases); and occurrence ofGBS EOS in infants without risk factors (2 cases).

res to predict intrapartum GBS colonization (2 cases); non-compliance with voluntary recommendations to administer chemoprophylaxis (2 cases); failure of chemoprophylaxis or ther- apy for intraamniotic infection to prevent neonatal infection (3 cases); and occurrence ofGBS EOS in infants without risk factors (2 cases). Conclusions: An education program for resident physicians regarding chemoprophylaxis for GBS EOS did not significantly reduce the absolute incidence ofdisease. Alternative strategies are needed that redress the causes offailure inherent in the current guidelines. Some cases ofGBS EOS are not preventable because the parturient does not have risk factors that indicate chemoprophylaxis. (C) 1994 Wiley-Liss, Inc. KEY WORDS Pregnancy complications; infections, pregnancy outcome, outcome assessment (health care), neonate, septicemia arly onset group B streptococcal sepsis (GBS EOS) is the most common single cause of neo- natal bacterial infection. In the past decade, the risk factors associated with GBS EOS were identi- fled and the effectiveness ofchemoprophylaxis dur- ing the intrapartum period was demonstrated. 2'3 Antepartum cultures are poor predictors of intra- partum colonization.4'5 Furthermore, a reliable Address correspondence/reprint requests to Dr. Jeffrey S. Greenspoon, Department of Obstetrics and Gynecology, Room 1738, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048. Presented at the 40th Annual Meeting of the American College of Obstetricians and Gynecologists, Las Vegas, NV, April 27-30, 1992. Clinical Study Received December 9, 1993 Accepted March 15, 1994 CHEMOPROPHYLAXIS FOR PREVENTION OF GBS EOS GREENSPOON ET AL. rapid diagnostic test for group B Streptococcus (GBS) colonization that is feasible to use during the intra- partum period is not yet available.4'6 Thus, it is not possible to identify patients colonized with GBS when they present in labor. Therefore, Minkoff and Mead7 have recommended that all pregnant patients with preterm labor (PTL) or preterm pre- mature rupture of membranes (PROM) whose GBS status is unknown receive chemoprophylaxis until a negative culture is obtained. Pregnant pa- tients with preterm PROM and/or PTL are easily identified. They are at risk for GBS EOS and will benefit from chemoprophylaxis.3 We assessed the absolute rate of GBS EOS before and after a spe- cific resident education effort was begun at our institution.

moprophylaxis until a negative culture is obtained. Pregnant pa- tients with preterm PROM and/or PTL are easily identified. They are at risk for GBS EOS and will benefit from chemoprophylaxis.3 We assessed the absolute rate of GBS EOS before and after a spe- cific resident education effort was begun at our institution. MATERIALS AND METHODS This study was conducted at Cedars-Sinai Medical Center, Los Angeles, CA, an urban, university- affiliated, community medical center serving an ethnically mixed population. During the time in- terval from January 1, 1988, to December 31, 1989, there were no established management guide- lines to prevent GBS EOS. From January 1, 1990, selective chemoprophylaxis was strongly recom- mended by the staff for patients with pregnancies complicated by PTL (<37 weeks) or preterm PROM. Term pregnancies complicated by pro- longed ROM (P-ROM) (> 18 h) were also candi- dates for chemoprophylaxis, although this was left to the discretion of the physician.7 Intrapartum fever which was thought to represent intraamniotic infection was an indication for antibiotic therapy with ampicillin and gentamicin or a therapeutically equivalent regimen. Compliance with the chemo- prophylactic regimen was voluntary. The resident education program consisted of a discussion of the risk factors for GBS EOS and the benefits of chemoprophylaxis during the daily work rounds whenever the management of a patient with pre- term PROM and/or PTL was discussed with the resident staff. Chemoprophylaxis consisted ofampi- cillin, 2 g q 6 h. Patients with penicillin allergy received erythromycin.7 We reviewed all identifiable cases of GBS EOS to determine the incidence during both time peri- ods. Obstetric data were reviewed to determine whether the incidence of GBS EOS decreased in patients with preterm delivery or preterm PROM. Cases of GBS EOS were identified by reviewing the ICDM-9 (International Classification of Dis- eases, Clinical Modification, 9th Revision) dis- charge diagnostic codes for GBS meningitis or GBS bacteremia. A computerized microbiologic data- base of all positive blood and cerebrospinal fluid cultures for GBS was reviewed to identify addi- tional cases. Medical records were available for all the cases identified.

eases, Clinical Modification, 9th Revision) dis- charge diagnostic codes for GBS meningitis or GBS bacteremia. A computerized microbiologic data- base of all positive blood and cerebrospinal fluid cultures for GBS was reviewed to identify addi- tional cases. Medical records were available for all the cases identified. Rates were compared using the program Confidence Interval Analysis.9 RESULTS During the 2-year period (1988-89) before the education program regarding GBS chemoprophy- laxis, GBS EOS occurred in 7/14,335 deliveries (0.05%) compared with the 2-year period (1990- 91) following the education program when there were 9/13,999 (0.064%) (observed difference be- tween proportions 0.0155%, 95% confidence in- terval [CI] for the difference between the propor- tions -0.071% to 0.04%, P not significant [NS]). Among preterm deliveries fewer than 37 weeks gestation, there were 5/1,331 (0.376%) cases of GBS EOS before and 3/1,297 (0.23%) after- ward (observed difference between proportions 0.14%, 95% CI -0.28% to 0.56%, P NS) (Table 1). The overall incidence of GBS EOS did not dif- fer between the 2 periods. The incidence of GBS EOS in PTL did not differ between the 2 time periods. However, if one considers cases delivered prior to 36 weeks gestation, then only case of GBS EOS occurred in preterm infants during the latter time period when chemoprophylaxis was con- sistently recommended. We did not determine the magnitude of compli- ance with chemoprophylaxis among patients with risk factors. Patients receiving expectant manage- ment for preterm PROM receive care in a special maternal-fetal medicine unit where the resident staff and perinatologist make rounds twice daily and review the management. We estimate that the com- pliance rate for chemoprophylaxis in this group approached 100%. In the 1st study period (1988-89), 5 of the 7 cases were complicated by preterm PROM. One could prospectively anticipate a prolonged (> 18 h) time of ruptured membranes because expectant management was attempted in all these cases. Two additional cases of GBS EOS occurred in term INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY CHEMOPROPHYLAXIS FOR PREVENTION OF GBS EOS GREENSPOON ET AL. TABLE I. Incidence of GBS EOS before and after the introduction of a protocol for chemoprophylaxis Period before Period after chemoprophylaxis chemoprophylaxis (1988-89) (1990--91) No. cases GBS EOS/No. deliveries No. cases GBS Rate EOS/No.

ICS AND GYNECOLOGY CHEMOPROPHYLAXIS FOR PREVENTION OF GBS EOS GREENSPOON ET AL. TABLE I. Incidence of GBS EOS before and after the introduction of a protocol for chemoprophylaxis Period before Period after chemoprophylaxis chemoprophylaxis (1988-89) (1990--91) No. cases GBS EOS/No. deliveries No. cases GBS Rate EOS/No. deliveries Rate Preterm (<37 weeks gestation) 5/I,331 Term 2/I 3,004 Total 7114,335 1/266 (0.376%) 3/I,297 1/432 (0.23%) I/6,502 (0.015%) 6/12,702 I/2,117 (0.047%) I/2,048 (0.05%) 9/I 3,999 I/I,533 (0.064%) pregnancies without risk factors. The incidence of GBS EOS in term pregnancy was low even though patients with risk factors did not usually receive chemoprophylaxis in the absence of a defined pro- tocol (Table 2). During the 2nd period (1990-91), after the introduction of'a policy ofchemoprophylaxis, there were 9 cases of GBS EOS. One case involved a 24-week gestation in which the precipitous delivery precluded the administration of chemoprophylaxis during the intrapartum period. The other 8 cases of GBS EOS involved pregnancies of 36 weeks gesta- tion or longer (Table 3). Three of the 8 had P-ROM (>18 h) and received oxytocin induction of labor for this indication. P-ROM could not be predicted with certainty at the onset of labor, but could have been anticipated in these 3 cases after 12 h ofROM (patients 3, 5, and 6, Table 3). One case of GBS EOS was associated with ROM of 14 h duration in a patient undergoing induction of labor for post-dates pregnancy (patient 9, Table 3). Two patients at term developed fever during labor: pa- tient 9 was treated with ampicillin and gentamicin 65 min prior to delivery and patient 6 had penicil- lin allergy and received cefoxitin and gentamicin 2 h prior to delivery (Table 3). Chemoprophylaxis is most effective if administered at least 4 h prior to delivery, l0 In case, an antepartum vaginal culture ob- tained 3 days prior to delivery was negative, al- though the neonate was heavily colonized at the time of delivery. The only risk factor in this case was preterm delivery at 36 weeks gestation (patient 2, Table 3). One patient had spontaneous labor after sponta- neous ROM and developed a fetal tachycardia. Ma- ternal fever was detected during the cesarean sec- tion for fetal distress. This case represents the association of intrapartum fever with GBS EOS (patient 8, Table 3). Two patients (4 and 7, Table 3) had no risk factors.

. One patient had spontaneous labor after sponta- neous ROM and developed a fetal tachycardia. Ma- ternal fever was detected during the cesarean sec- tion for fetal distress. This case represents the association of intrapartum fever with GBS EOS (patient 8, Table 3). Two patients (4 and 7, Table 3) had no risk factors. The 3 neonatal deaths during the 4-year study occurred in preterm infants who did not receive chemoprophylaxis (Tables 2, 3). DISCUSSION Randomized prospective studies have demonstrated that GBS EOS can be prevented by chemoprophy- laxis of colonized, high-risk parturients.2'3 The impact of selective chemoprophylaxis on the inci- dence ofGBS EOS in a community hospital has not been described outside prospective clinical trials. Garland and Fliegnerll achieved a reduction in GBS EOS by screening all their public patients at 32 weeks gestation and by treating every colonized parturient. A recent analysis demonstrated that uni- versal prenatal screening for GBS and chemopro- phylaxis of colonized women with labor complica- tions are likely to be cost-beneficial in the United States. 12 The best management strategy to prevent EOS GBS has been sought. 13 However, a complete con- sensus has not been achieved. 14 Recent guidelines from the American College of Obstetricians and Gynecologists (ACOG)is differ from those pro- mulgated by the Committee on Infectious Diseases and the Committee on Fetus and Newborn. 10 Spe- cifically, the ACOG regards screening for GBS as an option, whereas the pediatric group recommends screening of all pregnant women. The situation in which maternal GBS status is unknown and a risk factor for GBS EOS is present is addressed in a similar manner by both groups. The ACOG bulle- tin states that "it may be reasonable to use antibiotic chemoprophylaxis empirically." The pediatric rec- 2J 2 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY CHEMOPROPHYLAXIS FOR PREVENTION OF GBS EOS GREENSPOON ET AL. TABLE 2.

or for GBS EOS is present is addressed in a similar manner by both groups. The ACOG bulle- tin states that "it may be reasonable to use antibiotic chemoprophylaxis empirically." The pediatric rec- 2J 2 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY CHEMOPROPHYLAXIS FOR PREVENTION OF GBS EOS GREENSPOON ET AL. TABLE 2. Data on cases of GBS EOS from the period before the introduction of a protocol for chemoprophylaxis of GBS EOS (1988-89)a Gestational Age age Risk Intrapartum Patient (years) G, P (weeks) factors GBS culture Chemoprophylaxis Neonatal outcome 32 7, 3 31.2 Preterm P-PROM Positive 2 37 4, 3 32.5 Preterm P-PROM Not done 3 43 8, 4 34.2 Preterm P-PROM Positive 4 34 2, 0 35.4 Preterm P-PROM Negative S 40 3, 35.7 Preterm P-PROM Not done 6 32 3, 0 38.3 None Not done 7 16 I, 0 39.0 None Not done None None None None None None None Home, intact Died Home, intact Home, intact Home, intact Home, intact Home, intact 3 gravidity, P parity; P-PROM prolonged premature rupture of membranes (>18 h). ommendation for this situation is that "Chemopro- phylaxis may be appropriate." Although there is general agreement with the need for selective chemoprophylaxis, the feasibility and effectiveness of implementation have not been described in the community. A cost-benefit analysis by Strickland et al. 16 suggested that it would be cost-effective to provide chemoprophylaxis on the basis of a rapid test performed during labor. Their analysis assumed a very rapid turnaround time. It is not possible to reliably identify women who are GBS-colonized in the intrapartum period for 2 rea- sons. First, the diagnostic tests are not adequately reliable.4,6,17 Second, implementing a rapid diag- nostic test requires laboratory services around the clock, which is not feasible in many institutions. Minkoff and Mead7 recommended intrapartum treatment with ampicillin (2 g q 6 h for 24 h) ofall mothers who deliver preterm infants and who are either carriers of group B beta-hemolytic strepto- cocci or whose GBS carriage status is unknown. They7 did not recommend treating term patients with P-ROM. In our study, the incidence ofGBS EOS in both time periods was lower than that described in other studies. 18 This may be related to the fact that preg- nant women in the Los Angeles area and at this medical center have lower rates of GBS coloniza- tion (approximately 2.4-5.7 %) than described else- where in the United States (Greenspoon, unpub- lished data).

both time periods was lower than that described in other studies. 18 This may be related to the fact that preg- nant women in the Los Angeles area and at this medical center have lower rates of GBS coloniza- tion (approximately 2.4-5.7 %) than described else- where in the United States (Greenspoon, unpub- lished data). 17,19,20 The prevalence of GBS colonization was 22.8% of 5,586 pregnant women reported by Boyer et al. 19 and 18.6% of 7,742 pregnant women reported by Regan et al.2 The low incidence of GBS EOS increases the risk of a beta error in a study of this size. A larger study is needed to have the power to demonstrate the statis- tical significance of small decreases in incidence rates. In this study, chemoprophylaxis for preterm pregnancies at high risk for GBS EOS was admin- istered more consistently during the 2nd period. There was a non-significant decrease in the number of cases among very preterm neonates. The imple- mentation of an education program for chemopro- phylaxis did not cause a statistically significant de- crease in the rate of GBS EOS. The inability to demonstrate a benefit from chemoprophylaxis is due to the combination ofthe inadequate predictive value of antenatal cultures for intrapartum GBS colonization (patients and 2, Table 3); non-com- pliance in administering indicated chemoprophy- laxis (patients 3 and 5; Table 3); failure of chemo- prophylaxis or intraamniotic infection treatment to prevent neonatal infection (patients 6, 8, and 9, Table 3); and the occurrence of GBS EOS in infants without risk factors (patients 4 and 7, Table 3). Only case of GBS EOS occurred in pregnan- cies of fewer than 36 weeks gestation during the 2nd time interval when chemoprophylaxis was rec- ommended. This case involved a patient with a cervical cerclage who presented in active PTL at 24 weeks. She had a negative genital culture for GBS 5 weeks prior to admission. The patient delivered 25 min after arrival at the hospital. Boyer et al. 18 observed that delivery within h of antibiotic therapy did not prevent GBS EOS. Thus, this patientwouldnothavebenefitedfromchemoprophy- laxis because she delivered too rapidly (patient 1, Table 3). The other cases of GBS EOS involved pregnan- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 2l 3 CHEMOPROPHYLAXIS FOR PREVENTION OF GBS EOS GREENSPOON ET AL. TABLE :3.

therapy did not prevent GBS EOS. Thus, this patientwouldnothavebenefitedfromchemoprophy- laxis because she delivered too rapidly (patient 1, Table 3). The other cases of GBS EOS involved pregnan- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 2l 3 CHEMOPROPHYLAXIS FOR PREVENTION OF GBS EOS GREENSPOON ET AL. TABLE :3. Data on cases of GBS EOS from the period after the introduction of a protocol for chemoprophylaxis of GBS EOS (1990-91)a Gestational Age age Risk Intrapartum Chemoprophylaxis Patient (years) G, P (weeks) factors GB$ culture or therapy Neonatal outcome 34 5, 2 24.0 Preterm Not done None Died Negative 5 weeks before delivery 2 25 I, 0 36.0 Preterm Not done None Died Negative 3 days prior to delivery 3 25 2, 0 36.6 Preterm P PROM Not done None Home, intact 4 30 I, 0 37. None Not done None Home, intact 5 31 I, 0 38.0 P-PROM Not done None Home, intact 6 37 2, 39.0 P-PROM, fever Not done Cefoxitin, gentamicin (penicillin Home, intact allergy; 2 h prior to delivery) 7 28 2, 39.2 None Not done None Home, intact 8 33 2, 0 39.5 Fever during cesarean section Not done None Home, intact 9 27 2, 41.5 14 h ROM, fever Not done Ampicillin, gentamicin (65 min prior Home, intact to delivery) =G gravidity, P parity; P-PROM prolonged premature rupture of membranes (>18 h). cies at or near term (36 or more completed men- strual weeks). Some ofthese cases had P-ROM. In all cases, it would have been possible after 12 h to prospectively determine that patients were likely to have P-ROM and to initiate chemoprophylaxis dur- ing labor (patients 3, 5, and 6, Table 3). We emphasize the association ofthe induction of labor for PROM and GBS EOS in 3 ofthe 9 cases. Furthermore, a 4th case of GBS EOS was associ- ated with ROM of 14 h duration in a patient un- dergoing induction of labor for post-dates preg- nancy (patient 9, Table 3). Patients who require an induction of labor for spontaneous PROM may become candidates for chemoprophylaxis if they do not deliver within 18 h. It may be prudent to determine the GBS status of a patient who is likely to undergo induction oflabor within a few days and whose induction of labor may be complicated by P-ROM. Chemoprophylaxis is not of proven benefit in term pregnancy when the maternal GBS status is unknown and risk factors are absent. McDuffie et al.21 reported adverse perinatal outcomes associated with resistant Enterobacteriaceae after treatment with ampicillin or amoxicillin in 4 mothers and their 5 infants.

d by P-ROM. Chemoprophylaxis is not of proven benefit in term pregnancy when the maternal GBS status is unknown and risk factors are absent. McDuffie et al.21 reported adverse perinatal outcomes associated with resistant Enterobacteriaceae after treatment with ampicillin or amoxicillin in 4 mothers and their 5 infants. Further study of the potential ad- verse effects and their costs is justified. The inci- dence of GBS EOS in term pregnancy in both periods approaches the risk for fatal anaphylaxis due to penicillin therapy, which is approximately 1/10,000 cases. 2z None of the term infants in our study died or had ongoing disability at the time of discharge. CONCLUSIONS Our experience indicates that a voluntary policy of chemoprophylaxis for GBS did not decrease the rate ofGBS EOS. Even ifcompliance were 100%, cases of GBS EOS would continue to occur among some patients who become GBS-colonized after a negative antenatal GBS culture; among some pa- tients in whom appropriate chemoprophylaxis or therapy for intraamniotic infection is ineffective; and among GBS-colonized patients without risk factors. One strategy to decrease the incidence of GBS EOS is to offer chemoprophylaxis to all colo- nized patients, including those without risk factors. This approach should receive careful cost-benefit and cost-effectiveness analysis before it is recom- mended. The adverse perinatal outcomes due to ampicillin-resistant microorganisms should be in- cluded in any risk-benefit analysis.21 Thus, despite empiric chemoprophylaxis, GBS EOS will con- tinue to occur, albeit infrequently, among parturi- ents without risk factors. REFERENCES 1. Vollman JH, Smith WL, Ballard ET, Light IJ: Early onset group B streptococcal disease: Clinical, roentgeno- graphic, and pathologic features. J Pediatr 89:199-203, 1976. 214 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY CHEMOPROPHYLAXIS FOR PREVENTION OF GBS EOS GREENSPOON ET AL. 2. Boyer KM, Gadzala CA, Kelly PD, Gotoff SP: Selective intrapartum chemoprophylaxis ofneonatal group B strep- tococcal early-onset disease. III. Interruption of mother- to-infant transmission, j Infect Dis 148:810-816, 1983. 3. Boyer KM, GotoffSP: Prevention ofearly-onset neonatal group B streptococcal disease with selective intrapartum chemoprophylaxis. N Engl J Med 314:1665-1669, 1986. 4. Greenspoon JS, Wilcox JG, Kirschbaum TH: Group B Streptococcus: The effectiveness of screening and chemo- prophylaxis. Obstet Gynecol Surv 46:499-508, 1991. 5.

KM, GotoffSP: Prevention ofearly-onset neonatal group B streptococcal disease with selective intrapartum chemoprophylaxis. N Engl J Med 314:1665-1669, 1986. 4. Greenspoon JS, Wilcox JG, Kirschbaum TH: Group B Streptococcus: The effectiveness of screening and chemo- prophylaxis. Obstet Gynecol Surv 46:499-508, 1991. 5. Anthony BF, Okada DM, Hobel CJ: Epidemiology of group B Streptococcus: Longitudinal observations during pregnancy. J Infect Dis 137:524-530, 1978. 6. Yancey MK, Armer T, Clark P, Duff P: Assessment of rapid identification tests for genital carriage of group B streptococci. Obstet Gynecol 80:1038-1047, 1992. 7. Minkoff H, Mead P: An obstetric approach to the pre- vention ofearly-onset group B beta-hemolytic streptococ- cal sepsis. Am J Obstet Gynecol 154:973-977, 1986. 8. Mead PB: When to treat intra-amniotic infection (edito- rial). Obstet Gynecol 72:935-936, 1988. 9. Gardner SB, Winter PD, Gardner MJ: Confidence In- terval Analysis (CIA). Version 1.0. Br Med J 19-24, 1989. 10. Committee on Infectious Diseases and Committee on Fe- tus and Newborn: Guidelines for prevention of group B streptococcal (GBS) infection by chemoprophylaxis. Pedi- atrics 90:775-780, 1992. 11. Garland SM, Fliegner JR: Group B Streptococcus (GBS) and neonatal infections: The case for intrapartum prophy- laxis. Aust NZ J Obstet Gynaecol 31:119-122, 1991. 12. Mohle-Boetani JC, Schuchat A, Plikaytis BD, Smith JD, Broome CV: Comparison ofprevention strategies for neo- natal group B streptococcal infection: A population-based economic analysis. JAMA 270:1442-1448, 1993. 13. Gibbs RS, Hall RT, Yow MD, McCracken GH Jr, Nelson JD: Consensus: Perinatal prophylaxis for group B streptococcal infection. Pediatr Infect Dis J 11 179-183, 1992. 14. Peter G, Lepow ML, McCracken GH, Phillips CF (eds): Group B Streptococcal Infections. Report ofthe Commit- tee on Infectious Diseases. 22nd Ed. Elk Grove Village, IL: American Academy ofPediatrics, pp 447-450, 1991. 15. American College of Obstetrics and Gynecology: Group B Streptococcal Infections in Pregnancy. ACOG Techni- cal Bulletin No. 170, pp 1-5, 1992. 16. Strickland DM, Yeomans ER, Hankins GDV: Cost-ef- fectiveness of intrapartum screening and treatment for maternal group B streptococci colonization. Am J Obstet Gynecol 163:4-8, 1990. 17. GreenspoonJS, Fishman A, WilcoxJG, Greenspoon RL, Lewis W: Comparison of culture for group B Streptococ- cus versus enzyme immunoassay and latex agglutination rapid tests: Results in 250 patients during labor.

screening and treatment for maternal group B streptococci colonization. Am J Obstet Gynecol 163:4-8, 1990. 17. GreenspoonJS, Fishman A, WilcoxJG, Greenspoon RL, Lewis W: Comparison of culture for group B Streptococ- cus versus enzyme immunoassay and latex agglutination rapid tests: Results in 250 patients during labor. Obstet Gyneco177:97-100, 1991. 18. Boyer KM, Gadzala CA, Burd LI, Fisher DE, PatonJB, Gotoff SP: Selective intrapartum chemoprophylaxis of neonatal group B streptococcal early-onset disease. I. Ep- idemiologic rationale. J Infect Dis 148:795-801, 1983. 19. Boyer KM, Gadzala CA, Kelly PD, Burd LI, GotoffSP: Selective intrapartum chemoprophylaxis ofneonatal group B streptococcal early-onset disease. II. Predictive value of prenatal cultures. J Infect Dis 148:802-809, 1983. 20. Regan JA, Klebanoff MA, Nugent RP for the Vaginal Infection and Prematurity Study Group: The epidemiol- ogy of group B streptococcal colonization in pregnancy. Obstet Gynecol 77:604-610, 1991. 21. McDuffie RS, McGregor JA, Gibbs RS: Adverse peri- natal outcome and resistant Enterobacteriaceae after anti- biotic usage for premature rupture ofthe membranes and group B Streptococcus carriage. Obstet Gynecol 82:487- 489, 1993. 22. Goodman LS, Gilman AG (eds): Goodman and Gilman's The Pharmacological Basis ofTherapeutics. 8th Ed. New York: Pergamon Press, 1990. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:216-219 (I 994) (C) 1994 Wiley-Liss, Inc. Epidemiology of Sexually Transmitted Diseases Among Pregnant Adolescents Lisa N. Gittens, Rhonda R. Nichols, and Joseph J. Apuzzio Department of Obstetrics and Gynecology, New Jersey Medical School, Newark, NJ ABSTRACT Objective: The purpose of this study was to determine the epidemiology of sexually transmitted diseases (STDs) among pregnant adolescents. Methods: Charts of all patients (n 735) who attended the Maternal and Infant Care Clinic at University Hospital, Newark, NJ, between July 1, 1991, and June 30, 1992, were reviewed for STDs which included gonorrhea, chlamydia, syphilis, and human immunodeficiency virus (HIV). At the first prenatal visit, each registrant had endocervical specimens obtained to detect gonorrhea and chlamydia. A serum sample was obtained for syphilis screening. HIV testing was made avail- able to all patients and testing was done on a voluntary basis. The same STD screening that was done at the initial visit was repeated at 28 and 36 weeks. Results: Twenty-five percent ofpatients tested positive for one or more STDs. The mean patient age was 17.3 years. The mean gestational age at first visit was 19.5 weeks. The mean number of visits was 7.3. The following STDs were identified: 4.8% of patients tested positive for gonorrhea, 20.9% tested positive for chlamydia, and 1.7% tested positive for syphilis. Twenty-one percent of patients had a positive STD diagnosed at the initial visit. Another 4.8% of patients had an STD diagnosed at some time after the initial visit when the initial screen was negative for STDs. An additional 1% of patients who initially tested positive for an STD had subsequent screening which revealed another STD (different organism). Seven patients tested HIV positive. Sixty-one percent of patients with STDs agreed to HIV testing. One patient had HIV coexistent with another STD. Conclusions: Pregnant adolescents are at risk for multiple STDs. HIV testing should be offered. STD screening should be repeated in the third trimester in adolescent patients. (C) 1994 Wiley-Liss, Inc.

itive. Sixty-one percent of patients with STDs agreed to HIV testing. One patient had HIV coexistent with another STD. Conclusions: Pregnant adolescents are at risk for multiple STDs. HIV testing should be offered. STD screening should be repeated in the third trimester in adolescent patients. (C) 1994 Wiley-Liss, Inc. KEY WORDS Pregnancy, chlamydia, gonorrhea, HIV he number of sexually active adolescents has risen dramatically in the past 15 years. Studies in 1988 show that 25% of 15-year-old girls and 75% of 19-year-old girls have had sexual inter- course. 1,2 An estimated 1,014,680 teens became pregnant in 1987, with 50% of these pregnancies resulting in births. The teenage birth rate in 1989 (girls aged 15-19 years) was 49/1,000 among whites and 97/1,000 among minorities; the teenage birth rate in the United States is higher than in many developing countries.2'3 Pregnancies among adolescents, especially those under age 15 years, are complicated by adverse neonatal outcome.4,s Research has shown, however, that maternal age alone has little effect on poor outcome when other contributing risk factors such as inadequate maternal weight gain, poor nutrition, and substance abuse are controlled for. The pres- ence of a sexually transmitted disease (STD) repre- sents an additional but correctable risk for the preg- nant adolescent. Infections with herpes, syphillis, gonorrhea, and chlamydia may cause disease result- Address correspondence/reprint requests to Dr. Joseph Apuzzio, Department of Obstetrics and Gynecology, New Jersey Medical School, 185 South Orange Avenue, E506, Newark, NJ 07103. Received September 5, 1993 Clinical Study Accepted January 24, 1994 PREGNANT ADOLESCENTS AND STDs GITTENS ET AL. ing in preterm labor, chorioamnionitis, and sepsis in the mother and pneumonia, conjunctivitis, con- genital malformation, and sepsis in the neonate. 6'7 The range of perinatal morbidity associated with human immunodeficiency virus (HIV) infection is still being described. Depending upon the popula- tion studied, 8-25% of sexually active adolescents have endocervical infections with Chlamydia tra- chomatis and 0.4-12% have endocervical infection with Neisseria gonorrhoeae.8-11 A 0.7% incidence rate of syphilis among a group of 15-19-year-old indigent pregnant girls has been reported. 12 AS OF March 1990, 1,429 cases ofacquired immunodefi- ciency syndrome (AIDS) in adolescents have been reported to the Centers for Disease Control and Prevention.

vical infection with Neisseria gonorrhoeae.8-11 A 0.7% incidence rate of syphilis among a group of 15-19-year-old indigent pregnant girls has been reported. 12 AS OF March 1990, 1,429 cases ofacquired immunodefi- ciency syndrome (AIDS) in adolescents have been reported to the Centers for Disease Control and Prevention. There are no studies that review the epidemiology of STDs including HIV in the ado- lescent pregnant patient. To aid in targeting sites for intervention, we studied the epidemiology of STDs in an inner-city pregnant adolescent popula- tion. MATERIALS AND METHODS All patients who had at least one prenatal visit to the Maternal and Infant Care Clinic at University Hos- pital, Newark, NJ, and delivered at one institution between July 1, 1991, and June 30, 1992, were included in the study. As part of routine prenatal care, all patients were screened for N. gonorrhoeae, C. trachomatis, and syphilis at the first visit. Two endocervical specimens were obtained. The first was inoculated on Thayer Martin medium, incu- bated, and studied for the presence of N. gonor- rhoeae; the second was processed for chlamydia de- tection by immunoassay (Chlamydiazyme, Abbott Laboratories, Chicago, IL). A serum sample was obtained for Rapid Plasma Reagin (RPR). Fluo- rescent treponemal antibody testing (FTA) was per- formed on blood from all patients who tested RPR positive. The above-described STD screening was repeated at 28 and 36 weeks gestation. All patients with suspicious genital lesions had cultures for herpes simplex virus. At the first pre- natal visit, patients were counseled regarding HIV. Voluntary HIV screening by enzyme-linked im- munosorbent assay (ELISA) (Recombigen, Cam- bridge Biotech Corp., Worchester, MA) was per- formed on all patients who agreed to testing. A positive culture for gonorrhea or a positive Chlamy- diazyme was considered diagnostic ofthe respective disease. Patients were described as having syphilis if the RPR and FTA were positive (patients with history of infection and positive titers were ex- cluded from this group). All patients who tested HIV positive by ELISA had HIV confirmation by Western blot analysis (Novapath, Bio-Rad Labora- tories, Hercules, CA). Results of STD screening and demographic information regarding the pa- tient's age, parity, gestational age at time of first visit, and the number of prenatal visits were re- corded.

patients who tested HIV positive by ELISA had HIV confirmation by Western blot analysis (Novapath, Bio-Rad Labora- tories, Hercules, CA). Results of STD screening and demographic information regarding the pa- tient's age, parity, gestational age at time of first visit, and the number of prenatal visits were re- corded. Demographic data were reviewed to determine measures of central tendency and proportions that described the patient population. Results of STD screening were reviewed to determine percentages of disease in our population. Means and ranges were used where appropriate. RESULTS Maternal Characteristics A total of 735 adolescent patients enrolled in our clinic delivered between July 1, 1991, and June 30, 1992. Our patients were predominantly black and Hispanic females. Of the 735 enrolled pa- tients, 188 patients (25.2%) had an STD. The mean age for patients with STDs was 17.3 years (range 12-20 years). The mean number ofprenatal visits was 7.3 (range 1-13 visits). The mean gesta- tional age at the time of" the first visit was 19.5 weeks (range 6-35 weeks); 57 patients (30.3%) presented for initial visit in the first trimester, 90 patients (47.8%) presented initially in the second trimester, and 41 patients (21%) had their first visit in the third trimester. Twenty-one percent of patients had an STD diagnosed at the initial visit. Follow-up STD screening was done according to clinic protocol on all patients at 28 and 36 weeks. This rescreening, performed on all patients and not based upon the presence of symptoms or historical information, revealed that 4.8% had an STD diag- nosed at a follow-up visit after the initial STD screen was negative. One percent had a second STD diagnosed at the follow-up visit after the ini- tial screen had already identified one STD. Three percent of the patients had more than one STD. STD Characteristics Twenty-one percent of patients had chlamydia in- fection [95% confidence interval (CI) 18.0- 24.0%], 4.8% had infection with gonorrhea (95% INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 217 PREGNANT ADOLESCENTS AND STDs GITTENS ET AL. CI 3.3-6.3%), 1.7% new cases of syphilis were identified, and 0.6% had infection with herpes sim- plex virus. Seven patients tested HIV positive. Sixty-one percent (116/188) of patients with a known STD agreed to HIV testing. One patient had HIV coexistent with another STD (syphilis). The other 6 HIV-positive patients had no coexist- ent STD.

ases of syphilis were identified, and 0.6% had infection with herpes sim- plex virus. Seven patients tested HIV positive. Sixty-one percent (116/188) of patients with a known STD agreed to HIV testing. One patient had HIV coexistent with another STD (syphilis). The other 6 HIV-positive patients had no coexist- ent STD. DISCUSSION Pregnant adolescents in our population are at risk for multiple STDs, including HIV. ACOG guide- lines recommend STD screening at the initial and follow-up visits. While prenatal care has improved for mothers in all age groups in recent years, teen- agers continue to get the least adequate medical attention. A 1978 study reported that mothers un- der the age of 15 years are 2.5 times more likely than mothers aged 20-24 years to be without pre- natal care in the first trimester and nearly 4 times more likely to receive no care or to delay care until the third trimester.4 Our studies confirm these trends. Because more than half of adolescents pre- sented for the first visit later than the first trimester and because more than 20% of patients had an STD, a scheduled follow-up visit 1-2 weeks after initial presentation and emphatic encouragement regarding compliance with follow-up visits are in order. Repeated testing for STDs will identify those adolescents who continue to test positive because of noncompliance with therapy or continued exposure to the untreated asymptomatic partner. Almost 5% of patients had an STD diagnosed on repeat screen- ing after the initial screen was negative. The choice ofan individual partner and whether that partner resides in an area where the prevalence of STDs is high both contribute to an adolescent's risk. 13,14 An understanding ofthe adolescent's rea- soning process, her concept of monogamy, and knowledge of adolescent sexuality patterns, includ- ing those of the male partner, will provide addi- tional areas for intervention. The attempt to control disease transmission in pregnant adolescents is additionally impaired by the fact that a significant number of teenagers have never used a contraceptive method. Already preg- nant teenagers may not understand the rationale for the use ofa barrier contraceptive during their preg- nancy. In communities where the prevalence of STDs including HIV is high, all pregnant adoles- cents should be encouraged to use condoms if they remain sexually active during their pregnancy. The seroprevalence ofHIV in adolescents in our community is unknown.

le for the use ofa barrier contraceptive during their preg- nancy. In communities where the prevalence of STDs including HIV is high, all pregnant adoles- cents should be encouraged to use condoms if they remain sexually active during their pregnancy. The seroprevalence ofHIV in adolescents in our community is unknown. There are no reported studies that have reviewed the epidemiology of STDs including HIV in pregnant inner-city ado- lescents. We identified 7 HIV positive adolescents. None of these patients had obvious risk factors for HIV infection and thus may have acquired the infection through heterosexual contact. Since not all patients agreed to testing, the actual seroprevalence rate among pregnant adolescents in our community (at least 1%) is likely to be higher. The risk and possibility for heterosexual transmission of HIV in this patient group cannot be minimized. While others have suggested that the presence of an STD is a sufficient factor upon which to base decisions regarding HIV screening in adolescents, we noted that only of the 7 patients who tested HIV posi- tive had a coexistent STD. While it is true that 39% ofour patients who had STDs did not agree to HIV testing, the discovery of 6 HIV positive patients among the group ofpatients who tested negative for STDs makes the presence ofan STD an insufficient risk factor upon which to base decisions regarding HIV screening in our patient group. We recom- mend the availability ofHIV counseling and screen- ing to all pregnant adolescents, particularly those who reside in communities where the seropreva- lence of the disease is high. While many programs have been developed to meet the needs of pregnant women who test HIV positive, specific programs that address the needs of such infected adolescents are lacking. Ethical and legal issues regarding testing and informing partners, emotional differences in coping mecha- nisms, and age-related differences in cognitive func- tion make the diagnosis and treatment of HIV in- fection different in the adolescent than in the adult. While unified support groups specific for subsets ofthe adult population such as homosexuals, hemo- philiacs, and substance abusers exist, few such groups are available to adolescents. The develop- ment ofmultidisciplinary teams with specific atten- tion to the emotional and social needs of HIV- infected adolescents will hopefully assure better perinatal outcomes.

ets ofthe adult population such as homosexuals, hemo- philiacs, and substance abusers exist, few such groups are available to adolescents. The develop- ment ofmultidisciplinary teams with specific atten- tion to the emotional and social needs of HIV- infected adolescents will hopefully assure better perinatal outcomes. 218 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY PREGNANT ADOLESCENTS AND STDs REFERENCES 1. Centers for Disease Control: Premarital sexual experience among adolescent womenwUnited States 1970-1988. Morbidity Mortality Weekly 39:929-931, 1991. 2. Alan Guttmacher Institute: Teen Sexual and Reproduc- tive Behavior in the U.S. Facts in Brief. New York: Alan Guttmacher Institute, 1990. 3. Alan Guttmacher Institute: Teenage Pregnancy: The Problem That Hasn't Gone Away. New York: Alan Gutt- macher Institute, 1981. 4. McAnarney ER: Young age and adverse neonatal out- come. American Journal of Diseases of Children 141 1053-1059, 1987. 5. Felice M, Grandos JL, Ances IG, et al.: The young pregnant teenager: Impact ofcomprehensive prenatal care. J Adolescent Health Care 1:193-197, 1981. 6. Chlamydia trachomatis infections--Policy guidelines for prevention and control. Morbidity Mortality Weekly 34(Suppl):53-74, 1985. 7. Gravett MG, Nelson HP, DeRouen T, Critchlow C, Eschenbach D, Holmes K: Independent associations of bacterial vaginosis and Chlamydia trachomatis infection with adverse pregnancy outcome. JAMA 258:1899- 1903, 1986. GITTENS ET AL. 8. Sharer MA, Sweet RL, Ohm-Smith MJ: Microbiology of the lower genital tract in post-menarchal adolescent girls: Differences by sexual activity, contraception and presence ofnonspecific vaginitis. J Pediatr 107:974-981, 1985. 9. Hein K, Marks A, Cohen M: Asymptomatic gonorrhea: Prevalence in a population ofurban adolescents. J Pediatr 90:634, 1977. 10. Chacko MR, Lovchik JC: Chlamydia trachomatis infec- tion in sexually active adolescents: Prevalence and risk factors. Pediatrics 73:836-840, 1984. 11. Fraser GJ, Rettig PJ, Kaplan DW: Prevalence ofcervical Chlamydia trachomat# and Nelsseria gonorrhoeae in female adolescents. Pediatrics 71:333-336, 1983. 12. Mumford DM, Smith PB, Goldfarb JL: Prevalence of venereal disease in indigent pregnant adolescents. J Re- prod Med 19:83, 1977. 13. Aral SO, Soskoline V, Joesoef RM, O'Reilly K: Sex partner recruitment as risk factor for STD: Clustering of risky modes. Sexually Transmitted Dis 18:10-17, 1991. 14. O'Reilly KR, Aral S: Adolescents and sexual behavior.

JL: Prevalence of venereal disease in indigent pregnant adolescents. J Re- prod Med 19:83, 1977. 13. Aral SO, Soskoline V, Joesoef RM, O'Reilly K: Sex partner recruitment as risk factor for STD: Clustering of risky modes. Sexually Transmitted Dis 18:10-17, 1991. 14. O'Reilly KR, Aral S: Adolescents and sexual behavior. J Adolescent Health Care 6:262-270, 1985. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 219

Infectious Diseases in Obstetrics and Gynecology 1:220--227 (1994) (C) 1994 Wiley-Liss, Inc. Randomized Trial of Antibiotics in Addition to Tocolytic Therapy to Treat Preterm Labor D. Heather Watts, Marijane A. Krohn, Sharon L. Hillier, and David A. Eschenbach Departments of Obstetrics and Gynecology (D.H.W., M.A.K., S.L.H., D.A.E.) and Epidemiology (D.A.E.), University ofWashington, Seattle, WA ABSTRACT Objective: The objective ofthis study was to assess whether antibiotic therapy plus tocolysis given to women in preterm labor would prolong pregnancy compared with tocolysis alone. Methods: A randomized, double-blind trial of intravenous mezlocillin and oral erythromycin therapy vs. placebo was used in addition to tocolysis among women in preterm labor <34 weeks gestation with intact membranes. Amniocentesis was performed, and chorioamnionic membranes were examined histologically and cultured for microorganisms after delivery. Results: Clinical characteristics including gestational age at enrollment, frequency of contrac- tions, cervical Bishop's score, and white blood cell count on admission were similar in the 2 groups. Antibiotic therapy was well tolerated. No significant differences in the interval to delivery, birth weight, and neonatal outcomes were observed between the 2 groups. Women in the antibiotic group had a significantly lower incidence of postpartum infections compared with women in the placebo group. Patients with evidence of upper genital tract infection in either group had a significantly shorter interval to delivery, lower gestational age at delivery, lower mean birth weight, and increased neonatal hospitalization time. Conclusions: Lack of an antibiotic effect on the gestational age at delivery may be due to the low prevalence of upper genital tract infection among unselected women in preterm labor, to advanced preterm labor unresponsive to antibiotic therapy, or to an inability ofantibiotics given alone to inhibit the cytokine response. Further work is needed to identify markers of upper genital tract infection among women in preterm labor and to evaluate other potential therapeutic interventions. (C) 1994 Wiley-Liss, Inc.

d preterm labor unresponsive to antibiotic therapy, or to an inability ofantibiotics given alone to inhibit the cytokine response. Further work is needed to identify markers of upper genital tract infection among women in preterm labor and to evaluate other potential therapeutic interventions. (C) 1994 Wiley-Liss, Inc. KE WORS Antibiotics, prematurity, amniotic fluid infection reterm birth remains a major unsolved problem in obstetrics and infection has been suspected to play a role in some preterm deliveries. Upper gen- ital tract (amniotic fluid and chorioamnion) infec- tion has been identified among a higher proportion of women delivering preterm compared with those delivering at term. Amniotic fluid (AF) infection is present in up to 6 women presenting in preterm labor (PTL) below 28 weeks gestation. Chorio- amnion infection decreases from a rate of 80% be- low 26 weeks to approximately 10% at term,2 and histologic chorioamnionitis decreases rapidly from a rate of 55% below 28 weeks gestation to 5% from 29 weeks gestation to term. 3 Thus, the prevalence ofupper genital tract infection and inflammation is inversely related to gestational age, and infection appears to contribute to preterm birth in patients with PTL resistant to tocolysis. Address correspondence/reprint requests to Dr. D. Heather WaRs, Department ofObstetrics and Gynecology, University of Washington, 1959 NE Pacific Street, RH-20, Seattle, WA 98195. Presented at the annual meeting ofthe Infectious Disease Society for Obstetrics and Gynecology, Seattle, WA, August 1990. Clinical Study Received June 14, 1993 Accepted March 7, 1994 ANTIBIOTICS IN PRETERM LABOR WATTS ET AL. Despite the strong association between upper genital tract infection and preterm delivery, a cause- and-effect relationship between the 2 has not been established. Evidence of a cause-and-effect rela- tionship would be strengthened if a reduction of preterm delivery occurred among infected patients given antibiotics in a randomized double-blind fash- ion. To evaluate the potential contribution of infec- tion to preterm birth, we randomized women in PTL with intact membranes to antibiotics or pla- cebo in addition to tocolytic therapy. Attempts were made to enroll patients at high risk for infection and to examine the results in the infected vs. the uninfected group. SUBJECTS AND METHODS Women presenting with preterm contractions with intact membranes between October 1986 and Oc- tober 1987 were evaluated for eligibility for this study.

ytic therapy. Attempts were made to enroll patients at high risk for infection and to examine the results in the infected vs. the uninfected group. SUBJECTS AND METHODS Women presenting with preterm contractions with intact membranes between October 1986 and Oc- tober 1987 were evaluated for eligibility for this study. We attempted to enroll patients at increased risk for infection (<34 weeks getation), but with- out clinical evidence of infection (<38C tempera- ture), those with no known cause of labor, yet a high chance of actually being in PTL. To be in- cluded, women had to be between the ages of 15-45 years, have a singleton gestation without known maternal uterine or fetal anomalies, an estimated gestational age <34 weeks, intact membranes, an admission temperature <38C, contractions occur- ring at least every 10 min with a documented cervi- cal change, and a Bishop's score of at least 4 with at least cm dilatation or 50% effacement. Women allergic to penicillin or erythromycin or on anti- biotics within the previous 7 days, with cervical dilatation >4 cm, with placenta previa, vaginal bleeding more than bloody show, coagulation ab- normalities or placental abruption noted on ultra- sound, contraindications to tocolytic therapy, or difficulty in communication were excluded. Dur- ing the study period, 718 women presented to the University of Washington Medical Center with possible PTL and were screened for enrollment. Six hundred ten patients (85%) were excluded: pa- tients with gestational age above 34 weeks, 13%; preterm rupture of membranes, 29%; no labor, 20%; fetal or uterine anomalies, 5%; placenta pre- via or abruption, 7%; penicillin or erythromycin allergies, 4%; multiple gestation, 9%; recent anti- biotic use, 3%; cervix dilated more than 4 cm, 3%; maternal medical conditions, 3%; temperature >38C, 1%; and other conditions including non- English speaking or psychotic, 3%. In addition, 29 women refused participation. Of the remaining 79 women who were eligible for enrollment, 56 were approached and signed informed consent to partic- ipate in the study. All women underwent ultrasound for assessment of gestational age and fetal health. Women with adequate AF volume who consented underwent am- niocentesis for fetal lung maturity studies and mi- crobiologic cultures.

re eligible for enrollment, 56 were approached and signed informed consent to partic- ipate in the study. All women underwent ultrasound for assessment of gestational age and fetal health. Women with adequate AF volume who consented underwent am- niocentesis for fetal lung maturity studies and mi- crobiologic cultures. Aliquots of AF were trans- ported in a syringe to the clinical microbiology laboratory for routine bacterial culture and trans- ported immediately to the research microbiology laboratory or placed into a Port-a-Cul vial (Becton- Dickinson Co., Inc., Cockeysville, MD) where 100 Ixl ofAF was cultured as previously reported. Urine was obtained by bladder catheterization for urinalysis and culture. A speculum examination was performed to collect vaginal specimens and to exclude rupture of the fetal membranes. A vaginal swab was rolled onto a clear glass slide and ob- served microscopically for a ferning pattern to rule out ruptured membranes. The pI--I of the vaginal fluid was determined by pH paper. Vaginal fluid was also collected on cotton swabs for Gram stain and transported in Amies transport medium (Med- ical Media Lab, Boring, OR) for cultures ofgroup B Streptococcus and Escherichia coli as previously described.4 Cervical specimens were obtained on dacron swabs for Gram smear and inoculated onto Thayer-Martin media for Neisseria gonorrhoeae cul- tures and centrifuged onto McCoy cell monolayers for Chlamydia trachomatis cultures as previously indicated, s Vaginal and cervical smears were stained with crystal violet and Gram's iodine with a saffra- nin counterstain and evaluated for the presence of white blood cells (WBCs) and bacterial vaginosis according to published criteria. 6 Bishop's score was determined by digital examination of the cervix. After obtaining vaginal AF, urine, and cervical specimens for culture, we randomly assigned the women in a blinded fashion to either of 2 groups: 1) mezlocillin, 3 g intravenously (IV) every 6 h, and erythromycin ethylsuccinate, 333 mg orally every 8 h; or 2) identical placebos of each. The antibiotic regimen was chosen to inhibit the most common microorganisms isolated from AF and the chorioamnion isolates at our hospital, including anaerobic and facultative bacteria and Ureaplasma INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 221 ANTIBIOTICS IN PRETERM LABOR WATTS ET AL. urealyticum. 1'2'7 IV therapy was continued for 5 days and oral therapy for 10 days. Tocolytic ther- apy was administered to all women.

ion isolates at our hospital, including anaerobic and facultative bacteria and Ureaplasma INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 221 ANTIBIOTICS IN PRETERM LABOR WATTS ET AL. urealyticum. 1'2'7 IV therapy was continued for 5 days and oral therapy for 10 days. Tocolytic ther- apy was administered to all women. The choice of tocolytics was at the discretion of the resident and attending physician staff and included IV ritodrine or magnesium sulfate, intramuscular ritodrine, and/or oral indomethacin. Women whose PTL had initially been stopped on parenteral tocolytic ther- apy received oral terbutaline or ritodrine until de- livery or 36 completed weeks gestation. Be- thamethasone in standard doses was administered at the discretion of the attending and resident physi- cian staff. Women at less than 34 weeks gestation with recurrent episodes of contractions were re- treated with parenteral tocolytics unless the cervix was more than 4 cm dilated or the membranes were ruptured. Maternal obstetrical, laboratory, and delivery data were collected by interview and chart review. At delivery, the chorioamnion was cultured and examined histologically as previously described.2 Infant outcomes were recorded by a combination of chart review and interview of the mother. Respira- tory distress syndrome was diagnosed in the pres- ence of 1) tachypnea and intercostal or substernal retractions, 2) the need for supplemental inspired oxygen to maintain arterial oxygen pressure greater than 50 torr, and 3) bilateral diffuse air broncho- grams on chest radiograph. Confirmed neonatal sepsis was diagnosed in the presence of a positive blood or cerebrospinal fluid culture from the infant within 3 days of delivery. Categorical variables were compared using the chi-square test with Yates correction or two-tailed Fisher exact test as appropriate. Continuous vari- ables that were normally distributed were com- pared using the Student's t-test. Continuous vari- ables that were not normally distributed were compared using a median test. 8 Proportional haz- ard models developed by Cox were used to compare the rates (hazard) of preterm delivery among women receiving antibiotics and those receiving placebo.9 For the proportional hazards-model, the time-to-event was the number of days from enroll- ment until the patient delivered or reached 35 com- pleted gestational weeks; the outcome event was delivery before 35 completed weeks.

s (hazard) of preterm delivery among women receiving antibiotics and those receiving placebo.9 For the proportional hazards-model, the time-to-event was the number of days from enroll- ment until the patient delivered or reached 35 com- pleted gestational weeks; the outcome event was delivery before 35 completed weeks. The rate (the frequency of outcome events per unit time) of pre- term delivery was determined by a computer algo- rithm that includes, at any given time point, only those women still at risk for preterm delivery. Like- lihood ratio tests were used to verify the 95% confi- dence intervals (CI) for the risk ratio. Kaplan- Meier curves were performed to display the probability of remaining undelivered for women receiving antibiotics or placebo. 10 RESULTS Fifty-six women were enrolled in the study. Thirty were randomized to receive mezlocillin and eryth- romycin and 26 were randomized to receive pla- cebo. Characteristics ofwomen in each ofthe groups on admission are summarized in Table 1. The women in the 2 groups did not differ significantly in any of the characteristics listed in Table 1. Al- most twice as many women in the antibiotic vs. the placebo group had C-reactive protein (CRP) levels above 1.5 mg/dl, the level previously shown to be elevated in our hospital, 11 but this difference was not statistically significant. AF cultures were posi- tive at enrollment in 4 of 21 women randomized to receive antibiotics and of 15 women randomized to placebo, but only 64% of women underwent amniocentesis before randomization. Results of genital cultures were similar between the 2 groups. One patient in the antibiotic group had N. gonorrhoeae and patient in the placebo group had C. trachomatis in the cervix. Group B Streptococcus was isolated from 3 (11%) of 28 pa- tients in the antibiotic group and 2 (8%) of 24 in the placebo group. E. coli was present in (4%) of 24 patients in the antibiotic group and 0 of 22 patients in the placebo group. No patients in the antibiotic group and 2 patients in the placebo group had > 105 bacteria/ml ofurine. Vaginal Gram stain results in the antibiotic group were: normal12 (43%) of 28, intermediate13 (46%), and bacte- rial vaginosis3 (11%). Vaginal Gram stain re- sults in the placebo group were: normal12 (46%) of 26, intermediates9 (35%), and bacterial vagi- nosis5 (19%). Women in both groups received similar num- bers of doses of study medication A summary of drug dosing and side effects is listed in Table 2.

te13 (46%), and bacte- rial vaginosis3 (11%). Vaginal Gram stain re- sults in the placebo group were: normal12 (46%) of 26, intermediates9 (35%), and bacterial vagi- nosis5 (19%). Women in both groups received similar num- bers of doses of study medication A summary of drug dosing and side effects is listed in Table 2. Antibiotic therapy was well tolerated. Nausea and vomiting were as common in the placebo group as the antibiotic group and were likely related to toco- lytic and other multiple medications used among women in PTL. The increased diarrhea among women receiving antibiotics may have been related 222 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY ANTIBIOTICS IN PRETERM LABOR WATTS ET AL. TABLE I. Enrollment characteristics among women randomized to receive mezlocillin/erythromycin or placebo Mezlocillin/erythromycin Placebo (n 30) (n 26) Maternal age, mean _+ SD (years) Ethnicity: Caucasian Currently married Previous preterm birth/total with previous pregnancy >20 weeks Current smoker Drug use this pregnancy (excludes alcohol) Gestational age, mean ___SD (weeks) Gestational age, median (weeks) Bishop's score, mean ___SD Cervical dilatation, mean -+ SD (cm) Positive amniotic fluid culture Peripheral WBC count, mean __- SD (I,000/cm3) Maximum temperature before enrollment, mean --+ SD (C) CRP, median (mg/dl) >1.5 mg/dl Bacterial vaginosis on Gram stain 25.9 4.8 23.9 _+ 5.6 0.20 23 (77%) 19 (73%) 0.76 17 (57%) 14 (54%) 0.95 9/18 (50%) 7/14 (50%) 0.72 12/26 (47%) 10/24 (42%) 0.80 5/27 (19%) 5/24 (21%) 0.99 30. _+ 3. 30. +_ 3.2 0.98 31.0 31.25 0.95 7.0 2.3 6.3 _+ 2.7 0.30 2.1 -+ 1.6 2.1 _+ 1.2 0.95 5/22 (23%) 1/15 (7%) 0.37 12.3 -+ 7. 11.6 -+ 5.5 0.70 36.8 -+ 4.4 36.9 -+ 5.1 0.50 1.75 1.45 0.30 12/19 (63%) 6/16 (38%) 0.20 /8 ( %) s/6 ( 9%) o.so TABLE 2. Comparison of drug dosing, reason for study drug discontinuation, and side effects between the randomized treatment groupsa Mezlocillin/erythromycin (n 30) IV Oral IV Placebo (n 26) Oral No. doses received, mean -+ SD 11.9 -+ 7.0 15.5 12.3 11.3 -+ 7.4 Stopped drug before course completed 23 19 17 Reason for discontinuation Delivered 13 12 10 Refused further IV therapy 0 2 Discharged undelivered 6 5 Required other antibodies 0 Experienced side effectsb 3 5 0 Nausea/vomiting 2 Diarrhea 4 Rash Total with side effects 7 (20%) 16.2 -12.7 16 2 0 3 3 0 4(%) aThere are no significant differences in side effects or number of doses between the 2 groups. bOne patient in each group had more than one side effect.

elivered 6 5 Required other antibodies 0 Experienced side effectsb 3 5 0 Nausea/vomiting 2 Diarrhea 4 Rash Total with side effects 7 (20%) 16.2 -12.7 16 2 0 3 3 0 4(%) aThere are no significant differences in side effects or number of doses between the 2 groups. bOne patient in each group had more than one side effect. to either antibiotic. None of the women who devel- oped diarrhea had a positive stool culture for Clostridium difficile or a positive C. difficile toxin. No significantly different rates of adverse effects related to antibiotics were seen in infants. Outcome characteristics between the 2 groups in intent-to-treat analyses are shown in Table 3. Women receiving antibiotics did not differ from placebo-treated women in the median gestation at delivery, median birth weight, or median numbers ofdays from enrollment to delivery. Kaplan-Meier plots of interval from enrollment to delivery are shown in Figure 1. The hazard ratio for preterm delivery in the antibiotic compared with the pla- cebo group is 0.76; 95% CI 0.37-1.5. Women receiving antibiotics were less likely to develop post- partum infections or to need antibiotic treatment during or after labor compared with women in the placebo group (3/30 vs. 10/26, P 0.03). Posi- tive membrane cultures were present in 7 (32%) of 22 patients in the antibiotic and 9 (45%) of 20 patients in the placebo group (N.S.). The propor- tions with histologic chorioamnionitis did not dif- fer between the 2 treatment groups (Table 3). Infant outcomes are compared between the 2 treatment groups in Table 3. Gestational age esti- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 223 ANTIBIOTICS IN PRETERM LABOR WATTS ET AL. TABLE 3. Pregnancy and infant outcome according to treatment group Mezlocillin/erythromycin (n 30) Placebo (n 26) Gestational age at delivery (weeks) Obstetrical dates Mean -+ SD 33. 4.8 Median 33.8 Dubowitz score Mean 34.2 5.0 Median 35 Birth weight, mean -+ SD (g) 2,202.4 _851.3 Birth weight, median (g) 2,360 Median interval from enrollment to delivery (days) 15.5 No. undelivered >1 week 17 (57%) Maternal non-study antibiotic therapy Before delivery 3 After delivery Total 3 Histologic chorioamnionitis 17125 (68%) Infant hospital days Median 9 <10 16 10-60 8 >60 5 Infant respiratory distress syndrome 13 Infant antibiotic therapy 14 5-rain Apgar score, mean +- SD 7.4 +- 2.3 33.5 -4.1 0.8 33 0.9 34.8 -4.

17 (57%) Maternal non-study antibiotic therapy Before delivery 3 After delivery Total 3 Histologic chorioamnionitis 17125 (68%) Infant hospital days Median 9 <10 16 10-60 8 >60 5 Infant respiratory distress syndrome 13 Infant antibiotic therapy 14 5-rain Apgar score, mean +- SD 7.4 +- 2.3 33.5 -4.1 0.8 33 0.9 34.8 -4. 0.7 34.5 0.9 2,212 862.2 .97 2,280 0.9 15 0.8 3 (50%) 0.8 5 0.4 6 0.04 10 0.03 14/20 (70%) 0.9 12 0.8 II 0.4 II 3 8/25 0.5 9/25 0.6 7.2 +-- 2.8 0.8 aDoes not include one infant who died < day of age. bChi-square for trend. mates by Dubowitz and birth weights in the 2 groups were similar. The median hospital stay of the infant, need for antibiotic or oxygen therapy, incidence ofrespiratory distress syndrome, and pro- portion of infants with prolonged hospital stays did not differ between the 2 groups. Fourteen infants in the group whose mothers were randomized to antibiotics received antibiotics during their hospital stay. One infant was treated with a single dose of penicillin because ofa positive maternal culture for N. gonorrhoeae at delivery. The remainder received empiric courses of gentamicin and ampicillin after admission to the neonatal intensive care unit be- cause of respiratory distress which could not be differentiated from pneumonia. One infant in this group also received later treatment for a positive culture for coagulase-negative Staphylococcus related to an IV catheter infection. In the placebo group, 9 infants received empiric courses of antibiotics. In addition, infant received 2 courses of antibiotic therapy for coagulase-negative Staphylococcus bacte- remia related to IV catheter infections. No infants had complications related to maternal antibiotic therapy. One infant in each group died. In the antibiotic group, an infant weighing 1,015 g died at less than day of age of respiratory insufficiency related to prematurity. In the placebo group, an infant weighing 1,096 g at birth died on day 79 of life of respiratory complications from prematurity. Because antibiotics were not expected to have an effect on pregnancies without evidence ofinfection, outcomes were compared by therapy among subsets of women with or without the potential markers of upper genital tract infection. No difference in preg- nancy outcome was found between antibiotic and placebo groups among women with or without bac- teria in the AF, WBCs in the AF, CRP levels above 1.5 mg/dl, positive membrane cultures, or histologic chorioamnionitis (data not shown).

without the potential markers of upper genital tract infection. No difference in preg- nancy outcome was found between antibiotic and placebo groups among women with or without bac- teria in the AF, WBCs in the AF, CRP levels above 1.5 mg/dl, positive membrane cultures, or histologic chorioamnionitis (data not shown). In addition, since positive AF and membrane cul- tures2'3 are more commonly found in women de- livering at or below 30 weeks gestation, outcome by therapy was evaluated in women enrolled at less than 31 weeks. No difference in outcome with anti- biotic therapy was found in this subset. Antibiotics had no influence on the pregnancy outcome of pa- tients with markers of infection. Although antibiotics had no effect on the out- 224 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY ANTIBIOTICS IN PRETERM LABOR WATTS ET AL. 1.0 0.8 0.4 0.2 0.0 Antibiotic group Placebo group 0 10 20 30 40 50 60 70 80 90 100 Interval from Enrollment to Delivery (days) Fig. I. Probability of remaining undelivered until 35 weeks gestation by randomized treatment assignment. comes, even among patient groups at higher risk for infection, those patients with markers of infec- tion in either treatment group delivered earlier in gestation. Patients with compared with those with- out markers of infection (AF WBCs, AF bacteria, and CRP > 1.5 mg/dl) had a significantly shorter interval from enrollment to delivery, a lower gesta- tional age and birth weight at delivery, and longer neonatal hospital stays. The magnitude of differ- ence between those with and without infection or elevated CRP was large enough to represent sub- stantial clinical differences. Patients with compared with those without histologic chorioamnionitis did not have significantly different outcomes. DISCUSSION This study failed to demonstrate any prolongation of pregnancy among antibiotic-treated compared with placebo-treated women. Sample size calcula- tions demonstrate that with the current numbers, this study has greater than 80% power to detect an increase in the proportion of women remaining undelivered for more than week from 50% in the placebo group to 80% in the antibiotic group or a doubling of the interval from enrollment to deliv- ery in the antibiotic group. Thus, it is unlikely that we have failed to detect a significant benefit in the antibiotic group. Previous studies of antibiotic use among women with PTL with intact membranes have yielded vari- able results.

0% in the antibiotic group or a doubling of the interval from enrollment to deliv- ery in the antibiotic group. Thus, it is unlikely that we have failed to detect a significant benefit in the antibiotic group. Previous studies of antibiotic use among women with PTL with intact membranes have yielded vari- able results. The previous study most similar to this study evaluated IV ampicillin and oral erythromy- cin vs. placebo, and no difference in outcome oc- curred between the 2 groups. 12 Limited evalua- tions for possible upper genital tract infection were done in that study, but the proportion of women with upper genital tract infection was most likely low because ofa mean 34-day prolongation ofpreg- nancy in both groups. This interval was similar to the mean prolongation of pregnancy in patients in PTL without AF bacteria in this and 2 previous studies. 1,3 However, prolongation of pregnancy was found among women in PTL treated with clin- damycin compared with placebo, especially among those presenting before 33 weeks gestation. 14 Pro- longation of pregnancy also occurred in an un- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 225 ANTIBIOTICS IN PRETERM LABOR WATTS ET AL. blinded study comparing either oral erythromycin or oral ampicillin with no therapy among women in PTL. is In that study, 24% of patients had cervical C. trachomatis and 21% of patients had vaginal group B Streptococcus, rates of infection much higher than in the present study. Treatment of these infections may have accounted for the ob- served effect. Additionally, not all of the outcome data in that study appeared normally distributed and more accurate analysis would have been possi- ble using non-parametric statistics, is In 2 other reports, oral erythromycin vs. placebo in addition to tocolysis given to women in PTL failed to show any overall prolongation of pregnancy although some effect was demonstrated in those with cervical dilatation of at least cm. 6, 7 The lack of benefit of antibiotic therapy may be related to several factors. First, many patients en- rolled had no infection and would not be expected to benefit from antibiotics. Only 14% ofthose tested had AF infection, and among women not receiving antibiotics before delivery, only 41% had chorio- amnion infection and 65% had chorioamnion in- flammation.

y may be related to several factors. First, many patients en- rolled had no infection and would not be expected to benefit from antibiotics. Only 14% ofthose tested had AF infection, and among women not receiving antibiotics before delivery, only 41% had chorio- amnion infection and 65% had chorioamnion in- flammation. Fifty-five percent ofour patients were above 30 weeks gestation at enrollment, a group with only an 11% rate of AF infection and a low rate of membrane infection.2'3 Thus, the inclusion ofwomen without upper genital tract infection into either group would obscure any potential benefit from antibiotic therapy. Improved tests to identify women at high risk for both AF and membrane infection before delivery are needed to allow target- ing of antibiotic trials to assess better the impact on upper genital tract infection. Some women with upper genital tract infection could have labor that is too advanced at presentation to be inhibited by the antibiotic-tocolytic therapy used in this study. Magnesium sulfate and beta- mimetic agents were the primary tocolytics. Few patients received prostaglandin synthetase inhibi- tors. Elevated levels of prostaglandins in the AF have been found among women in PTL with AF infection. 8, 9 A combination of antibiotics and prostaglandin synthetase inhibitors might more ef- fectively inhibit PTL than antibiotic-tocolytic agents in patients with upper genital tract infection. However, in one report, pregnancy outcome was not influenced by a combination of antibiotics and indomethacin.2 Antibiotics given may fail to eliminate infection at a site critical to the stimulation of labor. A criti- cal site could be the decidua18 and this area was not sampled after antibiotic therapy in this study, al- though the rate of positive chorioamnion cultures did not differ between the 2 groups. Antibiotic levels in the decidua and chorioamnion may not be adequate to treat an established infection or may enhance the effects of infection on inciting PTL. Cytokines are released in response to AF infec- tion18,19 and histologic chorioamnionitis, 19 pre- sumably stimulated by bacterial products. Lysis of bacteria cell walls by antibiotics and release of li- popolysaccharide might even aggravate PTL by further stimulating the cytokine response.

f infection on inciting PTL. Cytokines are released in response to AF infec- tion18,19 and histologic chorioamnionitis, 19 pre- sumably stimulated by bacterial products. Lysis of bacteria cell walls by antibiotics and release of li- popolysaccharide might even aggravate PTL by further stimulating the cytokine response. Success- ful attempts to inhibit infected patients in PTL may require the down regulation of the cytokine re- sponse before antibiotic therapy by the use of corti- costeroids or cytokine antagonists.2 It was apparent that the markers evaluated (AF- WBC's, AF bacteria, CRP > 1.5 Ig/dl) identify patients at significant risk for delivery at a low gestational age and for prolonged infant hospital- ization. These markers identify a high-risk group with either infection, inflammation, or some other unidentified upper genital tract condition. Thus, despite the failure of antibiotics to prolong the ges- tation of patients in PTL, we reject the notion that infection is not related to preterm delivery. Further studies are necessary to prove a causal relationship between the two, and should be directed at the use of rapid tests to detect AF and chorioamnion infec- tion and the evaluation ofantibiotic therapy in com- bination with other therapies such as prostaglandin synthetase inhibitors and cytokine antagonists. In addition, studies are needed to evaluate whether women at increased risk of preterm delivery with upper genital tract infection can be identified and treated earlier in pregnancy to prevent PTL. ACKNOWLEDGMENTS This work was supported in part by a grant from Miles Pharmaceutical Co., Inc. REFERENCES 1. Watts DH, Krohn MA, Hillier SL, Eschenbach DA: The association of amniotic fluid infection with gesta- tional age and neonatal outcome among women in preterm labor. Obstet Gyneco179:351-357, 1992. 2. Hillier SL, MartiusJ, Krohn M, Kiviat N, Holmes KK, Eschenbach DA: A case-control study of chorioamnionic 226 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY ANTIBIOTICS IN PRETERM LABOR WATTS ET AL. infection and histologic chorioamnionitis in prematurity. N Engl J Med 319:972-978, 1988. 3. Russell P: Inflammatory lesions ofthe human placenta. I. Clinical significance of acute chorioamnionitis. Am J Di- agn Gynecol Obstet 127-137, 1979. 4. Martius J, Krohn MA, Hillier SL, Stamm WE, Holmes KK, Eschenbach DA: Relationships ofvaginal Lactobacil- lus species, cervical Chlamydia trachomatis, and bacterial vaginosis to preterm birth. Obstet Gynecol 71:89-95, 1988. 5.

Clinical significance of acute chorioamnionitis. Am J Di- agn Gynecol Obstet 127-137, 1979. 4. Martius J, Krohn MA, Hillier SL, Stamm WE, Holmes KK, Eschenbach DA: Relationships ofvaginal Lactobacil- lus species, cervical Chlamydia trachomatis, and bacterial vaginosis to preterm birth. Obstet Gynecol 71:89-95, 1988. 5. Stamm WE, Tam MR, Koester M, Cles L: Detection of Chlamydia trachomatis inclusions in McCoy cell cultures with fluorescein-conjugated monoclonal antibodies. J Clin Microbiol 17:666-668, 1983. 6. Nugent RP, Krohn MA, Hillier SL: Reliability of diag- nosing bacterial vaginosis is improved by a standardized method of Gram stain interpretation. J Clin Microbiol 29:297-301, 1991. 7. Hillier SL, Krohn MA, Kiviat NB, Watts DH, Eschen- bach DA: Microbiologic causes and neonatal outcomes associated with chorioamnion infection. Am J Obstet Gy- necol 165:955-961, 1991. 8. Siegel S: Nonparametric Statistics for the Behavioral Sci- ences. New York: McGraw-Hill, pp 111-116, 1956. 9. Cox DR: Regression models and life-tables (with discus- sion). J R Stat Soc B 34:187-200, 1972. 10. Kaplan EL, Meier P: Nonparametric estimation from incomplete observations. J Am Stat Assoc 53:457-481, 1958. 11. Watts DH, Krohn MA, Wener MH, Eschenbach DA: C-reactive protein in normal pregnancy. Obstet Gynecol 77:176-180, 1991. 12. Newton ER, Dinsmoor MJ, Gibbs RS: A randomized, blinded, placebo-controlled trial of antibiotics in idio- pathic preterm labor. Obstet Gyneco174:562-566, 1989. 13. Gravett MG, Hummel D, Eschenbach DA, Holmes KK: Preterm labor associated with subclinical amniotic fluid infection and bacterial vaginosis. Obstet Gyneco167:229- 237, 1986. 14. McGregor JA, French JI, Kyung S: Adjunctive clin- damycin therapy for preterm labor: Results of a double- blind, placebo-controlled trial. AmJ Obstet Gynecol 165: 867-875, 1991. 15. Morales WJ, Angel JL, O'Brien WF, Knuppel RA, Finazzo M: A randomized study of antibiotic therapy in idiopathic preterm labor. Obstet Gynecol 72:829-832, 1988. 16. McGregor JA, French JI, Reller LB, Todd JK, Ma- kowski EL: Adjunctive erythromycin treatment for idio- pathic preterm labor: Results of a randomized, double- blinded, placebo-controlled trial. Am J Obstet Gynecol 154:98-103, 1986. 17. Winkler M, Baumann L, Ruckhaberle KE, Schiller EM: Erythromycin therapy for subclinical intrauterine infec- tions in threatened preterm deliverymA preliminary re- port. J Perinat Med 16:253-256, 1988. 18.

abor: Results of a randomized, double- blinded, placebo-controlled trial. Am J Obstet Gynecol 154:98-103, 1986. 17. Winkler M, Baumann L, Ruckhaberle KE, Schiller EM: Erythromycin therapy for subclinical intrauterine infec- tions in threatened preterm deliverymA preliminary re- port. J Perinat Med 16:253-256, 1988. 18. Romero R, Brody DT, Oyaryum E, Major M, Woy K, Hobbins JC, Durum SK: Infection and labor. III. Inter- leukin-l: A signal for the onset of parturition. Am J Obstet Gynecol 160:1117-1123, 1989. 19. Hillier SL, Witkin SS, Krohn MA, Watts DH, Kiviat NB, Eschenbach DA: The relationship of amniotic fluid cytokines and prostaglandin E with preterm delivery, amniotic fluid infection, histologic chorioamnionitis, and chorioamnion infection. Obstet Gynecol 81:941-948, 1993. 20. Newton ER, Shields L, Ridgway LE III, Berkus MD, Elliott BD: Combination antibiotics and indomethacin in idiopathic preterm labor: A randomized double-blind clinical trial. Am J Obstet Gynecol 165:1753-1759, 1991. 21. Romero R, Tartabzovsky B: The natural interleukin-1 receptor antagonist prevents interleukin-l-induced pre- term delivery in mice. Am J Obstet Gynecol 167:1041- 1045, 1992. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 2.27

Infectious Diseases in Obstetrics and Gynecology 1:228-234 (1994) (C) 1994 Wiley-Liss, Inc. Trichomonas vaginalis: Diagnosis and Clinical Characteristics in Pregnancy R. Phillip Heine, James A. McGregor, Elisa Patterson, Ruth Parker, Deborah Draper, Janice French, and Ward Jones Department ofObstetrics and Gynecology, University ofColorado Health Sciences Center, Denver, CO ABSTRACT Objective: The objectives of this study were to 1) determine the prevalance and characterize the symptomatology of Trichomonas vaginalis (TV) infection in pregnant women on entry into prenatal care in an inner-city population; 2) compare conventional microscopic methods vs. culture tech- niques in diagnosing TV in both symptomatic and asymptomatic pregnant patients; and 3) correlate wet mount microscopic and microbiologic characteristics ofvarying manifestations oftrichomonia- SiS. Methods: One thousand two hundred sixty patients in an inner-city population were tested at entry into prenatal care for TV by saline wet mount and culture techniques. Other tests for lower genital tract infection were also performed. Vaginal symptoms were ascertained through standard- ized questioning prior to examination. Standard microscopic and microbiologic data were also obtained for analysis. Wet mounts were systematically examined and considered negative if no TV was identified in 10 high powerfields (HPFs). Cultures were inspected from days 4 to 7 or until positive results were obtained. Results were analyzed using McNemar's test for correlated propor- tions, chi-squared test, or Fisher exact test where appropriate. Results: Culture and wet mount results were available in 1,175 patients. TV infection was documented by one or both techniques in 110/1,175 (9.4%). Culture methods detected 105/110 (94.5%) ofall patients while wet mount detected 90/110 (73%) (P < 0.001). Vaginal symptoms were present in only 20/110 patents (18.2%). Among asymptomatic patients, culture detected 94% while wet mount detected 70% (P < 0.001). Among symptomatic patients, wet mount and culture were both effective and diagnosed 85% and 95% ofinfections, respectively (P not significant).

110 (73%) (P < 0.001). Vaginal symptoms were present in only 20/110 patents (18.2%). Among asymptomatic patients, culture detected 94% while wet mount detected 70% (P < 0.001). Among symptomatic patients, wet mount and culture were both effective and diagnosed 85% and 95% ofinfections, respectively (P not significant). Patients with TV were more likely to have increased vaginal fluid white blood cells (WBCs) and more severe vaginal flora disruption than uninfected controls. Subgroup analysis revealed wet mount-positive/ culture-positive patients were more likely to have vaginal flora disruption, as evidenced by decreased lactobacilli and elevated vaginal pH, than wet mount-negative/culture-positive subjects. Coexistent infection rates were similar regardless ofwet mount status. Elevated vaginal fluid WBCs were more common among patients with symptoms. Conclusions: 1) Screening pregnant women for TV based solely on symptomatology is ineffective in this population; 2) culture techniques detected more infections than conventional microscopic evaluation; and 3) significant increases in vaginal fluid WBCs and altered vaginal flora are found in both symptomatic and asymptomatic TV, suggesting that both infestations have the potential to adversely affect pregnancy outcome. Studies on the influence of TV on pregnancy outcomes are ongoing. (C) 1994 Wiley-Liss, Inc. KEY WORDS Trichomoniasis, vaginitis, pathogenesis, prematurity, pregnancy espite advances in perinatal and neonatal care, preterm birth (PTB) remains the major cause ofneonatal morbidity and mortality. The causes of PTB are multifactorial; however, considerable evi- dence shows that common reproductive tract infec- tions and/or associated inflammation play roles in Address correspondence/reprint requests to Dr. James A. McGregor, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Box B198, Denver, CO 80262. Received December 20, 1993 Clinical Study Accepted March 15, 1994 TV CHARACTERISTICS IN PREGNANCY HEINE ET AL. preterm labor (PTL), PTB, and premature rup- ture of membranes (PROM) in significant num- bers of women. 2-4 Understanding possible causal pathogenic factors such as reproductive tract infec- tion can allow for development of etiologic-based interventions to prevent at least some instances of PTB.

REGNANCY HEINE ET AL. preterm labor (PTL), PTB, and premature rup- ture of membranes (PROM) in significant num- bers of women. 2-4 Understanding possible causal pathogenic factors such as reproductive tract infec- tion can allow for development of etiologic-based interventions to prevent at least some instances of PTB. Case-controlled and cohort studies have linked vaginal infections associated with Trichomonas vag- inalis (TV) to decreased gestational age at delivery and PROM.s'6 Several smaller studies did not show these associations. 7'8 Recent results of a large mul- ticenter (Vaginal Infection in Prematurity [VIP]) study, which included multivariate analysis to cor- rect for potential confounding variables, confirm significant associations between TV infection and PROM, PTB, and low birth weight in diverse populations of women in the United States.9 Fur- ther research is warranted to delineate and charac- terize possible causal roles of TV infection in the pathogenesis of prematurity. Recognition, treat- ment, and understanding of TV pathobiology dur- ing pregnancy can be important steps in the incre- mental reduction of PTB and PROM. The purposes of this study were to 1) determine the prevalence and characterize symptomatology of TV in women in an inner-city population on entry into prenatal care; 2) compare culture to standard clinic-based microscopic wet mount techniques for the diagnosis ofTV in symptomatic and asymptom- atic women; and 3) correlate microbiologic and microscopic characteristics of TV in symptomatic and asymptomatic pregnant women. MATERIALS AND METHODS Patients One thousand two hundred sixty women entering prenatal care at Denver General Hospital (Denver, CO) between July 1990 and September 1991 were enrolled in an integrated program for prematurity prevention. Prior to pelvic examination, lower re- productive tract symptoms were ascertained by stan- dardized questions regarding 1) abnormal quantity or character of discharge, 2) vaginal or vulvar burning or irritation, and 3) urinary complaints. To correlate presentation and findings of TV with an uninfected control group, we analyzed as a con- trol the next enrolled patient with no evidence of cervical or vaginal infection with TV, Chlamydia trachomatis, bacterial vaginosis (BV), Neisseria gon- orrhoeae, or yeast species. This study was approved by the hospital's Investigational Review Board.

ings of TV with an uninfected control group, we analyzed as a con- trol the next enrolled patient with no evidence of cervical or vaginal infection with TV, Chlamydia trachomatis, bacterial vaginosis (BV), Neisseria gon- orrhoeae, or yeast species. This study was approved by the hospital's Investigational Review Board. Microbiology At initial pelvic examination, specimens were ob- tained for various microbiologic, microscopic, and enzymatic assessments. A cotton-tipped swab was placed on the posterior fornix and cultured for TV (InPouch TV, Biomed Diagnostics, Santa Clara, CA). We previously demonstrated the InPouch TV to be comparable to the use ofDiamond's media culture for identification of TV. 10 Another cotton- tipped swab from the upper lateral vaginal wall was tested for vaginal pH (ColorpI-Iast Indicator Strips, pH 4-7, EM Science, Cherry Hills, NJ) and rolled onto a glass slide for Gram's stain evaluation for BV. 11 This swab was then placed into 50 ll of normal saline at room temperature for microscopic and amine test examinations. An endocervical sam- ple was collected with a cotton-tipped swab and inoculated onto Thayer Martin agar (Remel, Den- ver, CO) for recovery of N. gonorrhoeae. A sepa- rate endocervical sample was tested for C. tracho- matis by enzyme immunoassay (Pathfinder Chlamydia EIA Swab Collection System, Kalles- tad, Chaska, MN) using standard criteria. Further vaginal fluid specimens were collected from the posterior vaginal fornix and frozen at -70C for measurement of vaginal fluid enzymes. Results of these tests will be reported elsewhere. Cultures of N. gonorrhoeae and TV were held in a candle jar at room temperature and transported to the lab within 4 h of collection. Cultures for TV were examined from days 4 to 7 for characteristic protozoan motility and morphologic characteris- tics. Cultures for N. gonorrhoeae were incubated at 35C with 5% carbon dioxide and the organism was identified by standard microbiologic techniques. 12 Microscopy The vaginal sample in normal saline was examined immediately by wet mount slide for evidence of motile trichomonads, yeast pseudohyphae or bud forms, clue cells, lactobacillus morphotypes, and white blood cells (WBCs). At least 10 high power fields (HPFs) were examined by experienced clini- cians. A drop offluid was placed in 10% KOH and evaluated for release ofamine odor, i.e., whifftest. The fluid was again examined for yeast forms.

east pseudohyphae or bud forms, clue cells, lactobacillus morphotypes, and white blood cells (WBCs). At least 10 high power fields (HPFs) were examined by experienced clini- cians. A drop offluid was placed in 10% KOH and evaluated for release ofamine odor, i.e., whifftest. The fluid was again examined for yeast forms. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 229 TV CHARACTERISTICS IN PREGNANCY TABLE I. Symptoms associated with TV infection Symptomatic All Symptom N patients (%) patients (%) Discharge 8 40 7 Pruritus 8 40 7 Odor 6 30 5 Dysuria 5 Burning 3 15 3 A wet mount was considered positive for TV when motile trichomonads were visualized. BV was diagnosed when 2 of the following 3 occurred: >20% of epithelial cells were clue cells, vaginal fluid pI--I > 4.7, and/or a whifftest was positive. 13 In presence of TV, the diagnosis of BV was made only if >20% clue cells were present. As we previ- ously obtained good correlation between clinic- based and Gram's stain diagnosis of BV, only a clinic-based diagnosis of BV was evaluated in this study. 14 WBCs were considered elevated if >5 were visualized on wet mount in each HPF exam- ined; less than lactobacillus morphotype per HPF was considered decreased. Statistical Analysis McNemar's test for correlated proportion was used to compare the wet mount with culture for diagno- sis of TV. Chi-squared or Fisher exact test was utilized to compare various microscopic and micro- biologic findings between groups. RESULTS Culture and wet mount results were available in 1,175 patients. TV infection was documented in 9.4% (110/1,175). BV and yeast were present in 32.9% and 17.8%, respectively. C. trachomatis was isolated in 7.9% of patients; culture of N. gonor- rhoeae was positive in only 11 women (0.9%). De- mographic information in regard to age, parity, and racial distribution of those infected with TV was similar to the entire population with the excep- tion of a 50% increase in prevalence among the African-American population (19% vs. 30%). The mean age ofsubjects was 25 years; two thirds ofthe patients were multiparous. Symptoms elicited at study entry are listed in Table 1. Only 18.2% (20/110) of TV-infected women presented with complaints. This was not significantly different from the 11% with symp- HEINE ET AL. TABLE 2.

rican population (19% vs. 30%). The mean age ofsubjects was 25 years; two thirds ofthe patients were multiparous. Symptoms elicited at study entry are listed in Table 1. Only 18.2% (20/110) of TV-infected women presented with complaints. This was not significantly different from the 11% with symp- HEINE ET AL. TABLE 2. Proportion of women presenting with symptomatic T in each pregnancy trimester TV TV wet Weeks symptomatic mount- gestation N patients (%) positive (%) 0-14 19 33* 71 14--28 51 24* 78 28+ 30 4 84 *P < 0.05 compared with symptomatic patients at 28+ weeks gestation. Wet mount comparisons in the different trimesters were not significantly different. TABLE 3. Comparison of wet mount vs. culture in the diagnosis of T both with and without symptomsa Diagnosis (% positive) All symptomatic asymptomatic (N II0) (N 20) (N 90) Wet mount 73* 85 70* Culture 95 95 94 aWet mount was inferior in asymptomatic patients but equivalent in symptomatic patients. *P < 0.001 compared with culture diagnosis. toms in the uninfected control group. Discharge and pruritus were the most common symptoms; however, they occurred in fewer than in 10 women with TV. When comparing gestational age at examination, symptomatic patients were signifi- cantly more likely to present in the 1st and 2nd trimesters than in the 3rd trimester (Table 2). In fact, only 4% (1/30) with documented 3rd trimes- ter infection presented with vaginal complaints, while 33% and 24% presented with symptoms in the st and 2nd trimesters, respectively. Comparison of diagnostic methods revealed that culture was superior to wet mount in all subjects diagnosed with TV (Table 3). Analysis of sub- groups based on symptomatology demonstrated that culture and wet mount were not different in symp- tomatic patients (85% vs. 95%), while culture was superior (94% vs. 70%) in asymptomatic patients. Diagnostic methods were equivalent in all 3 trimes- ters (Table 2). The microscopic and microbiologic findings characterizing TV infection are presented in Table 4. Symptomatic women were more likely to have 230 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY TV CHARACTERISTICS IN PREGNANCY HEINE ET AL. TABLE 4.

ents. Diagnostic methods were equivalent in all 3 trimes- ters (Table 2). The microscopic and microbiologic findings characterizing TV infection are presented in Table 4. Symptomatic women were more likely to have 230 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY TV CHARACTERISTICS IN PREGNANCY HEINE ET AL. TABLE 4. Results (%) of vaginal microscopic and microbiologic findings in varying manifestations of TV and an uninfected control groupa TV TV Wet mount Wet mount @ TV symptomatic asymptomatic culture culture No infection (N 10) (N 20) (N 90) (N 74) (N 30) (N 10) VVBC > 5/HPF 50** 76** 44*'** 54** 43* 26 Lactcobacilli 75** 71 ** 75** 81 ** 60*'** 32 < I/HPF Vaginal pH 79** 75** 71"* 79** 43"** 17 >4.5 Whiff test 39** 52** 36** 40** 27** 3 Coexistent infection Any 63 57 63 60 57 BV 43 52 40 48 30 Yeast 24 10 28 21 27 Chlarnydia 9 0 I0 I0 3 aSymptomatic patients were more likely to have elevated vaginal WBCs compared with asymptomatic patients. Wet mount-positive patients were more likely to have decreased lactobacilli and an elevated vaginal pH compared with wet mount-negative patients. Patients with any TV infection were more likely to have elevated vaginal fluid WBCs and altered vaginal flora compared with uninfected controls. bHigh power field (400). *P < 0.05 comparison within groups (asymptomatic vs. symptomatic, wet mount vs. wet mount e). Other comparisons between groups were not significantly different. **P < 0.05 comparison to no infection group. All comparisons reached significance. increased vaginal WBCs compared with asymp- tomatic patients. Vaginal flora disruption, as evalu- ated by decreased lactobacilli, altered vaginal pH, or positive amine test, was not different between symptomatic and asymptomatic women. Although the prevalence of coexistent infections was not sig- nificantly different, a trend toward increased C. trachomatis or yeast infections was observed in the asymptomatic group (P 0.10). Both symptom- atic and asymptomatic patients were more likely to exhibit increased density ofvaginal WBCs and dis- rupted vaginal flora compared with uninfected con- trol women. Wet mount-positive/culture-positive patients had a similar prevalence of increased vaginal WBCs compared with wet mount-negative/culture-positive patients. However, wet mount-positive women were more likely to have disruption of the vaginal flora, as evidenced by a decrease in lactobacilli and an increase in vaginal pH.

et mount-positive/culture-positive patients had a similar prevalence of increased vaginal WBCs compared with wet mount-negative/culture-positive patients. However, wet mount-positive women were more likely to have disruption of the vaginal flora, as evidenced by a decrease in lactobacilli and an increase in vaginal pH. Coexistent infection rates were similar, but there was a trend toward an increase in BV in wet mount-positive vs. wet mount- negative women (48% vs. 30%, P 0.15). Both wet mount-positive and wet mount-negative pa- tients were more likely to have increased vaginal WBCs and vaginal flora disruption compared with uninfected controls. The contributions of TV vs. effects from coex- istent infections on vaginal pathology are shown in Table 5. Coexistent TV infection with BV created the most severe disruption of vaginal flora. TV alone exhibited significant microscopic microflora disruption compared with controls; however, TV alone or in combination with yeast rarely produced a positive whiff test. Furthermore, infection with yeast species did not significantly alter WBCs or lactobacillus outcomes; however, the sample size was small. DISCUSSION In this study, 9.4% of inner-city Denver, CO, pregnant women presenting for prenatal care were infected with TV. Only 18.2% of TV-infected women complained of vaginitis-related symptoms prior to pelvic examination. For diagnosis, culture was superior to wet mount in this mostly asympt- omatic population. Importantly, when compared with uninfected women, the occurrence of infection with TV either alone or in combination with other studied infections was associated with increased numbers of vaginal fluid WBCs and altered vagi- nal flora. Furthermore, symptomatic TV patients were more likely than asymptomatic patients to have increased vaginal fluid WBCs, while wet mount- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 231 TV CHARACTERISTICS IN PREGNANCY HEINE ET AL. TABLE 5.

ted with increased numbers of vaginal fluid WBCs and altered vagi- nal flora. Furthermore, symptomatic TV patients were more likely than asymptomatic patients to have increased vaginal fluid WBCs, while wet mount- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 231 TV CHARACTERISTICS IN PREGNANCY HEINE ET AL. TABLE 5. Vaginal microscopic and microbiologic findings for women with TV alone, TV with coexistent infections, and an uninfected control groupa Coexistent infections No infection TV alone Any BV Yeast (N 33) (N 65) (N 38) (N 14) (N II0) WBC > 5/HPFs 55** 53** 68** 43 26 Lactobacilli 55** 83*'** 97"** 50 32 < I/HPF Vaginal pH 45** 80"** 97*'** 36 17 >4.5 Whiff test 3 51" 79*'** 0 3 aPatients with BV or any infection were significantly more likely to have decreased lactobacilli, increased vaginal pH, and a positive whiff test compared with those with "IV infection alone. Patients with TV alone or coinfected with BV were more likely to have elevated vaginal fluid WBCs and altered vagina flora compared with controls. TV alone did not exhibit a positive whiff test. TV combined with yeast was not different from controls. *P < 0.05 compared with TV infection alone. Comparisons not noted were not significantly different. **P < 0.05 compared with the no infection group. Comparisons not noted were not significantly different. positive/culture-positive patients had more vaginal flora disruption than their wet mount-negative/ culture-negative counterparts. Microscopic analy- sis also revealed that coexistent infection ofTV with BV was associated with the greatest disruption of vaginal flora. The TV prevalence rate in this study is consistent with the 12.6% rate obtained in the large multi- center VIP study.9 Similar populations have docu- mented TV infestation rates as low as 3% and as high as 48%. 15,16 Our findings appear to be repre- sentative of infection rates in many urban popula- tions. The increased prevalence among African- American women is consistent with previous reports. 17 Other prior reported associations such as infection in older women18 and/or coexistent infec- tion with N. gonorrhoeae were not noted. 19 HOW- ever, we had an extremely low prevalence of N. gonorrhoeae. Importantly, TV was more commonly identified than either N. gonorrhoeeae or C. tra- chomatis, for which we routinely screen in our ob- stetric population. Two prior reports describe symptoms in 62- 91% ofTV-infected pregnant women.

oeae were not noted. 19 HOW- ever, we had an extremely low prevalence of N. gonorrhoeae. Importantly, TV was more commonly identified than either N. gonorrhoeeae or C. tra- chomatis, for which we routinely screen in our ob- stetric population. Two prior reports describe symptoms in 62- 91% ofTV-infected pregnant women. 2'21 Neither ofthese studies rigorously or systematically assessed symptoms prior to diagnosis. One previous study involving pregnant women evaluated symptoms by severity; fewer than 20% of patients with TV had "marked" symptoms, the majority being classified as "slight.''22 In each ofthese studies, symptomatol- ogy was elicited after identification of TV. Our documentation of symptoms prior to vaginal exam- ination and the commonly held belief that some vaginal symptomatology is normal in pregnancy could explain such divergent results. This latter suggestion is supported by our findings ofincreased symptomatic infections in the 1st and 2nd trimes- ters, although symptoms in the 1st trimester were present in only 33% of women. Overall, in our population, TV-ascribable symptoms were uncom- mon and could not be used to identify women for selective examination. Reported sensitivities of wet mount in the diag- nosis of TV vary from 35% to 82%.23,24 Our overall sensitivity approaches the optimal rate; smears were reviewed by practiced clinicians who visualized 10 or more HPFs prior to reaching a negative or positive diagnosis. In clinical practice, numerous examiners with varying levels of exper- tise utilizing nonstandardized techniques will likely identify fewer instances of TV infection. Even un- der our optimized research conditions, culture was superior to wet mount in women presenting with asymptomatic TV infection. The classic microscopic finding ofelevated num- bers ofvaginal fluid WBCs associated with TV was confirmed in this study. The degree of abnormal- ity, however, was different from prior work. Pre- vious work revealed increased vaginal WBCs in approximately 75% of patients infected with TV,25 while only half of our patients exhibited elevations. A ratio of WBCs to epithelial cells of > was previously used to define elevation, while the present study used absolute numbers. Such differ- ences are probably not clinically relevant, but for research purposes, absolute numbers are undoubt- 232 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY TV CHARACTERISTICS IN PREGNANCY HEINE ET AL. edly more reproducible.

previously used to define elevation, while the present study used absolute numbers. Such differ- ences are probably not clinically relevant, but for research purposes, absolute numbers are undoubt- 232 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY TV CHARACTERISTICS IN PREGNANCY HEINE ET AL. edly more reproducible. We suggest that future studies use cell counting chambers to more accu- rately measure inflammatory cells in vaginal fluid. In this study, both symptomatic and asymptom- atic TV was associated with increased vaginal fluid WBC numbers. Subgroup analysis revealed that symptomatic patients were more likely than their asymptomatic counterparts to have elevated vaginal fluid WBCs. This suggests that immunologic acti- vation and inflammation are important for devel- opment of symptoms. Strain-variable antigenicity is a potential explanation; however, experiments evaluating virulent vs. avirulent strains have shown no demonstrable differences to date.26 More likely explanations are the increased production ofneutro- phil chemoattractant factors exhibited by virulent TV strains in vitro27 and increased concentrations of microorganisms. Whatever the etiology, host response likely participates in possible TV-related adverse pregnancy outcomes. The majority of previous investigations concur with the association of TV and altered vaginal flora. s, 16 TV infection alone was associated with decreased levels of lactobacilli; however, coinfec- tion with BV appeared largely responsible for ele- vated vaginal pH and positive amine test associated with TV. Our study showed that fewer than 50% of patients with TV alone or TV with yeast had a vaginal pH of greater than 4.5, and only of 47 had a positive amine test. Clearly, BV should be suspected in TV patients with altered vaginal flora. Prior teaching that these conditions are mutually exclusive appears incorrect. Future investigations regarding TV or BV associations with adverse preg- nancy outcome should account for effects of com- bined infections. Wet mount-positive/culture TV-positive patients demonstrated greater vaginal flora disruption than did wet mount-negative/culture-positive patients; however, the numbers of patients with increased vaginal fluid WBCs were similar. Previous work suggests that decreased parasitic burden is responsi- ble for wet mount negativity.

nt-positive/culture TV-positive patients demonstrated greater vaginal flora disruption than did wet mount-negative/culture-positive patients; however, the numbers of patients with increased vaginal fluid WBCs were similar. Previous work suggests that decreased parasitic burden is responsi- ble for wet mount negativity. 19 Therefore, our findings suggest that vaginal floral disruption is associated with trichomonad numbers, while host responsiveness as mnifested by increased numbers of vaginal fluid WBCs is less directly inoculum- related. These findings have relevance to studies evaluat- ing untoward effects of TV infection during preg- nancy. Research studies linking TV with PTB and PROM used microbiologic methods to identify in- fection; in contrast, most clinicians caring for preg- nant women use symptoms or grossly abnormal vaginal discharge to identify TV using wet mount microscopy. Obviously, the use of microbiologic- based methods can lead to identification of more women at risk for TV-associated pregnancy mor- bidity; future work will show whether symptomatic or wet mount-positive women are at greater risk than presumably more lightly infected wet mount- negative women. We speculate that 1) densities of infecting microorganisms, 2) individual infesting strains' abilities to produce virulence factors, and 3) nature ofhost responses may all contribute to the pathogenesis of TV-related adverse pregnancy out- come. In this study, both wet mount-positive and wet mount-negative TV-infected women demon- strated increased numbers of vaginal fluid WBCs. This suggests that even low density infections are associated with possibly damaging local host per- turbations, which could increase risks ofpregnancy morbidity. If host response is the most important determinant of TV-associated morbidity, then uni- versal screening and treatment may be most appro- priate. In summary, TV was common among pregnant women receiving publicly supported antenatal care in Denver, CO. Under optimized research condi- tions, wet mount diagnosis was inferior to culture for identification of TV except in the minority of patients who were symptomatic. Because symptoms were so infrequent, a program involving screening of symptomatic patients will be ineffective. The optimal testing method among culture or wet mount requires correlation of varying manifestations with pregnancy outcome.

e for identification of TV except in the minority of patients who were symptomatic. Because symptoms were so infrequent, a program involving screening of symptomatic patients will be ineffective. The optimal testing method among culture or wet mount requires correlation of varying manifestations with pregnancy outcome. TV alone, or in combination with other infections, regardless of symptomatol- ogy or wet mount positivity, created considerable disruption ofthe vaginal flora and elevated vaginal WBCs. This suggests that all manifestations of TV have the potential to create complications in preg- nancy, and screening methods that identify the ma- jority of cases appear justified. REFERENCES 1. Lee K, Paneth N, Gartner LM, Pearlman MA, Gruss L: Neonatal mortality: An analysis of the recent improve- ment in the United States. Am J Public Health 70:15- 21, 1980. 2. McGregor JA: Prevention of preterm birth: New initia- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 2]] TV CHARACTERISTICS IN PREGNANCY HEINE ET AL. tives based on microbial-host interactions. Obstet Gyne- col Surv 43:1-14, 1988. 3. Romero R, Mazor M, Wu YK, et al.: infection in the pathogenesis of preterm labor. Semin Perinatol 12:262- 279, 1988. 4. McGregor JA, French JI, Seo K: Adjunctive clindamy- cin therapy for preterm labor: Results of a double-blind, placebo-controlled trial. Am j Obstet Gynecol 165:867- 875, 1991. 5. Hardy PH, Nell EE, Spence MR, et al.: Prevalence of six sexually transmitted disease agents among pregnant inner-city adolescents and pregnancy outcome. Lancet 2:333-337, 1984. 6. Minkoff H, Grunebaum AN, Schwarz RH, et al.: Risk factors for prematurity and premature rupture of mem- branes: A prospective study of the vaginal flora in preg- nancy. Am J Obstet Gynecol 150:965-972, 1984. 7. Mason PR, Brown ML: Trichomoniasis in pregnancy. Lancet 1025-1026, 1990. 8. Ross SM, Van Middlekoop A: Trichomonas infection in pregnancy: Does it affect perinatal outcome? S Afr Med J 63:566-567, 1983. 9. Cotch MF: Vaginal Infections in Prematurity Study Group. Carriage of Trichomonas vaginalis is associated with adverse pregnancy outcome. Presented at the 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Abstract 681, Atlanta, Georgia, October 21-24, 1990. 10. Draper DL, Parker R, Patterson E, et al.: Detection of T. vaginalis in pregnant women with the InPouch TV culture system. J Clin Microbiol 31(4):1016-1018, 1993. 11.

outcome. Presented at the 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Abstract 681, Atlanta, Georgia, October 21-24, 1990. 10. Draper DL, Parker R, Patterson E, et al.: Detection of T. vaginalis in pregnant women with the InPouch TV culture system. J Clin Microbiol 31(4):1016-1018, 1993. 11. Nugent RP, Krohn MA, Hillier SL: Reliability of diag- nosing bacterial vaginosis is improved by a standardized method of Gram's stain interpretation. J Clin Microbiol 29:297-301, 1991. 12. Lennette EH, Balows A, Housler WJJr, Shadomy MG (eds): Manual of Clinical Microbiology. 4th Ed. Ameri- can Society for Microbiologists, 1985. 13. Krohn MA, Hillier SL, Eschenbach DA: Comparison of methods for diagnosing bacterial vaginosis among preg- nant women. J Clin Microbiol 27:1266-1271, 1989. 14. McGregor JA, French JI, Jones w, et al.: Association of cervicovaginal infections with increased vaginal fluid phospholipase A activity. Am J Obstet Gynecol 167: 1558-1594, 1992. 15. deLouvou J, Hurley R, Stanley VC: Microbial flora of the lower genital tract during pregnancy. J Clin Pathol 28:731-735, 1975. 16. Kite EK, Hesseltine HC, Goldstein L: A study of the bacterial flora of the normal and pathologic vagina and uterus. Am j Obstet Gynecol 53:233-240, 1947. 17. Cotch MF, Pastorek JG, Nugent RP, et al.: Demo- graphic and behavioral predictors of Trichomonas vagina- lis infection among pregnant women. Obstet Gynecol 6:1067-1092, 1991. 18. Burch TA, Ries CW, Reardon LV: Epidemiologic stud- ies of human trichomoniasis. Am J Trop Med Hyg 8:312-318, 1959. 19. Fouts AC, Kraus SJ: Trichomonas vaginalis: Reevaluation of its clinical presentation and laboratory diagnosis. J Infect Dis 141; 137-143, 1980. 20. Bramley M: Study of female babies entering confinement with vaginal trichomoniasis. Br J Vener Dis 52:58-62, 1976. 21. Robinson SC, Michandani G: Observation on vaginal trichomoniasis. Am J Obstet Gynecol 91:1005-1012, 1965. 22. Wisdom AR, Dunlop EMC: Trichomoniasis. Study of the disease and its treatment in women. Br J Vener Dis 41:90-96, 1965. 23. Laughlin M, Tidwell B, Finley R, Cleary J, Lushbaugh W: A double blind placebo-controlled troal ofsingle-dose intravaginal vs. single-dose oral metronidazole in the treat- ment of Trichomonas vaginalis. Presented at the 32nd In- terscience Conference on Antimicrobial Agents and Che- motherapy, Abstract 225, Anaheim, California, October 11-14, 1992. 24.

leary J, Lushbaugh W: A double blind placebo-controlled troal ofsingle-dose intravaginal vs. single-dose oral metronidazole in the treat- ment of Trichomonas vaginalis. Presented at the 32nd In- terscience Conference on Antimicrobial Agents and Che- motherapy, Abstract 225, Anaheim, California, October 11-14, 1992. 24. McMillian A: Laboratory diagnostic methods and cryo- preservation of trichomonads. In Honigberg BM (ed): Trichomonads Parasitic in Humans. New York: Spring Hill, pp 297-310, 1990. 25. Rein MF: Clinical manifestations ofurogenital trichomo- niasis in women. In Honigberg BM (ed): Trichomonads Parasitic in Humans. New York: Spring Hill, pp 225- 234, 1990. 26. Su-Lin KE, Honigberg BM: Antigenic analysis of Trich- omonas vaginalis strains by quantitative fluorescent anti- body methods. Z Parasitenkd 69:162-181, 1983. 27. Mason PR, Forman L: Polymorphonuclear cell chemo- taxis to secretions of pathogenic and nonpathogenic Trich- omonas vaginalis. J Parasitol 68:457-462, 1982. 2]4 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:235-241 (1994) (C) 1994 Wiley-Liss, Inc. Human Papillomaviruses and Papillomatosis Lesions of the Female Lower Genital Tract Yu-Liang Fu, Yao-Xiong Hu, Han-Liang Ling, Zhen-Zhong Ye, Tian Liang, Mei-Gui Zhang, Yun-Ke Liu, Biao Kang, Yuan-Ji Luo, Shu-Ying He, and Yong-Jian Lian Guangzhou Maternal and Neonatal Hospital (Y.-L.F., Y.-K.L., S.-Y.H., Y.-J.Li.), Guangzhou Medicinal Institute (Y.-X.H., T.L., B.K., Y.-J.Lu.), Sun Yat Sen University ofMedical Sciences (H.-L.L.), and Guangzhou Medical College (Z.-Z. Y, M.-G.Z.), Guangzhou, China ABSTRACT Objective: The objective of this study was to determine whether human papillomavirus (HPV) infections are involved in the development ofpapillomatosis lesions ofthe lower female genital tract. Methods: A total of 616 biopsy specimens of genital papillomatous lesions (307 nodular and 309 papular types) from 598 patients were anaylyzed for the presence of HPV DNA sequences by polymerase chain reaction (PCR). These specimens were also examined by histopathological as- sessment for characteristic HPV-associated cytological changes, by immunohistochemical staining for HPV-associated antigen, and by electron microscopy for the presence ofvirions. Results: HPV DNA sequences were found in 97.9% (140 of 143 cases) and 1.1% (1 of91 cases) of the nodular and papular papillomatosis cases tested, respectively. In 18 patients who had both types ofpapillomatosis lesions, HPV DNA was invariably found only in nodular tissues. HPV-associated antigen, koilocytosis, and virions were found in 53.6% (98 of 183 cases), 70.5% (129 of 183 cases), and 5.9% (5 of85 cases) ofnodular papillomatosis lesions tested, respectively. Conclusions: These data suggest that nodular papillomatosis was closely associated with HPV infection, but that papular papillomatosis ofthe lowerfemale genital tract may have an etiology other than HPV infection. (C) 1994 Wiley-Liss, Inc. KEY WORS Condyloma acuminatum, pseudocondyloma, human papillomavirus, PCR uman papillomaviruses (HPVs) are found fre- quently in the female lower genital tract where they induce different lesions depending on the HPV types involved.

genital tract may have an etiology other than HPV infection. (C) 1994 Wiley-Liss, Inc. KEY WORS Condyloma acuminatum, pseudocondyloma, human papillomavirus, PCR uman papillomaviruses (HPVs) are found fre- quently in the female lower genital tract where they induce different lesions depending on the HPV types involved. The association between the pres- ence of different types of I--IPV and clinical lesions is better established for the cervix than for the vulva. For example, vulvar vestibular papilloma- tosis is one type of vulvar lesion whose origin and clinical significance remain controversial. Fried- rich2 attributed the vestibular papillae present in asymptomatic women as anatomic and/or functional variants of normal vulvar epithelium. Pekham et al.3 and Friedrich4 considered vestibular papil- lomatosis a condition reactive to phylogosis caused by non-viral agents. However, recent descriptions ofso-called "subclinical" HPV infections by Grow- don et al. 5 and by Campion et al.6 raised the ques- tion of whether all these vestibular papillae were indeed related to HPV infections.7-1 Vulvar condyloma acuminatum, on the other hand, is clearly caused by infection with certain types of HPV that are mainly transmitted through sexual contact. 2-s Inquiries into the natural his- tory and establishment of an unequivocal diagnosis Address correspondence/reprint requests to Dr. Yu-Liang Fu, Associated Chief, Guangzhou Maternal and Neonatal Hospi- tal, 402 Renminzong Road, Guangzhou 510180, China. Basic Science Article Received September 14, 1993 Accepted January 15, 1994 VULVAR HPV INFECTIONS FU ET AL. ofHPV infection in vulvar vestibular papillomato- sis have been hampered by the lack of an in vitro propagation system for the virus and by the poor correlation between HPV serology and disease ac- tivity. In view of the rapid spreading of HPV infection in recent years, both in Western coun- tries7'12-18 and in China, 19,20 a reliable method for the detection of HPV infection and differential di- agnosis between condyloma acuminata and vulvar vestibular papillomatosis would help patient evalu- ation and management. In this paper, we report results of clinical and laboratory examinations ofpapillomatosis lesions of the lower female genital tract.

ble method for the detection of HPV infection and differential di- agnosis between condyloma acuminata and vulvar vestibular papillomatosis would help patient evalu- ation and management. In this paper, we report results of clinical and laboratory examinations ofpapillomatosis lesions of the lower female genital tract. The polymerase chain reaction (PCR) technique, which was used to am- plify HPV DNA sequences and to determine the presence of HPV DNA sequences, was supple- mented with histopathological assessments of HPV infections by immunohistochemical staining of HPV-associated antigen, microscopy (both light and electron microscopes), colposcopy, clinical eval- uation, and follow-up. SUBJECTS AND METHODS Patients and Specimens Between January 1990 and August 1992, a total of 616 vulvar papillomatosis biopsies (307 nodular and 309 papular types) were obtained from 598 patients, including 18 patients who had both nodu- lar and papular types of vulvar papillomatosis, be- fore any therapeutic management was implemented. The ages of these patients were between 14 and 48 years. Laboratory tests were performed and results were analyzed in a double-blinded fashion. Determination of HPV DNA Sequences by PCR Portions of freshly frozen tissues were treated with proteinase K (Sigma Chemicals, St. Louis, MO). DNA was extracted by phenol and chloroform and purified by alcohol precipitation. Purified DNA was then used as template for PCR amplification of HPV DNA sequences. Consensus primers MY09 and MYll commercially obtained from Perkin- Elmer Cetus (Norwalk, CT) are capable of ampli- fying DNA from genital HPV types 6, 11, 16, 18, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 57, and 59 and from at least another 25 yet unchar- acterized HPV types. 18 Dermal HPV types 1, 5, 8,26, 27, 41, 47, and 48 are also amplified with TABLE I. Clinical and laboratory findings on nodular and papular papillomatosis lesions in the lower female genital tract* Test item Condyloma acuminatum Pseudocondyloma (nodular type (papular type lesions) lesions) HP DNA (types 6, 97.9% I. I% II, 16, 18, and 33) (140/143) (I/91) HPV-associated 53.6% 0.9% antigen (98/183) (2/225) HPV virions 5.9% 0 (s/ss) (0/3) History of multiple 80.8% 7. I% sexual partners (248/307) (22/309) *Results were presented as percentage of cases tested positive. Differ- ences in all test items (except HP virions) between these two groups of genital papillomatosis were statistically significant (P < 0.01 ). these primers.

virions 5.9% 0 (s/ss) (0/3) History of multiple 80.8% 7. I% sexual partners (248/307) (22/309) *Results were presented as percentage of cases tested positive. Differ- ences in all test items (except HP virions) between these two groups of genital papillomatosis were statistically significant (P < 0.01 ). these primers. Sequences of the oligonucleotide primers were CGTCCMARRGAWACTGATC and GCMCAGGWCATAAYAATGG (where M=A+C, R=A+G, W=A+T, Y= C + T). DNA samples, deoxyribonucleoside tri- phosphates, and primers were heated in buffer to 95C for 5 min before Taq DNA polymerase (Per- kin-Elmer Cetus) was added to the reaction mix- ture and reaction started in a thermocycler (Model 480, Perkin-Elmer Cetus). The temperatures of the reaction mixture were cycled 3 5 times through 95C denaturation (50 sec), 50C annealing (50 sec), and 72C extension (1 min) with a 5-min incubation at 72C at the end. Positive and negative control DNA were always included in every PCR assay. Portions of the amplified reaction mixture were separated by electrophoresis in 2% agarose gel with pGEM-3 DNA digested with a mixture of restriction endonucleases HinJI, RsaI, and SinI used as size standards. The HPV positivity was deter- mined by the presence of 450 base pairs of ampli- fied HPV DNA on visual inspection under ultravi- olet light after staining with ethidium bromide. 11 The types of HPV were determined by hybridiza- tion of amplified DNA with a radioactively labeled type-specific internal oligonucleotide as probe. The sensitivities of detection for HPV by our PCR method were determined by amplifying either a serial dilution of purified cloned HPV DNA of known concentrations or DNA from CaSki and HeLa cells. 236 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY VULVAR HPV INFECTIONS FU ET AL. AtBIC Fig. I. Gross features of nodular papillomatosis lesions in bunched grapes (A), flat warty (B), and polyps (C) appearances. Detection of HPV-Associated Antigen by Avidin Biotin Complex (ABC) Immunochemical Staining Method ABC reagents were purchased from Vector Labora- tories, Inc. (Burlingame, CA) and diluted 1:100 before use. Rabbit antisera directed against L gene product of HPV (DAKO B580) and biotin-labeled sheep anti-rabbit IgG antibodies were both diluted 1:200 before use.

vidin Biotin Complex (ABC) Immunochemical Staining Method ABC reagents were purchased from Vector Labora- tories, Inc. (Burlingame, CA) and diluted 1:100 before use. Rabbit antisera directed against L gene product of HPV (DAKO B580) and biotin-labeled sheep anti-rabbit IgG antibodies were both diluted 1:200 before use. The presence of intranuclear brownish-yellow granules after development with diaminobenzidine (DAB) was considered an indi- cation of HPV-associated antigen.21 Microscopic Investigations Light microscopic examinations of koilocytotic changes of HPV-infected cells were performed af- ter routine hematoxylin-eosin (H&E) staining. 12 Koilocytes are intermediate or mature squamous cells characterized by a large perinuclear cavity. Near the cavity, which has sharply defined bor- ders, the cytoplasm is dense and often amphophilic. Nuclei may be single or multiple. The nuclear membrane is not apparent. In the fully developed koilocyte, the chromatin is often smudged. These cells often contain no nucleoli or inclusion bodies. Electron microscopic studies of HPV virions were carried out with ultrathin sections of tissue slices prepared by standard procedures and examined in a JEM (Tokyo, Japan) 100CXII transmission elec- tron microscope after being negatively stained with phosphotungstic acid. 22 Clinical Management and Follow-Up No treatment was given to those asymptomatic pa- tients with papular papillomatosis. All patients were examined every 2 weeks for at least 3 months. RESULTS The clinical and laboratory features and character- istics of nodular and papular types of lesions of the lower female genital tract were quite different. As shown in Table 1, the differences in HPV preva- lence, presence of HPV-associated antigen, koilo- cytotic changes, and history of multiple sexual part- ners between these two types of papillomatosis were all statistically significant (P < 0.01). Of the 307 women with nodular papillomatosis, 271 (88.3%) had multicentric involvement of the lower genital tract (vagina, cervix, and perineum), 25 (8.1%) had lesions confined to the cervix, and 11 (3.6%) had lesions confined to the vagina. Gross appearances of these nodular papillomatosis cases INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 237 VULVAR HPV INFECTIONS FU ET AL. Fig. 2. Clinical features of papular papillomatosis. A: Teardrop pattern; I: finger-like papillae usually 5-8 mm in. length; and C: patterned vascular architectures under colposcope.

ss appearances of these nodular papillomatosis cases INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 237 VULVAR HPV INFECTIONS FU ET AL. Fig. 2. Clinical features of papular papillomatosis. A: Teardrop pattern; I: finger-like papillae usually 5-8 mm in. length; and C: patterned vascular architectures under colposcope. consisted of those shaped like polyps, chicken- crowns, or bunched grapes found in lower genital tract and perineum areas (272 cases); fuzzy-ball appearances found in the vestibular area (8 cases), and flat warty appearances found in the cervix (20 cases) (Fig. 1). The gross appearances of the 309 cases ofpapular papillomatosis were mostly soft and smooth teardrops or roughened and raised mucosal surfaces (Fig. 2A) or finger-like (Fig. 2B) projec- tions with patterned vascular architectures on col- poscopy (Fig. 2C). Among 309 cases of the papu- lar lesions, 279 (90.3%) were found on the inside mucosal surface of the labia minora and 30 (9.7%) were found in the vagina. HPV DNA sequences of one or more types of HPV could be detected in 140 of" 143 (97.9%) cases of nodular papillomatosis lesions tested by the PCR method (Fig. 3). Repeated testing of the 3 HPV negative cases was not possible because no papillomatosis tissue was left after the first biopsy. On the other hand, only ofthe 91 (1.1%) papular papillomatosis tissue tested positive for HPV DNA with the PCR method, and this single case became negative when tested again a week later. Four ofthe HPV DNA negative cases were tested negative 2 or 3 times upon patients' requests within a 6-month period. Noteworthy were the HPV DNA PCR test results on specimens obtained from 18 patients who were found to have both nodular and papular types of papillomatosis on the mucosal surface of their labia minora or lower vagina. HPV DNA se- quences were found in all 18 nodular portions but in none of the papular portions of the lesions. Two women with sparse and very minute lesions of un- certain classification who tested positive for HPV DNA sequences had developed nodular lesions when examined again 2 weeks later. HPV-associated antigen could be stained by the immunohistochemical method in 98 of 183 cases (53.6%) of nodular lesions tested but in only 2 of 225 cases (0.9%) of papular type of papillomatosis lesion examined with the same method (Fig. 4). No characteristic koilocytotic changes were seen in the papular papillomatosis tissues.

iated antigen could be stained by the immunohistochemical method in 98 of 183 cases (53.6%) of nodular lesions tested but in only 2 of 225 cases (0.9%) of papular type of papillomatosis lesion examined with the same method (Fig. 4). No characteristic koilocytotic changes were seen in the papular papillomatosis tissues. Other microstruc- tural features in the cells of the nodular papilloma- tosis included the disappearance of cellular or- ganelles, enlargement ofmitochondria, and vacuole formation in the nuclei. Electron microscopy re- 238 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY VULVAR HPV INFECTIONS FU ET AL. 1 2 3 4 5 6 7 8 9 Fig. 3. Agarose gel electrophoresis of amplified HPV DNA using consensus primers and under conditions described in "Subjects and Methods." pGEM-3 DNA digested with a mix- ture of restriction endonucleases Hinfl, Rsal, and Sinl was used as size standards in the 2 outside lanes, and the sizes are (from top to bottom) 2,645, 1,605, 1,198, 676, 517, 460, 396; 350, 222, 179, 126, 75, 65, 51, and 36 base pairs. Lanes I,:2= Amplification of DNA from purified cloned HPV types 6 and DNA, respectively. Lanes :3,4: Amplification of nod- ular and papular papillomatosis tissues from 2 patients, re- spectively. Lanes 5,6: Amplification of DNA from nodular and papular portions of papillomatosis tissues of a single patient, respectively. Lane 7: Amplification of 50 ng of hu- man DNA with the HPV consensus primers MY09 and MY I. Lanes 8,9: Amplification of CaSki and HeLa cells, respec- tively, which are known to contain HPV types 16 and 18, respectively. vealed a grid pattern and round HPV virion-like structure of 40-50 Ixm in diameter in 5 of 85 cases (5.9%) of nodular papillomatosis lesions examined (Fig. 5). No such virion-like structure could be found in 23 papular papillomatosis tissues. All 7 cases of papular papillomatosis from pa- tients who were pregnant regressed spontaneously within 1-42 days (mean 14 days) after pregnancies were terminated by abortion or induced or term delivery, but none ofthe 8 cases ofpregnant women with nodular papillomatosis regressed spontaneously during the same period after termination of preg- nancy. None of the 89 patients with asymptomatic and untreated papular papillomatosis developed nodular papillomatosis during follow-up of 3 months to 2 years (mean 4 months). No case of genital warts could be documented among sexual partners of patients with papular papillomatosis.

me period after termination of preg- nancy. None of the 89 patients with asymptomatic and untreated papular papillomatosis developed nodular papillomatosis during follow-up of 3 months to 2 years (mean 4 months). No case of genital warts could be documented among sexual partners of patients with papular papillomatosis. A history of multiple sexual partners was admitted by 248 of 307 (80.8%) patients with nodular papillo- rnatosis, but by only 22 of 309 (7.1%) patients with papular papillomatosis. Furthermore, 23 of 309 patients with papular papillomatosis were young virginal women in their 20s with intact hymen upon physical examinations. DISCUSSION Research in the pathogenesis ofHPV in the past has concentrated on the carcinogenic nature of HPV and on the development of condyloma acumina- tum. Relatively little is known so far on whether HPV infection is really involved in the develop- ment of vestibular papillae or papular papillomato- sis of the lower female genital tract. The term "pseudocondyloma" based on HPV-associated anti- gen studies and the term "condyloma-like vulvar lesions" based on pathophysiological studies on koilocytotic cells have been used synonymously to describe these lesions. 10 Various other terminolo- gies have also been found in the literature to de- scribe these papular papillomatosis lesions which include "subclinical HPV infections," "precondy- loma," and condyloma,v-9 There is no consensus on the possible origin and etiology of these lesions, INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 239 VULVAR HPV INFECTIONS FU ET AL. Fig. 4. Immunohistochemical staining of HPV L gene antigen by ABC method. Presence of HPV L gene antigen is indicated by granules inside nuclei that stained brownish-yellow with DAB. Fig. 5. HPV virions under electron microscope. 330,410. although it has been reported that patients with vulvar micropapillomatosis are not significantly more frequently infected with HPV than controls.23 The PCR method used in this study has a lower limit of detection of approximately 50-500 viral genomes equivalent (based on HPV types 6, 11, 16, and 18 results) depending on the type of I-IPV. Our PCR results indicated that the HPVs present in almost all nodular lesions but were either present in extremely small quantity (estimated to be less than copy per 3,000 cells) or, more likely, not 240 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY present in papular lesions at all.

depending on the type of I-IPV. Our PCR results indicated that the HPVs present in almost all nodular lesions but were either present in extremely small quantity (estimated to be less than copy per 3,000 cells) or, more likely, not 240 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY present in papular lesions at all. Because of its technically demanding and labor-intensive nature, electron microscopy has probably only very limited use in the diagnosis of HPV infections. In summary, HPV DNA sequences, I-IPV-asso- ciated antigen, koilocytotic changes, and virions can be found very frequently in nodular papilloma- tosis lesions ofthe lower female genital tract. These lesions can be transmitted efficiently by sexual con- tact. On the other hand, our results also clearly suggest that papular papillomatosis lesions very rarely contain HPV DNA sequences. We believe that these HPV DNA results, when taken together with other laboratory tests carried out in this study, suggest that prudence should be exercised in inter- preting the nature and origin of papular papilloma- tosis lesions, especially in asymptomatic women without other clinical indications. ACKNOWLEDGMENTS The authors gratefully acknowledge the encourage- ment and support of Dr. MengI-Iao Fan of the Public Health Bureau of Guangzhou. REFERENCES 1. Howley P: Role ofthe human papillomaviruses in human cancer. Cancer Res (Suppl) 51"5019s-5022s, 1991. 2. Friedrich EG: The vulvar vestibule. J Reprod Med 28: 773-777, 1983. 3. Pekham BM, Maki DG, Patterson JJ, Hafez GR: Focal VULVAR HPV INFECTIONS FU ET AL. vulvitis: A characteristic syndrome and cause of dyspare- unia. Am J Obstet Gynecol 154:855-864, 1986. 4. Freidrich EG: Vulvar vestibulitis syndrome. J Reprod Med 82:110-114, 1987. 5. Growdon WA, Fu YS, Lebherz TB, Rapkin A, Mason GD, Parks G: Pruritic vulvar squamous papillomatosis: Evidence for human papillomavirus etiology. Obstet Gy- necol 66:564-568, 1985. 6. Campion MJ, Cuzick J, McCance DJ, Singer A: Pro- gressive potential of mild cervical atypia: Prospective cy- tological, colposcopic and virological study. Lancet 2:237, 1986. 7. Sobel JD: Vulvovaginal infections: Current concepts in diagnosis and therapy. In Reid R (ed): The Biology and Management of Human Papillomavirus Associated Dis- eases. New York: Academy Professional Information Ser- vices, pp 108-148, 1990. 8. Cecchini S, Grazzini G, Iossa A, et al: Subclinical vulvar papillomavirus infection. J Reprod Med 36:143-146, 1991. 9.

concepts in diagnosis and therapy. In Reid R (ed): The Biology and Management of Human Papillomavirus Associated Dis- eases. New York: Academy Professional Information Ser- vices, pp 108-148, 1990. 8. Cecchini S, Grazzini G, Iossa A, et al: Subclinical vulvar papillomavirus infection. J Reprod Med 36:143-146, 1991. 9. Costa S, Rotala A, Terzano P, et al.: Is vestibular papil- lomatosis associated with human papillomavirus? J Med Virol 35:7-13, 1991. 10. Nuovo GJ, O'Connell M, Blanco JS, Levine RU, Silver- stein SJ: Correlation ofhistology and human papillomavi- rus DNA detection in condyloma acuminatum and condy- loma-like vulvar lesions. Am J Surg Pathol 13:700-706, 1989. 11. Cone R, Beckmann A, Aho M, et al.: Subclinical mani- festations ofvulvar human papillomavirus infection. IntJ Gynaecol Pathol 10:26-35, 1991. 12. Sehgal VN, Koranne RV, Srivastava SB: Genital warts. Current status. Int J Dermatol 28:75-85, 1989. 13. Beutner KR, Becker TM, Stone KM: Epidemiology of human papillomavirus infections. Dermatol Clin 9:211- 218, 1991. 14. Howley PM, Schlegel R: The human papillomavirus. An overview. Am J Med 85:155-158, 1988. 15. World Health Organization: Genital human papilloma- virus infection and cancer. Memorandum from a WHO meeting. Bull WHO 65:817-827, 1987. 16. Patsner B, Baker DA, Orr JW Jr: Human papillomavi- rus genital tract infections during pregnancy. Clin Obstet Gynecol 33:258-267, 1990. 17. Hatch KD: Vulvovaginal human papillomavirus infec- tions: Clinical implications and management. Am J Ob- stet Gynecol 165:1183-1188, 1991. 18. Bauer HM, Ting Y, Greer CE, et al.: Genital human papillomavirus infections in female university students as determined by a PCR-based method. JAMA 265:472- 477, 1991. 19. National Sexually Transmitted Disease Surveillance Working Team: Brief report and analysis on the preva- lence of sexually transmitted diseases from national sur- veillance stations between 1987 and 1990. Beijing, China: Ministry ofHealth, China 54:2-5, 1991. 20. Fu YL, ChunJZ, Chiu TM, Sun CK: Clinical analysis of 5,905 cases of sexually transmitted diseases in Guang- zhou. Chin J Obstet Gynecol 25:262-265, 1990. 21. Wilbur DC, Reichman RC, Stoler MH: Detection of infection by human papillomavirus in genital condyloma. A comparison study using immunochemistry and in situ nucleic acid hybridization. Am J Clin Pathol 89:505- 510, 1988. 22. Lee FK, Nahmias AJ, Stagno S: Rapid diagnosis of cy- tomegalovirus infection in infants by electron micros- copy.

n RC, Stoler MH: Detection of infection by human papillomavirus in genital condyloma. A comparison study using immunochemistry and in situ nucleic acid hybridization. Am J Clin Pathol 89:505- 510, 1988. 22. Lee FK, Nahmias AJ, Stagno S: Rapid diagnosis of cy- tomegalovirus infection in infants by electron micros- copy. N Engl J Med 299:1266-1270, 1978. 23. Bergeron C, Ferenczy A, Richart R, Guralinick M: Mi- copapillomatosis labialis appears unrelated to human pap- illomavirus. Obstet Gynecol 76:281-286, 1990. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 241

Infectious Diseases in Obstetrics and Gynecology 1:242-245 (1994) (C) 1994 Wiley-Liss, Inc. Pregnancy Outcome in Swiss-Webster Mice Infected With Chlamydia trachomatis Bryan T. Oshiro, Jack M. Graham, Jorge D. Blanco, Ibrahim M. Seraj, and Karen D. Bishop Department ofObstetrics, Gynecology, and Reproductive Sciences, University ofTexas Health Science Center, Houston, TX (B.T.O., J.M.G., J.D.B., K.D.B.), and Department of Obstetrics and Gynecology, Loma Linda University, Loma Linda, CA (I.M.S.) ABSTRACT Objective: The objective ofthis study was to observe pregnancy outcomes in mice infected transvag- inally with Chlamydia trachomatis. Methods: Pregnant mice were inoculated transvaginally with either C. trachomatis (CT) or sterile calf serum (CON) on pregnancy day 4. Pregnancy outcomes as well as genital tract histology and culture were compared. Statistical analysis was performed using Fisher's exact test and Student's t-test. Results: Twenty-four of 26 CT mice had positive uterine cultures for C. trachomatis. Inflamma- tion occurred in 9 (34.6%) (P 0.002, 95% confidence interval 1.7-3.5) and intrauterine fetal demise occurred in 5 (19.2%) (P 0.05, 95% confidence interval 1.6-2.9) ofCT mice. No mice in the CON group (0/24) had positive uterine cultures, developed inflammation, or experienced intrau- terine fetal demise. Conclusions: Lower genital tract chlamydial infection is associated with intrauterine fetal demise in Swiss-Webster mice. (C) 1994 Wiley-Liss, Inc. KEY WORDS Chlamydia trachomatis, fetal demise, Swiss-Webster mice hlamydia trachomatis is recognized as the most common sexually transmitted organism, with over 4 million cases reported annually. C. tra- chomatis is responsible for causing inclusion con- junctivitis and pneumonia in the neonate and is the leading cause of preventable blindness in the world.2 Although C. trachomatis has clearly been shown to cause mucopurulent cervicitis,3 acute salpingi- tis,4 and neonatal disease, its effect on pregnancy outcome remains controversial. Martin and col- leagues5 reported that the incidence of" prematurity and perinatal death was higher in women with a C. trachomatis infection identified before 20 weeks.

learly been shown to cause mucopurulent cervicitis,3 acute salpingi- tis,4 and neonatal disease, its effect on pregnancy outcome remains controversial. Martin and col- leagues5 reported that the incidence of" prematurity and perinatal death was higher in women with a C. trachomatis infection identified before 20 weeks. In contrast, other authors6'7 have not found an associ- ation between C. trachomatis infection and adverse pregnancy outcome. The Swiss-Webster mouse has been used exten- sively to study the effects of C. trachomatis on the female genital tract. 8-1 Rank et al. 11 have re- ported infecting mice transvaginally with C. tra- chomatis. However, to our knowledge, an animal model has not been developed to study the effects of C. trachomatis lower genital tract infection on preg- nancy outcome. To determine whether adverse pregnancy out- come is associated with C. trachomatis genital tract infection in an animal model, we studied the effect of lower genital tract C. trachomatis infection in pregnant Swiss-Webster mice. Address correspondence/reprint requests to Dr. Bryan T. Oshiro, Department ofPerinatology, McKay-Dee Hospital Center, 3939 Harrison Boulevard, Ogden, UT 84409. Received November 15, 1993 Basic Science Article Accepted March II, 1994 PREGNANCY OUTCOME IN MICE INFECTED WITH CT OSHIRO ET AL. SUBJECTS AND METHODS Mice Seventy-six 6-8-week-old female Swiss-Webster mice (Sasco, Inc., Omaha, NE)with unlimited access to food and water were placed 2 per cage with a male of established fertility over a 24-h period. The mice were then separated into individual cages. Pregnancy was confirmed by the presence of a cop- ulation plug and later by palpation of a pregnant uterus. Twenty-six non-pregnant mice were re- moved from the study. This experiment was ap- proved by the Animal Welfare Committee of the University ofTexas Health Science Center at Hous- ton, and all practices were in accordance with this committee's guidelines. Chlamydia The mouse pneumonitis strain of C. trachomatis ATCC BR-123 was reconstituted to a concentration of approximately 106 inclusion-forming units/ml in sterile calfserum as described by Blanco et al. 10 Experimental Design The female mice were separated into 2 groups: a group for chlamydial (CT) inoculation and a con- trol group (CON). The inoculation technique used in this experiment has been reported by Rank et al. 1 On pregnancy day 4, the CT group was inoc- ulated transvaginally with chlamydia suspended in 50 I1 of sterile calf serum.

he female mice were separated into 2 groups: a group for chlamydial (CT) inoculation and a con- trol group (CON). The inoculation technique used in this experiment has been reported by Rank et al. 1 On pregnancy day 4, the CT group was inoc- ulated transvaginally with chlamydia suspended in 50 I1 of sterile calf serum. The CON group was similarly inoculated with 50 I1 of sterile calf se- rum. On pregnancy day 7, lower genital tract cul- tures were obtained on all mice using a dacron- tipped aluminum swab which was placed in chlamydia transport media and immediately frozen at -70C. The mice were observed daily for signs of clinical infection or preterm delivery. The mice were sacrificed and a laparotomy was performed on the day ofdelivery. Ifdelivery did not occur by the normal date of confinement (day 21), the observa- tion period was extended to pregnancy day 24 at which time all undelivered mice were sacrificed and explored. The abdominal cavity and reproduc- tive tract were grossly examined for evidence of inflammation and the uterus was removed. The distal most portion of the uterus was transected and placed in chlamydia transport media and frozen at -70C. The remainder of the uterus was placed in 10% formalin. The pups were counted and weighed. Culture The lower genital tract culture swab specimens were allowed to thaw at room temperature and vortexed for 15 min. The uterine tissue was also thawed, then morcelated and vortexed. The supernatants were removed and placed on McCoy cells which were previously washed with Mg2+, Ca2+, and phosphate-buffered saline. The cells were then cen- trifuged for h at 3,400g and incubated at 37C for 48 h. A second-pass culture was performed on all samples. The culture technique is standard and has been described elsewhere. 2 The cells were stained with a chlamydia-specific fluorescent anti- body stain (Bartels Diagnostic Division, McGaw Park, IL) and examined by indirect fluorescent microscopy in a blinded fashion. Histology The formalin-fixed tissue was placed in paraffin blocks, sliced, stained with hematoxylin and eosin, and mounted on glass slides. The examiner was blinded as to whether the specimens originated from CT or CON animals. The tissue was classified as inflamed if plasma cells or a polymorphonuclear infiltrate were present on examination ofthe slides. Statistical Analysis Statistical analysis was performed by Fisher's exact test and Student's t-test where appropriate. P < 0.05 was considered statistically significant.

m CT or CON animals. The tissue was classified as inflamed if plasma cells or a polymorphonuclear infiltrate were present on examination ofthe slides. Statistical Analysis Statistical analysis was performed by Fisher's exact test and Student's t-test where appropriate. P < 0.05 was considered statistically significant. RESULTS Twenty-six mice in the CT group and 24 mice in the CON group became pregnant and were in- cluded in the study. All 26 (100%) of the CT mice had positive lower genital tract swab cultures for chlamydia on day 7. Uterine tissue cultures were positive in 24/26 (92.3%) of CT mice. Inflamma- tion of the uterine tissues and intrauterine fetal death were significantly increased in the CT mice (Table 1). In the 5 pregnancies in which fetal death occurred, all of the pups in the litters were dead. Placental histology could not be determined in each pregnancy affected by intrauterine fetal demise be- cause of the various stages of resorption of the pregnancy products, but extensive inflammation INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 243 PREGNANCY OUTCOME IN MICE INFECTED WITH CT OSHIRO ET AL. TABLE I. Adverse pregnancy outcome data in mice infected with C. trachomatis 95% CT CON Relative Confidence (n- 26) (n 24) P risk interval Inflammation 9 (35%) 0 (0%) 0.002 2.4 1.7-3.5 Fetal deaths 5 (19%) 0 (0%) 0.05 2.2 1.6-2.9 TABLE 2. Delivery data in mice with liveborn pupsa CT CON (n 21) (n= 24) Significance No. live pup-mouse 9.0 -+ 1.7 8.9 -+ 2.2 NS Pup weight (g) 1.59 0.24 1.59 -+ 0.15 NS Pregnancy days 21.6 -+ I.I 21.2 -+ 1.6 NS aValues are mean --+ SD. NS not significant. was observed in the placentas that were examined. In 4 of the pregnancies in which fetal death oc- curred, the gestational sacs appeared to be intact. Because of advanced resorption of pregnancy prod- ucts in the 5th pregnancy, the status of the sacs could not be determined. There were no instances of preterm delivery in the CT group, and none of the mice with intrauterine fetal death delivered their pups by the end ofthe normal confinement period. The fetal deaths occurred only in the group ofmice with uterine inflammation. None ofthe mice in the CON group had positive cultures, evidence of an inflammatory reaction in uterine tissues, or intrau- terine fetal deaths. Table 2 shows the pregnancy outcome data in mice with liveborn pups.

e normal confinement period. The fetal deaths occurred only in the group ofmice with uterine inflammation. None ofthe mice in the CON group had positive cultures, evidence of an inflammatory reaction in uterine tissues, or intrau- terine fetal deaths. Table 2 shows the pregnancy outcome data in mice with liveborn pups. No sig- nificant difference was found in the mean duration of pregnancy, the mean number of live pups born per mouse, or the mean weight ofthe liveborn pups between the CT and the CON groups if fetal death did not occur. DISCUSSION The role of C. trachomatis in adverse pregnancy outcomes is controversial. In humans, several stud- ies have not shown an association between C. tra- chomatis infection and adverse pregnancy out- comes. 6'7'13-18 However, others have implicated C. trachomatis in premature rupture of the mem- branes, preterm birth, low birth weight, intraam- niotic infection, and stillbirth, s' 19-24 One of the first studies to suggest that C. tra- chomatis was associated with adverse pregnancy out- comes was performed by Martin and colleagues5 in 1982. In their study, 18 of 268 (6.7%) pregnant women screened before 20 weeks gestation were positive for C. trachomatis. Twenty-eight percent ofthe chlamydia-positive patients delivered prema- turely compared with 6% of the chlamydia-nega- tive patients. Most significantly, the perinatal mor- tality in the infected group was 33% compared with 0.4% in the uninfected group. Gravett et al. 19 found that chlamydial infection of the cervix was associated with low birth weight, premature rup- ture ofthe membranes, and preterm birth. Martius et al.2 and The Johns Hopkins Study Group for Cervicitis and Adverse Pregnancy Outcomes21 also found that cervical infection with C. trachomatis was significantly associated with preterm birth. Chlamydial infections of the cervix may ascend and infect the membranes and fetus. Thorp et al.22 reported a case of fetal death attributed to an in- traamniotic chlamydial infection across intact mem- branes. Although chlamydia was never cultured from the fetus, the formalin-fixed lung tissue was highly positive for fluorescent antibody against chlamydia. Other authors23'24 have also reported in utero chlamydial infection across intact mem- branes. In this study, we found that lower genital tract infection with chlamydia caused infection ofuterine tissues across intact membranes and furthermore was significantly associated with intrauterine de- mise in Swiss-Webster mice.

her authors23'24 have also reported in utero chlamydial infection across intact mem- branes. In this study, we found that lower genital tract infection with chlamydia caused infection ofuterine tissues across intact membranes and furthermore was significantly associated with intrauterine de- mise in Swiss-Webster mice. We also found that, in contradistinction to humans, intrauterine or in- traamniotic chlamydial infection in mice did not result in premature rupture of the membranes or preterm delivery. Rather, the affected pregnancies were retained past their normal term of gestation. With the exception of the study by Martin and colleagues,s human studies have not found an in- crease in stillbirth associated with chlamydial infec- tion in pregnancy. Many other studies looking at the effect of C. trachomati in pregnant women have had small sample sizes and did not specifically look at stillbirths as an outcome variable. We have shown that C. trachomati is capable of causing intrauterine fetal death in Swiss-Webster mice. These results may not correlate to adverse preg- nancy outcomes in humans, but it would be inter- esting to determine if C. trachomatis is a significant 244 INFECTIOU& DISEASES IN OBSTETRICS AND GYNECOLOGY PREGNANCY OUTCOME IN MICE INFECTED WITH CT OSHIRO ET AL. cause of stillbirth in human pregnancy as we have demonstrated in the Swiss-Webster mice. REFERENCES 1. Centers for Disease Control: Chlamydia trachomatis infec- tions: Policy guidelines for prevention and control. MMWR 34(Suppl 3):53s-74s, 1985. 2. Schachter J, Hanna L, Hill EC, Massad S, Sheppard CW, ConteJE, Meyer KF: Are chlamydial infections the most prevalent venereal disease? JAMA 231:1252-1255, 1975. 3. Brunham RC, Paavonen J, Stevens CE, et al.: Mucopu- rulent cervicitis: The ignored counterpart in women of urethritis in men. N Engl J Med 311:1-6, 1984. 4. Sweet RL: Chlamydial salpingitis and infertility. Fertil Steril 38:530-533, 1982. 5. Martin DH, Koutsky K, Eschenbach DA, et al.: Prema- turity and perinatal mortality in pregnancies complicated by maternal Chlamydia trachomat# infections. JAMA 247: 1585-1588, 1982. 6. Harrison HR, Alexander ER, Weinstein L, et al.: Cer- vical Chlamydia trachomatis and mycoplasmal infections in pregnancy. Epidemiology and outcomes. JAMA 250: 1721-1727, 1983. 7. Sweet RL, Landers DV, Walker C, SchachterJ: Chlamy- dia trachomatis infection and pregnancy outcome. Am J Obstet Gynecol 156:824-833, 1987. 8.

arrison HR, Alexander ER, Weinstein L, et al.: Cer- vical Chlamydia trachomatis and mycoplasmal infections in pregnancy. Epidemiology and outcomes. JAMA 250: 1721-1727, 1983. 7. Sweet RL, Landers DV, Walker C, SchachterJ: Chlamy- dia trachomatis infection and pregnancy outcome. Am J Obstet Gynecol 156:824-833, 1987. 8. Swenson CE, Donegan E, Schachter J: Chlamydia tra- chomatis-induced salpingitis in mice. J Infect Dis 148: 1101-1107, 1983. 9. Patton DL, Landers DV, Schachler J: Experimental Chlamydia trachomatis salpingitis in mice: Initial studies on characterization of the leukocyte response to chlamy- dial infection. J Infect Dis 159:1105-1110, 1989. 10. Blanco JD, Patterson RM, Ramzy I, Turner T: Clin- damycin and ibuprofen effects on chlamydial salpingitis in mice. Sex Trans Dis 16:192-194, 1989. 11. Rank RG, Ramsey KH, Hough AJ: Antibody-mediated modulation ofarthritis induced by chlamydia. AmJ Pathol 132:372-381, 1988. 12. Ripa KT, Mardh P-A: Cultivation of Chlamydia trachom- atis in cycloheximide-treated McCoy cells. J Clin Micro- biol 6:328-331, 1977. 13. Heggie A, Lumicao GG, Stuart LA, Gyves MT: Chlamydia trachomatis infection in mothers and infants: A prospective study. Am J Dis Child 135:507-511, 1981. 14. Berman SM, Harrison HR, Boyce WT, Haffner WJ, Lewis M, Arthur JB: Low birth weight, prematurity, and post-partum endometritis. Association with prenatal cervical Mycoplasma hominis and Chlamydia trachomatis infection. JAMA 257:1189-1194, 1987. 15. Ross SM, Windson IM, Robins-Browne RM, Ballard RC, Adhikari M, Fenn DB: Microbiological studies dur- ing the perinatal period: An attempt to correlate selected bacterial and viral infection with intrauterine deaths and preterm labor. S Afr Med J 66:598-603, 1984. 16. FitzSimmons J, Callahan C, Shanahan B, Jungkind D: Chlamydia infection in pregnancy. J Reprod Med 31:19- 22, 1986. 17. Hardy PH, Nell EE, Spence MR, et al.: Prevalence of six. sexually transmitted disease agents among pregnant inner-city adolescents and pregnancy outcome. Lancet 2:333-337, 1984. 18. Thompson S, Lopez B, Wong K-H, et al.: A prospective study of chlamydia and mycoplasma infections during pregnancy: Relation to pregnancy outcome and maternal morbidity. In Mardh P-A, et al. (eds): Chlamydia Infec- tions. Amsterdam: Elsevier Biomedial Press, pp 15-18, 1982. 19.

ancy outcome. Lancet 2:333-337, 1984. 18. Thompson S, Lopez B, Wong K-H, et al.: A prospective study of chlamydia and mycoplasma infections during pregnancy: Relation to pregnancy outcome and maternal morbidity. In Mardh P-A, et al. (eds): Chlamydia Infec- tions. Amsterdam: Elsevier Biomedial Press, pp 15-18, 1982. 19. Gravett, MG, Nelson HP, DeRouen T, Critchlow C, Eschenbach DA, Holmes KK: Independent associations of bacterial vaginosis and Chlamydia trachomatis infection with adverse pregnancy outcome. JAMA256:1899-1903, 1986. 20. Martius J, Krohn M, Hillier S, et al.: Relationships of vaginal Lactobacillus species, cervical Chlamydia trachom- atis, and bacterial vaginosis to preterm birth. Obstet Gy- necol 71:89-95, 1988. 21. The Johns Hopkins Study Group for Cervicitis and Ad- verse Pregnancy Outcomes: Association ofChlamydia tra- chomatis and Mycoplasma hominis with intrauterine growth retardation and preterm delivery. Am J Epidemiol 129: 1247-1251, 1989. 22. ThorpJMJr, Katz VL, Fowler LJ, KurtzmanJT, Bowes WA Jr: Fetal death from chlamydial infection across in- tact amniotic membranes. Am J Obstet Gynecol 161: 1245-1246, 1989. 23. Givner LB, Rennels MB, Woodward CL, Huang S-W: Chlamydia trachomatis infection in infant delivered by cesarean section. Pediatrics 68:420-421, 1981. 24. La Scolea LJ, Paroski JS, Burzynski L, Faden HS: Chlamydia trachomatis infection in infant, delivered by cesarean section. Clin Pediatr 23:118-120, 1984. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 245

Infectious Diseases in Obstetrics and Gynecology 1:246-248 (1994) (C) 1994 Wiley-Liss, Inc. Fatal Herpetic Hepatitis in Pregnancy Stephen Chatelain, D. Edward Neumann, and Samuel M. Alexander Maternal Fetal Medicine Section, Woman's Hospital, Baton Rouge (S.C., D.E."N.), and Department of Obstetrics and Gynecology, Louisiana State University School ofMedicine, New Orleans (S.M.A.), LA ABSTRACT Background: Disseminated herpetic infections during pregnancy have been reported in the literature. Case: This case presentation describes a pregnant patient who presented with fever, elevated liver enzymes, and upper abdominal tenderness and succumbed from fulminant herpetic hepatitis. Conclusion: Early diagnosis and treatment are essential because ofthe high mortality rate. (C) 1994 Wiley-Liss, Inc. KEY WORDS Herpes simplex virus, thrombocytopenia, pregnancy CASE REPORT he patient was a 20-year-old white female, G P0, with an intrauterine pregnancy at 32 weeks gestation by ultrasound examination performed in the referring hospital on October 14, 1991. The patient was seen by her primary physician in the emergency room at the referring hospital week prior to transfer. She was diagnosed with a urinary tract infection and was treated with Septra. The patient continued to experience a febrile course and was admitted to the referring hospital where her temperature was found to be 39.6C. Her blood pressure was 90/60 and her pulse was 98. The patient's medical history was negative for diabetes mellitus, hypertension, asthma, hepatitis, and thyroid and kidney disease. She denied the use of tobacco, alcohol, and illicit drugs. The initial physical examination revealed a pa- tient who was not icteric and not dehydrated. Right upper quadrant tenderness was found. Mild uter- ine tenderness was also noted. Occasional uterine contractions were noted. Her cervix was cm di- lated and 25% effaced with intact membranes. Initial laboratory values showed a white blood cell (WBC) count of 4,000 with 64% polyps, 18% bands, and 12% lymphocytes. The platelet count was 124,000, and hematocrit 36.8%.

r- ine tenderness was also noted. Occasional uterine contractions were noted. Her cervix was cm di- lated and 25% effaced with intact membranes. Initial laboratory values showed a white blood cell (WBC) count of 4,000 with 64% polyps, 18% bands, and 12% lymphocytes. The platelet count was 124,000, and hematocrit 36.8%. Liver en- zymes showed an ALT of 1,062, ALP of 204, AST of 2,031, and lactate dehydrogenase (LDH) of 1,918. A liver ultrasound was obtained and did not show any gallstones. Hepatitis B surface anti- gen was negative. The patient's prenatal laboratory results showed her to be human immunodeficiency virus (HIV) negative. Differential diagnosis in- cluded hemolytic uremic syndrome, acute fatty liver, and hepatitis. The patient was treated with magnesium sulfate tocolysis for the uterine contractions and was trans- ferred to the tertiary care hospital on December 22, 1991. On her arrival at the tertiary care hospital, her temperature was 38.3C, her pulse was 108, and her blood pressure was 110/60. A repeat phys- ical examination showed the lungs to be clear. Ab- dominal palpation showed definite right upper quadrant tenderness. The lower abdomen and uterus were non-tender to palpation. The patient Address correspondence/reprint requests to Dr. Samuel M. Alexander, Mercy-Baptist Hospital, 4429 Clara Street, Suite 540, New Orleans, LA 70115. Received November 23, 1993 Obstetrics Case Report Accepted March 4, 1994 FATAL HERPETIC HEPATITIS IN PREGNANCY CHATELAIN ET AL. was alert and oriented. Her cervix was cm di- lated, long, and posterior. Laboratory examination showed the WBC count to be 2,800, platelets 86,000, PT and PTT within normal limits, fibrinogen 386, and bleeding time 4.5 min. A urinalysis showed 2 + ketones but no evidence of bile, blood, nitrites, or leukocyte es- terases. Two to 4 WBCs were noted with a rare red blood cell (RBC). Repeat chemistry panel showed an AST of 4,053 and an LDH of 12,255. Biliru- bin was 1.0. Bacterial cultures were obtained ofthe urine, blood, cervix, and amniotic fluid. An internal medicine consultation was requested soon after the patient's arrival at the tertiary care hospital. The assessment of the patient was 1) fever of unknown origin; 2) epigastric pain, rule out gastri- tis, gastric reflux, and hepatic enlargement; 3) thrombocytopenia; and 4) elevated liver enzymes. The patient was started on ceftriaxone and tobramy- cin. Magnesium sulfate was continued at 2 g/h. TORCH titers were ordered.

e assessment of the patient was 1) fever of unknown origin; 2) epigastric pain, rule out gastri- tis, gastric reflux, and hepatic enlargement; 3) thrombocytopenia; and 4) elevated liver enzymes. The patient was started on ceftriaxone and tobramy- cin. Magnesium sulfate was continued at 2 g/h. TORCH titers were ordered. Twelve hours after her admission, the patient's physical examination had not changed. The patient complained of hun- ger and thirst. She was no longer contracting. The cervix was unchanged. The uterus was non-tender. Ultrasound examination showed the fetus to be consistent with 32 weeks gestation. A liver and spleen ultrasound did not show enlargement of ei- ther organ. The chest X-ray was also normal. Re- peat laboratory data showed the WBC count to be 3,200, platelets 90,000, uric acid 5.9, total biliru- bin 1.2, amylase <30, LDH 18,574, AST 5,498, and ALT 2,254. Gram stain of the amniotic fluid was returned as negative for organisms and WBCs. The differential diagnosis was expanded to include possible cholangitis. The patient was started on in- travenous metronidazole for coverage of anaerobic bacteria. A gastroenterological consultation was ob- tained. The impression was that the patient had common bile duct stones with cholangitis, rule out pancreatitis, rule out hepatitis. IgG TORCH titers were returned with a positive titer for cytomegalo- virus. IgM titers were not returned initially. All bacterial cultures were negative. The patient remained on continuous fetal moni- tor. Approximately 30 h after admission, the pa- tient developed persistent late decelerations. There were no uterine contractions noted by the patient; however, contractions were picked up by the fetal monitor. The patient was treated with 3 doses of subcutaneous terbutaline but persisted in having contractions. A cesarean section was planned due to the persistent late decelerations. A coagulation sur- vey and CBC were obtained prior to surgery. Plate- lets were 27,000 and PT and PTT were both mark- edly elevated. Bleeding time was now 10 min. The patient was transfused with 2 units of fresh frozen plasma and 10 units of platelets. An emergency cesarean section was performed under general anes- thesia with delivery ofa viable female infant weigh- ing approximately 1,800 g, Apgar 6/8. The pla- centa showed evidence of retroplacental hematoma consistent with placental abruption. Postpartally, the PT and PTT remained ele- vated. Fibrinogen was 177 and platelets 53,000.

n section was performed under general anes- thesia with delivery ofa viable female infant weigh- ing approximately 1,800 g, Apgar 6/8. The pla- centa showed evidence of retroplacental hematoma consistent with placental abruption. Postpartally, the PT and PTT remained ele- vated. Fibrinogen was 177 and platelets 53,000. Four hours postoperatively, the patient began to have a large amount ofvaginal bleeding. The uter- ine fundus was firm on examination. Repeat coagu- lation studies showed prolonged PT and PTT, fi- brinogen of90, and platelets of40,000. The patient was transfused with 10 units of platelets and unit offresh frozen plasma. She became disoriented and developed labored respirations. Arterial blood gases showed a pH of 7.27, PCO2 of 18, po2 of 89, bicarbonate of 8, oxygen saturation of 97% with base excess of 16.3. The impression at this time was metabolic acido- sis secondary to sepsis vs. hepatitis. Antibiotics were continued. The patient was transfused with 8 units of packed RBCs, 9 units of fresh frozen plasma and 10 units of platelets, but continued to bleed profusely. Twenty-four hours postoperatively, the patient became unresponsive to pain, and her urine output dropped to 0. She experienced a grand mal seizure and her pupils were noted to be fixed and dilated. Two successive electroencephalograms were read as flat line. The patient's ventilator support was removed on December 27, 1991. TORCH IgM titers were returned only after the patient had become unresponsive and were found to be positive for herpes simplex virus (HSV) type I and type II. A repeat I--IIV was negative. Epstein-Barr virus IgM was also negative. The patient was negative for leptospirosis. An autopsy was performed and was limited to the thorax and abdomen at the family's request. Autopsy findings were consistent with herpetic hep- atitis with fulminant hemorrhagic hepatitis necro- sis. Figure demonstrates hepatic necrosis with INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 247 FATAL HERPETIC HEPATITIS IN PREGNANCY CHATELAIN ET AL. Fig. I. Hepatic necrosis with herpetic inclusions. herpetic inclusions. Herpetic inclusions were also identified in the endocervical glandular epithelium. DISCUSSION Fourteen cases of herpetic hepatitis have been re- ported in the literature.

ICS AND GYNECOLOGY 247 FATAL HERPETIC HEPATITIS IN PREGNANCY CHATELAIN ET AL. Fig. I. Hepatic necrosis with herpetic inclusions. herpetic inclusions. Herpetic inclusions were also identified in the endocervical glandular epithelium. DISCUSSION Fourteen cases of herpetic hepatitis have been re- ported in the literature. Herpetic hepatitis is a difficult diagnosis to make because of confusion with pregnancy-specific hepatic disorders, such as HELLP syndrome (hemolysis, elevated liver func- tion tests, low platelets) and acute fatty liver of pregnancy. Herpetic hepatitis is extremely rare, with a maternal mortality rate of 43%. The high mortality rate may be due to a defect in the cell- mediated response in the pregnant patient. There are several distinguishing characteristics ofherpetic hepatitis as stated in the literature. These include onset of disease in the 3rd trimester, a prodromal illness, vulvar or oropharyngeal vesicu- lar lesions, primary HSV infection, and anicteric presentation. The patient in this case had the above presentations; however, she had no obvious vesicu- lar lesions. The postmortem examination did show evidence of an endocervical herpetic infection. Ofthe 14 patients reported in the literature, 6 o them died. Four patients treated with parenteral acyclovir survived and 2 ofthe patients treated with the antiviral agent vidarabine also survived; how- ever, 5 of 7 patients who did not receive antiviral therapy died. Parenteral acyclovir has been shown to be efficacious in the treatment of disseminated HSV. Not only has acyclovir improved maternal morbidity rates, but it also has been shown to im- prove neonatal HSV infection. 1,z Herpetic hepatitis is a difficult diagnosis to make. Disseminated herpes has a high mortality rate in pregnant patients. 1,2 It is essential to make a diagnosis in order to institute therapy. HSV hepa- titis should be included in the differential diagnosis of hepatic dysfunction in the 3rd trimester. The pregnant patient with a primary HSV lesion should be closely watched for any evidence ofdisseminated disease. Diagnosis can be made by liver biopsy to confirm the presence ofherpetic intranuclear inclu- sion bodies. Once the diagnosis is made, acyclovir can be initiated to significantly reduce maternal mortality. REFERENCES 1. Klein NA, Mabie WC, Shaver DC, Latham PS, Adamec TA, Pinstein ML, Reilly C: Herpes simplex virus hepati- tis. Gastroenterology 100:239-244, 1991. 2.

m the presence ofherpetic intranuclear inclu- sion bodies. Once the diagnosis is made, acyclovir can be initiated to significantly reduce maternal mortality. REFERENCES 1. Klein NA, Mabie WC, Shaver DC, Latham PS, Adamec TA, Pinstein ML, Reilly C: Herpes simplex virus hepati- tis. Gastroenterology 100:239-244, 1991. 2. Wertheim RA, Brooks BJ, Rodriquez FH, Lesesne HR, Jennette JC: Fatal herpetic hepatitis in pregnancy. Obstet Gynecol 62:38--42, 1983. 248 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:249-251 (1994) (C) 1994 Wiley-Liss, Inc. Ovarian Abscess Following Therapeutic Insemination Bradford A. Kolb, Lane Mercer, Albert J. Peters, and Ralph Kazer Department ofObstetrics and Gynecology, Northwestern University Medical School, Chicago, IL ABSTRACT Background: Artificial insemination is a commonly performed procedure for the treatment ofvarious forms of infertility. Infectious complications have only rarely been noted as a complication of intrauterine insemination (IUI). Case: In this presentation, we report the first case of an ovarian abscess following IUI with the husband's semen. Despite treatment with triple antibiotics, an oophorectomy was required. Surgical as well as pathological evaluation confirmed the diagnosis ofan ovarian abscess. Following surgery, the patient responded well to antibiotic therapy. Conclusion: Since pelvic infections are ascending processes, the violation of the natural cervical barrier with IUI can theoretically place the patient at increased risk for this complication. While few advocate routine cultures ofsemen samples, the clinician must be acutely alert to potential infectious morbidity following this procedure. Early diagnosis and intervention are necessary to minimize morbidity and optimize treatment. (C) 1994 Wiley-Liss, Inc. KEY WORDS Infertility, infection, ovary, intrauterine insemination rtificial insemination is a commonly performed procedure for the treatment of" various forms of infertility. It can be defined as the mechanical deposition of semen in the vagina, cervical canal, or uterine cavity. Although rare, infectious mor- bidity can be associated with this procedure. The transmission ofthe human immunodeficiency virus (HIV) by artificial insemination with donor semen has been reported. ,2 Other studies have indicated donor semen samples can contain ureaplasm, Chlamydia trachomatis, and hepatitis B.2 In spite of these reports, pelvic inflammatory disease has only rarely been noted as a complication of artificial insemination.3 Recently, Sable et al.4 reported the first case ofa ruptured tubo-ovarian abscess (TOA) following intrauterine insemination (IUI).

contain ureaplasm, Chlamydia trachomatis, and hepatitis B.2 In spite of these reports, pelvic inflammatory disease has only rarely been noted as a complication of artificial insemination.3 Recently, Sable et al.4 reported the first case ofa ruptured tubo-ovarian abscess (TOA) following intrauterine insemination (IUI). Since pelvic infections are ascending infections, the viola- tion of the natural cervical barrier with IUI can theoretically place the patient at increased risk for infectious morbidity. A case of an ovarian abscess following IUI with the husband's sperm is reported and the possible pathophysiological mechanism of this is discussed. CASE REPORT A 3 5-year-old female, GP0010 with the diagnosis ofstage-1 endometriosis presented for reproductive assistance due to infertility. After a 2-year attempt to conceive with timed intercourse, the patient un- derwent IUI using her husband's sperm following empiric controlled ovulation hyperstimulation with clomiphene citrate. The patient's gynecologic his- tory was not remarkable except for stage-1 endo- metriosis diagnosed by laparoscopy 2 years prior to seeking reproductive assistance. The fresh semen sample was washed in a Tyrode culture medium containing 0.1% bovine serum albumin, centri- fuged, resuspended, and filtered over wet glass wool. No preinsemination cultures were performed. The freshly prepared sample was deposited intra- Address correspondence/reprint requests to Dr. Lane Mercer, Department of Obstetrics and Gynecology, Northwestern University Medical School, 333 East Superior Street, #420, Chicago, IL 60611-3095. Gynecological Case Report Received July 27, 1993 Accepted January 8, 1994 OVARIAN ABSCESS FOLLOWING IUI KOLB ET AL. uterinely via a small catheter after washing the cervix with sterile Tyrode's solution. Five days later, the patient presented with complaints of a mild vaginal discharge and lower abdominal dis- comfort, greater on the left side. Physical examina- tion revealed uterine and left adnexal tenderness; however, her temperature was not elevated. Fur- thermore, pelvic sonography failed to reveal any significant findings. She was subsequently given doxycycline, 100 mg twice a day, for a possible pelvic infection, although the source of her symp- toms was believed to be mostly likely due to a ruptured ovarian cyst. The patient reported having some initial relief with antibiotic therapy. Six days later, the patient complained of increased abdomi- nal pain and fever of 39.6C.

e, 100 mg twice a day, for a possible pelvic infection, although the source of her symp- toms was believed to be mostly likely due to a ruptured ovarian cyst. The patient reported having some initial relief with antibiotic therapy. Six days later, the patient complained of increased abdomi- nal pain and fever of 39.6C. Physical examination revealed a tender 4 4-cm left adnexal mass and a white blood cell (WBC) count of 11.0 103/Ixl. Transvaginal sonography demonstrated a 4.5 4.4-cm left adnexal mass with uniform ho- mogeneous low-grade internal echoes within the ovary. A urine pregnancy test was negative. The patient was hospitalized with the diagnosis of pelvic inflammatory disease and treated with cefotetan, 2 g IV q 12 h. Due to the patient's persistent fever, antibiotic therapy was changed on hospital day 3 to ampicillin (2 g IV q 6 h), gentam- icin (100 mg IV q 8 h), and clindamycin (900 mg IV q 8 h). Blood and cervical cultures were without growth at 72 h. A repeat ultrasound on hospital day 7 revealed the cystic left adnexal mass to have increased to 4.5 6.0 cm, and to be bilobed, hyperechoeic, and homogeneous. A presumptive diagnosis of a pelvic abscess was made, and laparotomy was per- formed on the following day. Percutaneous catheter drainage was considered but due to its experimental nature and high rate of complications as well as patient desire for definative treatment, surgical drainage was performed. Dense pelvic adhesions encasing the left adnexa and obliterating the cul-de- sac were noted. The ovary and distal portion of the fallopian tube were friable with a brownish-green appearance, although the endosalpinx was only min- imally involved. Upon manipulation ofthe ovary a thick yellow-white exudate was expressed. Since the overlying tube appeared necrotic and friable, a sal- pingo-oophorectomy was performed. Gross and mi- croscopic evaluation of the ovarian tissues revealed marked edema and hemorrhage, while the distal portion of the fallopian tube which was adherent to the ovary displayed extensive perisalpingitis. Fluid from the abscess was collected by aspiration into a syringe and transported to the laboratory. Gram stain from the abscess cavity revealed the presence of neutrophils; however, no bacteria were present. All cultures were plated within 2 h of sampling.

was adherent to the ovary displayed extensive perisalpingitis. Fluid from the abscess was collected by aspiration into a syringe and transported to the laboratory. Gram stain from the abscess cavity revealed the presence of neutrophils; however, no bacteria were present. All cultures were plated within 2 h of sampling. The specimen was plated onto 5% sheep blood agar, Columbia CNA agar, Mac Conkey agar, chocolate agar, and modified Thayer-Martin agar and into a thioglycollate broth for the recovery of aerobic or- ganisms. For anaerobic isolates, the specimen was plated onto laked horse blood agar and 5% laked sheep blood with 50 Ig/ml gentamicin and into enriched thioglycollate broth. All intraoperative cultures of the peritoneum and abscess cavity, both aerobic and anaerobic, were negative for growth. Postoperatively, the patient's condition rapidly im- proved while on antibiotic therapy. She was dis- charged home on postoperative day 4 with no fur- ther antibiotics. Urethral cultures of the patient's husband were negative for Neisseria gonorrhoeae or C. tracho- matis. DISCUSSION Pelvic inflammatory disease is an ascending infec- tio from the cervix, subsequently affecting the fallopian tubes and, if untreated, the ovaries. Al- though inciting organisms have been well identi- fied, the bacteria most commonly isolated from pelvic infections are mixed aerobic and anaerobic species consistent with normal vaginal flora. It is believed that the cervix with its mucous barrier may protect against the ascent of potentially patho- genic bacteria, s The patient reported herein underwent thera- peutic IUI with her husband's sperm, thereby by- passing the normal physiologic barriers to infection of the upper genital tract. Based on the failure to find pathogenic organisms in the urethra of her partner, it is more likely that contamination with vaginal flora rather than direct infection by the husband's semen resulted in this episode of pelvic infection. While the patient was empirically started on doxycycline when she first presented with ab- dominal tenderness, a broader spectrum of cover- 2S0 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY OVARIAN ABSCESS FOLLOWING IUI KOLB ET AL. age reflecting the natural vaginal flora may have helped to minimize infectious morbidity. Ovarian abscess is a rare complication of pelvic infection and differs from the much more common TOA in that the ovarian stroma is the primary site of infection.

OBSTETRICS AND GYNECOLOGY OVARIAN ABSCESS FOLLOWING IUI KOLB ET AL. age reflecting the natural vaginal flora may have helped to minimize infectious morbidity. Ovarian abscess is a rare complication of pelvic infection and differs from the much more common TOA in that the ovarian stroma is the primary site of infection. While peritubal inflammation may be present, the endosalpinx is only minimally in- volved. A TOA, by contrast, involves the ovary by secondary spread from the infected tube. Most com- monly, an ovarian abscess occurs after rupture of a follicular ovarian cyst with subsequent seeding of bacteria into the stigmata or colonization of the ovarian stroma. 6 The onset of an ovarian abscess is insidious and usually presents as pain and low- grade fever with acute peritonitis occurring from week to 6 months following initial contamination. Unfortunately, there are few findings other than operative diagnosis that help in the differentiation between an ovarian abscess and a TOA. This pa- tient, having undergone controlled ovarian hyper- stimulation with clomiphene citrate in conjunction with timed insemination, may have been at in- creased risk for an ovarian infection due to the more extensive serosal disruption associated with this approach. In any patient undergoing a transcervical proce- dure at the time of" ovulation in whom nondescript symptoms of pelvic infection occur, an ovarian ab- scess should be considered. The treatment of an ovarian abscess, once suspected, is surgical inter- vention. In any patient undergoing IUI who has risk factors for sexually transmitted disease or a history ofthe same, antibiotic prophylaxis might be instituted. However, the side effects must be con- sidered and antibiotics are not universally recom- mended. Generally, a history of salpingitis should be viewed as a contraindication to IUI. Appropriate diagnostic modalities include evi- dence of systemic infection such as the sedimenta- tion rate of C-reactive protein and ultrasound or computerized tomography for the presence of a fluid-filled cavity within the ovary. As with many abscesses, there may be little evidence of acute ele- vation of WBC count until rupture or overwhelm- ing sepsis occurs. As antibiotic therapy is almost always unsuccessful, surgical intervention is the hallmark of therapy.30ophorectomy with preser- vation of other reproductive structures can be ac- complished if the diagnosis is made early and accu- rately.

of acute ele- vation of WBC count until rupture or overwhelm- ing sepsis occurs. As antibiotic therapy is almost always unsuccessful, surgical intervention is the hallmark of therapy.30ophorectomy with preser- vation of other reproductive structures can be ac- complished if the diagnosis is made early and accu- rately. Ovarian abscess following IUI is a rare compli- cation. The combination of a transcervical proce- dure and controlled ovarian hyperstimulation may render this complication more likely. The clinician must be alert to the potential infectious morbidity following this procedure and the importance of early diagnosis and intervention in minimizing morbidity. REFERENCES 1. Hummel WP, Talbert LM: Current management of a donor insemination program. Fertil Steril 51:919-930, 1989. 2. Leiva JL, Peterson EM, Wetkowski M, de la Maza LM, Stone SC: Microorganisms in semen used for artificial insemination. Obstet Gynecol 65:669-672, 1985. 3. Chong AP, Taymor ML: Sixteen year's experience with ther- apeutic donor insemination. Fertil Steri126:791-795, 1975. 4. Sable DB, Yanushpolsky EH, Fox JH: Ruptured pelvic abscess after intrauterine insemination: A case report. Fer- til Steril 59:679-680, 1993. 5. Sparks RA, Purrier BG, Watt PJ, Elstein M: Bacteriolog- ical colonization of uterine cavity: Role of tailed intrauter- ine contraceptive device. Br Med J 282:1189, 1981. 6. Wetchler SJ, Dunn LJ: Ovarian abscess. Report of a case and review ofthe literature. Obstet Gynecol Surv 40:476- 485, 1985. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:252-255 (1994) (C) 1994 Wiley-Liss, Inc. Group A Streptococcal Puerperal Sepsis: Historical Review and 1990s Resurgence Lawrence Nathan and Kenneth J. Leveno Department ofObstetrics and Gynecology, University of Texas Southwestern Medical School, Dallas, TX ABSTRACT There appears to be a resurgence ofpuerperal sepsis due to a historically important pathogen, group A I-hemolytic streptococcus. (C) 1994 Wiley-Liss, Inc. Kv WORDS Group A streptococcus, puerperal fever, sepsis dramatic decline in the prevalence of serious infection caused by group A streptococci has been observed throughout most of the 20th cen- tury, and Streptococcus pyogenes is currently an un- common cause of maternal morbidity and mortal- ity. However, recent reports1- have suggested a resurgence ofvirulent group A streptococci causing sepsis, severe soft-tissue invasion, toxic shock-like syndrome, disseminated intravascular coagulation, and death. In view of the apparent reemergence of classic childbed sepsis due to this organism, we have written this review to emphasize the return of a historically important pathogen in the annals of puerperal infection. In 1772, John Leake4 first recognized that puerperal fever was contagious. Later that century, Alexander Gordon5 of Aberdeen suggested that puerperal fever was a communicable disease. In 1843, Oliver Wendell Holmes6 described the con- tagiousness ofpuerperal fever as "a momentous fact which is no longer to be considered as a subject for trivial discussion He became convinced that childbed fever was contagious when he witnessed the death of a physician who, prior to his death, performed an autopsy on a woman with puerperal fever and attended several other parturients with similar infection. A controversy ensued and contin- ued for many years between Holmes and American obstetricians Hugh Lennox Hodge and Charles Meigs, who vehemently opposed the theory that doctors could spread this deadly disease to their patients.

woman with puerperal fever and attended several other parturients with similar infection. A controversy ensued and contin- ued for many years between Holmes and American obstetricians Hugh Lennox Hodge and Charles Meigs, who vehemently opposed the theory that doctors could spread this deadly disease to their patients. In the mid-19th century, Ignaz Semmelweis7 noticed that puerperal fever was significantly more common in of the 2 maternity divisions of the Vienna Lying-In Hospital; patients attended by medical students were more frequently afflicted with childbed fever than were patients cared for by midwives. Semmelweis7 was therefore convinced that childbed fever could be spread from person to person and insisted that students wash their hands in chlorine solution before examining women in labor. Remarkably, maternal mortality decreased from 5% to 1.3% after implementation ofthis mea- sure. Eighteen years later, Pasteur demonstrated that the disease described by Holmes6 and Semmel- weis7 was caused by the same hemolytic streptococ- cus that was responsible for erysipelas, scarlet fe- ver, and surgical wound infections. 8 Although obstetrical texts from the beginning of the 20th century contain descriptions of what is now considered puerperal sepsis due to group A Address correspondence/reprint requests to Dr. Lawrence Nathan, Department of Gynecology and Obstetrics, Emory University School ofMedicine, 69 Butler Street, S.E., Atlanta, GA 30303. Review Aicle Received February 8, 1994 Accepted April 13, 1994 GROUP A STREPTOCOCCUS NATHAN AND LEVENO streptococci, similar descriptions are not available in modern texts. 9-11 For example, J. Whitridge Williams, 12 in the 1st edition of Williams Obstetrics in 1903, described S. pyogenes as the most frequent cause ofepidemic and fatal puerperal infection. He distinguished these infections from those caused by anaerobic bacteria by emphasizing that the latter were characterized by "putrefaction" (suppuration).

hitridge Williams, 12 in the 1st edition of Williams Obstetrics in 1903, described S. pyogenes as the most frequent cause ofepidemic and fatal puerperal infection. He distinguished these infections from those caused by anaerobic bacteria by emphasizing that the latter were characterized by "putrefaction" (suppuration). Williams12 observed that "the local changes of vir- ulent (aerobic) streptococcal infections are compar- atively slight, the process rapidly spreading through the lymphatics or veins past the uterus and giving rise to a peritonitis or a general systemic infection." He13 continued, "In a certain number of cases the infection is so virulent that the organisms do not have a chance to become localized to any one organ, and both they and their toxins are found in abun- dance in the circulating blood, with very slight implication (involvement) of the uterus." In the 1930s, Fry advanced our understanding ofpuerperal infection with his careful studies ofthe morbid anatomy in fatal infections. 14 He showed that each of the more common organisms tended to produce its own pathologic lesions. Fry's work gave rise to a better clinical understanding of these com- mon infections, particularly of aerobic and anaero- bic puerperal infections. The characteristic find- ings in aerobic streptococcal infections included extreme invasiveness such that the organisms rap- idly spread through the uterine wall with hardly any inflammatory reaction to halt them, quickly reaching the peritoneal cavity and pelvic tissues. Such aerobic streptococcal infections were also char- acterized by early onset and relatively severe sys- temic illness, yet unobtrusive localized clinical find- ings. Specifically, high pyrexia and rapid pulse rate were typically most prominent, while other clinical findings, with the exception of paralytic ileus, were lacking. Fry also observed innumerable loci ofbac- teria within the uterine wall that he concluded es- caped into the bloodstream so that a sustained septi- cemia resulted. In contrast, anaerobic streptococcal puerperal infections were characterized by promi- nent clinical findings that included putrid lochia, suppurative wounds, and pelvic abscesses. Puer- peral women with anaerobic infections were typi- cally not overwhelmed at the outset, as is seen with aerobic infections. Anaerobic infections typically progressed to abscess formation, which often in- cluded sites as distant as the lung.

ings that included putrid lochia, suppurative wounds, and pelvic abscesses. Puer- peral women with anaerobic infections were typi- cally not overwhelmed at the outset, as is seen with aerobic infections. Anaerobic infections typically progressed to abscess formation, which often in- cluded sites as distant as the lung. Pelvic throm- bophlebitis along with thromboembolism was the proposed mechanism for lung involvement. Also in the 193 0s, Rebecca C. Lancefieldl5 re- ported that streptococci could be differentiated into several groups. Lancefield'sis group A [3-hemolytic streptococcus is now recognized as the organism responsible for a variety of human diseases includ- ing puerperal sepsis and thought to be the organism responsible for the epidemics of puerperal infec- tions in the past. In the early 20th century, epidemics of group A streptococcal puerperal infection occurred with variable frequency and intensity. The death rate in Britain from puerperal infection was 1-2/1,000 between 1925 and 1935,16 while an epidemic in New York's Sloan Hospital described by Watson17 had a fatality rate of 36%. Over the subsequent 2-3 decades, aerobic streptococcal puerperal infections became a rarity that even antedated the availability of sulfa and penicillin. This decline was presumed to be related to development of knowledge on the contagiousness of aerobic streptococci resulting in the use of aseptic techniques during parturition. Moreover, part of the improvement in maternal infections was attributed to the better management of labor and elimination of those procedures that contributed to prolonged labor. 18 In the preantibiotic era, obstetricians were con- cerned about gram-positive aerobes such as group A [-hemolytic streptococci (S. pyogenes) and anaer- obes (Peptostreptococcus) such as S. putridus. 19 All these organisms shared a remarkable susceptibility to penicillin, and the introduction of penicillin into clinical practice in the mid-1940s greatly reduced clinical concerns about infections due to these patho- gens.2 Indeed, contemporary views on the patho- genesis ofpuerperal infections changed significantly in the postantibiotic period with group A [3-he- molytic streptococci being infrequently recovered from women with postpartum metritis, and most such infections occurred only after cesarean sec- tion.21 At our institution, most puerperal infections follow cesarean section, are typically polymicrobial (2.5 bacterial species/infection), and are associated with suppuration.

tic streptococci being infrequently recovered from women with postpartum metritis, and most such infections occurred only after cesarean sec- tion.21 At our institution, most puerperal infections follow cesarean section, are typically polymicrobial (2.5 bacterial species/infection), and are associated with suppuration. 22 The most frequent bacteria iso- lated in these infections are Peptostreptococcus, Bac- teroides, and Enterobacteriaceae. Indeed, group A [-hemolytic streptococci were isolated from only 2% of women developing puerperal infection fol- lowing cesarean section, and heretofore we have not INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY GROUP A STREPTOCOCCUS NATHAN AND LEVENO witnessed septic shock due to this organism. Dur- ing the 1960s, '70s, and early '80s, puerperal in- fections due to group A streptococci became spo- radic with only minor geographic epidemics that were limited and controlled23-26 such that group A streptococcus was considered an infrequent cause of serious puerperal infections. For example, in 1981, Blanco and colleagues27 reported that group A streptococcus was isolated from 3.3% of patients with puerperal endometritis. Beginning in the mid-1980s, some investigators suggested that aerobic streptococci were reemerg- ing as a cause of life-threatening soft-tissue infec- tions. Stevens and co-workers reported an out- break of 20 group A [3-hemolytic streptococcal infections in the Rocky Mountain region. These infections were remarkable because of the severity of the soft-tissue destruction and associated life- threatening systemic toxicity. The mortality rate was 30% despite the median age of the patients being 36 years with no underlying evidence of immune incompetence. They postulated that the historical disappearance of serious streptococcal in- fections was partially correlated with the disappear- ance of type A exotoxin produced by S. pyogenes. The exotoxin, also called scarlet fever toxin, is be- lieved to cause life-threatening systemic effects due to group A [3-hemolytic streptococci. Other inves- tigators2'28-31 have also observed a resurgence of S. pyogenes infections with complications, including rheumatic fever, sepsis, and toxic shock-like syn- drome. Cleary and co-workers2 investigated the association of the return of scarlet fever toxin, sys- temic toxicity, and the possibility that a new highly virulent clone of S. pyogenes had emerged.

d a resurgence of S. pyogenes infections with complications, including rheumatic fever, sepsis, and toxic shock-like syn- drome. Cleary and co-workers2 investigated the association of the return of scarlet fever toxin, sys- temic toxicity, and the possibility that a new highly virulent clone of S. pyogenes had emerged. Using restriction enzyme methodology and gene probes, they studied isolates from patients with sepsis and compared these with S. pyogenes isolates not associ- ated with sepsis. They observed that streptococcal strains from patients with sepsis are a unique clone capable of producing exotoxin A, which is pro- posed to explain the apparent return of life-threat- ening group A [3-hemolytic streptococci. Importantly, group A streptococci have increas- ingly been associated with life-threatening infec- tions on obstetric services on both sides of the At- lantic. In Europe, virulent group A [3-hemolytic streptococci causing sepsis, soft-tissue infection, toxic shock syndrome, disseminated intravascular coagulation, and maternal death have been re- ported,-6 while in the United States, toxic shock due to this organism has been described in San Antonio, TX,37 and Chapel Hill, NC.8 Recently, Silver and associates described 2 patients with life- threatening puerperal infection due to group A [3-hemolytic streptococcus. Both women presented with bacteremia and shock, failed aggressive medi- cal intervention, and required hysterectomy. Fi- nally, we9 reported 2 pregnancies complicated by group A [3-hemolytic streptococcal sepsis after al- most 2 decades without such infections at our hospi- tal, during which time almost 200,000 women have been delivered. It therefore appears that puerperal sepsis caused by group A [3-hemolytic streptococcus, recently considered to be of historic interest only, has re- sumed importance and should be returned to ob- stetric concepts of contemporary causes of severe puerperal infection. The recent increase in both the frequency and virulence of group A streptococcal infections serves notice that this pathogen is with us today. REFERENCES 1. Stevens DL, Tanner MH, Winship J, et al.: Severe group A streptococcal infections with a toxic shock like syndrome and scarlet fever toxin A. N Engl J Med 321: 1-7, 1989. 2. Cone LA, Woodard DR, Schlievert PM, Tomory GS: Clinical and bacteriologic observations of a toxic shock- like syndrome due to Streptococcus pyogenes. N Engl J Med 317:146-193, 1987. 3.

l.: Severe group A streptococcal infections with a toxic shock like syndrome and scarlet fever toxin A. N Engl J Med 321: 1-7, 1989. 2. Cone LA, Woodard DR, Schlievert PM, Tomory GS: Clinical and bacteriologic observations of a toxic shock- like syndrome due to Streptococcus pyogenes. N Engl J Med 317:146-193, 1987. 3. Silver RM, Heddleston LN, McGregor JA, Gibbs RS: Life-threatening puerperal infection due to group A strep- tococci. Obstet Gynecol 79:894-896, 1992. 4. LeakeJ: Practical Observations on Child-Bed Fever; Also on the Nature and Treatment of Uterine Haemorrhages, Convulsions, and Such Other Acute Diseases as Are Most Fatal to Women During the State ofPregnancy. London: J Walter, 1772. 5. Gordon A: A Treatise on the Epidemic Puerperal Fever ofAberdeen. London: CG & J Robinson, 1795. 6. Holmes OW: The contagiousness of puerperal fever. N Engl J Med Surg 1:503-530, 1843. 7. Semmelweis IP: Childbed fever. (Excerpts from: The etiology, the concept and the prophylaxis of childbed fe- ver, 1861.) Rev Infect Dis 3:808-811, 1981. 8. Watson BP: Puerperal infection. Am J Obstet Gynecol 40:584-588, 1940. 9. Cunningham FG, MacDonald PC, Gant NF, Leveno KJ, Gilstrap LC (eds): Williams Obstetrics. 19th Ed. Norwalk, CT: Appleton & Lange, 1993. 10. Ledger WJ (ed): Infection in the Female. 2nd Ed. Phila- delphia: Lea & Febiger, 1986. 254 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY GROUP A STREPTOCOCCUS NATHAN AND LEVENO 11. Gilstsrap LC III, Faro S (eds): Infections in Pregnancy. New York: Alan R. Liss, Inc., 1990. 12. Williams JW (ed): Pathology of the puerperium. In: Williams Obstetrics. 1st Ed. New York: D. Appleton & Co., p 764, 1903. 13. Williams JW (ed): Pathology of the puerperium. In: Williams Obstetrics. 1st Ed. New York: D. Appleton & Co. p 780, 1903. 14. Gibberd GF: Puerperal sepsis, 1930-1965. J Obstet Gy- naecol Br Commonw 73:1-10, 1966. 15. Lancefield RC: A serological differentiation of human and other groups of hemolytic streptococci. J Exp Med 57:571-595, 1933. 16. Rubenstein A: Subtle poison: The puerperal fever contro- versy in Victorian Britain. Hist Stud 20:420-438, 1983. 17. Watson BP: An outbreak of puerperal sepsis in New York City. AmJ Obstet Gynecol 16:157-179, 1928. 18. Douglas RG, Davis IF: Puerperal infection. Etiologic, prophylactic and therapeutic considerations. Am J Obstet Gynecol 51:352-371, 1946. 19. Schwarz OH, Dieckmann WJ: Puerperal infection due to anaerobic streptococci. Am J Obstet Gynecol 13:467- 485, 1977. 20.

in New York City. AmJ Obstet Gynecol 16:157-179, 1928. 18. Douglas RG, Davis IF: Puerperal infection. Etiologic, prophylactic and therapeutic considerations. Am J Obstet Gynecol 51:352-371, 1946. 19. Schwarz OH, Dieckmann WJ: Puerperal infection due to anaerobic streptococci. Am J Obstet Gynecol 13:467- 485, 1977. 20. Ledger WJ: A historical review ofpelvic infections. AmJ Obstet Gynecol 158:687-693, 1988. 21. Ledger WJ: Hospital-acquired obstetric infections. In Zuspan FP (ed): Infection in the Female. 1st Ed. Phila- delphia: Lea & Febiger, p 206, 1977. 22. Gilstrap LC III, Cunningham FG: The bacterial patho- genesis of infection following cesarean section. Obstet Gynecol 53:545-549, 1979. 23. JewettJF, Rerd DE, Salon LE, Easterday CL: Childbed fevermA continuing entity. JAMA 206:344-350, 1968. 24. Ledger WJ, Headington JT: Group A beta hemolytic streptococcus. An important cause of serious infections in obstetrics and gynecology. Obstet Gynecol 39:474-482, 1972. 25. Ogden E, Amstey MS: Puerperal infection due to group A beta hemolytic streptococcus. Obstet Gynecol 52:53- 55, 1978. 26. McGregor J, Ott A, Villard M: An epidemic of "child- bed fever." Am J Obstet Gynecol 150:385-388, 1984. 27. Blanco JD, Gibbs RS, Castaneda YS: Bacteremia in ob- stetrics: Clinical course. Obstet Gynecol 56:621-625, 1981. 28. Bartter T, Dascal A, Carroll K, Curley FJ: "Toxic strep syndrome": A manifestation of group A streptococcal in- fection. Arch Intern Med 148:1421-1424, 1988. 29. Bisno AL: Group A streptococcal infections and acute rheumatic fever. N Engl J Med 325:783-793, 1991. 30. Veasy LG, Wiedmeier SE, Orsmond GS, et al.: Resur- gence of acute rheumatic fever in the intermountain area ofthe United States. N Engl J Med 316:421-427, 1987. 31. Stollerman GM: Changing group A streptococci. The reappearance of streptococcal "toxic shock." Arch Intern Med 148:1268-1270, 1988. 32. Cleary PP, Kaplan EL, Handley JP, Wlazlo A, Kim MH, Hauser AR, Schlievert PM: Clonal basis for resur- gence ofserious Streptococcuspyogenes disease in the 1980s. Lancet 339:518-521, 1992. 33. Martens PR, Mullie A, Goessens L: A near-fetal case of puerperal sepsis. Anaesth Intensive Care 19:108-110, 1991. 34. Acharya U, Lamont CAR, Cooper K: Group A beta- haemolytic streptococcus causing disseminated intravascu- lar coagulation and maternal death. Lancet 1:595, 1988. 35. Kavi J, Wise R: Group A beta-haemolytic streptococcus causing disseminated intravascular coagulation and mater- nal death. Lancet 1:993-994, 1988.

991. 34. Acharya U, Lamont CAR, Cooper K: Group A beta- haemolytic streptococcus causing disseminated intravascu- lar coagulation and maternal death. Lancet 1:595, 1988. 35. Kavi J, Wise R: Group A beta-haemolytic streptococcus causing disseminated intravascular coagulation and mater- nal death. Lancet 1:993-994, 1988. 36. Swingler GR, Bigrigg MA, Hewitt BG, McNulty CAM: Disseminated intravascular coagulation associated with group A streptococcal infection in pregnancy. Lancet 1:1456-1457, 1988. 37. Whitted RW, Yeomans ER, Hankins GDV: Group A [3-hemolytic streptococcus as a cause of toxic shock syn- drome. J Reprod Med 35:558-560, 1990. 38. Dotters DJ, Katz VL: Streptococcal toxic shock associated with septic abortion. Obstet Gynecol 78:549-550, 1991. 39. Nathan L, Peters MT, Ahmed AM, Leveno KJ: The return oflife-threatening puerperal sepsis caused by group A streptococci. AmJ Obstet Gynecol 169:571-572, 1993. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 255

Infectious Diseases in Obstetrics and Gynecology 1:257-258 (1994) (C) 1994 Wiley-Liss, Inc. Trichomonas vaginalis in Pregnancy Trichomonas vaginalis is a common cause of vaginitis in both the pregnant and nonpregnant patient. This protozoan is usually susceptible to metronidazole, but there is reluctance to administer this antibiotic to pregnant patients. The presence of T. vaginalis usually indicates that the vaginal ecosystem has become unbalanced. The microflora tends to shift from a lactobacilli-dominant ecosystem to one domi- nated, most likely, by anaerobes. This imbalance in the ecosystem may be the factor that is associated with preterm labor or premature rupture of the membranes. The presence of trichomonads may not be a significant factor. In addition, the abundance of bacteria provides a food source for trichomonads, which may present an opportunity of temporary treat- ment in the first trimester of pregnancy. Metronidazole remains the only treatment of T. vaginalis vaginitis. The use of metronidazole gel is not appropriate regardless of the trimester of pregnancy. At best, the gel would provide temporary relief; however, the gel is not effective in the treatment of T. vaginalis vaginitis. Moreover, it does not reach significant tissue or blood levels to treat infection of Bartholin's glands, Skene's glands, or bladder involvement. In addition to the potential for interruption ofthe pregnancy, the presence of T. vaginalis should alert the physician to the possible presence of other sexually transmitted organisms. T. vaginalis may cause a severe cervicitis, which may result in vaginal bleeding. Thus, a patient with an abnormal vaginal discharge and a decrease in hydrogen concentration (pH > 5) should be evaluated for T. vaginalis as well as bacterial vaginosis. The treatment of T. vaginalis in pregnancy becomes a worrisome problem. Although metronidazole has proved to be relatively safe, there is great reluctance to administer this agent to pregnant women, especially in the first trimester. Anti- microbial trials are not conducted in pregnant women for obvious reasons. Perhaps the following approach can be taken in the first trimester of pregnancy.

though metronidazole has proved to be relatively safe, there is great reluctance to administer this agent to pregnant women, especially in the first trimester. Anti- microbial trials are not conducted in pregnant women for obvious reasons. Perhaps the following approach can be taken in the first trimester of pregnancy. The initial treatment can be focused on reducing the number ofbacteria because they will most likely consist ofgram-negative facultative anaerobes and gram-negative and gram- positive obligate anaerobes. If the whiff test is positive, than an agent such as clindamycin cream can be utilized. If the whiff test is negative, amoxicillin/ clavulanate (Augmentin, Beecham Laboratories, Bristol, TN), 500 mg t.i.d, for 10 days, can be utilized. If there is strong suspicion that either Neisseria gonor- rhoeae or Chlamydia trachomatis or both are present, Augmentin should be effec- tive. Following the completion of treatment, the patient's vaginal ecosystem should be re-evaluated. The goal is not the eradication of T. vaginalis but a significant reduction in associated bacteria. Once the patient enters the second trimester, oral metronidazole can be administered. Editorial Received July 7, 1994 Accepted July 7, 1994 TRICHOMONAS VAGINALIS IN PREGNANCY FARO The obstetrician could use assurance in the treatment of T. vaginalis vaginitis in pregnancy. Therefore, it would be most helpful to hear from the infectious-disease specialists regarding their suggestions or recommendations in the treatment of T. vaginalis vaginitis in pregnancy. Sebastian Faro, M.D., Ph.D. Editor-in-Chief INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:259-264 (1994) (C) 1994 Wiley-Liss, Inc. Subcutaneous Tissue: To Suture or Not to Suture at Cesarean Section Van R. Bohman, Larry C. Gilstrap III, Susan M. Ramin, Bertis B. Little, Rigoberto Santos-Ramos, Kenneth G. Goldaber, Jody Dax, and Kenneth J. Leveno Department of Obstetrics and Gynecology, University ofTexas Southwestern Medical School, Dallas, TX ABSTRACT Objective: The null hypothesis for this investigation was that there was no difference in the fre- quency ofwound disruption between women who had their subcutaneous tissues approximated with suture and those who did not during cesarean section. Methods: During alternating months, consecutive women delivered by cesarean section either did (N 716) or did not (N 693) have their subcutaneous tissues closed with suture. All data were analyzed using chi square, Student's t-test, Fisher's exact probability test, analysis of variance, or logistic regression. Results: A 32% decrease in the frequency ofwound disruption was observed when subcutaneous tissues were brought into apposition with suture at cesarean section (P 0.03). Conclusions: Closure of Scarpa's and Camper's fascia with suture during cesarean section signif- icantly decreased the frequency ofwound disruption in this population. (C) 1994 Wiley-Liss, Inc. KEy woPs Wound disruption, infection, wound closure pproximately in 4 women in the United States are delivered by cesarean section, and it is well established that operative abdominal delivery is as- sociated with a significant risk of infection com- pared with vaginal delivery. Another major com- plication is wound disruption, which may be associated with infections, hematomas, seromas, pulmonic complications, host immunocompromise, and poor nutritional status.

established that operative abdominal delivery is as- sociated with a significant risk of infection com- pared with vaginal delivery. Another major com- plication is wound disruption, which may be associated with infections, hematomas, seromas, pulmonic complications, host immunocompromise, and poor nutritional status. 1-12 These risks are in- creased with preexisting operative site infection, breaks in sterile technique,7 prolonged preopera- tive admissions1 that may result in colonization with resistant microbes, prolonged operative dura- tion,2'8 use of electrocautery,8 obesity,3'9 advanced age, inadequate host immunocompetence, 10 increased abdominal pressure,9 liver disease, malnutrition, ste- roid use, malignancies, and introduction of foreign material or devitalized tissues to the healing site.7' Wound breakdown leads to a prolonged hospital stay with added discomfort and may add significantly to the already overburdened health care dollar. There is no consensus of opinion regarding whether or not it is advantageous to approximate Scarpa's and Camper's fascia with suture ligatures. Approximating these tissue layers with suture oblit- erates dead space and helps approximate the skin edges, but also adds foreign material to the healing site. The purpose ofthe present investigation was to compare the frequency of wound disruption be- tween cesarean section wounds that did and did not have Scarpa's and Camper's fascia approximated with suture. Address correspondence/reprint requests to Dr. Larry C. Gilstrap III, Department ofObstetrics and Gynecology, University ofTexas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, TX 75235-9032. Received December 21, 1993 Clinical Study Accepted April 25, 1994 SUBCUTANEOUS WOUND CLOSURE BOHMAN ET AL. SUBJECTS AND METHODS All pregnancies delivered by cesarean section at Parkland Memorial Hospital from April 1, 1991, through September 30, 1991, were assigned, on an alternating-month basis, to have Scarpa's and Camper's fascia closed or not closed with inter- rupted 000 plain catgut suture. Either a vertical or transverse (pfannenstiel) skin incision was utilized. All uterine incisions were closed with #1 chromic suture, bladder flaps with 00 chromic suture, pari- etal peritoneum with 00 chromic suture, and rectus fascia with 0 polydioxanone suture. The skin edges were approximated with surgical staples (Visi Stat skin stapler, Edward Weck & Company, Inc., Research Triangle Park, NC).

erine incisions were closed with #1 chromic suture, bladder flaps with 00 chromic suture, pari- etal peritoneum with 00 chromic suture, and rectus fascia with 0 polydioxanone suture. The skin edges were approximated with surgical staples (Visi Stat skin stapler, Edward Weck & Company, Inc., Research Triangle Park, NC). Staples were re- moved 3-5 days after the operation, and the opera- tive site was secured with adhesive tapes (shur- strips, Inman Leibelt Corporation, Arlington, TX). Amnionitis was defined as labor complicated by maternal fever (>38C) or an infant with a foul odor at delivery. Puerperal endometritis was de- fined as a fever (>3 gC) with uterine and parame- trial tenderness. The Quetelet's index, a derived value that represents weight/unit height, was calcu- lated with the following formula: weight (kg)/ height (m). 1 Obesity was defined as a Quetelet's index value >25. Superficial wound disruption was diagnosed when tissues above the rectus fascia did not remain in apposition during the puerperal period and in- volved the entire length of the incision. Fascial dehiscence was diagnosed when the integrity of the rectus fascia was lost. Operative time longer than h was considered prolonged. Excessive blood loss was defined as a 10-volume % decrease in the he- matocrit between admission and the 1st postopera- tive morning. Perioperative antimicrobial prophy- laxis was used and typically included cefazolin, cefotetan, or ampicillin/sulbactam. Prolonged rupture of membranes was defined as longer than 24h. Each patient's clinical history and progress were reviewed, computerized, and statistically analyzed utilizing chi square, Fisher's exact probability test, Student's t-test, analysis of variance, and logistic regression models using SAS software (SAS Insti- tute, Inc., Cary, NC). Probabilities <0.05 were considered significant. RESULTS A total of 7,670 women were delivered during the study period and 1,428 (18.6%) received cesarean sections. Nineteen women with cesarean sections were excluded because of incomplete data (N 15), or laparotomy was performed for indi- cations other than cesarean section (N 4). In the remaining 1,409 pregnancies, 1,184 (84%) had a vertical skin incision and 225 (16%) had a low transverse, pfannenstiel-type incision. Six hundred seven (51%) of the patients with a vertical incision and 109 (48%) ofthose with a pfannenstiel incision had closure of the subcutaneous tissue.

cesarean section (N 4). In the remaining 1,409 pregnancies, 1,184 (84%) had a vertical skin incision and 225 (16%) had a low transverse, pfannenstiel-type incision. Six hundred seven (51%) of the patients with a vertical incision and 109 (48%) ofthose with a pfannenstiel incision had closure of the subcutaneous tissue. There were no significant differences in demographic charac- teristics between the suture and non-suture groups (Table 1) or vertical vs. transverse skin incision. Skin staples were used to approximate the skin edges in 1,369 (97%) patients. Selected pregnancy outcomes are summarized in Table 2. The relative frequency of indications for these cesarean sections and type of skin incision did not differ between the 2 study groups. Similarly, the 2 study groups were comparable with respect to prolonged operative time, excessive blood loss, pu- erperal infection, and fascial dehiscence. However, although not significant, rectus fascia dehiscence (N 3) was limited to the non-subcutaneous su- ture group. Superficial wound disruption occurred in a total of 96 (7%) women. Superficial wound disruption was significantly decreased in those women with suture approximation of Scarpa's and Camper's fascia prior to stapling the skin. Specifically, 59 (9%) of the non-suture closures disrupted compared with 40 (6%, P 0.03) women with sutures placed into Scarpa's and Camper's fascia. Ninety-one (7.7%) of 1,184 with a vertical skin incision vs. 5 (2.2%) of 225 with a pfannenstiel incision (P 0.004) had a superficial wound dehiscence. The incidence of superficial wound disruption was not significantly affected by the choice of anti- microbial utilized for prophylaxis. Four hundred forty-nine women received a single 2 g intravenous (IV) dose of cefazolin for prophylaxis and had a wound disruption rate of 7.3% (N 33); 447 re- ceived a single 2 g IV dose of cefotetan and had a wound disruption rate of 5.3% (N 24); and 177 received a single IV dose of 2 g ampicillin/1 g sulbactam and had a wound disruption rate of 8.5% (N 15). 260 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY SUBCUTANEOUS WOUND CLOSURE BOHMAN ET AL. TABLE I.

N 33); 447 re- ceived a single 2 g IV dose of cefotetan and had a wound disruption rate of 5.3% (N 24); and 177 received a single IV dose of 2 g ampicillin/1 g sulbactam and had a wound disruption rate of 8.5% (N 15). 260 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY SUBCUTANEOUS WOUND CLOSURE BOHMAN ET AL. TABLE I. Pregnancy demographics in 1,409 women with and without subcutaneous sutures at cesarean section Subcutaneous suture Demographic Yes No factor (N 716) (N 693) Comparison Age (years) 25 6 25 -+ 6 NS Gravidity 3 2 3 -+ 2 NS Parity 2 - 2 -+ NS Obesity 29 7 29 6 NS Operative time (min) 48 -+ 18 49 -+ 19 NS Rupture of membranes (h) 5 +- 14 6 28 NS Race Hispanic 331 (46%) 301 (43%) NS Black 215 (30%) 235 (34%) NS White 153 (21%) 141 (20%) NS Other 17 (2%) 16 (2%) NS Labor 416 (58%) 403 (58%) NS Prolonged rupture of membranes 93 (I 3%) 100 (14%) NS Diabetes 34 (4%) 21 (3%) NS Amnionitis 52 (7%) 58 (8%) NS aDifferences in the mean compared with Student's t-test; differences in the frequency compared with chi square; obesity Quetelet's index value >25 [weight (kg)/height (m)]; prolonged rupture ofmembranes >24 h; amnionitis fever >38C in labor and/or foul-smelling infant at delivery; NS not significant. bMean -SD. CNumber with percentage in parentheses. TABLE 2. Pregnancy outcomes in 1,409 women with and without subcutaneous sutures at cesarean sectiona Subcutaneous suture Yes No Outcome (N 716) (N 693) Comparison Indication for cesarean section Repeat 326 (46%) 300 (43%) NS Dystocia 166 (23%) 177 (26%) NS Abnormal presentation 115 (16%) 96 (14%) NS Fetal distress 95 (I 3%) 105 (I 5%) NS Other 14 (2%) 15 (2%) NS Skin incision type Pfannenstiel 109 (15%) 116 (17%) NS Vertical 607 (85%) 577 (83%) NS Prolonged operative time 109 (15%) 118 (17%) NS Excessive blood loss 32 (5%) 34 (5%) NS Endometritis 166 (23%) 169 (24%) NS Fascial dehiscence 0 (0%) 3 (0.4%) NS Superficial wound disruption 40 (6%) 59 (9%) 0.03 aFrequencies compared with chi square; prolonged operative time >1 h; excessive blood loss 10-volume % decrease in hematocrit; NS not significant. bNumber with percentage in parentheses. CFisher's exact probability test. Thirty-three (33%) of the 99 open wounds had bacterial colonization, and 12 other wounds were clinically infected because of cellulitis or purulent drainage but no bacteria were isolated.

ss 10-volume % decrease in hematocrit; NS not significant. bNumber with percentage in parentheses. CFisher's exact probability test. Thirty-three (33%) of the 99 open wounds had bacterial colonization, and 12 other wounds were clinically infected because of cellulitis or purulent drainage but no bacteria were isolated. Staphylococ- cus epidermidis was present in 19 (58%) of open wounds, Staphylococcus aureus in 7 (21%), with Streptococcus pyogenes, Streptococcus agalactiae, En- terococci, gamma hemolytic streptococci, Coryne- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 261 SUBCUTANEOUS WOUND CLOSURE BOHMAN ET AL. TABLE 3. Preoperative pregnancy demographics in 99 women with wound disruption compared with 1,310 women without this complicationa Demographic Disrupted Intact factor (N 99) (N 1,310) Comparison Age (years) 24 -+ 6 25 -+ 6 NS Gravidity 3 -+ 2 3 -+ 2 NS Parity 2 _+ 2 -+ NS Obesity 32 _+ 7 20 -+ 6 <0.001 Operative time (min) 42 -+ 18 48 -+ 18 NS Rupture of membranes (h) 8 +- 21 6 +- 22 NS Race Hispanic 38 (38%) 595 (45%) NS Black 43 (43%) 407 (31%) NS White 16 (16%) 278 (21%) NS Other 2 (2%) 30 (2%) NS Labor 68 (68%) 751 (57%) NS Prolonged rupture of membranes 24 (24%) 169 (I 2%) 0.002 Diabetes 4 (4%) S (4%) NS Amnionitis 13 (I 3%) 97 (7%) 0.041 aDifferences in the mean compared with Student's t-test; differences in the frequency compared with chi square; obesity Quetelet's index value >25 [weight (kg)/height (m)]; prolonged rupture of membranes >1 h; amnionitis fever >38C and/or foul-smelling infant at delivery; NS not significant. bMean +- SD. CNumber with percentage in parentheses. dFisher's exact probability test. bacterium sp., Pseudomonas sp., Acinetobacter sp., Escherichia coli, Proteus mirabilis, and C#robacter sp. comprising the remaining organisms. Sixteen women had disrupted wounds that were colonized with 2-4 different organisms. Logistic regression analysis was used to deter- mine those potential preoperative factors signifi- cantly associated with superficial wound disrup- tion. Shown in Table 3 are selected preoperative demographic factors in the 99 women with superfi- cial wound disruption compared with the 1,310 women with intact wounds. Obesity (P < 0.001), prolonged membrane rupture (P 0.002), and amnionitis in labor (P 0.04) were significantly increased in women subsequently developing su- perficial wound disruption. Other factors such as prolonged operative time, excessive blood loss, and diabetes were not predictive of wound disruption.

ct wounds. Obesity (P < 0.001), prolonged membrane rupture (P 0.002), and amnionitis in labor (P 0.04) were significantly increased in women subsequently developing su- perficial wound disruption. Other factors such as prolonged operative time, excessive blood loss, and diabetes were not predictive of wound disruption. Shown in Table 4 are intraoperative and puerperal characteristics in those women with wound disrup- tions compared with those with intact wounds. Un- sutured Scarpa's and Camper's fascia (P 0.03), endometritis (P 0.001), and cesarean sections performed for dystocia (P 0.03) were signifi- cantly associated with wound disruption, while no association was found with excessive blood loss or prolonged operative time. DISCUSSION We found the usual forerunners of wound disrup- tion in our patient population but they were incon- sistent in predicting wound complications. Obesity and preexisting operative site infection, i.e., am- nionitis, increased the risk for development of wound complication, similar to the findings ofother investigators.3 Prolonged operative time, excessive blood loss at surgery, and diabetes have also been reported to be associated with an increased risk of wound disruption in some studies2'8'1 but were not predictors in the present investigation. A clean wound is one in which the alimentary, respiratory, and genitourinary tracts were not en- tered and in which aseptic technique was main- 14 tained and no apparent inflammation was present. A clean-contaminated wound is one in which a mi- nor break in technique occurred or in which the respiratory, gastrointestinal, or genitourinary tracts were entered but with no significant spillage. 14 Wound disruption has been reported to occur in 2-4% of clean operations and 10-20% of those classified as clean-contaminated. 1,2,10 Cesarean sec- tions are usually classified as clean or clean-contam- inated procedures and are associated with a widely divergent frequency of wound complications. The clinical wound infection rate after cesarean section 262 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY SUBCUTANEOUS WOUND CIOSURE BOHMAN ET AL. TABLE 4.

arean sec- tions are usually classified as clean or clean-contam- inated procedures and are associated with a widely divergent frequency of wound complications. The clinical wound infection rate after cesarean section 262 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY SUBCUTANEOUS WOUND CIOSURE BOHMAN ET AL. TABLE 4. Intraoperative and puerperal characteristics in 99 women with wound disruption compared with 1,310 women without this complicationa Disrupted Intact Characteristic (N 99) (N 1,310) Comparison Indication for cesarean section Repeat 40 (40%) 586 (45%) NS Dystocia 35 (35%) 308 (24%) 0.03 Fetal distress 13 (I 3%) 187 (14%) NS Abnormal presentation 9 (9%) 202 (I 5%) NS Other 2 (2%) 27 (2%) NS Prolonged operative time II (I I%) 216 (16%) NS Excessive blood loss 5 (5%) 61 (4%) NS Endometritis 39 (39%) 296 (22%) <0.001 Fascial dehiscence 3 (3%) 0 (0%) NS Subcutaneous suture 40 (5%) 59 (8%) 0.03 aFrequencies compared with chi square; prolonged operative time >1 h; excessive blood loss > 10-volume % decrease in hematocrit; NS not significant. bNumber with percentage in parentheses. CFisher's exact probability test. ranges from 4.8 to 14%, 1-5'1s while 89% of dis- rupted wounds were colonized with pathogens.6 Fascial dehiscence has been reported in 0.6- 12.5% of all disrupted cesarean wotlrlds,4'6'9' 15,16 resulting in a 2-3% maternal mortality.9' 16 Fascial dehiscence occurred in only 0.2% (N 3) of our study population. All 3 patients with fascial dehis- cence were in the group who did not have suture closure of their subcutaneous tissues. In our population, only obesity, endometritis, and non-sutured closure of the subcutaneous tissues were significantly predictive of wound disruption by logistic regression. Although it does not seem possible to reduce patient obesity at the time of cesarean section, the frequency of metritis can be reduced by prophylactic antibiotics. Importantly, closure of Scarpa's and Camper's fascia during ce- sarean section (in patients with a vertical skin inci- sion) with interrupted 000 plain catgut suture is easily and rapidly performed with marked benefit to the patient, i.e., less dehiscence. These results may not apply to patients with a pfannenstiel inci- sion, since the rate of dehiscence is significantly lower than with the vertical incision. There were only 5 (2.2%) dehiscences in patients with this latter incision.

easily and rapidly performed with marked benefit to the patient, i.e., less dehiscence. These results may not apply to patients with a pfannenstiel inci- sion, since the rate of dehiscence is significantly lower than with the vertical incision. There were only 5 (2.2%) dehiscences in patients with this latter incision. We conclude that there is significant benefit in approximating Scarpa's and Camper's fascia with suture at the time of cesarean section when surgical staples are used to approximate the skin. REFERENCES 1. Ortona L, Federico G, Fantoni M, et al.: A study on the incidence of postoperative infections and surgical sep- sis in a university hospital. Infect Control 8:320-324, 1987. 2. Cruse PJE, Foord R: A five-year prospective study of 23,649 surgical wounds. Arch Surg 107:206-210, 1973. 3. Pelle H, Jepson OB, Larsen SO, et al.: Wound infection after cesarean section. Infect Control 7:456-461, 1986. 4. Gibbs RS, Blanco JD, St. Clair PJ: A case control study of wound abscess after cesarean section. Obstet Gynecol 62:498-501, 1983. 5. Moir-Bussy BR, Hutton RM, Thompson JR: Wound infection after cesarean section. J Hosp Infect 5:359- 370, 1984. 6. Emmons SL, Krohn M, Jackson M, et al.: Development of wound infections among women undergoing cesarean section. Obstet Gynecol 72:559-564, 1988. 7. Polk HC Jr, Simpson CJ, Simmons BP, Alexander JW: Guidelines for prevention of surgical wound infection. Arch Surg 118:1213-1217, 1983. 8. Schwartz RI--I: Prevention of wound infections--Chang- ing perceptions. Infect Med Dis Lett Obstet Gynecol 12:34--36, 1990. 9. Helmkamp BF: Abdominal wound dehiscence. Am J Obstet Gynecol 128:803-807, 1977. 10. Charles D: Historical perceptions of wound infections. Infect Med Dis Lett Obstet Gynecol 12:28-33, 1990. 11. Mead PB: Abdominal wound infections. In Schaefer G, Graber EA (eds): Complications in Obstetrics and Gyne- cologic Surgery. Hagerstown: Harper & Row, chap 10, 1991. 12. Cruse PJ, Foord R: The epidemiology of wound infec- tion. Surg Clin North Am 60:27-41, 1980. 13. Roche AF, Seirvogel RM, Chumlea WC, Webb P: Grad- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 263 SUBCUTANEOUS WOUND CLOSURE BOHMAN ET AL. ing body fatness from limited anthropometric data. Am J Clin Nutr 34:2831-2838, 1981. 14. Olson M, O'Connor M, Schwartz ML: Surgical wound infections. A 5-year prospective study of 20,193 wounds at the Minneapolis VA Medical Center. Ann Surg 199: 253-259, 1984. 15.

YNECOLOGY 263 SUBCUTANEOUS WOUND CLOSURE BOHMAN ET AL. ing body fatness from limited anthropometric data. Am J Clin Nutr 34:2831-2838, 1981. 14. Olson M, O'Connor M, Schwartz ML: Surgical wound infections. A 5-year prospective study of 20,193 wounds at the Minneapolis VA Medical Center. Ann Surg 199: 253-259, 1984. 15. Poole GV Jr: Mechanical factors in the abdominal wound closure: The prevention of fascial dehiscence. Surgery 97:631-639, 1985. 16. Mowat J, Bonnar J: Abdominal wound dehiscence after caesarean section. Br Med J 2:256-257, 1971. 2ga4 INFECTIOUS DISEASES IN OBSVI'ETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:265-268 (1994) (C) 1994 Wiley-Liss, Inc. Microbiology of Bartholin's Duct Abscess Anu Mattila, Ari Miettinen, and Pentti K. Heinonen Departments ofClinical Microbiology (A.M., A.M.) and Obstetrics and Gynecology (P.K.H.), University Hospital, and Department ofClinical Medicine (P.K.H.), University of Tampere, Tampere, Finland ABSTRACT Objective: The aim of the study was to determine the currently most frequent microbial findings in Bartholin's duct abscess. Methods: Computerized records of microbial findings of 249 cases of Bartholin's duct abscess were retrospectively studied. Results: In 129 cases, only 1 microbe and, in 117 cases, >1 microbe were recovered. In 3 cases, the flora was recorded as normal for the lower genital tract. Ofall bacteria isolated, 252 were aerobic or facultative and 108 were anaerobic or microaerophilic. Aerobic or facultative bacteria alone caused 142 (57%) of the 249 cases, Escherichia coli being the most frequent isolate in this group. Anaerobic or microaerophilic bacteria alone caused 33 cases (13%), Bacteroides species and Prevo- tella species being most frequently identified. Both aerobic or facultative and anaerobic or mi- croaerophilic bacteria were isolated in 70 cases (28%). Candida albicans alone caused 1 case of Bartholin's duct abscess. The sexually transmitted pathogens Neisseria gonorrhoeae and Chlamydia trachomatis were both involved in only 2 cases. Conclusions: Bartholin's duct abscess was mainly caused by opportunistic bacteria, and sexually transmitted pathogens were only rarely involved in its pathogenesis. Since potentially pathogenic bacterial species were also frequently isolated, the use of antibiotics to complement the surgical treatment of Bartholin's duct abscess seems advisable, especially in patients with systemic symp- toms. (C) 1994 Wiley-Liss, Inc. KEY WORDS Bartholinitis, bacterial etiology, anaerobic bacteria artholinitis is a common infection and involves populations far beyond the groups at risk for sexually transmitted diseases. In acute Bartholin's duct abscess, incision and drainage are considered the primary treatment.

ymp- toms. (C) 1994 Wiley-Liss, Inc. KEY WORDS Bartholinitis, bacterial etiology, anaerobic bacteria artholinitis is a common infection and involves populations far beyond the groups at risk for sexually transmitted diseases. In acute Bartholin's duct abscess, incision and drainage are considered the primary treatment. However, antibiotics are frequently included in the treatment in addition to surgical procedures, and the regimen is usually chosen based on empiric knowledge. Previous studies on the bacteriology of Bartho- lin's duct abscess have emphasized the significance of gonococcus. 1-4 It has been reported to be in- 1--4 volved in approximately 1/ or more of cases. Anaerobic bacteria have also been reported to be often involved.2'5'6 During the last decade, Chlamydia trachomatis has been recognized as an important genital pathogen, and it has also been identified in Bartholin's duct abscess.3'7 However, there are only a few reports on C. trachomatis caus- ing bartholinitis,3'7'8 and the incidence of chla- mydial bartholinitis has not been thoroughly stud- ied. Unusual etiologic agents causing bartholinitis, and even septic shock arising from Bartholin's duct abscess, have also been reported.9-11 The aim of this study was to determine which microbes are currently the most common pathogens in Bartholin's abscess so that the antibiotic regimens Address correspondence/reprint requests to Dr. Anu Mattila, Department of Clinical Microbiology, Tampere Universi.ty Hospital, P.O. Box 2000, FIN-33521 Tampere, Finland. Clinical Study Received February 24, 1994 Accepted April 27, 1994 MICROBIOLOGY OF BARTHOLINITIS MATTILA ET AL. could be correctly directed against the most liable pathogens even before definite identification of the causing agent. SUBJECTS AND METHODS Tampere University Hospital, Tampere, Finland, has kept computerized records of its microbial lab- oratory findings since 1969. To see which mi- crobes are the most significant in Bartholin's duct abscess at the moment, we studied the records ret- rospectively from the beginning of 1983 to the end of 1989. During that time, the microbiological sampling and laboratory techniques followed an identical scheme. Positive microbial findings were recorded from 220 women with bartholinitis during this time, 24 of whom suffered from or more recurrences. Altogether, positive microbiological findings were available from 249 cases of Bartholin's duct ab- scess. The abscesses were defined on a clinical basis.

owed an identical scheme. Positive microbial findings were recorded from 220 women with bartholinitis during this time, 24 of whom suffered from or more recurrences. Altogether, positive microbiological findings were available from 249 cases of Bartholin's duct ab- scess. The abscesses were defined on a clinical basis. The samples were taken and studied according to general principles of diagnostic laboratory meth- ods. The surface of the abscess was thoroughly cleansed with povidone-iodine and 70% alcohol. The content of the abscess was aspirated percutane- ously or at the time of incision of the abscess with a needle attached to a plastic syringe of 0.5-20 ml. A Gram-stained smear was also made from each aspi- rate. If the interval between the aspiration of the abscess and inoculation of the cultures was fewer than 2 h, the syringe was capped and carried to the laboratory. If the interval between sampling and inoculation was longer than 2 h, the aspirate was injected into a transport medium for anaerobic sam- ples (Portagerm(R), BioM6rieux, Charbonni6res- les-Bains Cedex, France). Alternatively, purulent material was collected on cotton-wool or dacron swabs and transported in Stuart tubes. Specimens for the culture of C. trachomatis were instantly placed into the transport medium, refrigerated at /4C, and transported to the laboratory in 24 h. If the specimens for C. trachomatis could not be cul- tured in 24 h, they were stored deep frozen at -70C until cultured. Swabs or aliquots of each aspirate were inocu- lated onto chocolate agar, Columbia agar contain- ing 7% horse blood, and into a thioglycollate tube, which in addition to thioglycollate medium also contained vitamin K (0.1 Ixg/ml), NaHCO3 (1 mg/ml), and 5% Fildes enrichment. These cultures were incubated at +37C for 48 h and examined for identifying aerobic and facultative organisms. For the isolation of anaerobic organisms, swabs or aliquots of each aspirate were inoculated onto fastidious anaerobe agar containing 5% horse blood. These cultures were incubated at +37C for 48 h in an anaerobic jar with an atmosphere of 80% nitro- gen, 10% carbon dioxide, and 10% hydrogen. Specimens for the culture of C. trachomatis were inoculated on cycloheximide-treated McCoy cells. These cultures were incubated for 3 days at +35C in an atmosphere containing 5% carbon dioxide, stained with iodine, and examined with a light mi- croscope.

mosphere of 80% nitro- gen, 10% carbon dioxide, and 10% hydrogen. Specimens for the culture of C. trachomatis were inoculated on cycloheximide-treated McCoy cells. These cultures were incubated for 3 days at +35C in an atmosphere containing 5% carbon dioxide, stained with iodine, and examined with a light mi- croscope. RESULTS Of all bacterial isolates recorded, 252 were aerobic or facultative and 108 were anaerobic or microaero- philic bacteria (Table 1). Candida albicans was iden- tified in 3 cases, and mixed microbial flora referred to as normal for the lower genital tract mucosa in 3 cases. With or without other specific isolates re- corded, mixed microbial flora, i.e., positive growth of several bacteria without any specific species that could be pointed out as the major agent, was re- corded in 45 cases. In 129 cases, only microbe was recorded. In 111 cases, the sole pathogen was an aerobic or a acultative microbe and, in 17 cases, an anaerobic or a microaerophilic microbe. In case, the sole pathogen was C. albicans. In 117 cases, >1 mi- crobes were found. On the average, 1.65 bacteria/ case were recorded, ranging from to 7. Alto- gether, 45 different bacterial species were found (Table 1). Aerobic or facultative species alone caused 142 (57%) of the 249 cases. Anaerobic or microaerophilic species alone caused 33 cases (13%) ofBartholin's duct abscess. Both aerobic or faculta- tive and anaerobic or microaerophilic species were found in 70 cases (28%). C. albicans was the only pathogenic agent in case, along with aerobic or facultative agents in case and both aerobic or facultative and anaerobic or microaerophilic agents in case. In 3 cases, the flora was referred to as normal for the lower genital tract mucosa. Most of the aerobic or facultative bacteria could be regarded as being opportunistic pathogens. The most frequently isolated microbe representing this group was Escherichia coli. The sexually transmit- 266 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY MICROBIOLOGY OF BARTHOLINITIS MATTILA ET AL. TABLE I.

act mucosa. Most of the aerobic or facultative bacteria could be regarded as being opportunistic pathogens. The most frequently isolated microbe representing this group was Escherichia coli. The sexually transmit- 266 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY MICROBIOLOGY OF BARTHOLINITIS MATTILA ET AL. TABLE I. Bacterial findings in 249 Bartholin's abscess cases of 220 patients Aerobic/Facultative Bacteria; N 252 Gram-negative Escherichia coli 77 Proteus group 9 Haemophilus influenzae 4 Klebsiella pneumoniae 4 Klebsiella oxytoca Salmonella enteritidis Gram-positive Staphylococcus species 58 Streptococcus agalactiae 19 Enterococcus faecalis 18 Streptococcus alfa-haemolyticus 15 Staphylococcus aureus 13 Streptococcus beta-haemolyticus 8 Streptococcus milleri 6 Streptococcus non-haemolyticus 5 Diplococcus pneumoniae 4 Corynebacterium species Micrococcus species Staphylococcus saprophyticus Streptococcus equinis Streptococcus mitis Streptococcus species Sexually transmitted pathogens Chlamydia trachomatis 2 Neisseria gonorrhoeae 2 Anaerobic/Microaerophilic Bacteria; N 108 Gram-negative Prevotella species 23 Bacteroides species 17 Bacteroides fragilis group 10 Fusobacterium species 3 Propionibacterium species 2 Veillonella species 2 Gardnerella vaginalis Porphyromonas asaccharolytica Gram-positive Peptostreptococcus species 2 Peptococcus species 19 Lactobacillus species 5 Microaerophilic streptococcus 4 aProteus group (N 9) includes here P. mirabilis (N-- 7) and Mor- ganella morgani (N 2). bStreptococcus beta-haemolyticus (N 7) includes here S. beta-haemolyti- cus group G (N 4), S. beta-haemolyticus group F (N 2), S. beta- haemolyticus group A (N I), and S. beta-haemolyticus group C (N ). cprevotella species (N 23) includes here P. bivia (N 16), P. melanino- genica (N 4), P. intermedia (N 2), and P. oris (N I). dVeillonella species (N 2) includes here V. parvula (N I), and anaer- obic gram-negative coccus (N I). ePeptococcus species (N 19) includes here Peptococcus species (N 9), P. asaccharolyticus (N 8), and anaerobic coccus (N 2). ted pathogens Neisseria gonorrhoeae and Chlamydia trachomatis were not found in significant numbers. Of the aerobic or facultative bacteria regarded as being commensals, the most frequent ones were coagulase-negative Staphylococcus species. Of the anaerobic or microaerophilic bacteria, the most fre- quent isolates were Bacteroides and Prevotella species.

and Chlamydia trachomatis were not found in significant numbers. Of the aerobic or facultative bacteria regarded as being commensals, the most frequent ones were coagulase-negative Staphylococcus species. Of the anaerobic or microaerophilic bacteria, the most fre- quent isolates were Bacteroides and Prevotella species. DISCUSSION The significance of polymicrobial infections with anaerobic bacteria involved has been documented in severe gynecological infections with abscess for- mation, such as pelvic inflammatory disease with tubo-ovarian abscess. 12 In contrast, albeit fre- quently isolated in conjunction with less severe gy- necological infections, such as endometritis, the pathogenic role of anaerobic bacteria in such infec- tions remains questionable. As a numerous part of vaginal flora, anaerobic bacteria often contaminate specimens taken vaginally, which makes it difficult to assess their pathogenic significance. With regard to bartholinitis, the contamination ofspecimens with vaginal flora is also possible, albeit less likely. In our study, anaerobic or microaerophilic bacteria were involved in 103 (41%) of the 249 cases with bartholinitis either alone or in conjunction with aerobic or facultative bacteria. Bacteroides and Pre- votella species were the most common anaerobic bacteria isolated, which is an accordance with pre- vious studies on the subject.5'6 According to the current results, the use of antibiotic regimens cov- ering anaerobic bacteria should be advantageous in the treatment of acute bartholinitis. E. coli was the most frequent facultative microbe causing Bartholin's duct abscess in our study. In addition to its role as the major cause of urinary tract infections, E. coli has been reported as an important cause of various infections in the female genital tract, including bartholinitis.2's'6'13-5 On the other hand, the pathogenic role of aerobic or facultative bacteria regarded as being commensals is questionable. The possibility of their being con- taminants from mucosal surfaces cannot be ex- cluded. The incidence of gonococcal infection has de- creased markedly in Scandinavian countries since the 1970s. Therefore, it is not surprising that the gonococcus is no longer a major pathogen in Bar- tholin's duct abscess in this population. In contrast, although the incidence of C. trachomatis has in- creased during the last decade, our study shows that C.

on has de- creased markedly in Scandinavian countries since the 1970s. Therefore, it is not surprising that the gonococcus is no longer a major pathogen in Bar- tholin's duct abscess in this population. In contrast, although the incidence of C. trachomatis has in- creased during the last decade, our study shows that C. trachomatis is of no special significance in caus- INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 267 MICROBIOLOGY OF BARTHOLINITIS MATTILA ET AL. ing Bartholin's duct abscess. C. trachomatis should be respected as a rare cause of bartholinitis and it is likely that the Bartholin's gland is not the primary site for chlamydial infection. The recommended treatment of Bartholin's duct abscess is incision and drainage. Opinions on the benefit of including antibiotics in the treatment are somewhat discordant. In our study, a considerable number ofBartholin's duct abscess cases were caused by bacteria regarded as being potential pathogens, some ofthem documented to cause even septic shock arising from bartholinitis, e.g., E. coli and Strepto- coccus beta-haemolyticus group A. 10,11 It would seem advisable to include antibiotics to avoid the spread of infection in the treatment of Bartholin's duct abscess in addition to surgical procedures, espe- cially in patients presenting with systemic symp- toms. The possibility of a sexually transmitted in- fection as a rare cause of Bartholin's duct abscess emphasizes the importance of obtaining bacterial and chlamydial specimens as a part of the routine treatment. REFERENCES 1. Rees E: Gonococcal bartholinitis. BrJ Vener Dis 43:150- 156, 1967. 2. Lee Y-H, Rankin JS, Alpert S, Daly AK, McCormack WM: Microbiological investigation of Bartholin's gland abscesses and cysts. Am J Obstet Gynecol 129:150-154, 1977. 3. Davies JA, Rees E, Hobson D, Karayiannis P: Isolation of Chlamydia trachomatis from Bartholin's ducts. Br J Vener Dis 54:409-413, 1978. 4. Cheetham DR: Bartholin's cyst: Marsupialization or aspi- ration? Am J Obstet Gynecol 152:569-570, 1985. 5. Wren MWD: Bacteriological findings in cultures ofclin- ical material from Bartholin's abscess. J Clin Pathol 30: 1025-1027, 1977. 6. Brook I: Aerobic and anaerobic microbiology of Bartholin's abscess. Surg Obstet Gynecol 169:32-34, 1989. 7. Saul HM, Grossman MB: The role of Chlamydia tra- chomatis in Bartholin's abscess. Am J Obstet Gynecol 158:576-577, 1988. 8. Bleker OP, Smalbraak DJC, Schutte MF: Bartholin's abscess: The role of Chlamydia trachomatis.

I: Aerobic and anaerobic microbiology of Bartholin's abscess. Surg Obstet Gynecol 169:32-34, 1989. 7. Saul HM, Grossman MB: The role of Chlamydia tra- chomatis in Bartholin's abscess. Am J Obstet Gynecol 158:576-577, 1988. 8. Bleker OP, Smalbraak DJC, Schutte MF: Bartholin's abscess: The role of Chlamydia trachomatis. Genitourin Med 66:24-25, 1990. 9. Morton BD, McCarthy LR: Bartholinitis--An unusual etiologic agent. Obstet Gynecol 55:97S-98S, 1980. 10. Carson GD, Smith LP: Escherichia coli endotoxic shock complicating Bartholin's gland abscess. CMA J 122: 1397-1398, 1980. 11. Shearin RS, Boehlke J, Karanth S: Toxic shock-like syndrome associated with Bartholin's gland abscess: Case report. Am J Obstet Gynecol 160:1073-1074, 1989. 12. Eschenbach DA, Buchanan TM, Pollock HM, et al.: Polymicrobial etiology of acute pelvic inflammatory dis- ease. N EnglJ Med 293:166-171, 1975. 13. Heinonen PK: Carbon dioxide laser in the treatment of abscess and cyst of Bartholin's gland. J Obstet Gynecol 10:535-537, 1990. 14. Brihmer C, Kallings I, Nord C-E, Brundin J: Salpingi- tis; aspects of diagnosis and etiology: A 4-year study from a Swedish capital hospital. Eur J Obstet Gynecol Reprod Biol 24:211-220, 1987. 15. Sweet RL, Draper DL, Schachter J, James j, Hadley WK, Brooks GF: Microbiology and pathogenesis ofacute salpingitis as determined by laparoscopy: What is the appropriate site to sample. Am J Obstet Gynecol 138: 985-989, 1980. 268 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:269-274 (1994) (C) 1994 Wiley-Liss, Inc. Prevalence of Hepatitis C Virus Antibody in Patients With Sexually Transmitted Diseases Attending a Harrisburg, PA, STD Clinic Robert L. Sautter, Sharon Jones, Daniel I. Weber, William D. LeBar, Daniel F. Heitjan, Mary Magdalene C. Kopreski, and Frederick D. Curcio Departments ofMicrobiology and Pathology (R.L.S.) and Obstetrics and Gynecology (D.I.W., F.D.C.), Harrisburg Hospital and Planned Parenthood ofthe Capital Region (S.J., D.I.W.), Harrisburg, and Department of Obstetrics and Gynecology (D.I.W., M.M.C.K., F.D.C.), Centerfor Biostatistics and Epidemiology (D.F.H.), and Department ofPathology (R.L.S.), Hershey Medical Center, The Pennsylvania State University, Hershey, PA; and Department ofMicrobiology and Pathology (W.D.L.), Citation Clinical Laboratory-Providence Hospital, Southfield, MI ABSTRACT Objective: The prevalence of hepatitis B and hepatitis C in a sexually transmitted disease (STD) clinic population was studied, along with the prevalence of various STD agents, in an attempt to identify possible STD markers for the hepatitis C virus and help delineate the role of hepatitis C as an STD. The hepatitis C antibody rates found in the STD clinic were also compared with those found among patients attending a local OB/GYN clinic and those enrolled in a blood donor program, all from the same geographical area. Methods: A total of 150 women attending an STD clinc were examined for each ofthe following agents: Chlamydia trachomatis, Neisseria gonorrhoeae, syphilis, hepatitis B surface antigen, hepatitis B core antibody, hepatitis B surface antibody, and hepatitis C virus antibody. Additionally, several patients who signed informed consent to be evaluated for human immunodeficiency virus (HIV) antibody were tested by an enzyme immunoassay (EIA) screen method. The prevalence of each agent was then compared with the other agents. Results: The overall prevalence rates detected were as follows: hepatitis B 16%, hepatitis C 4%, chlamydia 18.7%, gonorrhea 7.4%, syphilis 0.7%, and HIV 0%.

munodeficiency virus (HIV) antibody were tested by an enzyme immunoassay (EIA) screen method. The prevalence of each agent was then compared with the other agents. Results: The overall prevalence rates detected were as follows: hepatitis B 16%, hepatitis C 4%, chlamydia 18.7%, gonorrhea 7.4%, syphilis 0.7%, and HIV 0%. Hepatitis C antibody was detected in 4% of patients in the STD clinic, 0.76% of volunteer blood donors from central Pennsylvania, and 0% ofpatients studied from the Harrisburg Hospital (Harrisburg, PA) prenatal population. Conclusions: This screening study reveals an association between attending a Harrisburg, PA, area STD clinic and having an increased prevalence of hepatitis C antibody, but larger matched control studies will be needed to help clarify sexual transmission as a mode of transmission for the hepatitis C virus. (C) 1994 Wiley-Liss, Inc. KEY WORDS Incidence, non-A, non-B hepatitis, STD hepatitis is reported in 10-20% of patients receiving 3 or more units of blood. Many patients who contract hepatitis have no de- tectable antibody against type A or B hepatitis vi- ruses and are classified as having non-A, non-B hepatitis. Ninety percent of" post-transfusion hepa- Address correspondence/reprint requests to Dr. Robert L. Sautter, Departments ofMicrobiology and Pathology, Harrisburg Hospital, South Front Street, Harrisburg, PA 17101. Received March 7, 1994 Clinical Study Accepted May 12, 1994 HEPATITIS C IN CENTRAL PENNSYLVANIA SAUTTER ET AL. titis has now been attributed to non-A, non-B hepa- titis worldwide. 1,2 However, this type oftransmis- sion was recently estimated to account for as low as only 10-15% of patients with non-A, non-B hepa- titis.3 Recently, the isolation and cloning of a piece of DNA from non-A, non-B hepatitis virus and development of an assay for the antibody to hepati- tis C virus (HCV) made possible the detection of many patients with a non-A, non-B hepatitis and the examination oftransmission routes.4'5 Recently, a 2nd-generation test for the detection of antibody vs. HCV was licensed.6 This 2nd-generation test offers the advantage of increased sensitivity and specificity for the determination of HCV anti- body.s'6 Results have suggested that HCV is the major cause of transfusion-related non-A, non-B hepatitis,7 especially in those cases that develop chronicity.

tion of antibody vs. HCV was licensed.6 This 2nd-generation test offers the advantage of increased sensitivity and specificity for the determination of HCV anti- body.s'6 Results have suggested that HCV is the major cause of transfusion-related non-A, non-B hepatitis,7 especially in those cases that develop chronicity. 8 Additionally, HCV appears to be the major cause of a number of community-acquired non-A, non-B hepatitis for which no history of percutaneous exposure has been identified. 1,2,9 Studies investigating the possible sources of in- fection for non-A, non-B hepatitis or I-ICV without a history of percutaneous exposure have been con- tradictory to date. Several small case reports have been published recognizing possible transmission due to perinatal and conjugal relationships that fol- low patterns similar to transmission of hepatitis B, human immunodeficiency virus (HIV), and hu- man T-lymphotropic virus type I (HTLV 1). 10,11 In addition, other papers including studies relating HCV to patients with sexually transmitted diseases (STDs)6'12 and to heterosexual activity with more than partner have been published. 13 Contrary to these findings, other investigations have suggested only rare sexual transmission of HCV among ho- mosexuals3 and among sexual contacts of high-risk intravenous (IV)-drug abusers.2 To further delineate the possible method of spread for HCV, we studied the prevalence of hep- atitis B infection and hepatitis C infection in an STD clinic population and correlated other known STDs as possible markers for patients at high risk for hepatitis B and hepatitis C. SUBJECTS AND METHODS Subjects Women attending a Harrisburg area STD clinic were included in the study if they signed informed TABLE I. Demographic and clinical information collected on STD patients Today's date: Age: I. Race (circle one): Black White Hispanic Asian Other 2. Did you have a blood transfusion (received blood) between 1979 and May 1985? 3. Has there been any one year since 1980 during which you had more than 5 partners? 4. In the past 9 years, have you had sex with a person who was A. an IV drug abuser B. a hemophiliac C. a bisexual D. a prostitute 5. Have you ever had sex with anyone who (to your knowledge) had AIDS or was infected with the AIDS virus? 6. Have you ever had any of the following STDs? Gonorrhea Herpes Chlamydia Syphilis Genital warts Pelvic inflammatory disease 7. In the past 9 years, have you used IV drugs? 8. Have you ever had sex in exchange for money or drugs?

had sex with anyone who (to your knowledge) had AIDS or was infected with the AIDS virus? 6. Have you ever had any of the following STDs? Gonorrhea Herpes Chlamydia Syphilis Genital warts Pelvic inflammatory disease 7. In the past 9 years, have you used IV drugs? 8. Have you ever had sex in exchange for money or drugs? Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No consent and completed a questionnaire assessing their risk factors (Table 1). Black, Caucasian, and Hispanic individuals were included in the study. The prevalence of hepatitis C in the central Penn- sylvania blood donor population and the Harris- burg Hospital prenatal population was also evalu- ated. Laboratory Methods Chlamydia trachornatis Two direct antigen tests (enzyme immunoassay [EIA] methods, Chlamydiazyme, Abbott Labora- tories, Abbott Park, IL, and a research membrane filtration technique, Seradyn, Inc., Indianapolis, IN) and culture were performed on the specimens collected from the patients for the diagnosis of in- fection with C. trachomatis. In addition, the culture transport fluid was analyzed by direct immunoflu- orescence (DFA) for the presence of C. trachomatis antigen as previously described. 14 A specimen was considered positive for C. trachomatis ifthe culture was positive or if 2 ofthe 3 direct antigen tests were positive. All chlamydial procedures were performed according to the manufacturers' specifications. 270 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY HEPATITIS C IN CENTRAL PENNSYLVANIA SAUTTER ET AL. TABLE 2. Percentage of patients exhibiting multiple risk factors Total number of risk factors 0 35 37 2 17 3 6 4 4 5 0 6 Total 100 Hepatitis B Testing Auszyme, hepatitis BsAg (HBsAg), Corzyme, anti- hepatitis B core antigen (anti-HBc), and Ausab, anti-hepatitis Bs antigen (anti-HBs; Abbott Labo- ratories) were performed according to the manufac- turer's specifications. Only those specimens that were repeatedly reactive were classified positive. For statistical analysis, those patients who exhibited or all of the above markers without history of vaccination were considered to have evidence of hepatitis B infection at some time in the past. Hepatitis C Testing The presence of serum HCV (anti-HCV; Abbott Laboratories) antibody was measured according to the manufacturer's specifications. Both st- and 2nd-generation tests for the detection of antibody vs. HCV were used.

ation were considered to have evidence of hepatitis B infection at some time in the past. Hepatitis C Testing The presence of serum HCV (anti-HCV; Abbott Laboratories) antibody was measured according to the manufacturer's specifications. Both st- and 2nd-generation tests for the detection of antibody vs. HCV were used. Each reactive result was con- firmed in duplicate and sent for confirmatory test- ing. The confirmatory test performed was the Chi- ron HCV recombinant immunoblot assay (RIBA; Chiron Corporation, Berkeley, CA). Bacterial Culture and Syphilis Serology Gonococcal cultures were performed on Martin Lewis agar medium in a 5% carbon dioxide atmo- sphere. Standard bacteriologic techniques were used to identify the isolates. 15 Syphilis serology utilized 16 a standard Rapid Plasma Reagin (RPR) assay. Statistical Analysis The goal of our analyses was to determine whether there was a significant association between the STDs, i.e., whether presence of STD increased the chance of having another. Thus, every pair of STDs was tested for association using the Fisher- Irwin exact test (Table 2). 17 To determine whether the observed associations would hold up after con- TABLE 3. Total number of patients categorized by questionnaire and chart review for demographic data and risk characteristics Race Hispanic 4 Caucasian 49 Black 46 Asian 0 Other Total 100 Age (years) 13-20 45 21-30 37 31-40 14 41-50 3 51-54 Total 100 High-risk sexual practices Multiple sex partners 17 With IV-drug user 10 With hemophiliac 0 With bisexual 4 With prostitute With someone having AIDS For money or drugs 2 Previous medical conditions Blood transfusion 2 Gonorrhea 19 Syphilis 3 Herpes 3 Genital warts 19 Chlamydia 23 Pelvic inflammatory disease 6 trolling for STD risk status, we classified the women into high-risk and low-risk strata, then car- ried out a stratified analysis using the Cochran- Mantel-Haenszel (CMH) test. 18 We determined risk status by questionnaire and chart review (Table 3). Using these criteria, we called subjects "low risk" if they had no known behavioral or medical risk factors and classified those subjects with any risk factors as "high risk." Most computations were executed in the S-Plus language on a Sun SPARC- station workstation. 19 Exact tests were computed with StatXact, version 2.0 (Cytel, Cambridge, MA). RESULTS Demographic information on patients seen at the Harrisburg area STD clinic is presented in Table 3. Subjects ranged in age from 13 to 54 years.

t computations were executed in the S-Plus language on a Sun SPARC- station workstation. 19 Exact tests were computed with StatXact, version 2.0 (Cytel, Cambridge, MA). RESULTS Demographic information on patients seen at the Harrisburg area STD clinic is presented in Table 3. Subjects ranged in age from 13 to 54 years. The most frequent risk factors are presented as percent- ages in Table 3 and by prevalence in Table 2. The most prevalent risk factor was multiple sexual part- ners26 (17%), followed by sex with an IV-drug INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 271 HEPATITIS C IN CENTRAL PENNSYLVANIA SAUTTER ET AL. TABLE 4. Prevalence rates of 6 STDs with P values for tests of associationa Disease P value from test of association (prevalence) Hepatitis C Chlamydia Gonorrhea Syphilis HIV Hepatitis B (I 6.0%) 0.052 0.701 0.557 0.327 ND Hepatitis C (4.0%) 0.717 0.063 0.082 ND Chlamydia (I 8.7%) 0.034 0.189 ND Gonorrhea (7.4%) 0.151 ND Syphilis (0.7%) ND HV (0%) ap values are from I-sided Fisher exact tests of the hypothesis of no association between the diseases against the alternative of positive association. ND indicates insufficient data to test the hypothesis. Sample sizes are 149 for gonorrhea, 148 for syphilis, 27 for HI, and 150 for hepatitis B and C and chlamydia. abuser--15 (10%). Thirty-five patients admitted to having had a chlamydial infection at some time in the past. There were 53 "low-risk" and 97 "high- risk" subjects as defined in the previous section. Prevalence rates of 6 STDs are presented in Table 4. When the 1-sided Fisher-Irwin test was per- formed, associations between chlamydia and gonor- rhea (P 0.034) and between hepatitis B and hepatitis C (P 0.052) were found. After stratifi- cation using the CMH test, the chlamydia/ gonorrhea test (P 0.052) and the hepatitis B/hepatitis C test (P 0.086) approach significant positive associations. These results support conclu- sions reached using the Fisher-Irwin test mentioned earlier. The only statistically significant association following stratification was found between hepatitis C and syphilis (P 0.044). However, the associ- ation between these 2 diseases was negative. Seven patients were found to be repeatedly reac- tive by the HCV EIA procedure. Six of the 7 (85.7%) reactive EIA specimens were found to be positive for antibody to HCV by the 2nd-genera- tion HCV (EIA) procedure and by the RIBA. Of these 6 patients, 3 were also positive for hepatitis B core antibody.

es was negative. Seven patients were found to be repeatedly reac- tive by the HCV EIA procedure. Six of the 7 (85.7%) reactive EIA specimens were found to be positive for antibody to HCV by the 2nd-genera- tion HCV (EIA) procedure and by the RIBA. Of these 6 patients, 3 were also positive for hepatitis B core antibody. Of the subjects who were confirmed positive for HCV antibody, only (16%) had no risk factor as defined earlier. Three or 50% of the HCV-positive subjects had multiple risk factors with the most common risk factors being previous blood transfusion (50%), IV-drug abuse (33%), and multiple sexual partners (33%). No statistically significant associations were found for HCV-posi- tive subjects and their risk factors. Other STDs were detected in those patients positive for HCV; however, no statistically significant association was determined. The overall prevalence of hepatitis C in 3 populations (STD, prenatal, and blood donor) TABLE 5. Comparison of hepatitis C antibody in 3 Harrisburg patient populationsa Population studied Prevalence STD clinic 6/150 (4%) Blood donor 59/7,744 (0.76%) Prenatal 0/100 (0%) aAII positive HCV antibody tests were confirmed by RIBA. is presented in Table 5. They are significantly dif- ferent by the exact test on the 3 x 2 table (P 0.0005). Pairwise differences are significant for STD clinic vs. blood donor (P 0.0012) and STD clinic vs. prenatal (P 0.04). The blood donor and the prenatal groups are not significantly different. DISCUSSION HCV has been shown to be the causative agent of the majority of cases of post-transfusion hepati- tis, 1,20,). especially high-risk transfusion patients such as hemophiliacs,22-24 chronic renal patients, and those patients with recent cardiac surgery.24 In addition, the agent has been found in U.S. veter- ans,2s and implicated in maternal transmission, 11,26 sexual transmission, 12,13,27 and IV-drug abuse.28'29 The purpose of the present study was to explore the relationships between STDs and current or prior HCV infection and thus identify known STDs as possible markers for HCV. To discriminate the prevalence of HCV in the high-risk groups from that in the normal population, we studied the prev- alence of I--ICV in 2 low-risk patient populations in the Harrisburg area. Positive associations between STDs were found in the 150 STD patients studied. As expected, pa- 272 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY HEPATITIS C IN CENTRAL PENNSYLVANIA SAUTTER ET AL. tients positive for C.

, we studied the prev- alence of I--ICV in 2 low-risk patient populations in the Harrisburg area. Positive associations between STDs were found in the 150 STD patients studied. As expected, pa- 272 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY HEPATITIS C IN CENTRAL PENNSYLVANIA SAUTTER ET AL. tients positive for C. trachomatis were likely to be infected with N. gonorrhoeae. We also found hepa- titis B virus (HBV) and HCV to be associated with one another. The presence of hepatitis B markers (anti-HBc, anti-HBe, HBeAg) has been related to the presence of I-ICV in blood donors and chronic HCV carriers. 3'31 However, significant debate continues on the reliability of surrogate markers in blood donor populations for predicting the pres- ence of HCV.32-34 HBV and HCV seem to be transmitted concom- itantly in the United States and most of Europe, while Japan and selected countries in Europe show little or no association between transmission ofI--IBV and I-ICV. Differences in these associations could possibly be due to some unknown risk factors that are found in certain geographical locales and not in others. It has been suggested that several classes of HCV exist, with varying subtypes more prevalent in different countries. Geographical and/or genetic differences have yet to be explored as a method for interpreting transmission routes and prevalence rates. Studies have also suggested that heterosexual promiscuity and/or homosexual promiscuity with evidence of numerous prior STDs constitute sig- nificant risk factors for the transmission of both HBV and HCV. 12'13'27 Considerable debate over the role sexual practices have on the transmission of HCV can be found in the current literature. 3'9 In the present study, antibody to HCV was de- tected in 4% of patients attending a Harrisburg area STD clinic, in 0.76% of volunteer blood do- nors from central Pennsylvania, and in none of the patients studied from Harrisburg Hospital's pre- natal population. Hess et al.9 found similar results with 4.7% and 0.51% positive anti-HCV results from STD and blood donor patients, respectively.

attending a Harrisburg area STD clinic, in 0.76% of volunteer blood do- nors from central Pennsylvania, and in none of the patients studied from Harrisburg Hospital's pre- natal population. Hess et al.9 found similar results with 4.7% and 0.51% positive anti-HCV results from STD and blood donor patients, respectively. Additional studies in the current literature have shown that the positive rates for I--ICV in blood donors range between 0.5 and 1.5% 1,21,34,35 Posi- tive rates for sexual transmission of non-A, non-B hepatitis in heterosexual and homosexual popula- tions have ranged between 4.7 and 50%.9'12'13'27'35 Additional risk factors have included race, nation- ality, sex of the patient, multiple sexual partners, IV-drug abuse, preyious or concurrent positive tests for HBV and HIV, and evidence ofmultiple STDs. Our data differ slightly from a recently published review by Lynch-Salamon and Combs.35 The inci- dence reported in their review of the literature agrees with our data for blood donors and for those patients attending an STD clinic. However, the incidence of HCV positivity by risk groups in our study is lower than that previously reported.35 This is undoubtedly due to the small number of HCV- positive patients found in the Harrisburg area STD clinic. The present study shows that attending a Harrisburg area STD clinic is associated with an increased prevalence of I--ICV compared with 2 other low-risk populations in the same geographical area. However, we were unable to identify any specific disease among the known STDs that corre- lated statistically with the presence of HCV for use as a marker for HCV infection. Additional larger studies involving matched controls would be help- ful in order to help clarify the mode oftransmission of HCV. ACKNOWLEDGMENTS We thank the Harrisburg Hospital laboratory staff in the Department ofMicrobiology and Blood Bank as well as the staff of Planned Parenthood of the Capital Region for their technical assistance. This work was supported by a George Lafferty Founda- tion grant. Supplies for hepatitis screening were pro- vided by Abbott Laboratories (Abbott Park, IL). Additionally, we dedicate this paper to the late Dr. Frederick D. Curcio III, whose intellect, guid- ance, friendship, and compassion will be remem- bered by all those individuals who have benefited from knowing this extremely caring individual. REFERENCES 1.

ng were pro- vided by Abbott Laboratories (Abbott Park, IL). Additionally, we dedicate this paper to the late Dr. Frederick D. Curcio III, whose intellect, guid- ance, friendship, and compassion will be remem- bered by all those individuals who have benefited from knowing this extremely caring individual. REFERENCES 1. Stevens CE, Taylor PE, Pindyck J, et al.: Epidemiology of hepatitis C virus, a preliminary study in volunteer blood donors. JAMA 263:49-53, 1990. 2. Esteban JI, Viladomiu L, Gonzalez A, et al.: Hepatitis C virus antibodies among risk groups in Spain. Lancet 2:294-296, 1989. 3. Melbye M, Biggar RJ, Wantzin P, Krogsgard K, Ebbe- sen P, Becker NG: Sexual transmission of hepatitis C virus: Cohort study (1981-9) among European homosex- ual men. Br Med J 301:210-212, 1990. 4. Choo QL, Kuo G, Weiner AJ, Overby LR: Isolation ofa cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244:359-361, 1989. 5. Kuo G, Choo Q-L, Alter HJ, Citnick GL: An assay for circulating antibody to a major etiologic virus of human NANB hepatitis. Science 244:362-364, 1989. 6. Aach RD, Stevens CE, Hollinger FB, Moseley JW: Hepatitis C virus infection in post-transfusion. An analy- sis with first- and second-generation assays. N Engl J Med 325:1325-1329, 1991. INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 273 HEPATITIS C IN CENTRAL PENNSYLVANIA SAUTTER ET AL. 7. Marcellin P, Martinot-Peignoux M, Boyer N, et al.: Second-generation (RIBA) test for hepatitis C virus (let- ter). Lancet 337(8740):551-552, 1991. 8. Mosely JW, Aach RD, Hollinger FB, et al.: Non-A, non-B hepatitis and antibody to hepatitis C virus. JAMA 263:77-78, 1990. 9. Hess G, Massing A, Rossol S, et al.: Hepatitis C virus and sexual transmission. Lancet 2:987, 1989. 10. Kamitsukasa H, Harada H, Yakura M, et al.: Intrafa- milial transmission of hepatitis C virus (letter). Lancet 2:987, 1989. 11. Kuroki T, Nishiguchi S, Fukuda K, et al.: Mother-to- child transmission of hepatitis C virus. J Infect Dis 164: 427-428, 1991. 12. Tedder RS, Gilson RJC, Briggs M, et al.: Hepatitis C virus: Evidence for sexual transmission. Br Med J 302: 1299-1302, 1991. 13. Alter MJ, Coleman PJ, Alexander WJ, et al.: Importance of heterosexual activity in the transmission of hepatitis B and non-A, non-B hepatitis. JAMA 262:1201-1205, 1989. 14.

427-428, 1991. 12. Tedder RS, Gilson RJC, Briggs M, et al.: Hepatitis C virus: Evidence for sexual transmission. Br Med J 302: 1299-1302, 1991. 13. Alter MJ, Coleman PJ, Alexander WJ, et al.: Importance of heterosexual activity in the transmission of hepatitis B and non-A, non-B hepatitis. JAMA 262:1201-1205, 1989. 14. LeBar WD, Schubiner H, Jemal C, et al.: Comparison of the Kallestad Pathfinder EIA, cytocentrofuged direct fluorescent antibody, and cell culture for the detection of Chlamydia trachomatis. Diagn Microbiol Infect Dis 14: 17-20, 1991. 15. Morello JA, Janda W, Doern GV: Neisseria and Bran- hamella. In Balows A (ed): Manual of Clinical Microbi- ology. 5th ed. Washington, DC: American Society for Microbiology, pp 258-276, 1991. 16. Larsen SA, Bradford LL: Serodiagnosis of syphilis. In Rose NR (ed): Manual of Clinical Laboratory Immunol- ogy. 3rd ed. Washington, DC: American Society for Microbiology, pp 425-434, 1986. 17. Placket RL: The Analysis of Categorical Data. 2nd ed. New York: Macmillan, 1981. 18. Snedecor GW, Cochran WG: Statistical Methods. 9th ed. Ames: Iowa State University Press, 1989. 19. Statistical Sciences, Inc.: S Plus Version 2.3. Seattle: Sta- tistical Sciences, Inc., 1990. 20. Lee SH, Hwang SJ, Lu RH, Lai KH, Tsai YT, Lo KJ: Antibodies to hepatitis C virus in prospectively followed patients with posttransfusion hepatitis. J Infect Dis 163: 1354-1357, 1991. 21. Goldsmith MF: Blood bank officials hope donor altruism will pass new (anti-HCV) test. JAMA 262:1749-1750, 1990. 22. Widell A, Hansson BG, Bertorp E, et al.: Antibody to a hepatitis C virus related protein among patients at high risk for hepatitis B. Scand J Infect Dis 23:19-24, 1991. 23. Schulmn S, Grillner L: Antibodies against hepatitis C in a population of Swedish haemophiliacs and heterosexual partners. Scand J Infect Dis 22(4):393-397, 1990. 24. Brind AM, Codd AA, Cohen BJ, et al.: Low prevalence of antibody to hepatitis C virus in northeast England. J Med Viro132(4):243-248, 1990. 25. Kendrick V, Dunn B, Fink L, et al.: The presence of hepatitis C virus in veterans with and without abnormal liver function. AmJ Clin Pathol (Abstr Fall Meet No 79) 96:419, 1991. 26. Giovannini M, Tagger A, Ribero ML, et al.: Maternal- infant transmission of hepatitis C virus and HIV infec- tions: A possible interaction (letter). Lancet 335:1166, 1990. 27. Perillo RP: Potential importance of the sexual transmis- sion ofnon-A, non-B hepatitis. Hepatology 13:805-808, 1991. 28.

ents. Its use is not currently recommended in pregnancy unless dis- seminated disease exists. However, acyclovir has been used in hundreds of pregnant women without adverse fetal outcomes. 38'9 Its use has been pro- posed to attempt reduction in herpes recurrences and subsequent cesarean delivery for this indica- tion. Preliminary data have been promising in this regard;4'41 however, this therapy may still not prevent asymptomatic viral shedding and potential neonatal inection.42 Furthermore, the use of acy- clovir may delay the humoral response to I--ISV, thereby preventing or reducing passive immuniza- tion ofthe 'etus.4 At this time, acyclovir cannot be recommended except in research protocols and in cases of life-threatening disseminated illness. Of course, acyclovir will continue to be used in neo- nates and nonpregnant patients. Second, the management of premature rupture ofthe membranes (PROM) in the presence ofHSV lesions also remains an area of controversy. Case reports and case series have demonstrated the possi- bility of expectant management in preterm PROM.44'45 In the largest reported series of 18 patients, 3 patients suffered additional recurrences during their latency period. Eight patients were delivered by cesarean section for the presence of lesions at the time of indication for delivery. None INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY HERPES IN PREGNANCY COOK AND GALL of the 18 infants developed neonatal HSV infec- tions, although some of the mothers were treated with acyclovir antepartum.45 Based on these lim- ited data, it would seem reasonable to perform ce- sarean deliveries if" at or near term and otherwise to manage the remote-from-term patient expectantly unless delivery is indicated. There are still insuffi- cient data to recommend the antepartum use of acyclovir. Previously, it had been recommended to per- form a cesarean delivery within 4-6 h of rupture of the membranes at term. A vaginal delivery was allowed if ruptured beyond this period of time. This recommendation was based on 2 small patient series that demonstrated no infections if" ruptured for fewer than 4 h with significant infections de- spite cesarean delivery if ruptured beyond this point.46'47 Unfortunately, subsequent data have demonstrated infections even ifthe membranes were intact or ruptured fewer than 4 h.

o 79) 96:419, 1991. 26. Giovannini M, Tagger A, Ribero ML, et al.: Maternal- infant transmission of hepatitis C virus and HIV infec- tions: A possible interaction (letter). Lancet 335:1166, 1990. 27. Perillo RP: Potential importance of the sexual transmis- sion ofnon-A, non-B hepatitis. Hepatology 13:805-808, 1991. 28. van den Hoek JAR, van Haastrecht HJA, Goudsmit J, deWolfF, Coutinho RA: Prevalence, incidence, and risk factors of hepatitis C virus infection among drug users in Amsterdam. J Infect Dis 162:823-826, 1990. 29. Girardi E, Zaccarelli M, Tossini G, Puro V, Narciso P, Visco G: Hepatitis C virus infection to intravenous drug users: Prevalence and risk factors. Scand J Infect Dis 22(5):751-752, 1990. 30. Fattovich G, Tagger A, Brollo L, et al.: Hepatitis C virus infection in chronic hepatitis B carriers. J Infect Dis 163:400-402, 1991. 31. Koziol DE, Holland PV, Alling DW, et al.: Antibody to hepatitis B core antigen as a paradoxical marker for non-A, non-B hepatitis agents in donated blood. Ann Intern Med 104:488-495, 1986. 32. Ohto H, Nomura H, Ohmura K, Ishijima A, Okazaki S: Low overlap between anti-HCV and anti-HBc in Japa- nese. Transfusion 31:88-89, 1991. 33. Hetland G, Skaug K, Larsen J, Maland A, StrommeJH, Storvold G: Prevalence ofanti-HCV in Norwegian blood donors with anti-HBc or increased ALT levels. Transfu- sion 30:776-779, 1990. 34. Richards P, Holland P, Kuramoto K, Dourville C, Ran- dell R: Prevalence of antibody to hepatitis C virus in a blood donor population. Transfusion 31:109-113, 1991. 35. Lynch-Salamon DI, Combs CA: Hepatitis C in obstetrics and gynecology. Obstet Gynecol 79:621-629, 1992. 274 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

Infectious Diseases in Obstetrics and Gynecology 1:275-281 (1994) (C) 1994 Wiley-Liss, Inc. Genital Mycoplasmas in Placental Infections Andreas Stein, Lon Boubli, Bernard Blanc, and Didier Raoult Laboratoire de Microbiologie Clinique (A.S.) and Service de Gyn&ologie-Obsttrique (L.B., B.B.), H6pital de la Conception, and Facult de Mdecine, Unit des Rickettsies (D.R.), Marseille, France ABSTRACT Objective: The involvement of the genital mycoplasmas Ureaplasma urealyticum and Mycoplasma hominis in complications ofpregnancy has remained controversial especially because these microor- ganisms are frequent colonizers of the lower genital tract. Recovery of bacteria from the placenta appears to be the sole technique to represent a true infection and not vaginal contamination. Therefore, we investigated the presence of genital mycoplasmas, aerobic and anaerobic bacteria, and fungi in human placentas and evaluated their association with morbidity and mortality of pregnancy. Methods: We cultured placentas from 82 women with complicated pregnancies. One hundred placentas from women with uncomplicated pregnancies were evaluated as controls. When possible, placentas were examined histologically for presence ofchorioamnionitis. Results: Microorganisms were recovered from 52% of the placentas of complicated pregnancies and U. urealyticum was the microorganism isolated most frequently from the placenta. A significant association between positive mycoplasma culture ofthe placenta and complication ofpregnancy was found, and chorioamnionitis was positively related to isolation ofmycoplasmas. Conclusions: These data suggest that genital mycoplasmas are able to infect the human placenta where they can cause chorioamnionitis. This infection of the placenta by genital mycoplasmas is related to preterm birth and fatal outcome ofpregnancy. (C) 1994 Wiley-Liss, Inc. KEy woP,S Ureaplasma urealyticum, Mycoplasma hominis, morbidity of pregnancy, mortality of pregnancy he genital mycoplasmas Mycoplasma hominis and Ureaplasma urealyticum are colonizers of the female lower genital tract.

tal mycoplasmas is related to preterm birth and fatal outcome ofpregnancy. (C) 1994 Wiley-Liss, Inc. KEy woP,S Ureaplasma urealyticum, Mycoplasma hominis, morbidity of pregnancy, mortality of pregnancy he genital mycoplasmas Mycoplasma hominis and Ureaplasma urealyticum are colonizers of the female lower genital tract. 1-3 These organisms have been associated with various obstetrical complica- tions: mortality (early spontaneous abortion, late abortion, stillbirth, neonatal death)and morbidity (premature rupture of membranes, preterm birth, low birth weight).4 The involvement ofM. homin# and U. urealyticum in these pathologies has been determined by isolation of these bacteria in the female lower and upper genital tracts (vagina, uterus, and amniotic fluid).5-9 However, the high rate (10-60%) of genital colonization in the female population makes it difficult to appreciate the real pathogenic role of these 2 genital mycoplasmas in pregnancy.3'4'9 A study protocol was therefore de- signed to correlate isolation of these microorgan- isms directly from the placenta with morbidity and mortality of pregnancy. A sampling of the endo- metrial portion of the placenta from placentas of wornen with complicated pregnancies was cultured for genital mycoplasmas. A control group of pla- centas from uncomplicated pregnancies was simi- larly evaluated. All placentas were also examined for other bacterial and fungal pathogens. Placentas were examined histologically when possible and the patients' charts were reviewed to evaluate specific clinical parameters. Address correspondence/reprint requests to Dr. Didier Raou|t, Facult de Mdecine, Unit6 des Rickettsies, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France. Clinical Study Received February 2, 1994 Accepted May 12, 1994 GENITAL MYCOPLASMAS IN PLACENTAL INFECTIONS STEIN ET AL. TABLE I. Complicated pregnancies: maternal and fetal abnormalities indicating placenta sampling (abnormality could be associated) Maternal abnormalities N Fetal abnormalities N Maternal fever 40 Spontaneous abortion 12 Clinical evidence of infection 10 Stillbirth 6 Premature rupture of membranes 14 Preterm birth 13 Preterm labor 18 Low birth weight 4 SUBJECTS AND METHODS Women were enrolled between November 1991 and November 1992 from the Unit of Gynecology and Obstetrics of the University Hospital of Marseille, France. All women admitted to the unit were screened for eligibility.

remature rupture of membranes 14 Preterm birth 13 Preterm labor 18 Low birth weight 4 SUBJECTS AND METHODS Women were enrolled between November 1991 and November 1992 from the Unit of Gynecology and Obstetrics of the University Hospital of Marseille, France. All women admitted to the unit were screened for eligibility. Patients were selected as follows: women with documented uterine or fetal anomalies, placenta previa, abruptio placentae, or cervical cerclage were excluded from the study and women presenting with at least one maternal or fetal abnormality listed in Table were included in the study. The placentas of these subjects were sub- mitted to the laboratory for microbiological exami- nation and culture. Placentas from women with uncomplicated pregnancies and none of the fetal or maternal abnormalities listed in Table were eval- uated as controls. Samples of the maternal surface ofthe placenta were obtained with a sterile surgical blade by slicing a 1.0 1.0 1.0 cm portion of the depth ofthe placenta. The samples were ground with a sterile tissue grinder and examined follow- ing Gram staining. Aerobic and anaerobic cultures for bacteria and fungi were made. Mycoplasmas were cultured in both liquid and agar media. Bac- teria and fungi were identified according to the technique of Balows et al. 10 For culture of myco- plasmas, the placental tissue suspension was trans- ferred onto a Mycofast identification strip (Groupe Stago, International Mycoplasma, Signes, France) and selective A-7 agar medium (Groupe Stago, International Mycoplasma) following the manufac- turers' instructions. 11'12 Both the Mycofast strip and A-7 plates were incubated for 7 days at 37C in a COz atmosphere and were examined daily for the presence of U. urealyticum and/or M. homin#. Cul- ture confirmation and numeration of mycoplasma were based on the detection of the color changes in the Mycofast identification strip and on the colonial morphology in A-7 agar. All placentas were cul- tured on the same day of delivery without knowl- edge of the subject's status. When the attending physician requested it, the placenta was examined histologically without knowledge of the results of cultures or the status ofthe subject. The reasons for histopathological examination of the placenta in- cluded stillbirth, neonatal death, and complications of pregnancy, labor, or delivery.

s status. When the attending physician requested it, the placenta was examined histologically without knowledge of the results of cultures or the status ofthe subject. The reasons for histopathological examination of the placenta in- cluded stillbirth, neonatal death, and complications of pregnancy, labor, or delivery. Histological chorioamnionitis was defined as the demonstration of inflammatory cells, predominantly neutrophils, in the chorion and amnion. 13 The medical records of all women were re- viewed. Gestational age was estimated from the date of the mother's last menstruation, fundal height, and ultrasonography. Prematurity was de- fined as a gestational age of <35 weeks. Fatal out- come of pregnancy was defined as mortality due to early or late abortion, death in utero, or stillbirth. The distribution of the culture results of the microorganisms among the study groups was ex- amined. A X 2 test was used to evaluate the equality of proportions of positive cultures and presence of chorioamnionitis in the study groups and to corre- late ratios ofpositive culture, chorioamnionitis, pre- maturity, and fatal outcome of pregnancy. RESULTS In all, 82 placentas were obtained from women with complicated pregnancies and 100 placentas from women with none of the fetal or maternal abnormalities listed in Table were used as con- trols. The recovery of U. urealyticum, M. hominis, and other bacteria from the placenta in both groups is shown in Table 2. Microorganisms were recov- ered from 43 (52%) of the 82 placentas from com- plicated pregnancies. Twenty-two placentas (27%) contained genital mycoplasmas and 21 placentas (25%) other bacteria. Only 4 placentas (4%) of the control group grew microorganisms. The variation of the isolation rate of microorganisms among the study group and the control group was statistically significant (P < 10-8). In the study group, U. urealyticum was the microorganism isolated most frequently from the placenta (24%). These data show a significant association between positive my- coplasma cultures ofthe placenta and complications of pregnancy (P < 10-6). The results of the histopathological examination of the placenta are shown in Table 3. In all, 61 of 276 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY GENITAL MYCOPLASMAS IN PLACENTAL INFECTIONS STEIN ET AL. TABLE 2.

ion between positive my- coplasma cultures ofthe placenta and complications of pregnancy (P < 10-6). The results of the histopathological examination of the placenta are shown in Table 3. In all, 61 of 276 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY GENITAL MYCOPLASMAS IN PLACENTAL INFECTIONS STEIN ET AL. TABLE 2. Microorganisms recovered from placental cultures Study Control group group (N 82) (N 100) N % N % P Genital mycoplasmas Ureaplasma urealyticum 20 24 < 10-6 Mycoplasma hominis 2 2.4 Facultative bacteria Group B Streptococcus 3 3.7 Viridans streptococci 3 3.7 Enterococcus 3 3.7 Escherichia coli 5 6 Staphylococcus aureus 1.2 < 10-s Anaerobic bacteria Peptostreptococcus 4 4.9 Clostridium 1.2 2 2 Yeast Candida albicans 1.2 Total microorganisms 43 52 4 4 < 10-8 Sterile 39 48 96 96 aU. urealyticum was associated in placenta ofthe study group with group B streptococci and in another placenta with E. coil TABLE 3. Association of bacterial isolates from the placenta with histological chorioamnionitis Chorioamnionitis (placentas/placentas examined) N % Genital mycoplasmas Ureaplasma urealyticum I/I 6 69 Mycoplasma hominis Facultative bacteria Group B Streptococcus 2/2 I00 Viridans streptococci 3/3 100 Enterococcus 2/3 66 Escherichia coli 4/5 80 Staphylococcus aureus I/I 100 Anaerobic bacteria Peptostreptococcus I/2 50 Clostridium I/I 100 Total microorganisms 25/33 76 Sterile 9/28 32 the 82 placentas from complicated pregnancies were examined histopathologically. Of these, 34 placen- tas (56%) showed histopathological chorioamnioni- tis. Chorioamnionitis was seen in 69% of the 16 placentas from which mycoplasmas were isolated; 32% of the sterile placentas of the study group showed inflammation. This difference in occur- rence of chorioamnionitis was significant (P < 0.002). When comparing the sterile placen- tas, we noted that recovery of genital mycoplasmas or other bacteria of the placenta was significantly related to preterm birth and mortality of pregnancy (Table 4). In the group of placentas growing geni- tal mycoplasmas, 27% were associated with pre- term birth (P < 0.03) and 45% to fatal outcome of the pregnancy (P < 10-4). In the group of pla- centas infected with bacteria other than genital mycoplasmas, 24% were associated with preterm birth (_P < 0.05) and 33% with fatal outcome of pregnancy (P < 0.002). Recovery of genital my- coplasmas and other bacteria was significantly re- lated to preterm birth and mortality of pregnancy (Table 4).

the group of pla- centas infected with bacteria other than genital mycoplasmas, 24% were associated with preterm birth (_P < 0.05) and 33% with fatal outcome of pregnancy (P < 0.002). Recovery of genital my- coplasmas and other bacteria was significantly re- lated to preterm birth and mortality of pregnancy (Table 4). DISCUSSION The involvement of the genital mycoplasmas U. urealyticum and M. hominis in mortality and mor- bidity ofpregnancy was initially suggested by isola- tion of these bacteria in the female lower and upper genital tracts (vagina, uterus, amniotic fluid, and endometrium)5-9 or by serological diagnosis. 14,15 However, the role of genital mycoplasma infection in complications ofpregnancy has remained contro- versial especially because these microorganisms fre- quently colonize the lower genital tract of pregnant women who are not ill and who give birth to nor- mal, healthy infants. In fact, the high prevalence of genital colonization in the female population, which ranges from 10 to 60%, makes it difficult to accept that in some patients mycoplasmas are pathogenic, while in other there is no evidence of infection.2 A recent review on the role of U. urealyticum in pre- mature birth demonstrated that U. urealyticum in the lower genital tract is not directly associated with preterm birth, as preterm birth is related to a vari- ety ofrisk factors for prematurity (black race, young maternal age, low educational level, low income, smoking during pregnancy, history of marijuana and/or cocaine use, and separate, single marital status). The authors conclude that these factors may be interdependent and that no consistent cause-and- effect relationship exists between the presence of U. urealyticum in the lower genital tract of the mother and prematurity.9 This opinion is supported by the findings of 2 studies in which erythromycin was INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 277 GENITAL MYCOPLASMAS IN PLACENTAL INFECTIONS STEIN ET AL. TABLE 4. Relationship of preterm birth and fatal outcome (abortion, intrauterine decease, stillbirth) of the pregnancy to bacterial infection of the placenta Placentas growing Placentas growing bacteria genital mycoplasmas other than mycoplasmas Sterile placentas Total (N 22) (N 21) (N 39) (N 82) N % N % N % N % Preterm birth 6 27 S 24 2 5 13 16 Fatal outcome 10 45 7 33 2.5 18 22 used to treat women with genital colonization by mycoplasmas. In these 2 studies, it was demon- strated that erythromycin treatment in women with U.

mycoplasmas other than mycoplasmas Sterile placentas Total (N 22) (N 21) (N 39) (N 82) N % N % N % N % Preterm birth 6 27 S 24 2 5 13 16 Fatal outcome 10 45 7 33 2.5 18 22 used to treat women with genital colonization by mycoplasmas. In these 2 studies, it was demon- strated that erythromycin treatment in women with U. urealyticum in the lower genital tract had virtu- ally no impact upon low birth weight or prematu- rity. 6' 17 As there are case reports implicating U. urealyticum in clinical amnionitis, investigators tried to correlate the presence of mycoplasmas in the amniotic fluid with the morbidity ofpregnancy.7' 8 However, mycoplasmas are present in 50% of am- niotic fluid samples from both women with intra- amniotic infection and asymptomatic control women and U. urealyticum does not appear to be associated with clinically evident intra-amniotic mycoplasmal infection. 19 In fact, the recovery of bacteria from the pla- centa appears to be the sole technique to represent a true infection and not vaginal contamination,9 as bacteria from the typical vaginal flora are almost never recovered from placental specimens. Fur- thermore, the relationship between infection of the placenta and prematurity has been shown to be independent of the duration of labor, the presence of ruptured membranes, or the duration of rup- tured membranes, z These findings suggest that infection occurs before labor and is not a result of prolonged labor or rupture of membranes. So far, only 7 studies have examined the relationship be- tween colonization of the placenta and outcome of pregnancy. Naessens et al.2 cultured placentas for U. urealyticum but not M. homin# from 253 women and found no significant difference in the birth weight of infants whose placentas were colonized. Two other studies showed that the presence of U. urealyticum in the placenta was not related to low birth weight or prematurity.22'23 Embree et al.24 cultured placentas from a group of 446 "high-risk" pregnancies and 108 unselected deliveries of nor- mal full-term infants and found an association of the isolation of U. urealyticum with prematurity, lower birth weight, and intrauterine growth retar- dation. Kundsin et al.2s confirmed those data in a prospective study by isolating mycoplasmas more frequently from the placentas ofinfants who died in the perinatal period and from the placentas of those neonates admitted to the intensive case unit than from matched controls. Hillier et al.2 related both the isolation of U.