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Malaria affects between 300 and 600 million people annually and contributes towards more than 1 million deaths worldwide, with Plasmodium falciparum malaria causing more deaths [1–3]. The disease's clinical presentation ranges from asymptomatic parasitemia to febrile disease, presenting as uncomplicated malaria (UCM) or severe and often fatal illness classified either as cerebral malaria (CM) or severe malarial anemia (SMA) [3, 4]. Of the millions of African children who become ill with malaria annually, only 2 percent develop severe disease, and these are the ones who are at greatest risk of dying from the infection [3]. Although factors that determine whether P falciparum malaria infection develops into severe disease are not clearly known, current evidence suggests that some interplay between the parasite and host's immune response has a role [5–6]. Successful control and clearance of blood-stage malaria infection requires a well coordinated and timely antibody-mediated and cell-mediated immune response. Cell-mediated immunity involves different cell types that partly exert their effect through the release of proinflammatory cytokines such as interferon (IFN)-γ and tumor necrosis factor (TNF)-α [7, 8].

od-stage malaria infection requires a well coordinated and timely antibody-mediated and cell-mediated immune response. Cell-mediated immunity involves different cell types that partly exert their effect through the release of proinflammatory cytokines such as interferon (IFN)-γ and tumor necrosis factor (TNF)-α [7, 8]. If the proinflammatory response is left uncontrolled, instead of just achieving the parasite clearance and controlling the parasite replication [9], it can result in the development of immune-mediated pathology [10]. Immunity against severe malaria may depend upon the host's ability to regulate the magnitude and specific timing of the cell-mediated immune response, allowing the sequential induction of appropriate levels of proinflammatory and anti-inflammatory cytokines at crucial stages of the infection cycle [13, 14]. Regulatory T cells (Tregs), a subset of T cells, have been implicated in playing a role in the severity of malaria and also in the clearance of parasites in blood-stage infection in both mouse models and in human malaria [14, 15]. Depletion of Tregs protected mice from death when infected with a lethal strain of Plasmodium yoelii [16]. Both in vitro and in vivo depletion of Tregs significantly reversed the inhibition of interleukin (IL)-2 production, a proinflammatory cytokine, in Plasmodium berghei-infected mice [17].

in human malaria [14, 15]. Depletion of Tregs protected mice from death when infected with a lethal strain of Plasmodium yoelii [16]. Both in vitro and in vivo depletion of Tregs significantly reversed the inhibition of interleukin (IL)-2 production, a proinflammatory cytokine, in Plasmodium berghei-infected mice [17]. Studies in humans have shown that Treg levels increase during acute P falciparum malaria infection coinciding with increased parasite growth rate [21], which is thought to be essential in controlling proinflammatory responses in subsequent malaria infections, thereby preventing development of severe malaria [22]. A longitudinal study conducted in Kenya found an association between the percentage of Tregs and risk of subsequent clinical malaria upon reinfection [24]. A separate longitudinal study of healthy Gambian children and adults in high malaria transmission areas found that percentage and absolute numbers of Tregs increased significantly during the malaria season but were low in the dry season [27]. Taken together, the findings of these studies seem to suggest that pre-existing levels of Tregs may influence malaria susceptibility and severity. In a recent study, Gambian children presenting with acute UCM and SM were reported to have higher percentages and numbers of Tregs during convalescence compared with controls [14]. In addition, children living in endemic malaria areas would usually have suffered from UCM several times in the previous years before succumbing to CM [20].

, Gambian children presenting with acute UCM and SM were reported to have higher percentages and numbers of Tregs during convalescence compared with controls [14]. In addition, children living in endemic malaria areas would usually have suffered from UCM several times in the previous years before succumbing to CM [20]. We therefore conducted the current study to compare and contrast Tregs and cytokine levels in children presenting with UCM at a location with a high (Chikwawa) and a location with a low (Blantyre) malaria transmission rate.

, Gambian children presenting with acute UCM and SM were reported to have higher percentages and numbers of Tregs during convalescence compared with controls [14]. In addition, children living in endemic malaria areas would usually have suffered from UCM several times in the previous years before succumbing to CM [20]. We therefore conducted the current study to compare and contrast Tregs and cytokine levels in children presenting with UCM at a location with a high (Chikwawa) and a location with a low (Blantyre) malaria transmission rate. METHODS Study Site and Participants We conducted a prospective cohort study at Ndirande Health Centre (NHC) in Blantyre (area of low malaria transmission [32]) between March 2011 and July 2011 and at Chikwawa District Hospital (CDH) in Chikwawa (area of high malaria transmission [32]) between August 2011 and November 2011. We recruited 33 and 30 human immunodeficiency virus (HIV)-uninfected children aged between 6 and 60 months in Blantyre and Chikwawa, respectively, who presented with acute UCM. Participants were defined as having UCM when they were febrile at the time of recruitment, had a positive Rapid Diagnostic Test or positive malaria thick and thin slides, but had a Blantyre Coma Score of 5, and a hemoglobin (Hb) concentration above 5 dL/mL [4]. Children who were HIV-infected or presented with other comorbidities, infectious or noninfectious, were excluded from the study. Children with UCM were observed at 1 month and 3 months during convalescence. In addition, 30 healthy controls of ages that were in the same range as the study participants (ie, between 1 and 60 months) were recruited at both sites. Healthy controls were recruited from NHC and CDH under the following conditions: when they came for scheduled immunization and were not febrile; they had a temperature of 37°C or less when enrolled; they had not had clinical- or laboratory-confirmed malaria in the past 3 months; and they were HIV-uninfected and did not have any underlying illness at the time of recruitment. Thick and thin malaria slides were prepared from the blood sample of each healthy control on the day of recruitment to screen for malaria parasitemia.

not had clinical- or laboratory-confirmed malaria in the past 3 months; and they were HIV-uninfected and did not have any underlying illness at the time of recruitment. Thick and thin malaria slides were prepared from the blood sample of each healthy control on the day of recruitment to screen for malaria parasitemia. A 5 mL venous blood sample was collected from each participant at recruitment and during follow-up. Participants presenting with UCM were treated with coformulated tablets of 20 mg of artemether and 120 mg of lumefantrine (Coartem; Novartis), per Malawi Government guidelines as first-line treatment of UCM, before blood sample collection. Ethical approval for the study was obtained from College of Medicine Research and Ethics Committee, and written informed consent was obtained from the parent or guardian of every participant. Human Immunodeficiency Virus, Full Blood Count, and Malaria Parasite Testing Human immunodeficiency virus testing was performed using 2 rapid test kits; Determine (Abbott Laboratories, Japan) and Unigold (Trinity Biotch, Dublin). Where discordant results were obtained, polymerase chain reaction was used to confirm the results. Thick and thin blood smears on slides were prepared by standard methodology. Total white blood cell (WBC) counts, percentage, and absolute counts of lymphocytes were determined using an HmX hematological analyzer (Coulter).

ere discordant results were obtained, polymerase chain reaction was used to confirm the results. Thick and thin blood smears on slides were prepared by standard methodology. Total white blood cell (WBC) counts, percentage, and absolute counts of lymphocytes were determined using an HmX hematological analyzer (Coulter). Immunophenotyping Procedure Immunophenotyping of blood samples by flow cytometry and lymphocyte subset identification was performed as reported elsewhere [31]. A total of 200 µL of whole blood was stained with a cocktail of conjugated surface monoclonal antibodies (mAbs): CD4 peridinin chlorophyll 4 µL, CD25 phycoerythrin 4 µL, and CD127 fluorescein isothiocyanate 4 µL (all from Beckon Dickinson) were incubated for 15 minutes at room temperature in the dark. Erythrocytes were lysed with 2 mL 1:10 diluted FACS lysing solution (Becton Dickinson), incubated in the dark for 20 minutes. Cells were washed twice with 2 mL phosphate-buffered saline ([PBS] Sigma-Aldrich) and fixed with 500 µL 1:3 diluted Perm/Fix solution (Becton Dickinson), incubated for 20 minutes at room temperature in the dark. Cells were washed twice with 2 mL 1:10 diluted Perm Buffer (Beckon Dickinson). Permeabilized cells were stained with intracellular conjugated monoclonal antibody: 4 µL FoxP3 APC (Beckon Dickinson) for 30 minutes at room temperature in the dark and washed with 2 mL of Perm Buffer. Stained cells were resuspended in 350 µL 1% formaldehyde (Sigma-Aldrich)/PBS and analyzed on a CyAn flow cytometer (Beckman Coulter) within 24 hours. Figure 1 shows the gating strategy used for classic Tregs [33]. We defined CD4+ Tregs as a subset that expressed CD4+CD25hiFoxP3+CD127low. Figure 1. Gating strategy for CD4+CD25hiFoxP3+CD127low regulatory T cells that was used in this study. FITC, fluorescein isothiocyanate; PercP, peridinin chlorophyll protein.

hours. Figure 1 shows the gating strategy used for classic Tregs [33]. We defined CD4+ Tregs as a subset that expressed CD4+CD25hiFoxP3+CD127low. Figure 1. Gating strategy for CD4+CD25hiFoxP3+CD127low regulatory T cells that was used in this study. FITC, fluorescein isothiocyanate; PercP, peridinin chlorophyll protein. Quantification of Serum Cytokine Levels High protein binding 96-well enzyme-linked immunosorbent assay (ELISA) plates (Nunc-Immuno) were coated with monoclonal antibodies for IFN-γ, TNF-α, IL-10, and transforming growth factor (TGF)-β: 1-D1 K, TNF3/4, 9D7, and MT593 (all from MABTECH AB), respectively, at 2 µg/mL in PBS (Sigma-Aldrich) and adding 100 µL per well. Coated ELISA plates were incubated overnight at 4°C and washed twice with PBS adding 200 µL per well. Plates were blocked with PBS with 0.05% Tween 20 containing 0.1% bovine serum albumin (all from Sigma-Aldrich) (incubation buffer) and incubated at room temperature for 1 hour. Enzyme-linked immunosorbent assay plates were washed 5 times with PBS containing 0.05% Tween 20. A total of 100 µL of test serum and standards were added to appropriate wells, diluted in incubation buffer, and incubated at room temperature for 2 hours. Enzyme-linked immunosorbent assay plates were washed 5 times with PBS containing 0.05% Tween 20 and appropriate biotinylated mAbs for detection of serum IFN-γ, TNF-α, IL-10, and TGF-β: 7-B6-1-biotin, mAb TNF5-biotin, mAb 12G8-biotin, mAb MT517-biotin (MABTECH AB), respectively, at 1 µg/mL were added at 100 µL per well and incubated at room temperature for 1 hour. Enzyme-linked immunosorbent assay plates were washed 5 times with PBS containing 0.05% Tween 20 and 100 µL Streptavidin-ALP diluted 1:1000 (MABTECH AB) in incubation buffer added per well and incubated at room temperature for 1 hour. Enzyme-linked immunosorbent assay plates were washed 5 times with PBS containing 0.05% Tween 20 and 100 µL SIGMAFAST p-Nitrophenyl phosphate (Sigma-Aldrich) per well, and optical density was measured after 30 minutes of incubation using BioTek reader ELx800 (BioTek Instruments) at 405 nm. Concentrations of serum cytokines were determined using plotted standard curve.

on. Therefore, the observed high levels of IL-10 during acute infection in our study have been described as ideal and are consistent with results of other studies [13, 28]. However, our study was not able to replicate results of studies that have shown significantly higher than normal levels of TGF-β in acute UCM [21]. We had hypothesised that children presenting with acute UCM would have higher levels of the anti-inflammatory cytokines IL-10 and TGF-β, which would remain elevated compared with levels in healthy controls into convalescence. We had therefore anticipated that this predominantly anti-inflammatory profile would render the children more vulnerable to re-infection with malaria and to an increased probability of such re-infection developing into a more severe clinical form of malaria. In contrast, we found that at both sites, the levels of the cytokines IL-10 and TGF-β were much lower in convalescence compared with those observed during acute disease, and they were comparable to those levels observed in healthy controls. Although some participants at both sites were found to be asymptomatically parasitemic 1 month after treatment (14.8% in Blantyre and 12% in Chikwawa), and even more at the 3-month follow-up stage, none of these participants developed any symptomatic malaria during the 3-month period of convalescence.

hed 5 times with PBS containing 0.05% Tween 20 and 100 µL SIGMAFAST p-Nitrophenyl phosphate (Sigma-Aldrich) per well, and optical density was measured after 30 minutes of incubation using BioTek reader ELx800 (BioTek Instruments) at 405 nm. Concentrations of serum cytokines were determined using plotted standard curve. Spleen Size Grading Spleen size grading was performed using a protocol described previously [28]. In brief, the size of the spleen was examined while the child was resting supine with both hands at the side. The examiner left hand was used to support the left of the ribcage posterior laterally while the right hand was aligned with the fingertips parallel to the left costal margin. Palpation was done from the right lower quadrant towards the left costal margin, asking the patient to take a deep breath in and feeling for the movement of the spleen with the examiners’ fingers. A spleen that was felt was graded using the Hackett's grading system [28]. Essentially, the higher the spleen size grade the larger the spleen. Statistical Analyses Statistical analysis was performed using GraphPad Prism. A Mann-Whitney U test was performed between groups to detect statistically significant differences. A P value of < .05 was considered to show a statistically significant difference. Regression analysis was used to assess linear associations between the concentration of IL-10 and the percentage of Tregs at the 2 sites, with the Spearman's correlation value (r) of 1 indicating a strong correlation and anything lower showing a weaker correlation.

idered to show a statistically significant difference. Regression analysis was used to assess linear associations between the concentration of IL-10 and the percentage of Tregs at the 2 sites, with the Spearman's correlation value (r) of 1 indicating a strong correlation and anything lower showing a weaker correlation. RESULTS Participants' Demographic Data In Blantyre, 33 children presenting with UCM were recruited with a median age of 34.5 months (5.7–60). Twenty-seven of these children were observed at 1 month convalescence, and 26 children were successfully observed again after 3 months (Table 1). Of those not seen during the 2 follow-up visits, 5 withdrew from the study mainly because their parents or guardians did not want their children to continue participating in the study, and 1 died of causes other than malaria. In Chikwawa, 30 children presenting with acute UCM and with a median age of 26.9 months (6.1–51.3) were recruited; 5 withdrew from the study 1 month after recruitment, and 1 additional child withdrew by the second follow-up visit for the same reason as in Blantyre. Although all UCM participants recruited in Blantyre had positive parasitaemia on the day of recruitment, 4 participants were found to be parasitemic during convalescence by thick and thin malaria slides, whereas in Chikwawa, 3 participants were parasitemic after 3 months convalescence. Table 1. Demographic, Hematological, and Spleen Grade Data for the Study Participants in Blantyre and Chikwawa

on the day of recruitment, 4 participants were found to be parasitemic during convalescence by thick and thin malaria slides, whereas in Chikwawa, 3 participants were parasitemic after 3 months convalescence. Table 1. Demographic, Hematological, and Spleen Grade Data for the Study Participants in Blantyre and Chikwawa Study Participants' Parameters Blantyre Chikwawa Acute 1 Month 3 Months Control Acute 1 Month 3 Months Control Number of participants (%) 33 27 26 31 30 25 24 30 Median age in months (range) 34.5 (5.7–60) ND ND 16.5 (6.3–55.2) 26.9 (6.1–51.3) ND ND 24.8 (9.9–46.8) Loss to follow up (%) NA 2 of 33 (6) 0 of 27 (0) NA NA 5 of 30 (16.6) 1 of 25 (4) NA Number of deaths (%) NA 1 of 28 (3.5) 0 of 26 (0) NA NA 0 of 25 (0) 0 of 24 (0) NA Malaria positive slide or RDT (%) 33 of 33 (100) 4 of 27 (14.8) 4 of 26 (15.4) 0 of 31 (0) 30 of 30 (100) 3 of 25 (12) 3 of 24 (12.5) 0 of 30 (0) Median Hgb in g/dL (range) 9.2 (4.9–12.1) 11 (5.9–13.4) 11.6 (6.6–13.7) 10.1 (5.6–14) 8.9 (6.4–11.9) 10.7 (3.1–12.8) 11 (8.2–12.8) 10.7 (3.1–12.8) Median RBC ×106/μL (range) 3.9 (2.3–5) 4.2 (3.5–5.8) 4.4 (2.5–8.7) 4.5 (2.9–7.3) 3.5 (2.1–3.9) 4.1 (2.8–5) 4.4 (3.6–5.5) 4.5 (1.0–5.7) Median WBC ×103/μL (range) 9.4 (4.6–22.4) 9.4 (3.7–23) 7.2 (5.5–10.3) 8.5 (3.9–17.4) 7.7 (3.9–18.5) 9.6 (5.0–14.7) 8.2 (5.3–15.2) 9.1 (4–14.8) Number of spleen grade 0 (%) ND ND 21 of 26 (80.7) 27 of 31 (90) 10 of 30 (33.3) 22 of 25 (88) 24 of 24 (100) 28 of 30 (93.3) Number of spleen grade 1 (%) ND ND 2 of 26 (7.6) 3 of 31 (10) 16 of 30 (53.3) 2 of 25 (8) 0 of 24 (0) 2 of 30 (6.6) Number of spleen grade 2 (%) ND ND 3 of 26 (11.5) 0 of 31 (0) 4 of 30 (13.3) 1 of 25 (4) 0 of 24 (0) 0 of 30 (0) Abbreviations: Hgb, hemoglobin; NA, not applicable; ND, not done; RBC, red blood cells; RDT, rapid diagnostic test; WBC, white blood cells.

3 of 31 (10) 16 of 30 (53.3) 2 of 25 (8) 0 of 24 (0) 2 of 30 (6.6) Number of spleen grade 2 (%) ND ND 3 of 26 (11.5) 0 of 31 (0) 4 of 30 (13.3) 1 of 25 (4) 0 of 24 (0) 0 of 30 (0) Abbreviations: Hgb, hemoglobin; NA, not applicable; ND, not done; RBC, red blood cells; RDT, rapid diagnostic test; WBC, white blood cells. Acute Uncomplicated Malaria Was Characterized by Anemia and Splenomegaly Acute malaria was characterized by significantly lower median Hb levels as well as red blood cell counts compared with controls at both sites (P < .005). Both of these parameters normalized during convalescence (Table 1). A complete data set for spleen grades was only collected from the participants in Chikwawa because this assessment was initiated after recruitment had already been completed in Blantyre. The majority of the acute malaria cases had a spleen grade 1 (53%), followed by grade 0 (33%) with as high as 13% qualifying for grade 2. This is consistent with the hypothesis that acute malaria is associated with splenomegaly. By the second follow up, the spleen grades of all malaria cases had normalized, suggesting that the splenomegaly observed during acute malaria is transient. In addition, as expected, at both sites none of the controls had a spleen grade of 2, and the majority of the controls had a spleen grade of 0 (Table 1).

healthy controls. Although some participants at both sites were found to be asymptomatically parasitemic 1 month after treatment (14.8% in Blantyre and 12% in Chikwawa), and even more at the 3-month follow-up stage, none of these participants developed any symptomatic malaria during the 3-month period of convalescence. The observed lower IL-10 levels in healthy controls compared with acute disease is consistent with results found in Malian children [28], adding weight to the hypothesis that IL-10 is required to negate immune-mediated pathology associated with high levels of proinflammatory cytokines such as IFN-γ, TNF-α, and IL-2 [5, 9]. The positive correlation between Tregs and IL-10 levels we have shown in this study is also consistent with what other studies have shown, both in mice models [30] and in humans [14], suggesting that Tregs are one of the main producers of the anti-inflammatory cytokine IL-10. Although we did not find a similar correlation between Tregs and levels of TGF-β in acute UCM, others have shown that levels of this cytokine also increase significantly during acute disease [21].

nomegaly. By the second follow up, the spleen grades of all malaria cases had normalized, suggesting that the splenomegaly observed during acute malaria is transient. In addition, as expected, at both sites none of the controls had a spleen grade of 2, and the majority of the controls had a spleen grade of 0 (Table 1). Absolute Tregs Counts Were Higher During Convalescence at the High Transmission Site When lymphocytes were presented as a percentage of total WBCs (Figure 2A and B) and when they were presented as absolute counts (Figure 2C and D), acute UCM was characterized by lymphopenia, which normalized in convalescence. CD4+ T cell-specific lymphopenia in acute UCM was only significant when the subset was presented as absolute counts at both sites, and this too normalized during convalescence (Figure 2C and D). When presented as percentages of total CD4+ T cells, acute UCM was characterized by higher than normal levels of Tregs, although the difference between levels observed during acute malaria and those in controls was only significant in Chikwawa (P = .0342) (Figure 2A and 2B). Regulatory T cell percentages significantly decreased in Chikwawa from the high levels observed during acute infection to low levels during the second follow up (P = .0494). Although a similar trend was observed in Blantyre, the decrease in percentage Tregs during convalescence was not significant. Figure 2. Proportions of median values of lymphocytes, CD4+ T cells and regulatory T cells (Tregs) at different stages of infection in children recruited with uncomplicated malaria and in healthy controls presented as percentage for the Blantyre (A) and Chikwawa (B) sites and presented as absolute counts for the Blantyre (C) and Chikwawa (D) sites.

n values of lymphocytes, CD4+ T cells and regulatory T cells (Tregs) at different stages of infection in children recruited with uncomplicated malaria and in healthy controls presented as percentage for the Blantyre (A) and Chikwawa (B) sites and presented as absolute counts for the Blantyre (C) and Chikwawa (D) sites. However, when the number of cells expressing the Tregs phenotype was calculated using lymphocyte counts from the differential WBC, there were no differences in the Tregs counts between acute stage and any of the convalescence stages and the controls in Blantyre (Figure 2C), whereas in Chikwawa, Tregs counts were significantly higher (P < .0001 at both 1-month and 3-month stages) in convalescence compared with the cell counts in acute infection (Figure 2D). Compared between sites, healthy controls in Blantyre had significantly higher percentages (P = .0247) and absolute counts (P = .0210) of Tregs compared with the controls in Chikwawa (Table 2). Table 2. Comparison of the Medians (Range) of Various Parameters Between Participants Recruited in Blantyre and Those Recruited in Chikwawa

ween sites, healthy controls in Blantyre had significantly higher percentages (P = .0247) and absolute counts (P = .0210) of Tregs compared with the controls in Chikwawa (Table 2). Table 2. Comparison of the Medians (Range) of Various Parameters Between Participants Recruited in Blantyre and Those Recruited in Chikwawa Group %Lymphocytes Lymphocyte Count/μL % CD4+ T Cell CD4+ T-Cell Counts/μL %Tregs Tregs Cell Counts/μL IFN-γ (pg/mL) TNF-α (pg/mL) IL-10 (pg/mL) TGF-β (pg/mL) Blantyre (acute) 25.05 (5.71–60.90) 3240 (1000–11,090) 35.27 (18.41–51.15) 1213 (424–3636) 10.26 (3.79–53.13) 138 (71–499) 174 (64–1776) 571 (146–2948) 355 (56–1500) 205 (56–731) Chikwawa (acute) 34.05 (12.20–65.99) 2550 (1000–8800) 36.15(18.62–47.78) 1020 (478–3379) 10.32 (3.38–55.05) 107 (39–328) 162 (69–870) 574 (204–4286) 313 (70–947) 80 (14–173) P value 0.0928 0.3205 0.6916 0.3461 0.5985 0.1163 0.5433 0.7336 0.7432 < 0.0001 Blantyre (1 month) 44.73 (11.85–74.51) 4500 (1400–9100) 39.48 (24.23–51.07) 1733 (358–6829) 10.21 (3.99–25.37) 169 (43–697) 159 (69–985) 1431 (310–4508) 113 (68–787) 147 (41–396) Chikwawa (1 month) 43.71 (29.34–62.26) 4850 (2800–8700) 37.61 (19.94–52.10) 1566 (760–3430) 9.15 (3.56–17.69) 788 (205–1065) 100 (61–262) 656(166–4134) 77 (41–279) 101 (20–248) P value 0.4892 0.5417 0.1479 0.6911 0.3121 < 0.0001 0.0957 0.1447 0.0017 0.0014 Blantyre (3 months) 50.67 (19.98–65.90) 3800 (2500–7400) 38.45 (23.31–52.24) 1550 (769–2706) 9.605 (4.18–35.24) 134 (61–369) 174 (58–1318) 728 (212–3012) 87 (49–773) 180(81–731) Chikwawa (3 months) 52.40 (15.18–75.02) 4500 (1600–7500) 35.20 (25.12–43.45) 1689 (598–2713) 6.68 (2.75–19.77) 813 (452–1150) 101 (59–311) 679 (350–3478) 87 (46–279) 93(20–149) P value 0.7739 0.4062 0.1281 0.8331 0.0597 0 < 0.0001 0.2041 0.5288 0.6817 < 0.0001 Blantyre (controls) 52.81 (19.58–82.81) 4400 (2000–11,300) 37.02 (18.05–49.86) 1,640 (361–2,709) 9.99 (3.63–29.92) 166 (41–439) 217 (79–1,142) 783 (188–2,762) 88 (59–329) 232 (99–1088) Chikwawa (controls) 47.78 (23.17–61.73) 4550 (2300–8600) 32.78 (23.37–47.94) 1447 (701–2828) 6.72 (2.76–17.06) 134 (39–305) 105 (59–723) 654(254–2,992) 107 (46–256) 133 (9–760) P value 0.2135 0.6553 0.0548 0.6625 0.0247 0.0210 0.0448 0.8286 0.7681 0.0005 Abbreviations: IFN, interferon; IL, interleukin; TGF, transforming growth factor; TNF, tumor necrosis factor; Tregs, regulatory T cells.

7.94) 1447 (701–2828) 6.72 (2.76–17.06) 134 (39–305) 105 (59–723) 654(254–2,992) 107 (46–256) 133 (9–760) P value 0.2135 0.6553 0.0548 0.6625 0.0247 0.0210 0.0448 0.8286 0.7681 0.0005 Abbreviations: IFN, interferon; IL, interleukin; TGF, transforming growth factor; TNF, tumor necrosis factor; Tregs, regulatory T cells. The P values presented in bold are the ones that showed significant differences when specific parameters were compared between the two sites. Acute Uncomplicated Malaria Was Characterized by High Concentrations of Pro- and Anti-inflammatory Cytokines Participants presenting with acute malaria at both sites were characterized by high levels of IFN-γ compared with the controls (P = .2683 for Blantyre and P = .7485 for Chikwawa) (Figure 3). The higher IFN-γ levels then normalized by the first month in convalescence at both sites, but the decrease was only significant among Chikwawa participants (P = .2641 for Blantyre and P = .0488 for Chikwawa). Levels of TNF-α (Figure 3) and TGF-β (Figure 4) did not differ between the 4 groups. Figure 3. Concentrations of interferon (IFN)-γ and tumor necrosis factor (TNF)-α in serum of sera collected at different stages of uncomplicated malaria infection and in healthy controls in Blantyre (A) and Chikwawa (B). Figure 4. Concentrations of interleukin (IL)-10 and transforming growth factor (TGF)-β in sera collected from children presenting with uncomplicated malaria at different stages of infection and in controls in Blantyre (A) and in Chikwawa (B).

Acute Uncomplicated Malaria Was Characterized by High Concentrations of Pro- and Anti-inflammatory Cytokines Participants presenting with acute malaria at both sites were characterized by high levels of IFN-γ compared with the controls (P = .2683 for Blantyre and P = .7485 for Chikwawa) (Figure 3). The higher IFN-γ levels then normalized by the first month in convalescence at both sites, but the decrease was only significant among Chikwawa participants (P = .2641 for Blantyre and P = .0488 for Chikwawa). Levels of TNF-α (Figure 3) and TGF-β (Figure 4) did not differ between the 4 groups. Figure 3. Concentrations of interferon (IFN)-γ and tumor necrosis factor (TNF)-α in serum of sera collected at different stages of uncomplicated malaria infection and in healthy controls in Blantyre (A) and Chikwawa (B). Figure 4. Concentrations of interleukin (IL)-10 and transforming growth factor (TGF)-β in sera collected from children presenting with uncomplicated malaria at different stages of infection and in controls in Blantyre (A) and in Chikwawa (B). Serum from children presenting with acute malaria at both sites had significantly higher levels of IL-10 compared with that of controls (P < .0001 for both sites) (Figure 4), and these high levels decreased significantly to normal levels during follow-up period even as early as 1 month after recruitment (P = .0008 for Blantyre and P < .0001 for Chikwawa).

cute malaria at both sites had significantly higher levels of IL-10 compared with that of controls (P < .0001 for both sites) (Figure 4), and these high levels decreased significantly to normal levels during follow-up period even as early as 1 month after recruitment (P = .0008 for Blantyre and P < .0001 for Chikwawa). When compared between sites, at 1 month convalescence stage, study participants in Blantyre had significantly higher concentrations of IL-10 (P = .0017) and TGF-β (P = .0014) compared with participants in Chikwawa (Table 2). Study participants in Blantyre had significantly higher levels of TGF-β compared with study participants in Chikwawa at all stages (P < .0001 in acute and at 3-month stage, P = .0014 at 1-month stage). Healthy controls in Blantyre had significantly higher concentrations of IFN-γ (P = .0448) and TGF-β (P = .0005) compared with healthy controls in Chikwawa (Table 2). Percentage of Tregs Correlates Strongly With Interleukin-10 Levels in Acute Disease There was significant linear correlation between the IL-10 levels and the percentage of Tregs (Figure 5) during the acute infection at both sites (Chikwawa r = 0.40, P = .02, Blantyre r = 0.389, P = .049), suggesting that Tregs are an important producer of IL-10. Figure 5. Correlation of regulatory T cells and interleukin (IL)-10 in samples collected from Blantyre (A) and Chikwawa (B). r is the Spearman's correlation, and the 95% coefficient interval is provided in the brackets.

0, P = .02, Blantyre r = 0.389, P = .049), suggesting that Tregs are an important producer of IL-10. Figure 5. Correlation of regulatory T cells and interleukin (IL)-10 in samples collected from Blantyre (A) and Chikwawa (B). r is the Spearman's correlation, and the 95% coefficient interval is provided in the brackets. DISCUSSION We found that children recruited with acute UCM presented with lymphopenia and splenomegaly and higher than normal levels of IFN-γ, TNF-α, and IL-10, which normalized 1 month into convalescence. We also found that 15% of the children recruited with acute UCM were parasitemic during convalescence, but none of these participants developed any further form of symptomatic malaria. When Tregs were presented as percentages of the CD4+ T cells, we found that they were significantly higher in acute UCM compared with levels in healthy controls only at Chikwawa site. These then normalized in convalescence with the difference being significant only during the second follow up. Although a similar trend was observed in Blantyre, the differences were not significant between any stages.

ere significantly higher in acute UCM compared with levels in healthy controls only at Chikwawa site. These then normalized in convalescence with the difference being significant only during the second follow up. Although a similar trend was observed in Blantyre, the differences were not significant between any stages. However, when the levels of Tregs were presented as absolute counts, we found that these were just as low in acute malaria as in healthy controls at both sites (Figure 2C and D), consistent with the results of a study conducted in Peru Amazon that reported low levels of Tregs in asymptomatic individuals and in acute symptomatic malaria [30]. In Blantyre, the low transmission area, the Tregs counts did not differ between any of the groups and stages (Figure 2C); however, in Chikwawa, the high transmission area, the Tregs counts were significantly (P < .0001) higher in convalescence compared with acute disease (Figure 2D). This observation is consistent with the results of a study conducted in The Gambia [14], but it is different from our study. The Gambian study found no differences between acute and convalescent values when the Tregs levels were presented as percentages. In the case of the Tregs percentage results of our study, when the results of a few outliers were not included in the analysis, the percentages for acute UCM were similar to those observed in convalescence and in controls. However, revisiting the clinical and demographic characteristics of those few outliers did not isolate anything special about them compared with the other study participants.

a few outliers were not included in the analysis, the percentages for acute UCM were similar to those observed in convalescence and in controls. However, revisiting the clinical and demographic characteristics of those few outliers did not isolate anything special about them compared with the other study participants. Healthy controls in Blantyre were found to have significantly higher percentages and counts of Tregs compared with those from Chikwawa. Acute UCM participants in Blantyre had almost identical levels, but these ended up having significantly lower levels during convalescence (Table 2). The investigators of the 2 Gambian studies [14, 26] had argued that acute malaria triggers expansion of Tregs, which end up persisting for some weeks to maintain immune homeostasis during the contraction phase of the effector response. This could explain the higher than normal percentages and counts of Tregs in participants from the high transmission area of Chikwawa during convalescence.

te malaria triggers expansion of Tregs, which end up persisting for some weeks to maintain immune homeostasis during the contraction phase of the effector response. This could explain the higher than normal percentages and counts of Tregs in participants from the high transmission area of Chikwawa during convalescence. The high levels of the proinflammatory cytokines IFN-γ and TNF-α during acute UCM have been observed in other studies before [4, 28]. This is consistent with the current understanding that high levels of these proinflammatory cytokines are required for the clearance of parasitemia at the early stage of the infection [5]. Ideally, this predominantly proinflammatory environment is supposed to be followed by the release of high levels of anti-inflammatory cytokines such as IL-10 and TGF-β, although this could favor further parasite maturation and multiplication. Therefore, the observed high levels of IL-10 during acute infection in our study have been described as ideal and are consistent with results of other studies [13, 28]. However, our study was not able to replicate results of studies that have shown significantly higher than normal levels of TGF-β in acute UCM [21].

odels [30] and in humans [14], suggesting that Tregs are one of the main producers of the anti-inflammatory cytokine IL-10. Although we did not find a similar correlation between Tregs and levels of TGF-β in acute UCM, others have shown that levels of this cytokine also increase significantly during acute disease [21]. We did not expect to find any child having parasitemia during convalescence at both sites because we anticipated that the antimalarial treatment would clear all parasites from the infected children. Although a majority of the parents and guardians of the study participants reported to be using insecticide-treated nets during convalescence period, we did not determine whether this parasitemia resulted from new infection or was due to the children failing to completely clear the initial parasitemia. These results need to be replicated in another study, nevertheless such parasitaemia prevalence post treatment might justify administering an additional antimalarial prophylaxis in the affected individuals.

temia resulted from new infection or was due to the children failing to completely clear the initial parasitemia. These results need to be replicated in another study, nevertheless such parasitaemia prevalence post treatment might justify administering an additional antimalarial prophylaxis in the affected individuals. CONCLUSIONS Further studies need to be done to establish the role of Tregs in the transition from UCM to the more severe clinical forms of malaria, namely CM and SMA. In addition, by having larger sample sizes and observing the study participants for as long as 1 year or even 2 years, researchers can provide a better understanding of Tregs perturbations at times when the children are purely parasitemic and asymptomatic, when they develop UCM, and when they transition from UCM to the life-threatening CM. In addition, with malnutrition known to affect cytokine production and T-cell differentiation [34], subsequent studies will have to take nutrition status of the participants into consideration. Although anorexia nervosa, an extreme form of dietary calorie restriction, was shown not to affect Tregs and cytokine profile of the affected participants [35], knowing the nutrition status of study participants could also rule out nutrition as a confounder in future studies.

tus of the participants into consideration. Although anorexia nervosa, an extreme form of dietary calorie restriction, was shown not to affect Tregs and cytokine profile of the affected participants [35], knowing the nutrition status of study participants could also rule out nutrition as a confounder in future studies. Acknowledgments We thank Helen Mangochi for her contribution in recruiting the study participants, blood sample collection, and in observing participants in follow-up studies. We are also very grateful to all the children who participated in this study and to their guardians or parents for consenting on behalf of their children. Author contributions. W. L. M., L. S. K. W., C. A. M., M. E. M., and R. S. H. conceived the study. W. L. M., L. S. K. W., R. K., and T. S. N. performed the investigations. T. S. N. and W. L. M. analyzed the data. W. L. M. and T. S. N. wrote the report. All authors contributed to the study design and reviewed the report. Financial support. This work was supported by the Malaria Capacity Development Consortium, with funding from the Wellcome Trust and the Bill and Melinda Gates Foundation. Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest.

INTRODUCTION Streptococcus pneumoniae is a leading cause of respiratory tract infections and bacterial invasive disease [1]. Bacterial nasopharyngeal carriage precedes infection, and various studies have pointed toward a causal link between carriage and disease [2]. Children younger than 5 years are a population vulnerable to pneumococcal disease, and they form a reservoir for other age groups. Reduced carriage of S pneumoniae decreases exposure of unvaccinated individuals, which results in substantial indirect effects [2, 3]. S pneumoniae is one of the main bacterial pathogens in acute otitis media (AOM). In Finland, estimations of AOM incidence vary from 370 to 630 per 1000 child-years [4] to >1100 per 1000 child-years [5]. Clinical trials have shown efficacy of pneumococcal conjugate vaccines (PCVs) against vaccine-type AOM, but they have generally shown little or no efficacy against all-cause AOM (vaccine efficacy range, 1%–7%) [5–9]; except for one study showing efficacy of 34% for an 11-valent pneumococcal protein D–conjugated vaccine in children [10].

ve shown efficacy of pneumococcal conjugate vaccines (PCVs) against vaccine-type AOM, but they have generally shown little or no efficacy against all-cause AOM (vaccine efficacy range, 1%–7%) [5–9]; except for one study showing efficacy of 34% for an 11-valent pneumococcal protein D–conjugated vaccine in children [10]. The 10-valent pneumococcal polysaccharide non-typeable Haemophilus influenzae (NTHi) protein D–conjugated vaccine (PHiD-CV, or PCV10) [11–13] was licensed in the European Union in March 2009 (Synflorix™, GSK Vaccines). In addition to the recommended 3+1 schedule, PHiD-CV has increasingly been administered in a 2+1 schedule when given as part of routine infant immunization programs; however, efficacy/effectiveness data for the 2+1 schedule are limited. FinIP, a large cluster-randomized study in Finland, was the first clinical trial in Europe to document the effectiveness of PHiD-CV against invasive pneumococcal disease (IPD) and the impact on outpatient antimicrobial purchases when administered as a 2+1 or 3+1 schedule [14, 15]. Here, we present results from a nested study that evaluated the effectiveness of PHiD-CV, given on different schedules, against nasopharyngeal carriage as an indication of the potential to induce herd effects. This is the largest carriage assessment study with PHiD-CV to date. We also evaluated the effectiveness of PHiD-CV against AOM (in parallel to another clinical trial in Latin America [16]) and PHiD-CV immunogenicity and safety.

s, against nasopharyngeal carriage as an indication of the potential to induce herd effects. This is the largest carriage assessment study with PHiD-CV to date. We also evaluated the effectiveness of PHiD-CV against AOM (in parallel to another clinical trial in Latin America [16]) and PHiD-CV immunogenicity and safety. METHODOLOGY Study Design and Participants This phase III double-blind trial (ClinicalTrials.gov identifier NCT00839254), conducted between February 2009 and December 2011 in 15 study clinics in Finland coordinated by the Tampere University Vaccine Research Centre, was nested within the larger cluster-randomized FinIP study (ClinicalTrials.gov identifier NCT00861380), which assessed the effectiveness of PHiD-CV against IPD [14]. In addition to the FinIP effectiveness objectives, this study evaluated PHiD-CV immunogenicity, safety, and effectiveness against carriage and AOM. Children aged 6 weeks to 18 months who had not received a pneumococcal vaccine, a hepatitis A or B vaccine, or any investigational or nonregistered product and who had no contraindications to immunization were eligible for enrollment. Enrollment ended when PHiD-CV was introduced into the Finnish National Vaccination Program (NVP) (September 2010); before then, there had been limited PCV use. The study was conducted in accordance with Good Clinical Practice principles and the Declaration of Helsinki. The protocol was approved by an independent ethics committee. For each participant, written informed consent was obtained from each patient's parent(s) or legal guardian(s).

Children aged 6 weeks to 18 months who had not received a pneumococcal vaccine, a hepatitis A or B vaccine, or any investigational or nonregistered product and who had no contraindications to immunization were eligible for enrollment. Enrollment ended when PHiD-CV was introduced into the Finnish National Vaccination Program (NVP) (September 2010); before then, there had been limited PCV use. The study was conducted in accordance with Good Clinical Practice principles and the Declaration of Helsinki. The protocol was approved by an independent ethics committee. For each participant, written informed consent was obtained from each patient's parent(s) or legal guardian(s). The study is registered at ClinicalTrials.gov (NCT00839254) and available at http://www.gsk-clinicalstudyregister.com/study/112595?study_ids=112595#ps). Study Vaccines and Procedures Participants received the PHiD-CV or a control vaccine (hepatitis B [Engerix-B™] for children <12 months of age or hepatitis A [Havrix™ 720 Junior] for children ≥12 months of age [both provided by GSK Vaccines]). PHiD-CV contains 10 serotype-specific pneumococcal polysaccharides conjugated to H influenzae protein D, tetanus toxoid, or diphtheria toxoid [14].

ontrol vaccine (hepatitis B [Engerix-B™] for children <12 months of age or hepatitis A [Havrix™ 720 Junior] for children ≥12 months of age [both provided by GSK Vaccines]). PHiD-CV contains 10 serotype-specific pneumococcal polysaccharides conjugated to H influenzae protein D, tetanus toxoid, or diphtheria toxoid [14]. Participants received study vaccines according to an age-appropriate schedule: the 2+1 or 3+1 schedule for children aged 6 weeks to 6 months at enrollment (infant cohorts); the 2+1 schedule for children aged 7–11 months at enrollment (7- to 11-month catch-up cohort); or 2 doses for children aged 12–18 months at enrollment (12- to 18-month catch-up cohort) (Figure 1). Routine pediatric vaccines, such as the diphtheria, tetanus, acellular pertussis, and inactivated polio virus/H influenzae type B (DTaP–IPV/Hib) and human rotavirus vaccines, were coadministered at 3 and 5 months of age; the DTaP–IPV/Hib vaccine was also coadministered at 11–12 months. Figure 1. Study design. Syringes indicate vaccination; vials indicate blood sample acquisition. Abbreviations: HAV, hepatitis A vaccine; HBV, hepatitis B vaccine; M, months; NP, nasopharyngeal swab; PHiD-CV, 10-valent pneumococcal polysaccharide nontypeable Haemophilus influenzae protein D–conjugated vaccine; W, weeks.

Figure 1. Study design. Syringes indicate vaccination; vials indicate blood sample acquisition. Abbreviations: HAV, hepatitis A vaccine; HBV, hepatitis B vaccine; M, months; NP, nasopharyngeal swab; PHiD-CV, 10-valent pneumococcal polysaccharide nontypeable Haemophilus influenzae protein D–conjugated vaccine; W, weeks. Randomization Clusters were randomized (2:2:1:1: PHiD-CV 3+1, PHiD-CV 2+1, control 3+1, control 2+1) using a blocking scheme, stratified according to cluster size (below/above average), urbanity (urban/rural), and Tampere University Vaccine Research Centre trial enrollment. For nested study participants, individual randomization codes were used, aligned with cluster randomization based on place of residence. Outcomes Study outcomes included PHiD-CV effect on S pneumoniae colonization (including all pneumococcal serotypes, vaccine serotypes, non-vaccine/non–vaccine-related serotypes, and vaccine-related serotypes, particularly 6A and 19A) and other bacteria (NTHi, Moraxella catarrhalis, and Staphylococcus aureus). Study outcomes also included all-cause AOM and all-cause AOM with antimicrobial prescription. We assessed effectiveness in reducing the number of children reporting ≥1 AOM episode, and in reducing the occurrence of all AOM episodes. We also evaluated PHiD-CV safety and reactogenicity for all participants and immunogenicity for a subset of them (see Supplementary Methods).

se AOM with antimicrobial prescription. We assessed effectiveness in reducing the number of children reporting ≥1 AOM episode, and in reducing the occurrence of all AOM episodes. We also evaluated PHiD-CV safety and reactogenicity for all participants and immunogenicity for a subset of them (see Supplementary Methods). Carriage Assessment Study personnel collected nasopharyngeal samples from all participants using a pediatric rayon-tipped swab at the post–primary vaccination and post-booster visits (Figure 1). Pre-vaccination swabs were collected from the infant immunogenicity subset and from all children in the catch-up cohorts. All samples were transferred to STGG (skim milk, tryptone, glucose, and glycerol) transport medium [17] and stored below –65°C before transport to the laboratory at the National Institute for Health and Welfare in Oulu, Finland. A detailed description of culture, identification, and serotyping is provided in Supplementary Methods. AOM Assessment Parents were asked by automated text message every 2 weeks if their child had had a physician-confirmed AOM diagnosis. If there was no reply, a reminder message was sent after 24 hours; after 48 hours, the parents were contacted by a study nurse by telephone. If no contact could be made, AOM status was checked at the next study visit.

ed by automated text message every 2 weeks if their child had had a physician-confirmed AOM diagnosis. If there was no reply, a reminder message was sent after 24 hours; after 48 hours, the parents were contacted by a study nurse by telephone. If no contact could be made, AOM status was checked at the next study visit. For cases reported by the parents as physician-confirmed AOM, regardless of documentation in the medical records or other source documents, parents were asked to report AOM and antimicrobial prescriptions in an AOM questionnaire. Finland's national guidelines recommend antibiotics, when AOM diagnosis is certain [18], which are only available upon prescription. Statistical Analysis The encompassing FinIP study was powered to show significant differences (α = .05) in the rate of vaccine-type IPD between the PHiD-CV 3+1 and control groups in the infant cohort. The nested study reported here was not designed to draw any formal statistical conclusions, but it allowed descriptive assessments of the AOM, carriage, safety, and immunogenicity objectives without predefined success criteria. For carriage, assuming 1200 evaluable children per group and a 12.2% incidence rate of vaccine-type carriage in the control group, the study had 80% power to detect a vaccine effectiveness (VE) of 37%. For AOM, assuming 4500 evaluable children in the infant cohort (randomized 1:1:1) and an AOM incidence rate of 0.55 in the control group, the study had 80% power to detect a VE of 19.6%.

incidence rate of vaccine-type carriage in the control group, the study had 80% power to detect a vaccine effectiveness (VE) of 37%. For AOM, assuming 4500 evaluable children in the infant cohort (randomized 1:1:1) and an AOM incidence rate of 0.55 in the control group, the study had 80% power to detect a VE of 19.6%. Informative conclusions on statistical significance of the effectiveness were based on the positive lower limit of its non-adjusted 95% confidence interval (CI) and should be interpreted with caution linked to the descriptive character of the end points. Carriage and safety analyses were performed for the total vaccinated cohort (TVC), which comprised all children who received ≥1 vaccine dose according to treatment actually received. The percentage of participants with a positive nasopharyngeal sample and the 95% CI were calculated, as were VE, estimated as (1 – relative risk)*100, with the 95% CIs, derived using a conditional exact method. Across-visits results include the pre-vaccination visit.

ccine dose according to treatment actually received. The percentage of participants with a positive nasopharyngeal sample and the 95% CI were calculated, as were VE, estimated as (1 – relative risk)*100, with the 95% CIs, derived using a conditional exact method. Across-visits results include the pre-vaccination visit. We also evaluated cumulative acquisition, defined as the occurrence of bacterial pathogens or serotypes not detected at any of the previous time points; VE were calculated with 95% CIs (Supplementary Table 4). For the infant cohort, as pre-vaccination swabs were collected only from the immunogenicity subset, cumulative acquisition from pre- to 1 month post-primary vaccination was analyzed separately (Supplementary Table 4). For the full infant cohort, the first cumulative acquisition data from infants at the age of 11–12 months are presented, and 1 month post-primary vaccination (infant age 6 months) was the reference time point.

ive acquisition from pre- to 1 month post-primary vaccination was analyzed separately (Supplementary Table 4). For the full infant cohort, the first cumulative acquisition data from infants at the age of 11–12 months are presented, and 1 month post-primary vaccination (infant age 6 months) was the reference time point. AOM analyses were performed for the TVC for AOM effectiveness (excluding misrandomized children who did not receive the treatment assigned to their cluster). A new AOM episode was defined if it occurred ≥30 days after onset of the previous episode. We report results for AOM with level 1 diagnostic certainty: parent-reported physician-diagnosed AOM in the infant cohort. The number of participants in the catch-up cohorts was too low to obtain meaningful results. The analyzed follow-up time for the infant TVC started on the day of first vaccination and ended at the infants' last visit (planned at 18–22 months of age). A negative binomial model taking into account the cluster effect and stratification factors was used to derive VE against AOM as (1 – relative risk)*100 with 95% CIs, as detailed previously [14]. Blood samples were planned to be collected for the approximately 1500 first enrolled participants (immunogenicity subset). Immunogenicity analyses were performed on the according-to-protocol immunogenicity cohort, which comprised all evaluable subset participants (who met all eligibility criteria, complied with protocol-defined procedures/intervals, and met no elimination criteria) with results from ≥1 assay available.

unogenicity subset). Immunogenicity analyses were performed on the according-to-protocol immunogenicity cohort, which comprised all evaluable subset participants (who met all eligibility criteria, complied with protocol-defined procedures/intervals, and met no elimination criteria) with results from ≥1 assay available. Statistical analyses were performed using Statistical Analysis Systems (SAS Institute, Inc., Cary, North Carolina) version 9.22 or SAS Drug Development (SDD) and the StatXact-8.0 procedure (Cytel Software Corp, Cambridge, Massachusetts) on SAS.

unogenicity subset). Immunogenicity analyses were performed on the according-to-protocol immunogenicity cohort, which comprised all evaluable subset participants (who met all eligibility criteria, complied with protocol-defined procedures/intervals, and met no elimination criteria) with results from ≥1 assay available. Statistical analyses were performed using Statistical Analysis Systems (SAS Institute, Inc., Cary, North Carolina) version 9.22 or SAS Drug Development (SDD) and the StatXact-8.0 procedure (Cytel Software Corp, Cambridge, Massachusetts) on SAS. RESULTS Study Participants A total of 6178 infants and toddlers were enrolled in 50 clusters (Figure 2). Demographic characteristics were comparable between the PHiD-CV and control groups (Supplementary Table 1). The mean follow-up time was 18 months. The immunogenicity subset comprised 1635 children (855 infants and 780 toddlers; Figure 2). Figure 2. Participant flow chart. Because of an error in treatment number allocation, 3 children had 2 subject numbers allocated each; thus, the actual number of enrolled children was 6178 instead of 6181, corresponding to 5092 children instead of 5093 in the infant total vaccinated cohort for carriage/safety and 1082 instead of 1084 in the catch-up total vaccinated cohort for carriage/safety. Data for these children were recorded only once for the subject number corresponding to the time of participation. Abbreviations: AOM, acute otitis media; ATP, according-to-protocol; M, months; N, number of children in the specified group; PHiD-CV, 10-valent pneumococcal polysaccharide nontypeable Haemophilus influenzae protein D–conjugated vaccine; TVC, total vaccinated cohort; W, weeks.

corresponding to the time of participation. Abbreviations: AOM, acute otitis media; ATP, according-to-protocol; M, months; N, number of children in the specified group; PHiD-CV, 10-valent pneumococcal polysaccharide nontypeable Haemophilus influenzae protein D–conjugated vaccine; TVC, total vaccinated cohort; W, weeks. Because of a randomization error, 976 infants did not receive the treatment assigned to their cluster. These misrandomized infants were reallocated to the groups corresponding to the vaccination they actually received for the TVC for carriage/safety and immunogenicity according-to-protocol cohort (analyses per individual randomization) but were excluded from the TVC for AOM effectiveness (cluster-randomized analysis), which substantially affected the 2+1 PHiD-CV group for AOM assessment (371 misrandomized children) [14].

ly received for the TVC for carriage/safety and immunogenicity according-to-protocol cohort (analyses per individual randomization) but were excluded from the TVC for AOM effectiveness (cluster-randomized analysis), which substantially affected the 2+1 PHiD-CV group for AOM assessment (371 misrandomized children) [14]. Effect of PHiD-CV Vaccination on Nasopharyngeal Carriage of S pneumoniae Infant Groups The most prevalent pneumococcal serotypes in the control group were 6B, 19F, 23F, 6A, and 11A (Figure 3). PHiD-CV vaccination substantially reduced vaccine-serotype carriage. The highest VE were observed following the booster dose: 56.1% at 18–22 months of age in the 3+1 group and 37.9% at 14–15 months of age in the 2+1 group. This carriage reduction was mainly due to decreased carriage of serotypes 6B, 14, 19F, and 23F. With increasing age and the time elapsed after booster vaccination, a trend for increased carriage of non-vaccine/non–vaccine-related serotypes was observed in all the groups, with no major differences between the groups. Altogether, these changes resulted in a net reduction of overall pneumococcal carriage in infants who received pneumococcal vaccination according to either schedule; VE against carriage of all pneumococci increased with age, up to 28.3% and 15.0% for the 3+1 and 2+1 groups, respectively (Figures 4 and 5; Supplementary Table 2). The occurrence of the most common non-vaccine/non–vaccine-related serotypes with a prevalence of >3% is shown in Figure 3B. Of note, prevalence of serotypes 3 and 6C were low (maximum 0.5% and 1.4%, respectively, in the control group). Figure 3. Percentage of children with nasopharyngeal colonization across all visits (total vaccinated cohort for carriage). The occurrence of S pneumoniae serotypes in nasopharyngeal swabs across all visits (including baseline) is shown. No carriage for vaccine serotype 1 and 5 was observed. Abbreviations: M, months; PHiD-CV, 10-valent pneumococcal polysaccharide nontypeable Haemophilus influenzae protein D–conjugated vaccine; TVC, total vaccinated cohort.

f S pneumoniae serotypes in nasopharyngeal swabs across all visits (including baseline) is shown. No carriage for vaccine serotype 1 and 5 was observed. Abbreviations: M, months; PHiD-CV, 10-valent pneumococcal polysaccharide nontypeable Haemophilus influenzae protein D–conjugated vaccine; TVC, total vaccinated cohort. Figure 4. Percentage of infants with nasopharyngeal colonization (infant total vaccinated cohort for carriage). The percentages of infants, enrolled between 6 weeks and 6 months of age, colonized with S pneumoniae, NTHi, M catarrhalis, or S aureus were assessed at different ages: 3 months (before vaccination, only for a subset of infants), 6 months (1 month after primary vaccination), 11–12 months (before booster), 14–15 months (3 months after booster), and 18–22 months (7–12 months after booster). Mean values with 95% confidence intervals are shown. Vaccine-type S pneumoniae serotypes were 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F; non-vaccine/non–vaccine-related serotypes were any S pneumoniae serotype, excluding vaccine serotypes and excluding serotypes belonging to the same serogroup as vaccine serotypes. Abbreviations: m, months; PHiD-CV, p10-valent pneumococcal polysaccharide nontypeable Haemophilus influenzae protein D–conjugated vaccine.

; non-vaccine/non–vaccine-related serotypes were any S pneumoniae serotype, excluding vaccine serotypes and excluding serotypes belonging to the same serogroup as vaccine serotypes. Abbreviations: m, months; PHiD-CV, p10-valent pneumococcal polysaccharide nontypeable Haemophilus influenzae protein D–conjugated vaccine. Figure 5. Vaccine effectiveness against nasopharyngeal carriage at given time points (infant total vaccinated cohort for carriage). Vaccine efficacy against nasopharyngeal carriage of S pneumoniae, NTHi, M catarrhalis, and S aureus was assessed at different ages: 6 months (1 month after primary vaccination), 11–12 months (before booster), 14–15 months (3 months after booster), and 18–22 months (7–12 months after booster). Mean values with 95% confidence intervals are shown. Vaccine-type S pneumoniae serotypes were 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F; non-vaccine/non–vaccine-related serotypes were any S pneumoniae serotype, excluding the vaccine serotypes and any serotype that belonged to the same serogroup as the vaccine serotype. Abbreviation: PHiD-CV, 10-valent pneumococcal polysaccharide nontypeable Haemophilus influenzae protein D–conjugated vaccine.

, and 23F; non-vaccine/non–vaccine-related serotypes were any S pneumoniae serotype, excluding the vaccine serotypes and any serotype that belonged to the same serogroup as the vaccine serotype. Abbreviation: PHiD-CV, 10-valent pneumococcal polysaccharide nontypeable Haemophilus influenzae protein D–conjugated vaccine. For vaccine-related serotype 19A, the carriage prevalence was low, with a maximum colonization rate of 3.4% across all visits in the control group (Figure 3). Nevertheless, consistently positive VE against 19A carriage was observed at all post-vaccination time points in the 3+1 group, with a statistically significant VE at the 18- to 22-month time point and across all visits. Point estimates of VE against 19A were in the same range as VE against vaccine-type carriage at the 18- to 22-month time point. For the 2+1 group, a trend for reduction of serotype 19A carriage was observed, but no significant effectiveness was shown at any time point. Although the prevalence of vaccine-related serotype 6A was higher than that of serotype 19A, no consistent effect against 6A carriage was observed for either PHiD-CV schedule (Figure 5; Supplementary Table 2). Trends for VE against cumulative acquisition were similar to those against carriage occurrence at a given time point, with VE against cumulative acquisition of vaccine serotypes ranging across visits from 38.2% to 45.5% for the 3+1 group and from 35.8% to 40.4% for the 2+1 group. VE against cumulative acquisition of S pneumoniae, regardless of serotype, ranged from 14.8% to 16.4% for the 3+1 group and from 12.5% to 14.5% for the 2+1 group (Supplementary Table 3). VE against cumulative acquisition from pre- to one month post-primary vaccination, assessed for the immunogenicity subset with pre-vaccination swabs available, is presented in Supplementary Table 4.

serotype, ranged from 14.8% to 16.4% for the 3+1 group and from 12.5% to 14.5% for the 2+1 group (Supplementary Table 3). VE against cumulative acquisition from pre- to one month post-primary vaccination, assessed for the immunogenicity subset with pre-vaccination swabs available, is presented in Supplementary Table 4. Catch-Up Groups A trend toward VE against vaccine-type carriage was observed in the 7- to 11-month and 12- to 18-month catch-up cohorts (Supplementary Tables 5 and 6). Effect of PHiD-CV Vaccination on Carriage of Other Bacterial Pathogens NTHi carriage was low, with 10.6% of infants in the control group colonized at 18–22 months of age. Carriage of M catarrhalis or S aureus was more common. No differences in carriage of these pathogens were seen between the PHiD-CV and control groups (Figures 4 and 5).

accination on Carriage of Other Bacterial Pathogens NTHi carriage was low, with 10.6% of infants in the control group colonized at 18–22 months of age. Carriage of M catarrhalis or S aureus was more common. No differences in carriage of these pathogens were seen between the PHiD-CV and control groups (Figures 4 and 5). Effect of PHiD-CV Vaccination on AOM At least 1 AOM episode was reported for 63.0% (1163 of 1846) of the infants in the 3+1 group, 62.5% (589 of 942) in the 2+1 group, compared to 67.1% (892 of 1329) in the control group. VE in reducing the number of infants for whom ≥1 AOM episode was reported were 6.1% (95% CI, –2.7 to 14.1) for the 3+1 group and 7.4% (95% CI, –2.8 to 16.6) for the 2+1 group. VE in preventing all AOM episodes were 2.8% (95% CI, –9.5 to 13.9) for the 3+1 group and 10.2% (95% CI, –4.1 to 22.9) for the 2+1 schedule (Table 1). VE for both schedules combined were 6.7% (95% CI, –1.3 to 14.0) for reducing the number of infants for whom ≥1 AOM episode was reported and 6.4% (95% CI, –5.5 to 17.2) for preventing all AOM episodes (Table 2). Table 1. Vaccine Effectiveness Against Acute Otitis Media (Infant TVC for AOM Analysis)

1 schedule (Table 1). VE for both schedules combined were 6.7% (95% CI, –1.3 to 14.0) for reducing the number of infants for whom ≥1 AOM episode was reported and 6.4% (95% CI, –5.5 to 17.2) for preventing all AOM episodes (Table 2). Table 1. Vaccine Effectiveness Against Acute Otitis Media (Infant TVC for AOM Analysis) AOM Episodes Infant PHiD-CV Infant Controls (N = 1329; FU = 2012) 3+1 (N = 1846; FU = 2765) 2+1 (N = 942; FU = 1417) n n/FU VE (% [95% CI]) n n/FU VE (% [95% CI]) n n/FU ≥1 1163 421 6.1 (–2.7 to 14.1) 589 416 7.4 (–2.8 to 16.6) 892 443 ≥1, with antibiotics 1133 410 6.1 (–2.8 to 14.2) 579 409 6.4 (–4.0 to 15.8) 867 431 All 2753 996 2.8 (–9.5 to 13.9) 1375 970 10.2 (–4.1 to 22.9) 2033 1011 All, with antibiotics 2662 963 2.0 (–11.3 to 13.8) 1322 933 10.8 (–5.5 to 24.7) 1964 976 Analysis was performed on the total vaccinated cohort for acute otitis media effectiveness. Abbreviations: AOM, acute otitis media; CI, confidence interval; FU, sum of follow-up periods, expressed in years; N, total number of children in the specified cohort; n, number of children or episodes; n/FU, incidence of children with ≥1 AOM episode or incidence of all AOM episodes, expressed in 1000 child-years; PHiD-CV, 10-valent pneumococcal polysaccharide nontypeable Haemophilus influenzae protein D–conjugated vaccine; TVC, total vaccinated cohort; VE, vaccine effectiveness.

rt; n, number of children or episodes; n/FU, incidence of children with ≥1 AOM episode or incidence of all AOM episodes, expressed in 1000 child-years; PHiD-CV, 10-valent pneumococcal polysaccharide nontypeable Haemophilus influenzae protein D–conjugated vaccine; TVC, total vaccinated cohort; VE, vaccine effectiveness. The vast majority (>97%) of the infants with AOM received antimicrobial treatment. VE against AOM with antimicrobial prescription was in line with the corresponding overall effectiveness against AOM (Table 2). Table 2. Vaccine Effectiveness Against Acute Otitis Media for both Schedules Combined AOM Episodes Infant PHiD-CV (N = 2788; FU = 4182) Infant Controls (N = 1329; FU = 2012) n n/FU VE (% [95% CI]) n n/FU ≥1 1752 419 6.7 (–1.3 to 14.0) 892 443 All 4128 987 6.4 (–5.5 to 17.2) 2033 1011 Analysis was performed on the total vaccinated cohort for acute otitis media effectiveness. Abbreviations: AOM, acute otitis media; CI, confidence interval; FU, sum of follow-up periods, expressed in years; N, total number of children in the specified cohort; n, number of children or episodes; n/FU, incidence of children with ≥1 AOM episode or incidence of all AOM episodes, expressed in 1000 child-years; PHiD-CV, 10-valent pneumococcal polysaccharide nontypeable Haemophilus influenzae protein D–conjugated vaccine; VE, vaccine effectiveness.

children in the specified cohort; n, number of children or episodes; n/FU, incidence of children with ≥1 AOM episode or incidence of all AOM episodes, expressed in 1000 child-years; PHiD-CV, 10-valent pneumococcal polysaccharide nontypeable Haemophilus influenzae protein D–conjugated vaccine; VE, vaccine effectiveness. A post-hoc analysis comparing pre- and post-booster effectiveness suggested higher effectiveness post-booster in reducing the number of infants reporting ≥1 AOM episode, while VE against all episodes seemed to be lower post-booster (Table 3). Table 3. Vaccine Effectiveness Against Acute Otitis Media Before and After Booster Vaccination AOM Episodes Infant PHiD-CV Infant Controls (N = 1291; FU = 1992) 3+1 (N = 1783; FU = 2735) 2+1 (N = 917; FU = 1406) n n/FU VE (% [95% CI]) n n/FU VE (% [95% CI]) n n/FU ≥1, before booster 628 230 4.5 (–18.2 to 22.6) 327 233 7.9 (–13.6 to 26.0) 485 244 ≥1, after booster 423 155 8.6 (–5.7 to 20.9) 216 154 12.0 (–7.1 to 28.2) 347 174 All, before booster 1082 396 4.5 (–9.7 to 16.6) 549 390 10.2 (–8.1 to 26.1) 812 408 All, after booster 1640 560 1.9 (–11.6 to 14.3) 824 586 8.2 (–5.6 to 20.9) 1205 605 Analysis was performed on the total vaccinated cohort for acute otitis media analysis considering only infants with the full vaccination schedule (4 doses for the 3+1 or 3 doses for the 2+1 schedule).

549 390 10.2 (–8.1 to 26.1) 812 408 All, after booster 1640 560 1.9 (–11.6 to 14.3) 824 586 8.2 (–5.6 to 20.9) 1205 605 Analysis was performed on the total vaccinated cohort for acute otitis media analysis considering only infants with the full vaccination schedule (4 doses for the 3+1 or 3 doses for the 2+1 schedule). Abbreviations: AOM, acute otitis media; CI, confidence interval; FU, sum of follow-up periods, expressed in years; N, total number of children in the specified cohort; n, number of children or episodes; n/FU, incidence of children with ≥1 AOM episode or incidence of all AOM episodes, expressed in 1000 child-years; PHiD-CV, 10-valent pneumococcal polysaccharide nontypeable Haemophilus influenzae protein D–conjugated vaccine; VE, vaccine effectiveness. Immunogenicity Post-primay vaccination, for each of the vaccine serotypes, ≥79.3% of the infants who received the 3+1 schedule and ≥66.3% of those who received the 2+1 schedule had antibody concentrations of ≥0.2 µg/mL. Post-booster, these percentages were ≥94.7% and ≥96.9% for the 3+1 and 2+1 groups, respectively. Antibody geometric mean concentrations (GMCs) and opsonophagocytic assay (OPA) geometric mean titers (GMTs) were higher post-booster than post-primary vaccination, except for serotype 6B GMTs in the PHiD-CV 3+1 group, which remained in the same range. Antibody GMCs and OPA GMTs tended to be lower in the 2+1 group than in the 3+1 group for most serotypes, especially post-primary vaccination (Supplementary Tables 7 and 8).

Ts) were higher post-booster than post-primary vaccination, except for serotype 6B GMTs in the PHiD-CV 3+1 group, which remained in the same range. Antibody GMCs and OPA GMTs tended to be lower in the 2+1 group than in the 3+1 group for most serotypes, especially post-primary vaccination (Supplementary Tables 7 and 8). For each of the vaccine serotypes, the percentages of children with antibody concentrations of ≥0.2 µg/mL were ≥60.3% in the 7- to 11-month catch-up group and ≥86.2% in the 12- to 18-month catch-up group 1 month after dose 2 and ≥90.5% for the 7- to 11-month catch-up group post-booster. In the 7- to 11-month group, higher GMCs were observed post-booster than those post-primary vaccination, except the GMCs for serotype 4, which remained in the same range (Supplementary Table 7). Safety and Reactogenicity The PHiD-CV was well-tolerated and showed an acceptable safety profile (Supplementary Figure 1; Supplementary Table 9). Reactogenicity was expected to be higher for PHiD-CV than the control vaccines because of the known low reactogenicity of the hepatitis vaccine. Dose 2 of the infant 3+1 schedule was given without concomitant vaccinations and thus illustrates the reactogenicity of PHiD-CV vaccination alone.

e 1; Supplementary Table 9). Reactogenicity was expected to be higher for PHiD-CV than the control vaccines because of the known low reactogenicity of the hepatitis vaccine. Dose 2 of the infant 3+1 schedule was given without concomitant vaccinations and thus illustrates the reactogenicity of PHiD-CV vaccination alone. Serious adverse events considered by the investigator to be causally related to vaccination were reported for 4 infants in the PHiD-CV 3+1 group (sepsis with non-specified etiology in 1 infant, pyrexia in 1 infant, and convulsion in 2 infants), for none in the PHiD-CV 2+1 group, for 2 in the infant control groups (petit mal epilepsy in 1 infant and pyrexia in 1 infant), and for none in the catch-up groups (Supplementary Table 9). One fatal serious adverse event (sudden infant death, not considered vaccination related) was reported in the infant PHiD-CV 2+1 group.

none in the PHiD-CV 2+1 group, for 2 in the infant control groups (petit mal epilepsy in 1 infant and pyrexia in 1 infant), and for none in the catch-up groups (Supplementary Table 9). One fatal serious adverse event (sudden infant death, not considered vaccination related) was reported in the infant PHiD-CV 2+1 group. DISCUSSION In this cluster-randomized study, nasopharyngeal carriage of vaccine-type pneumococci and their acquisition was reduced after PHiD-CV vaccination. Effectiveness against vaccine-type pneumococcal carriage was observed with both the 3+1 and 2+1 infant schedules, although no VE was observed 1 month after the primary vaccination (infant age 6 months) for the 2+1 schedule. VE for the 3+1 and 2+1 schedules were at similar ranges 6 months post-primary vaccination (age 11–12 months) and 3 months post-booster (age 14–15 months). At the 18- to 22-month time point, VE continued to increase only for those who received the 3+1 schedule and tended to be higher than for those who received the 2+1 schedule. Our results suggest that PHiD-CV vaccination of children may induce herd protection against vaccine-type disease. This hypothesis can be supported by indirect carriage effects observed in FinIP [19], reports showing a decline in vaccine-type carriage [20] and vaccine-type IPD [21–24] across all age groups after PHiD-CV vaccination of children, and preliminary data showing decreases in pneumonia in unvaccinated children not eligible for the national vaccination program [25].

carriage effects observed in FinIP [19], reports showing a decline in vaccine-type carriage [20] and vaccine-type IPD [21–24] across all age groups after PHiD-CV vaccination of children, and preliminary data showing decreases in pneumonia in unvaccinated children not eligible for the national vaccination program [25]. The reduction in vaccine-type carriage was mainly a result of decreased carriage of the most prevalent serotypes, 6B, 14, 19F, and 23F. Post-vaccination, antibody levels against serotypes 6B and 23F were low, consistent with previous reports [10, 13]. Nevertheless, we observed reduced carriage of both serotypes. Furthermore, 100% PHiD-CV effectiveness against serotype 6B IPD has been reported [14], but the antibody levels needed to confer protection against IPD may be lower than those for nasopharyngeal carriage. We also noted reduced carriage of vaccine-related serotype 19A with the 3+1 PHiD-CV schedule, although the carriage rates were low and, thus, the CIs were large. This finding may fit with observations after PHiD-CV infant immunization showing decreased serotype 19A IPD [26–29]. We observed positive trends but no significant effectiveness against 19A colonization in the 2+1 group.

with the 3+1 PHiD-CV schedule, although the carriage rates were low and, thus, the CIs were large. This finding may fit with observations after PHiD-CV infant immunization showing decreased serotype 19A IPD [26–29]. We observed positive trends but no significant effectiveness against 19A colonization in the 2+1 group. No consistent effect on the carriage of vaccine-related serotype 6A was observed for the 2+1 schedule or for the 3+1 PHiD-CV schedule, although this serotype had a higher prevalence than 19A, and a reduction of 6A IPD after introduction of the PHiD-CV into the Finnish NVP was reported recently [29]. Similarly, some of the early PCV7 trials did not find a clear impact of vaccination on 6A carriage [30], whereas dramatically decreased 6A carriage was observed after widespread adoption of PCV7 [31], in addition to significant herd protection against serotype 6A IPD [32, 33]. Increases in non–vaccine-serotype carriage were limited and observed only at later study visits. Similar trends were noted in previous PHiD-CV studies [34, 35]. In contrast, PCV7 studies showed early and pronounced replacement [36]. Although the degree of replacement in nasopharyngeal carriage may be relative to the degree of vaccine-type reduction, replacement is also affected by changes in the entire microbiome, which, in addition to being under vaccine pressure, are also affected by viral coinfections, antibiotic selection of serotypes commonly resistant to antibiotics, clonal mutants quickly spreading, secular trends in serotype prevalence, etc.

ype reduction, replacement is also affected by changes in the entire microbiome, which, in addition to being under vaccine pressure, are also affected by viral coinfections, antibiotic selection of serotypes commonly resistant to antibiotics, clonal mutants quickly spreading, secular trends in serotype prevalence, etc. We found no impact of PHiD-CV vaccination on NTHi carriage, which is consistent with previous PHiD-CV reports [37, 38]. The 11-valent PHiD-CV predecessor vaccine had a 38.6% (95% CI, –6.3 to 64.6) reduction in NTHi carriage 3 months after a booster, but the difference between groups had disappeared by 12 months post-booster [35]. No significant effectiveness in reducing AOM rates was observed for the 3+1 or 2+1 regimen. Nevertheless, a low but consistent trend for effectiveness in reducing AOM was observed for each PHiD-CV vaccination regimen. No major differences between the 3+1 and 2+1 schedules were seen; however, this study was not designed to detect schedule differences. PHiD-CV VE against hospital-treated pneumonia [39] and against outpatient purchases of antimicrobial drugs [15] were similar for both schedules.

ed for each PHiD-CV vaccination regimen. No major differences between the 3+1 and 2+1 schedules were seen; however, this study was not designed to detect schedule differences. PHiD-CV VE against hospital-treated pneumonia [39] and against outpatient purchases of antimicrobial drugs [15] were similar for both schedules. A possible limitation is the collection of information about physician-diagnosed AOM from the parents. Nevertheless, Finland has well-established diagnostic and management guidelines for AOM [18], and the observed incidences were similar to those reported in a Finnish PCV7 efficacy study [5]. Moreover, most participants with AOM received an antimicrobial prescription (>97%), which is in line with national guidelines that recommend antibiotic treatment when an AOM diagnosis is certain [18]. The AOM study end point could be regarded as an antimicrobial-treated AOM end point. Last, because this study was part of a cluster-randomized study, the observed effectiveness against carriage and AOM could be higher than the vaccine's efficacy as a result of herd protection within the vaccinated clusters. However, vaccination uptake rates per cluster were low to moderate (8%–61%) [14], and carriage assessment was completed within 2 years after start of the study, which thus limits the possibility of observing a herd effect on the vaccine recipients.

efficacy as a result of herd protection within the vaccinated clusters. However, vaccination uptake rates per cluster were low to moderate (8%–61%) [14], and carriage assessment was completed within 2 years after start of the study, which thus limits the possibility of observing a herd effect on the vaccine recipients. PHiD-CV administered according to different age-appropriate schedules resulted in an acceptable safety and immunogenicity profile. Antibody GMCs and OPA GMTs were higher after the 3-dose than the 2-dose primary schedule; these differences diminished post-booster vaccination. CONCLUSIONS The observed effectiveness against nasopharyngeal carriage of vaccine-type pneumococci indicates the potential of PHiD-CV to induce a direct effect and herd protection against vaccine-type pneumococcal disease. Only a limited increase in the carriage of non–vaccine-type pneumococcal serotypes was seen at later time points, which resulted in overall decreased carriage of all pneumococci. After infant PHiD-CV vaccination, we noted a trend toward decreased numbers of parent-reported physician-diagnosed AOM episodes. PHiD-CV had an acceptable safety profile.

CONCLUSIONS The observed effectiveness against nasopharyngeal carriage of vaccine-type pneumococci indicates the potential of PHiD-CV to induce a direct effect and herd protection against vaccine-type pneumococcal disease. Only a limited increase in the carriage of non–vaccine-type pneumococcal serotypes was seen at later time points, which resulted in overall decreased carriage of all pneumococci. After infant PHiD-CV vaccination, we noted a trend toward decreased numbers of parent-reported physician-diagnosed AOM episodes. PHiD-CV had an acceptable safety profile. Supplementary Material Supplementary Data Acknowledgments We thank the children and their parents for their participation in the study. We also acknowledge the investigators (Anitta Ahonen, Iina Volanen, Tiina Karppa, Tiina Korhonen, Pia-Maria Lagerstrom-Tirri, and Ville Peltola) and their clinical teams for their contributions to the study and their support and care of the participants/patients. We thank the global and regional clinical teams of GSK Vaccines (Sophie Ledant, Severine Fanchon, and Markku Pulkkinen) for their contributions to the study and the scientific writers (Mireille Venken, Kristel Vercauteren, and Liliana Manciu) for clinical protocol and clinical report writing. We also thank the scientists involved in the analysis of the carriage results (Mervi Mannila, Teija Jaakkola, Aili Hökkä, Anne Hautala, Annika Saukkoriipi, Anu Ojala, Eeva-Liisa Korhonen, Eila Salminen, Hannele Huumonen, Marika Loukkola, Sara Kuusiniemi, Taina Poikela, Terhi Äijälä, Tiina Nokela, Tuula Heiskanen, Thierry Pascal, Koen Maleux, Dominique Wauters, and Luc Gagnon), and we acknowledge Joke Vandewalle (XPE Pharma & Science on behalf of GSK Vaccines) for drafting the manuscript and Bart van Heertum (XPE Pharma & Science on behalf of GSK Vaccines) for manuscript coordination.

Terhi Äijälä, Tiina Nokela, Tuula Heiskanen, Thierry Pascal, Koen Maleux, Dominique Wauters, and Luc Gagnon), and we acknowledge Joke Vandewalle (XPE Pharma & Science on behalf of GSK Vaccines) for drafting the manuscript and Bart van Heertum (XPE Pharma & Science on behalf of GSK Vaccines) for manuscript coordination. Financial support. GlaxoSmithKline Biologicals SA was the funding source and was involved in all stages of the study conduct and analysis. GlaxoSmithKline Biologicals SA also took responsibility for all costs associated with the development and publishing of this article. Potential conflicts of interest. T. V. declares that he received payment from the GlaxoSmithKline group of companies and other vaccine manufacturers for board membership, consultancy, and attending meetings; the institution of T. K. received grants from the GlaxoSmithKline group of companies; L. S., A. S., S. S., D. B., M. M., M. T., and P. L. are employees of the GlaxoSmithKline group of companies; M. H. is a consultant for Chiltern International for the GlaxoSmithKline group of companies; T. P. was a GlaxoSmithKline group of companies employee during the study; L. S., D. B., M. M., and P. L. declare stock and stock options ownership in the GlaxoSmithKline group of companies; and S. S. declares shares ownership in the GlaxoSmithKline group of companies. A. F. and I. S. declare no conflicts of interest.

; T. P. was a GlaxoSmithKline group of companies employee during the study; L. S., D. B., M. M., and P. L. declare stock and stock options ownership in the GlaxoSmithKline group of companies; and S. S. declares shares ownership in the GlaxoSmithKline group of companies. A. F. and I. S. declare no conflicts of interest. Author contributions. D. B., M. M., L. S., A. S., and T. P. coordinated the clinical aspects of the study; D. B., A. F., I. S., A. S., M. M., M. H., and T. K. were involved in data collection; D. B., A. F., L. S., S. S., A. S., P. L., T. P., T. V., M. T., and T. K. planned and designed the study and interpreted the results; M. M. and M. H. interpreted the results; P. L. and M. T. performed the statistical analyses; and all the authors critically reviewed the different drafts of the manuscript and approved the final version. Supplementary Data Supplementary materials are available at the Journal of The Pediatric Infectious Diseases Society online (http://jpids.oxfordjournals.org). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Neisseria meningitidis serogroup B (MnB) causes most meningococcal disease in Europe and approximately one third of cases in the United States [1, 2]. MnB infection is more common among adolescents and young adults than the general adult population [2]. MnB polysaccharide vaccines are poorly immunogenic, likely due to cross-reacting epitopes of MnB capsular polysaccharide with polysialic acid structures on human neuronal cells [3]. Vaccines produced from MnB outer membrane vesicles have shown clinical efficacy in children older than 4 years, against MnB strains bearing antigens homologous to those in the vaccine, but not against strains expressing antigens heterologous to the vaccine [4–6]. Therefore, alternative approaches based on conserved MnB protein antigens have been pursued to provide protection against diverse strains causing endemic and epidemic invasive MnB disease. A recombinant, multicomponent MnB vaccine (Bexsero, Novartis Vaccines, Siena, Italy) has been licensed in the European Union, Canada, Australia [7–9], and, more recently, in the United States [10]. Another multicomponent vaccine candidate, bivalent rLP2086 (Trumenba®), was recently approved in the United States to prevent invasive meningococcal disease caused by MnB in individuals 10 to 25 years of age [11]. The target antigen for this vaccine, LP2086, was identified using a combined biochemical and functional immunologic screening approach [12].

didate, bivalent rLP2086 (Trumenba®), was recently approved in the United States to prevent invasive meningococcal disease caused by MnB in individuals 10 to 25 years of age [11]. The target antigen for this vaccine, LP2086, was identified using a combined biochemical and functional immunologic screening approach [12]. LP2086 is a bacterial, surface-exposed virulence factor that binds human factor H, also known as factor H binding protein (fHBP) [13]. In a comprehensive survey of 1837 MnB strains isolated from patients with invasive disease in the United States and Europe, the LP2086 gene was present in all isolates [14], although in rare cases, invasive disease-causing MnB strains may lack a functional LP2086 gene [15]. LP2086 protein sequences can be grouped into 2 genetically and immunologically distinct subfamilies, designated A (30% of isolates) and B (70% of isolates) [14]. The bivalent vaccine contains recombinant lipidated proteins from each LP2086 subfamily (bivalent rLP2086), because this formulation was shown to be most effective at inducing broadly protective serum bactericidal antibodies. In phase 1 and 2 studies, immune sera elicited by bivalent rLP2086 demonstrated bacterial killing activity, measured in serum bactericidal assays using human complement (hSBAs), against diverse MnB strains bearing fHBP variants of both subfamilies and heterologous in sequence to those in the vaccine [16–19]. For immunogenicity evaluations, 4 MnB test strains were selected to reflect the epidemiology, breadth, and global distribution of MnB strains. Each hSBA test strain expresses an fHBP variant that is different from vaccine components, thus allowing an objective assessment of functional and protective bactericidal immune responses against invasive disease-associated strains in circulation [20].

the epidemiology, breadth, and global distribution of MnB strains. Each hSBA test strain expresses an fHBP variant that is different from vaccine components, thus allowing an objective assessment of functional and protective bactericidal immune responses against invasive disease-associated strains in circulation [20]. This study evaluated the immunogenicity, tolerability, and safety of bivalent rLP2086 administered in 3-dose or 2-dose regimens in healthy adolescents aged 11 to <19 years at enrollment. The immune response measured by hSBA was evaluated 1 month after dose 2 or 3 and included the percentage of subjects achieving hSBA titers ≥1:8, a more conservative indicator of potential seroprotection than the recognized correlate of protection, an hSBA titer ≥1:4 [21, 22]. Safety assessments included the incidence of local reactions, systemic events, and adverse events (AEs).

ter dose 2 or 3 and included the percentage of subjects achieving hSBA titers ≥1:8, a more conservative indicator of potential seroprotection than the recognized correlate of protection, an hSBA titer ≥1:4 [21, 22]. Safety assessments included the incidence of local reactions, systemic events, and adverse events (AEs). METHODS Study Participants and Design This phase 2, multicenter, randomized, single-blind study was conducted between March 3, 2011 and August 30, 2013, in healthy adolescents 11 to <19 years of age from the Czech Republic, Denmark, Finland, Germany, Poland, Spain, and Sweden. Subjects were judged healthy by the investigator and agreed to practice an effective form of contraception. Key exclusion criteria were previous vaccination with any MnB vaccine; previous anaphylactic reaction to any vaccine or vaccine-related component; history of culture-proven N meningitidis or Neisseria gonorrhoeae disease; pregnancy or nursing; current chronic use of systemic antibiotics; bleeding diathesis or condition that would contraindicate intramuscular injection; known or suspected disease of the immune system or receipt of immunosuppressive therapy; any neuroinflammatory or autoimmune condition; and receipt of any blood products, including immunoglobulin, within 6 months before the first study vaccination.

athesis or condition that would contraindicate intramuscular injection; known or suspected disease of the immune system or receipt of immunosuppressive therapy; any neuroinflammatory or autoimmune condition; and receipt of any blood products, including immunoglobulin, within 6 months before the first study vaccination. Subjects were randomized 3:3:3:2:1 corresponding to a 0,1,6-month, 0,2,6-month, 0,6-month, 0,2-month, and 0,4-month bivalent rLP2086 dosing schedule, respectively (groups 1–5) (Table 1; Figure 1). The unequal randomization schedule reflects the comparisons of interest for this study. The first 3 groups (randomized 3:3:3) were assigned to meet the hypothesis testing objectives; the remaining 2 groups (randomized 2:1) were descriptive and were used to explore different rLP2086 dosing schedules. Subjects were randomized using an interactive voice- or web-based response system. Enrollment was stratified by age (11 to <14 and 14 to <19 years) to ensure that all ages were adequately represented in the study. Each subject received 4 study injections, with administration of 2 or 3 doses of bivalent rLP2086 according to the aforementioned dosing schedule; saline was administered when bivalent rLP2086 was not scheduled. Participants were blinded to their assigned dose regimen. All laboratory staff were blinded to subject, visit, and treatment (ie, randomization group), but investigators and sponsor knew the allocation of subjects throughout the study. This study was conducted in accordance with the Declaration of Helsinki [23] and the International Conference on Harmonisation Guidelines for Good Clinical Practice [24] (ClinicalTrials.gov: NCT01299480). Table 1. Study Design

n group), but investigators and sponsor knew the allocation of subjects throughout the study. This study was conducted in accordance with the Declaration of Helsinki [23] and the International Conference on Harmonisation Guidelines for Good Clinical Practice [24] (ClinicalTrials.gov: NCT01299480). Table 1. Study Design rLP2086, mo Visit 1* (Month 0) Visit 2 (Month 1) Visit 3* (Month 2) Visit 4* (Month 3) Visit 5 (Month 6) Visit 6* (Month 7) Visit 7 (Month 12) Injection 1 Injection 2 Injection 3 Blood Draw Only Injection 4 Blood Draw Only Telephone Contact Only 0,1,6 rLP2086 rLP2086 Saline rLP2086 0,2,6 rLP2086 Saline rLP2086 rLP2086 0,2 rLP2086 Saline rLP2086 Saline 0,4 Saline Saline rLP2086 rLP2086 0,6 rLP2086 Saline Saline rLP2086 Abbreviations: fHBP, factor H binding protein; hSBA, serum bactericidal assay using human complement; rLP2086, bivalent rLP2086 vaccine. *Blood draw at visits 1, 3, 4, and 6 for hSBA performed with strains expressing fHBP variants A22, A56, B24, and B44. Figure 1. Subject disposition. *Values in this row used as denominators for percentages. Vaccine and Placebo Subjects received 1 intramuscular dose of bivalent rLP2086 or saline in the upper deltoid muscle. Each 0.5-mL dose of bivalent rLP2086 contained 60 µg each of rLP2086 A (A05) and B (B01) subfamily proteins produced in Escherichia coli and formulated with aluminum phosphate in an isotonic buffer in prefilled syringes. Saline (0.9% sodium chloride) was administered as a 0.5-mL dose.

aline in the upper deltoid muscle. Each 0.5-mL dose of bivalent rLP2086 contained 60 µg each of rLP2086 A (A05) and B (B01) subfamily proteins produced in Escherichia coli and formulated with aluminum phosphate in an isotonic buffer in prefilled syringes. Saline (0.9% sodium chloride) was administered as a 0.5-mL dose. Immunogenicity Assessments Blood samples for hSBA were obtained at 0, 2, 3, and 7 months (Table 1). Functional bactericidal antibodies were assessed against 4 MnB test strains expressing fHBP variants that are heterologous in sequence compared with the vaccine components; 2 strains express fHBP subfamily A variants (A22 and A56), and 2 strains express fHBP subfamily B variants (B24 and B44). The fHBP variants represent 4 of the 6 major fHBP variant subgroups that account for >90% of invasive meningococcal disease isolates circulating in the United States and Europe combined [20]. As an indication of the diversity of these 4 MnB test strains, the multilocus sequence type clonal complex (ie, the genetic background) of each strain represents 1 of the 4 most prevalent clonal complexes in circulating invasive MnB strains (Table 2) [20]. Each of these 4 MnB test strains have detectable fHBP surface expression levels representative of their respective fHBP variant group, as measured in a flow cytometry assay adapted from McNeil et al [25] using the anti-fHBP broadly cross-reactive monoclonal antibody MN86-994-11-1. Table 2. Genotypic Characteristics of N meningitidis serogroup B Test Strains

etectable fHBP surface expression levels representative of their respective fHBP variant group, as measured in a flow cytometry assay adapted from McNeil et al [25] using the anti-fHBP broadly cross-reactive monoclonal antibody MN86-994-11-1. Table 2. Genotypic Characteristics of N meningitidis serogroup B Test Strains Strain ID fHBP Variant (fHBP Peptide IDa) Homology,b % fHBP Subgroup ST Clonal Complex PorA Subtype PMB2001 A56 (187) 98.1 N1C2 cc213 P1.22,14 PMB2707 B44 (15) 91.6 N4/N5 cc269 P1.19-1,10-4 PMB80 A22 (19) 88.9 N2C2 cc41/44 P1.21,16 PMB2948 B24 (1) 86.2 N6 cc32 P1.12-1,13-1 Abbreviations: fHBP, factor H binding protein; ID, identification; ST, sequence type. afHBP peptide ID is from http://pubmlst.org/neisseria/fHbp/. bAmino acid identity to the fHBP subfamily matched vaccine component: A05 for the strains expressing fHBP subfamily A variants, or B01 for the strains expressing fHBP subfamily B variants. Within fHBP subfamily, the percentage amino acid identity is >83%.

Strain ID fHBP Variant (fHBP Peptide IDa) Homology,b % fHBP Subgroup ST Clonal Complex PorA Subtype PMB2001 A56 (187) 98.1 N1C2 cc213 P1.22,14 PMB2707 B44 (15) 91.6 N4/N5 cc269 P1.19-1,10-4 PMB80 A22 (19) 88.9 N2C2 cc41/44 P1.21,16 PMB2948 B24 (1) 86.2 N6 cc32 P1.12-1,13-1 Abbreviations: fHBP, factor H binding protein; ID, identification; ST, sequence type. afHBP peptide ID is from http://pubmlst.org/neisseria/fHbp/. bAmino acid identity to the fHBP subfamily matched vaccine component: A05 for the strains expressing fHBP subfamily A variants, or B01 for the strains expressing fHBP subfamily B variants. Within fHBP subfamily, the percentage amino acid identity is >83%. The primary immunogenicity endpoints were the proportion of subjects receiving 3 doses of bivalent rLP2086 (vaccination at 0, 1, 6 and 0, 2, 6 months) who achieved hSBA titer ≥1:8 for each of the 4 MnB test strains 1 month after the third dose of bivalent rLP2086. An hSBA titer ≥1:8 is a more conservative indicator of seroprotection than a titer ≥1:4, which is the recognized correlate of protection against meningococcal disease [21, 22] and addresses the ability of the hSBA to determine precisely and accurately a positive from a negative seroresponse. The hypothesis-testing secondary endpoint was the proportion of subjects receiving 2 doses of bivalent rLP2086 at 0,6 months who achieved hSBA titer ≥1:8 for each of the 4 MnB test strains 1 month after the second dose of bivalent rLP2086. Other secondary immunogenicity endpoints included assessment of geometric mean titers (GMTs) for each of the 4 MnB test strains and the proportion of responders with hSBA titers ≥1:8 for all groups at each sampling point. Responses after 1 dose of bivalent rLP2086 were only available for the 0,4-month dosing group.

86. Other secondary immunogenicity endpoints included assessment of geometric mean titers (GMTs) for each of the 4 MnB test strains and the proportion of responders with hSBA titers ≥1:8 for all groups at each sampling point. Responses after 1 dose of bivalent rLP2086 were only available for the 0,4-month dosing group. Safety Assessments Subjects were observed for ≥20 minutes (or longer per local practice) after vaccination for immediate reactions, which were documented as AEs. Reactogenicity data were collected by electronic diary (e-diary) for 7 days after each injection and included solicited local reactions and systemic events and use of antipyretic medications. Injection-site redness and swelling were measured by subjects using calipers provided, and measurements were subsequently graded as mild, moderate, or severe as defined prospectively by study protocol. Injection-site pain and systemic symptoms of headache, fatigue, chills, vomiting, diarrhea, muscle pain, and joint pain were graded by the subjects as mild, moderate, or severe. Temperature was measured at bedtime; fever was defined as temperature ≥38.0°C (100.4°F). Only the highest fever measurement recorded among measurements taken during any single 24-hour period was reported. Unsolicited AEs were collected from signing of informed consent through 1 month after the last injection (bivalent rLP2086 or saline). Serious AEs (SAEs) were collected throughout the study.

°C (100.4°F). Only the highest fever measurement recorded among measurements taken during any single 24-hour period was reported. Unsolicited AEs were collected from signing of informed consent through 1 month after the last injection (bivalent rLP2086 or saline). Serious AEs (SAEs) were collected throughout the study. Statistical Analysis The evaluable immunogenicity population included subjects who received all doses of bivalent rLP2086 as randomized, had no major protocol violation or use of prohibited vaccines, had blood drawn before dose 1 and 1 month after the last dose of bivalent rLP2086, and had a valid and determinate assay result for the proposed analysis. The safety population included all participants who received at least 1 injection (bivalent rLP2086 or saline) and had any safety data available. Immunogenicity endpoints, defined as the proportion of subjects responding with an hSBA titer ≥1:8, were summarized along with the exact 2-sided 95% confidence interval ([CI] or Clopper-Pearson confidence limit) for the proportion; the exact CI for the proportion was computed using the F distribution. The response rates in subjects receiving 3 versus 2 doses of bivalent rLP2086 were compared with 50% at a significance level of 1.25%, using a 1-sided exact test based on binomial distribution. GMTs were computed with 2-sided 95% CIs.

he proportion; the exact CI for the proportion was computed using the F distribution. The response rates in subjects receiving 3 versus 2 doses of bivalent rLP2086 were compared with 50% at a significance level of 1.25%, using a 1-sided exact test based on binomial distribution. GMTs were computed with 2-sided 95% CIs. RESULTS Subjects A total of 1714 subjects were enrolled and 1713 were randomized; 1 subject received saline without randomization and withdrew from the study (Figure 1). A total of 1712 subjects received at least 1 dose of bivalent rLP2086 or saline, and 1550 (90.5%) completed the study. Most subjects were white (99.0%) and non-Hispanic (98.5%). At the time of first injection, 63.4% of subjects were 14–18 years of age; mean age of the total population was 14.4 years (Table 3). Demographic characteristics were similar across dosing regimens. Table 3. Demographics, Safety Population Bivalent rLP2086 Dosing Schedule 0,1,6 mo n = 426 0,2,6 mo n = 414 0,6 mo n = 451 0,2 mo n = 277 0,4 mo n = 144 Total N = 1712 Sex, n (%) Female 212 (50) 217 (52) 227 (50) 135 (49) 79 (55) 870 (51) Male 214 (50) 197 (48) 224 (50) 142 (51) 65 (45) 842 (49) Race, n (%) White 423 (99) 408 (99) 448 (99) 272 (98) 144 (100) 1695 (99) Age at first injection, y, n (%) 11 to <14 155 (36) 154 (37) 164 (36) 101 (37) 53 (37) 627 (37) 14 to <19 271 (64) 260 (63) 287 (64) 176 (64) 91 (63) 1085 (63) Mean (SD) 14.4 (2.22) 14.4 (2.23) 14.4 (2.17) 14.4 (2.25) 14.3 (2.11) 14.4 (2.20) Abbreviation: SD, standard deviation.

9) 448 (99) 272 (98) 144 (100) 1695 (99) Age at first injection, y, n (%) 11 to <14 155 (36) 154 (37) 164 (36) 101 (37) 53 (37) 627 (37) 14 to <19 271 (64) 260 (63) 287 (64) 176 (64) 91 (63) 1085 (63) Mean (SD) 14.4 (2.22) 14.4 (2.23) 14.4 (2.17) 14.4 (2.25) 14.3 (2.11) 14.4 (2.20) Abbreviation: SD, standard deviation. Immunogenicity A total of 1450 subjects comprised the evaluable immunogenicity population. One month after dose 3, the proportion of subjects with hSBA titers ≥1:8 after 3 doses of bivalent rLP2086 administered at 0,1,6 months was 91.7%, 99.4%, 89.0%, and 88.5% for MnB test strains expressing vaccine-heterologous fHBP variants A22, A56, B24, and B44, respectively. For subjects vaccinated at 0,2,6 months, 95.0%, 98.9%, 88.4%, and 86.1% had hSBA titers ≥1:8 (Figure 2). There were no significant differences in immunogenicity among subjects who received bivalent rLP2086 on the 0,1,6-month and 0,2,6-month schedules. Figure 2. Percentage of subjects with hSBA titers ≥1:8 against N meningitidis serogroup B test strains A22, A56, B24, and B44 at baseline and 1 month after injection with bivalent rLP2086 or saline. Errors shown are 95% confidence intervals. hSBA, human serum bactericidal antibody assay using human complement.

hedules. Figure 2. Percentage of subjects with hSBA titers ≥1:8 against N meningitidis serogroup B test strains A22, A56, B24, and B44 at baseline and 1 month after injection with bivalent rLP2086 or saline. Errors shown are 95% confidence intervals. hSBA, human serum bactericidal antibody assay using human complement. For fHBP subfamily A strains, among those receiving 2 doses of bivalent rLP2086, >90% of subjects had hSBA titers ≥1:8 for A22 and >98% of subjects had hSBA titers ≥1:8 for A56 1 month after the second dose. For fHBP subfamily B strains, >69% of subjects had hSBA titers ≥1:8 for B24 and >70% for B44 (Figure 2). The study design allowed assessment of hSBA responses at 1 month after 1 dose of bivalent rLP2086 (0,4-month group only). After the first dose, 55.9%, 67.6%, 56.9%, and 23.8% of subjects had hSBA titers ≥1:8 for A22, A56, B24, and B44, respectively. Prevaccination hSBA responses to all 4 MnB test strains were low, ranging from 5.2% (B44) to 28.1% (A22).

of hSBA responses at 1 month after 1 dose of bivalent rLP2086 (0,4-month group only). After the first dose, 55.9%, 67.6%, 56.9%, and 23.8% of subjects had hSBA titers ≥1:8 for A22, A56, B24, and B44, respectively. Prevaccination hSBA responses to all 4 MnB test strains were low, ranging from 5.2% (B44) to 28.1% (A22). Postvaccination GMTs increased with each dose of bivalent rLP2086 and were highest among subjects receiving 3 doses of bivalent rLP2086. For subjects vaccinated at 0,1,6 months, GMTs 1 month after dose 3 were 55.1, 152.9, 29.1, and 40.3 for A22, A56, B24, and B44, respectively. For subjects vaccinated at 0,2,6 months, GMTs were 56.3, 155.6, 25.6, and 35.0 (Figure 3). For fHBP subfamily A strains, among subjects receiving 2 doses of bivalent rLP2086, GMTs ranged from 37.1 to 48.4 for A22, and 104.9 to 125.6 for A56 one month after the second dose. For the fHBP subfamily B strains, GMTs ranged from 14.7 to 20.6 for B24 and 17.8 to 22.5 for B44. After 2 doses of bivalent rLP2086, GMTs trended numerically higher among subjects with a longer interval between doses; subjects with a 6-month interval between doses (0,6-month group) had higher GMTs to all 4 MnB test strains compared with subjects having a 4-month (0,4-month), 2-month (0,2- and 0,2,6-month), or 1-month (0,1,6-month) interval between doses (Figure 3). After 1 dose of bivalent rLP2086 (0,4-month group), GMTs were 16.0, 26.8, 12.6, and 6.8, respectively, for A22, A56, B24, and B44. Figure 3. Geometric mean titers (GMTs) against N meningitidis serogroup B test strains A22, A56, B24, and B44 at baseline and 1 month after injection with bivalent rLP2086 or saline.

re 3). After 1 dose of bivalent rLP2086 (0,4-month group), GMTs were 16.0, 26.8, 12.6, and 6.8, respectively, for A22, A56, B24, and B44. Figure 3. Geometric mean titers (GMTs) against N meningitidis serogroup B test strains A22, A56, B24, and B44 at baseline and 1 month after injection with bivalent rLP2086 or saline. Safety The frequency of local reactions was higher after bivalent rLP2086 administrations compared with saline; pain at the injection site was the most common local reaction (Figure 4). Across dosing schedules, most cases of pain after bivalent rLP2086 and saline administration were mild or moderate. Severe pain was reported by ≤9.9% of subjects who received bivalent rLP2086 and ≤0.3% subjects who received saline, for each of the 4 injections. Other common local reactions included redness and swelling, which were mild or moderate in severity (Figure 4). The mean duration of all local reactions after each dose of bivalent rLP2086 was 2.1 to 3.2 days; 4 local reactions, all of which were pain at the injection site, had a duration greater than 14 days, but no subjects withdrew from the study due to pain. Figure 4. Local injection-site reactogenicity (recorded by electronic diary [e-diary]). Data have been aggregated across groups to show reactogenicity after each dose.

local reactions, all of which were pain at the injection site, had a duration greater than 14 days, but no subjects withdrew from the study due to pain. Figure 4. Local injection-site reactogenicity (recorded by electronic diary [e-diary]). Data have been aggregated across groups to show reactogenicity after each dose. Across all injections, the most common systemic events were headache and fatigue. The majority of systemic events were mild or moderate in severity. Severe headache was reported by ≤1.6% of subjects after either bivalent rLP2086 or saline. Severe fatigue was reported by ≤3.6% of subjects after either injection (Figure 5). In total, 3 subjects withdrew due to a systemic event. Figure 5. Systemic reactogenicity (recorded by electronic diary [e-diary]). Data have been aggregated across groups to show reactogenicity after each dose. *Fever: 38.0–38.4°C = mild; 38.5–38.9°C = moderate; and ≥39°C = severe. Fever ≥38°C within 7 days of vaccination was infrequent, reported by 1.7%–4.3% and 1.5%–2.1% of bivalent rLP2086 or saline recipients, respectively. Fever ≥39°C was infrequent (<1% across groups). The median duration of fever was 1 day after bivalent rLP2086 or saline administration. Across all 4 injections, 13.6%–16.2% and 7.5%–9.1% of subjects used antipyretic medication after bivalent rLP2086 and saline, respectively.

rLP2086 or saline recipients, respectively. Fever ≥39°C was infrequent (<1% across groups). The median duration of fever was 1 day after bivalent rLP2086 or saline administration. Across all 4 injections, 13.6%–16.2% and 7.5%–9.1% of subjects used antipyretic medication after bivalent rLP2086 and saline, respectively. During the vaccination phase, 35.5%–37.5% of subjects reported ≥1 AE across the 5 dosing groups. Most events were mild or moderate in severity. The most common AE was nasopharyngitis (5.5% − 10.1%). Eleven subjects experienced severe AEs considered to be related to bivalent rLP2086, including headache, injection-site pain, pyrexia, vomiting, and injection-site swelling. Two subjects reported SAEs considered to be related to bivalent rLP2086; 1 subject experienced vertigo, chills, and headache after dose 3, and another experienced pyrexia and vomiting after dose 1. Overall, there were no differences in the incidence of SAEs between bivalent rLP2086 and saline recipients or between the 3-dose and 2-dose schedules and no increase in AEs with subsequent dosing. Nineteen subjects (1.1%) withdrew due to an AE. Of these, 9 AEs were considered to be related to study vaccination and included injection-site pain (n = 4), headache (n = 2), migraine, fatigue, and vertigo (n = 1 each). No deaths were reported.

3-dose and 2-dose schedules and no increase in AEs with subsequent dosing. Nineteen subjects (1.1%) withdrew due to an AE. Of these, 9 AEs were considered to be related to study vaccination and included injection-site pain (n = 4), headache (n = 2), migraine, fatigue, and vertigo (n = 1 each). No deaths were reported. DISCUSSION Meningococcal B infection can lead to severe and debilitating disease. Vaccination against non-B meningococcal serogroups (ACWY) has proven efficacious in all age groups, particularly in adolescents who are at high risk for meningococcal disease [26]. The need for an effective MnB vaccine is underscored by recent outbreaks on US college campuses and by endemic infections worldwide [27–29]. The low basal immunity to meningococcal B strains in this study emphasizes the vulnerability of adolescents to infection and disease caused by N meningitidis serogroup B.

se [26]. The need for an effective MnB vaccine is underscored by recent outbreaks on US college campuses and by endemic infections worldwide [27–29]. The low basal immunity to meningococcal B strains in this study emphasizes the vulnerability of adolescents to infection and disease caused by N meningitidis serogroup B. In this study, all 5 bivalent rLP2086 vaccination regimens, regardless of schedule or frequency, were immunogenic and well tolerated in healthy adolescents. The 2-dose and 3-dose regimens elicited robust immune responses. Considering that hSBA titers ≥1:4 are an accepted correlate for protection against invasive meningococcal disease [21, 22] and that seroprotective responses in this study were defined using more stringent criteria of hSBA titers ≥1:8, bivalent rLP2086 has demonstrated a substantial and broad immune response after 2 doses and a more robust response after 3 doses, when administered across a range of dosing schedules. This study also allowed assessment of hSBA responses after 1 dose of bivalent rLP2086. After a single vaccination, increased seroprotective responses compared with baseline against all MnB test strains were observed.

2 doses and a more robust response after 3 doses, when administered across a range of dosing schedules. This study also allowed assessment of hSBA responses after 1 dose of bivalent rLP2086. After a single vaccination, increased seroprotective responses compared with baseline against all MnB test strains were observed. Among recipients receiving 3 doses of bivalent rLP2086, the timing of the second vaccination, whether 1 or 2 months after initial vaccination, had no discernable effect on immunogenicity. Nonetheless, the ability to elicit robust responses after 2 doses 1 month apart may be valuable during outbreaks when timely protection is critical. Immune responses after 2 doses of bivalent rLP2086 were substantial and indicative of a broad immune response to the MnB test strains that were selected to represent epidemiologically and antigenically relevant invasive MnB strains. Immunogenicity generally increased in the 2-dose schedules when there was a longer interval between the first and second dose, as seen by the higher GMTs and generally higher proportion of subjects with hSBA titers ≥1:8 after dose 2 in the 0,6-month dosing regimen compared with the other dosing schedules. Similar observations were described for quadrivalent human papillomavirus vaccination [30, 31]. In these studies, increasing the interval between doses 2 and 3 from 4 to 10 months resulted in higher antibody titers against all 4 human papillomavirus types examined.

sing regimen compared with the other dosing schedules. Similar observations were described for quadrivalent human papillomavirus vaccination [30, 31]. In these studies, increasing the interval between doses 2 and 3 from 4 to 10 months resulted in higher antibody titers against all 4 human papillomavirus types examined. Similar to other clinical studies of bivalent rLP2086 in adolescents and adults [18, 19, 32, 33], the majority of safety events observed in this study were local reactions and systemic events that were mild or moderate in severity, transient, and without potentiation at subsequent dosing. Approximately two thirds of subjects did not report any AEs during the study. Overall, 90.6% of subjects were able to complete their vaccination series, indicating that vaccinations were well tolerated in adolescents and that any safety events, such as injection-site pain, were not an impediment to vaccination. Safety events were not increased in subjects receiving a third dose of bivalent rLP2086. CONCLUSIONS In summary, 2 or 3 doses of bivalent rLP2086 were immunogenic and well tolerated. The 2-dose regimens provided substantial hSBA responses against diverse MnB strains expressing fHBP variants heterologous to vaccine antigen. However, the 3-dose regimens yielded the highest seroconversion rates against subfamily B strains and a higher level of hSBA antibodies as measured by GMTs. The convenience of a 2-dose schedule and the potential benefit of higher level of protective antibodies after a 3-dose schedule should be carefully considered for future MnB immunization schedules.

ed the highest seroconversion rates against subfamily B strains and a higher level of hSBA antibodies as measured by GMTs. The convenience of a 2-dose schedule and the potential benefit of higher level of protective antibodies after a 3-dose schedule should be carefully considered for future MnB immunization schedules. Acknowledgments We thank all the parents and guardians of all participants and the many investigators and study staff for their individual contributions to this study. Deborah M. Campoli-Richards, BSPharm, RPh, and Susan E. DeRocco, PhD (Complete Healthcare Communications, Inc.) provided assistance in preparing and editing the manuscript. The study was registered with ClinicalTrials.gov, number NCT00657709. Financial support. This study was funded by Pfizer Inc.

Acknowledgments We thank all the parents and guardians of all participants and the many investigators and study staff for their individual contributions to this study. Deborah M. Campoli-Richards, BSPharm, RPh, and Susan E. DeRocco, PhD (Complete Healthcare Communications, Inc.) provided assistance in preparing and editing the manuscript. The study was registered with ClinicalTrials.gov, number NCT00657709. Financial support. This study was funded by Pfizer Inc. Potential conflicts of interest. T. V. received consulting fees and support for meetings, travel, or accommodation expenses from GlaxoSmithKline; and he is a consultant and speaker for Merck, Sanofi Pasteur-Merck Sharp & Dohme (SPMSD), MedImmune, Novartis, and Pfizer. J. D.-D. participated in advisory boards and speaker bureaus for GlaxoSmithKline, SPMSD, and Pfizer, for which payment is received; and he was a principal investigator in clinical trials for GlaxoSmithKline and SPMSD. J. W. was the principal investigator of clinical trials sponsored by GSK, Novartis, Wyeth, and Pfizer; and he has received travel grants to participate in scientific conferences and was paid for lectures. J. B., J. E., Q. J., K. U. J., T. R. J., S. L. H., R. E. O., L. J. Y., J. L. P., and G. C. are full-time employees of Pfizer Inc. L. O. has served as a principal investigator for trials sponsored by Pfizer, GSK, and Sanofi-Pasteur MSD. C.-E. F. has been principal investigator for clinical trials for GlaxoSmithKline, SPMSD, Wyeth, Pfizer, SmithKline Beecham, and MSD.

., R. E. O., L. J. Y., J. L. P., and G. C. are full-time employees of Pfizer Inc. L. O. has served as a principal investigator for trials sponsored by Pfizer, GSK, and Sanofi-Pasteur MSD. C.-E. F. has been principal investigator for clinical trials for GlaxoSmithKline, SPMSD, Wyeth, Pfizer, SmithKline Beecham, and MSD. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

An estimated 3.2 million children are living with human immunodeficiency virus (HIV) [1], most of whom live in sub-Saharan Africa. Approximately 199,000 children were newly infected in 2013. Scale-up of antiretroviral therapy (ART) provision is likely to contribute to improvements in survival amongst children with HIV [2]. However, in sub-Saharan Africa, only 22% of HIV-positive children are receiving ART, lagging behind 39% coverage in adults [1]. Prevention of vertical infection through Prevention of Mother-to-Child Transmission (PMTCT) program is effective, but worldwide coverage requiring ART has been suboptimal. An estimated 1%–3% of all 10-year-olds in Southern Africa are HIV-infected, long-term survivors, with 68% of eligible women receiving ART as PMTCT in 2013 compared with 33% 5 years previously [1]. In a hospital-based study from Zimbabwe, advanced HIV infection was the single most common cause of admission and death in adolescents [3].

3% of all 10-year-olds in Southern Africa are HIV-infected, long-term survivors, with 68% of eligible women receiving ART as PMTCT in 2013 compared with 33% 5 years previously [1]. In a hospital-based study from Zimbabwe, advanced HIV infection was the single most common cause of admission and death in adolescents [3]. With longer survival, lung effects of HIV become more prominent. Human immunodeficiency virus-infected children in sub-Saharan Africa are subject to frequent pulmonary infections [4], and they commonly develop chronic cough in older childhood. In a Zimbabwean study of 116 HIV-infected adolescents receiving HIV care, dyspnoea was often disabling, and resting hypoxia or desaturation at submaximal exercise was present in 40% of patients [5]. Chest radiographs were abnormal in two thirds of patients, characterized by ring and tramline opacities, whose presence is unrelated to clinical symptoms. High-resolution computerized tomography (HRCT) scans suggested small airways disease as the most common cause of infection; however, bronchodilator response was not assessed [6]. Despite this, data on symptoms of chronic lung disease (CLD) and lung function testing, particularly in developing countries, are lacking. We describe the burden and clinically useful phenotypes of CLDs, and we assess the bronchodilator response with inhaled beta-agonist therapy in HIV-infected children aged 8 to 16 receiving HIV care in Blantyre, Malawi.

With longer survival, lung effects of HIV become more prominent. Human immunodeficiency virus-infected children in sub-Saharan Africa are subject to frequent pulmonary infections [4], and they commonly develop chronic cough in older childhood. In a Zimbabwean study of 116 HIV-infected adolescents receiving HIV care, dyspnoea was often disabling, and resting hypoxia or desaturation at submaximal exercise was present in 40% of patients [5]. Chest radiographs were abnormal in two thirds of patients, characterized by ring and tramline opacities, whose presence is unrelated to clinical symptoms. High-resolution computerized tomography (HRCT) scans suggested small airways disease as the most common cause of infection; however, bronchodilator response was not assessed [6]. Despite this, data on symptoms of chronic lung disease (CLD) and lung function testing, particularly in developing countries, are lacking. We describe the burden and clinically useful phenotypes of CLDs, and we assess the bronchodilator response with inhaled beta-agonist therapy in HIV-infected children aged 8 to 16 receiving HIV care in Blantyre, Malawi. METHODS Participants Participants were recruited from outpatient HIV clinics in Queen Elizabeth Central Hospital, Blantyre between July and December 2011. The first 3 eligible patients per day were included. Patients were not eligible for recruitment if they (1) resided outside urban Blantyre, (2) were currently taking tuberculosis (TB) treatment, (3) had Kaposi's sarcoma, (4) reported acute respiratory symptoms (≤1 week of any one or more of fever, purulent sputum, pleuritic chest pain), or (5) required emergent hospitalization.

s were not eligible for recruitment if they (1) resided outside urban Blantyre, (2) were currently taking tuberculosis (TB) treatment, (3) had Kaposi's sarcoma, (4) reported acute respiratory symptoms (≤1 week of any one or more of fever, purulent sputum, pleuritic chest pain), or (5) required emergent hospitalization. At baseline, we assessed medical history, symptoms, quality of life, and functional status by standardized verbal questionnaires administered in the local language. Examination included assessment of finger clubbing, growth, and World Health Organization (WHO) clinical staging of HIV disease. CD4 count was performed, and TB smear and culture were done in all participants who could spontaneously expectorate. Participants performed a 200 meter submaximal walk test, unless contraindicated due to resting hypoxia (SpO2 <92%) or tachypnoea (>24/minutes). Within 2 weeks, participants had spirometry unless there was evidence of TB or acute respiratory illness [7]. Chest radiographs were reported by 2 independent clinicians using a standardized scoring system [6], with discrepancies resolved by consensus.

ted due to resting hypoxia (SpO2 <92%) or tachypnoea (>24/minutes). Within 2 weeks, participants had spirometry unless there was evidence of TB or acute respiratory illness [7]. Chest radiographs were reported by 2 independent clinicians using a standardized scoring system [6], with discrepancies resolved by consensus. Quality of Life Assessment In the absence of a disease-specific tool, quality of life was assessed using the Cystic Fibrosis Questionnaire-Revised ([CFQ-R] for 6- to 13-year-olds and their carers in parallel). These incorporate 9 quality of life domains (physical, school, vitality, emotion, social, body image, eating, treatment burden, health perception) and 3 symptom domains (respiratory, digestion, weight). Translation was done by 2 independent translators, collation by consensus, and back-translation for accuracy [8].

l). These incorporate 9 quality of life domains (physical, school, vitality, emotion, social, body image, eating, treatment burden, health perception) and 3 symptom domains (respiratory, digestion, weight). Translation was done by 2 independent translators, collation by consensus, and back-translation for accuracy [8]. Spirometry and Radiology Spirometry was performed according to ATS/ERS guidelines [9] by experienced nursing staff and a respiratory physician. Forced exhalation following maximal inspiration was recorded while seated using an EasyOne World spirometer (ndd, Zurich, Switzerland). Up to 8 trials were recorded, and these were assessed by 2 clinicians independently for quality. The best forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) values from 3 admissible traces were included for analysis. Primary reference values were taken from the Global Lung Initiative [10], using the 5th centile of the reference population as the lower limit of normal ([LLN] 1.64 standard deviation [SD] below the mean). Results of FEV1, FVC, FEV1:FVC, and forced expiratory flow at 25%–75% are reported as residual SDs (z-scores). Participants with FEV1 and FEV1:FVC ratio of less than the LLN were classified as having obstructive spirometry. Where FVC was reduced below LLN and FEV1:FVC was not, we recorded “reduced FVC” because we were unable to measure total lung capacities. In participants with abnormal spirometry, testing was repeated after nebulized salbutamol (2.5 mg via face mask). Reversibility was defined as improvement of ≥12% in best FEV1 or FVC [9]. Participants meeting this criterion were prescribed inhaled salbutamol via metered dose inhaler with an Aerochamber device (GSK, UK), 200 µg at least twice a day, and additionally when symptomatic. These participants returned for clinical reassessment after 4 weeks of treatment.

ovement of ≥12% in best FEV1 or FVC [9]. Participants meeting this criterion were prescribed inhaled salbutamol via metered dose inhaler with an Aerochamber device (GSK, UK), 200 µg at least twice a day, and additionally when symptomatic. These participants returned for clinical reassessment after 4 weeks of treatment. Secondary interpretation used a locally derived reference range [11], with LLN of 80% predicted. Results are presented as “percentage of normal” given uncertain population standard errors. Laboratory Methods CD4 counts were determined by flow cytometry (BD FACSCount, Franklin Lakes, NJ). Participants with cough were asked to provide 2 sputum specimens. Concentrated decontaminated sputum specimens were examined with auramine and cultured using Mycobacterial Growth Indicator Tubes (MGIT; BD, Franklin Lakes, NJ). Positive mycobacterial cultures were confirmed by Ziehl-Neelsen staining and speciated using the Hain assay (Hain LifeScience GmbH, Germany). Ethical Approval Ethical approval was obtained from the College of Medicine Research Ethics Committee, Malawi (P.02/11/1039) and the London School of Hygiene and Tropical Medicine Ethics Committee (approval #5964). Informed written or witnessed thumbprint participant assent and parental or guardian consent were required for recruitment. Analysis Univariable associations of abnormal lung function were assessed using logistic regression, with predictors at P < .10 taken forward to a multivariable model in addition to age and sex.

Ethical Approval Ethical approval was obtained from the College of Medicine Research Ethics Committee, Malawi (P.02/11/1039) and the London School of Hygiene and Tropical Medicine Ethics Committee (approval #5964). Informed written or witnessed thumbprint participant assent and parental or guardian consent were required for recruitment. Analysis Univariable associations of abnormal lung function were assessed using logistic regression, with predictors at P < .10 taken forward to a multivariable model in addition to age and sex. Exploratory analysis was used to compare different prototype definitions of CLD, including 2-way associations between individual variables, aiming for a definition that could be applied in outpatient clinics where investigations are limited to oximetry and symptom screening. We assessed univariable relationships, looking for a clinically useful phenotype. Multivariable logistic regression was then used to identify independent variables independently associated with these phenotypes. A hierarchical approach to modeling (2 levels: distal and proximal) was used to account for factors that affect the lungs indirectly and directly, respectively [12]. Distal (indirect) factors were as follows: stunting, orphanhood, and variables selected a priori for inclusion including sex, age, ART, and CD4 count. Proximal (direct) factors included symptoms, physiological observations, and radiographic and spirometric abnormality. Participants with positive Mycobacterium tuberculosis culture were excluded from case definition analyses. Statistical analyses were conducted with Stata version 12 (StataCorp, College Station, TX).

t. Proximal (direct) factors included symptoms, physiological observations, and radiographic and spirometric abnormality. Participants with positive Mycobacterium tuberculosis culture were excluded from case definition analyses. Statistical analyses were conducted with Stata version 12 (StataCorp, College Station, TX). RESULTS The flow and baseline characteristics of the 160 participants are shown in Figure 1 and Table 1, respectively. All children in the study were black African, with a mean age of 11.1 years (SD = 2.1). One hundred fourteen (71.7%) were established on ART, and a further 46 (28.3%) had an established diagnosis of HIV but were not eligible for ART according to national guidelines. Median CD4 counts in these groups were 698 cells/µL and 406 cells/µL, respectively (P < .001, Wilcoxon rank-sum test). Perinatal acquisition of HIV was assumed after a systematic review of participants' risk factors: 16 (10.3%) participants had received blood transfusion; 11 (6.9%) participants had previous surgery; 53 (33.1%) participants had injections outside the healthcare setting, including escarification; 3 (1.9%) participants reported sexual abuse; none reported other sexual activity. Eighty-nine (56%) participants had no risk factors other than maternal orphanhood or known HIV infection, although reported factors for other transmission routes were higher than in the Zimbabwe study [13]. Previous respiratory complaints were common: 30 (18.8%) participants had been treated for TB; 20 (12.5%) participants had been treated for asthma; and 13 (8%) participants were treated for chest infection requiring hospitalization. Household air pollution was common. Seventy-six (47.5%) and 117 (73.1%) of households used biomass fuel as the predominant energy source for lighting and cooking, respectively. Passive smoking was reported in 23 (14.5%) participants. Table 1. Demographic and Clinical Characteristics

infection requiring hospitalization. Household air pollution was common. Seventy-six (47.5%) and 117 (73.1%) of households used biomass fuel as the predominant energy source for lighting and cooking, respectively. Passive smoking was reported in 23 (14.5%) participants. Table 1. Demographic and Clinical Characteristics Characteristic n (%) unless stated Age, median years (IQR) 11.1 (9.5–12.4) Sex, female (%) 80 (50.0%) Age at HIV diagnosis, median years (IQR) 7.9 (5.8–9.8) Child aware of HIV diagnosis,a n (%) 65 (41.1) CD4 count,c median cells/µL (IQR) 572 (370–876) Taking cotrimoxazole prophylaxis, n (%) 159 (99.4%) On ART,a n (%) 114 (71.7%) Duration of ART,b median years (IQR) 3.5 (1.3–4.6) Chest infection in preceding year, n (%) 30 (18.8%) Cough, n (%) 60 (37.5) Sputum produced, n (%) 32 (20.0) Wheezing in last 12 months, n (%) 13 (8.1) Breathlessness (NYHA class), n (%) 0 85 (53.1) 1 9 (5.6) 2 11 (6.9) 3 9 (5.6) 4 46 (28.8) Stunted [HFA z ≤2], n (%) 89 (55.6%) WFH z-score, mean (SD) −0.82 (±1.09) Finger clubbing, n (%) 34 (22.1) Resting pulse rate, median min−1 (IQR) 87.0 (76.0–98.5) Resting tachypnoea [>24/min], n (%) 57 (35.6) Resting hypoxia [SpO2 <92%], n (%) 33 (20.6) Normoxemia but desaturates >4%, n (%) 29 (18.1) Abbreviations: ART, antiretroviral therapy; HFA, height for age; HIV, human immunodeficiency virus; IQR, interquartile range; NYHA, New York Heart Association breathlessness scale; SD, standard deviation; WFH, weight for height. aContinuous data are represented as median (IQR). bMissing data n = 1. cUnknown n = 15; data unavailable n = 3.

Characteristic n (%) unless stated Age, median years (IQR) 11.1 (9.5–12.4) Sex, female (%) 80 (50.0%) Age at HIV diagnosis, median years (IQR) 7.9 (5.8–9.8) Child aware of HIV diagnosis,a n (%) 65 (41.1) CD4 count,c median cells/µL (IQR) 572 (370–876) Taking cotrimoxazole prophylaxis, n (%) 159 (99.4%) On ART,a n (%) 114 (71.7%) Duration of ART,b median years (IQR) 3.5 (1.3–4.6) Chest infection in preceding year, n (%) 30 (18.8%) Cough, n (%) 60 (37.5) Sputum produced, n (%) 32 (20.0) Wheezing in last 12 months, n (%) 13 (8.1) Breathlessness (NYHA class), n (%) 0 85 (53.1) 1 9 (5.6) 2 11 (6.9) 3 9 (5.6) 4 46 (28.8) Stunted [HFA z ≤2], n (%) 89 (55.6%) WFH z-score, mean (SD) −0.82 (±1.09) Finger clubbing, n (%) 34 (22.1) Resting pulse rate, median min−1 (IQR) 87.0 (76.0–98.5) Resting tachypnoea [>24/min], n (%) 57 (35.6) Resting hypoxia [SpO2 <92%], n (%) 33 (20.6) Normoxemia but desaturates >4%, n (%) 29 (18.1) Abbreviations: ART, antiretroviral therapy; HFA, height for age; HIV, human immunodeficiency virus; IQR, interquartile range; NYHA, New York Heart Association breathlessness scale; SD, standard deviation; WFH, weight for height. aContinuous data are represented as median (IQR). bMissing data n = 1. cUnknown n = 15; data unavailable n = 3. Figure 1. Study flowchart. Flow diagram illustrates participant retention and quality of spirometry throughout the study. CXR, chest x-ray.

Characteristic n (%) unless stated Age, median years (IQR) 11.1 (9.5–12.4) Sex, female (%) 80 (50.0%) Age at HIV diagnosis, median years (IQR) 7.9 (5.8–9.8) Child aware of HIV diagnosis,a n (%) 65 (41.1) CD4 count,c median cells/µL (IQR) 572 (370–876) Taking cotrimoxazole prophylaxis, n (%) 159 (99.4%) On ART,a n (%) 114 (71.7%) Duration of ART,b median years (IQR) 3.5 (1.3–4.6) Chest infection in preceding year, n (%) 30 (18.8%) Cough, n (%) 60 (37.5) Sputum produced, n (%) 32 (20.0) Wheezing in last 12 months, n (%) 13 (8.1) Breathlessness (NYHA class), n (%) 0 85 (53.1) 1 9 (5.6) 2 11 (6.9) 3 9 (5.6) 4 46 (28.8) Stunted [HFA z ≤2], n (%) 89 (55.6%) WFH z-score, mean (SD) −0.82 (±1.09) Finger clubbing, n (%) 34 (22.1) Resting pulse rate, median min−1 (IQR) 87.0 (76.0–98.5) Resting tachypnoea [>24/min], n (%) 57 (35.6) Resting hypoxia [SpO2 <92%], n (%) 33 (20.6) Normoxemia but desaturates >4%, n (%) 29 (18.1) Abbreviations: ART, antiretroviral therapy; HFA, height for age; HIV, human immunodeficiency virus; IQR, interquartile range; NYHA, New York Heart Association breathlessness scale; SD, standard deviation; WFH, weight for height. aContinuous data are represented as median (IQR). bMissing data n = 1. cUnknown n = 15; data unavailable n = 3. Figure 1. Study flowchart. Flow diagram illustrates participant retention and quality of spirometry throughout the study. CXR, chest x-ray. It is worth noting that 91 (56.9%) participants had 1 or more of the following symtoms: cough; moderate or severe dyspnoea New York Heart Association ([NYHA] grade III and IV); wheeze. Of those with previous pulmonary TB, 6 (3.8%) had received more than 2 courses. In the preceding year, 34 (21.3%) had used antibiotics for a lower respiratory tract infection: 17 (10.6%) had received multiple courses.

toms: cough; moderate or severe dyspnoea New York Heart Association ([NYHA] grade III and IV); wheeze. Of those with previous pulmonary TB, 6 (3.8%) had received more than 2 courses. In the preceding year, 34 (21.3%) had used antibiotics for a lower respiratory tract infection: 17 (10.6%) had received multiple courses. Quality of Life Participants reported high quality of life in 6 domains (median scores of 88.9–100.0 for emotional, eating, body image, treatment burden, respiratory, and digestion), where 100 is the maximum score. Social and physical activity domains had the lowest median scores of 57.1 (interquartile range [IQR], 47.6–57.1) and 83.3 (IQR, 55.6–100.0), respectively.

y of life in 6 domains (median scores of 88.9–100.0 for emotional, eating, body image, treatment burden, respiratory, and digestion), where 100 is the maximum score. Social and physical activity domains had the lowest median scores of 57.1 (interquartile range [IQR], 47.6–57.1) and 83.3 (IQR, 55.6–100.0), respectively. Developing a Case Definition and Phenotypes of Chronic Lung Disease An association matrix was used to investigate potential case definitions (Supplementary Table 1). Two patients with active TB were excluded from this analysis. Two candidate phenotypes were postulated: one characterized by cough (37.5%; 95% confidence interval [CI], 30.0%–45.1%) and the other by hypoxia or desaturation at submaximal exercise (38.8%; 95% CI, 31.1–46.4%). Although these might be expected to commonly coexist, only 22 (13.8%; 95% CI, 8.4%–19.1%) participants had both. There was lack of agreement between these 2 variables over that expected by chance alone (expected agreement 52.8%, observed agreement 51.3%, kappa = −3.3%). Therefore, we further characterized those 2 proposed phenotypes (CLD cough and CLD hypoxia) (see Figure 3). Table 2 summarizes univariable and multivariable analysis of risk factors for chronic cough and hypoxia or desaturation individually. Table 2. Risk Factors for Chronic Lung Disease Defined by Presence of Cough and Hypoxia

, we further characterized those 2 proposed phenotypes (CLD cough and CLD hypoxia) (see Figure 3). Table 2 summarizes univariable and multivariable analysis of risk factors for chronic cough and hypoxia or desaturation individually. Table 2. Risk Factors for Chronic Lung Disease Defined by Presence of Cough and Hypoxia Characteristic Presence of Cough Presence of Hypoxia or Desaturation Univariate OR (95% CI) P Multivariatea OR (95% CI) P Univariate OR (95% CI) P Multivariatea OR (95% CI) P Distal (Indirect) Factors Sex, female 0.72 (0.38–1.38) .322 1.24 (0.65–2.35) .514 Age, years 0.99 (0.84–1.16) .883 1.00 (0.81–1.23) .98 0.97 (0.82–1.14) .68 0.95 (0.79–1.13) .54 Orphaned (1+ parent died) 0.65 (0.34–1.24) .192 1.36 (0.71–2.6) .348 HFA, z-score 0.85 (0.64–1.12) .25 0.90 (0.68–1.18) .451 WFH, z-score 0.96 (0.71–1.30) .798 0.91 (0.67–1.22) .515 ART prescribed 0.91 (0.45–1.84) .788 0.79 (0.34–1.83) .59 0.64 (0.32–1.28) .207 0.83 (0.35–1.96) .67 Age at which ART started, years 0.94 (0.82–1.08) .372 0.92 (0.8–1.06) .227 CD4c <100 1.93 (0.53–7.04) .32 4.03 (0.99–16.39) .051 3.87 (0.91–16.42) .066 100–199 1.93 (0.53–7.04) .32 1.72 (0.47–6.30) .41 200–349 1.50 (0.52–4.31) .45 0.79 (0.26–2.41) .67 >349 1.00 – 1.00 – Proximal (Direct) Factors Previous TB 1.49 (0.69–3.22) .316 1.51 (0.7–3.24) .295 Smoker in household 0.44 (0.15–1.25) .104 0.49 (0.18–1.33) .145 NYHA grade 3 or 4 1.65 (0.84–3.24) .148 0.77 (0.39–1.52) .45 Wheeze 11.47 (2.44–53.83) <.001 6.94 (1.38–34.95) .019 0.44 (0.12–1.66) .198 CFQ-R physical domain 0.98 (0.97–0.99) <.001 0.98 (0.97–1.00) .017 1.00 (0.99–1.01) .623 Clubbing 1.19 (0.54–2.63) .666 2.28 (1.04–4.99) .038 1.28 (0.50–3.25) .60 Respiratory rate, >25/min at rest 0.98 (0.50–1.93) .948 3.00 (1.52–5.92) .001 2.39 (1.06–5.38) .032 Pulse rate, beats/min 1.01 (0.99–1.03) .185 1.00 (0.98–1.01) .585 CXR abnormalityb 3.00 (1.15–7.85) .023 3.43 (1.01–11.65) .048 1.04 (0.4–2.7) .941 Abnormal spirometry 2.61 (1.29–5.28) .007 2.09 (0.94–4.66) .072 1.65 (0.83–3.28) .153 Abbreviations: ART, antiretroviral therapy; CI, confidence interval; CFQ-R, Cystic Fibrosis Questionnaire-Revised; CXR, chest x-ray; HFA, height for age; NYHA, New York Heart Association breathlessness scale; OR, odds ratio; TB, tuberculosis; WFH, weight for height.

.28) .007 2.09 (0.94–4.66) .072 1.65 (0.83–3.28) .153 Abbreviations: ART, antiretroviral therapy; CI, confidence interval; CFQ-R, Cystic Fibrosis Questionnaire-Revised; CXR, chest x-ray; HFA, height for age; NYHA, New York Heart Association breathlessness scale; OR, odds ratio; TB, tuberculosis; WFH, weight for height. aAdjusted for priori variables: age, sex, and being on ART and significant distal and proximal variables. bDefined here as consolidation, volume loss, or lymphadenopathy because other findings were nondiscriminatory. cComparator population is those with CD4 >349. Participants diagnosed with pulmonary tuberculosis are not included in this analysis. Chronic lung disease cough was not associated with any indirect factors, but it was significantly associated with wheeze (odds ratio [OR] = 11.47; 95% CI, 2.44–53.83), abnormal chest radiograph (OR = 3.00; 95% CI, 1.15–7.85), and abnormal spirometry (OR = 2.61; 95% CI, 1.29–5.28). Participants with higher levels of exercise tolerance as measured by CFQ-R physical domain had reduced odds of CLD cough (OR = 0.98; 95% CI, 0.97–0.99). After multivariable analysis, wheeze (OR = 6.94; 95% CI, 1.38–34.95; P = .019), CFR-Q physical domain (OR = 0.98; 95% CI, 0.97–1.00; P = .017), and abnormal chest radiograph (OR = 3.43; 95% CI, 1.01–7.85; P = .048) remained significant.

CFQ-R physical domain had reduced odds of CLD cough (OR = 0.98; 95% CI, 0.97–0.99). After multivariable analysis, wheeze (OR = 6.94; 95% CI, 1.38–34.95; P = .019), CFR-Q physical domain (OR = 0.98; 95% CI, 0.97–1.00; P = .017), and abnormal chest radiograph (OR = 3.43; 95% CI, 1.01–7.85; P = .048) remained significant. Chronic lung disease hypoxia had more limited univariate predictors: only finger clubbing (OR = 2.28; 95% CI, 1.04–4.99) and respiratory rate (OR = 3.00; 95% CI, 1.52–5.92). Only resting tachypnoea remained significant in multivariable modeling adjusted for a priori variables. There was weak evidence for the association of CD4 <100 cell/µL and CLD hypoxia (OR = 3.87; 95% CI, 0.99–16.39; P = .051) compared with children having a CD4 count of 350 or more. Spirometry Spirometry results for 145 participants are summarized in Table 3 and Figure 2A. Median FEV1 and FVC were reduced compared with international reference ranges (1.31 SD and 0.89 SD below expected, respectively). Categorically, 90 (62.1%) participants had normal spirometry, 26 (17.9%) participants had obstructive defects, and 29 (20.0%) participants had reduced FVC. Fewer individuals were classified as having abnormalities using local compared with international reference range (43 vs 55). Within our cohort, FEV1 z-score did not significantly decline with age (Figure 2B; r2 = 0.026; P = .054). Table 3. Spirometric Indices*

tive defects, and 29 (20.0%) participants had reduced FVC. Fewer individuals were classified as having abnormalities using local compared with international reference range (43 vs 55). Within our cohort, FEV1 z-score did not significantly decline with age (Figure 2B; r2 = 0.026; P = .054). Table 3. Spirometric Indices* Baseline Spirometry (n = 145) GLI Reference [10] Local Reference FEV1 −1.31 (−2.10 to −0.27)† 92.2 (79.5 to 104.6)‡ FVC −0.89 (−1.91 to −0.18)† 93.9 (81.8 to 104.2)‡ FEV1/FVC −0.27 (−1.21 to 0.35)† 87.9 (82.1 to 91.6)§ FEF25-75% −0.69 (−1.63 to 0.38)† Not available No abnormality, n (%) 90 (62.1%) 102 (70.3%) Obstruction, n (%) 26 (17.9%) 18 (12.4%) Reduced FVC, n (%) 29 (20.0%) 25 (17.2%) Reversibility Testing FEV % Change Reversible, n (%) Reduced FVC pattern (n = 26) 2.6 (−3.6 to 9.5) 8 (30.8%) Obstructive pattern (n = 21) 3.3 (−4.3 to 12.1) 7 (33.3%) All (n = 47) 2.7 (−4.3 to 10.1) 15 (31.9%) Abbreviations: FEF25–75%, forced expiratory flow at 25%–75%; FEV1, forced expiratory volume in 1 second; FVC, forced vital capacity; GLI, Global Lung Initiative; IQR, interquartile range. *Continuous data presented as median (IQR) due to skewed distributions. Includes all traces meeting ATS criteria (grades A and B), n = 145 at baseline, n = 47 for reversibility testing. †Median z-score (IQR). ‡Median percentage of predicted (IQR). §Median percentage (IQR).

Baseline Spirometry (n = 145) GLI Reference [10] Local Reference FEV1 −1.31 (−2.10 to −0.27)† 92.2 (79.5 to 104.6)‡ FVC −0.89 (−1.91 to −0.18)† 93.9 (81.8 to 104.2)‡ FEV1/FVC −0.27 (−1.21 to 0.35)† 87.9 (82.1 to 91.6)§ FEF25-75% −0.69 (−1.63 to 0.38)† Not available No abnormality, n (%) 90 (62.1%) 102 (70.3%) Obstruction, n (%) 26 (17.9%) 18 (12.4%) Reduced FVC, n (%) 29 (20.0%) 25 (17.2%) Reversibility Testing FEV % Change Reversible, n (%) Reduced FVC pattern (n = 26) 2.6 (−3.6 to 9.5) 8 (30.8%) Obstructive pattern (n = 21) 3.3 (−4.3 to 12.1) 7 (33.3%) All (n = 47) 2.7 (−4.3 to 10.1) 15 (31.9%) Abbreviations: FEF25–75%, forced expiratory flow at 25%–75%; FEV1, forced expiratory volume in 1 second; FVC, forced vital capacity; GLI, Global Lung Initiative; IQR, interquartile range. *Continuous data presented as median (IQR) due to skewed distributions. Includes all traces meeting ATS criteria (grades A and B), n = 145 at baseline, n = 47 for reversibility testing. †Median z-score (IQR). ‡Median percentage of predicted (IQR). §Median percentage (IQR). Figure 2. Spirometry results overview. Graphs illustrating the degree and distribution of spirometric abnormality. (A) Boxes represent 25th and 75th percentiles, whiskers represent 10th and 90th percentiles, with outliers shown as individual dots. Upper limit of normal (ULN) and lower limit of normal (LLN) drawn by dashed line at +1.64 standard deviation (SD) and −1.64 SD from the mean, respectively. (B) Forced expiratory volume (FEV1) z-score for all participants as a function of age. Linear regression model is shown as a solid line. There is a nonsignificant tendency to reducing FEV1 with increasing age in this cohort (r2 = 0.026; P = .054). Similar results for forced vital capacity obtained (results not shown).

(B) Forced expiratory volume (FEV1) z-score for all participants as a function of age. Linear regression model is shown as a solid line. There is a nonsignificant tendency to reducing FEV1 with increasing age in this cohort (r2 = 0.026; P = .054). Similar results for forced vital capacity obtained (results not shown). Figure 3. Proposed CLD phenotypes. Proportional areas diagram illustrating the proposed CLD phenotypes, “cough” and “hypoxia”, and their overlap with individuals with abnormal spirometry. Percentages indicate the proportion of the entire study population for which spirometry data were available. Fifty-five participants had abnormal spirometry, 47 of whom adequately completed postbronchodilator testing. Median change in FEV1 was 2.9% (IQR, −4.3 to 9.2). Reversibility threshold of 12% increase was met in 7 (33.3%) of those with obstructive abnormalities and 8 (30.8%) with reduced FVC. When reviewed 4 weeks afterwards, despite being given salbutamol, only 2 participants continued to use their inhaler, and none reported symptomatic improvement.

s 2.9% (IQR, −4.3 to 9.2). Reversibility threshold of 12% increase was met in 7 (33.3%) of those with obstructive abnormalities and 8 (30.8%) with reduced FVC. When reviewed 4 weeks afterwards, despite being given salbutamol, only 2 participants continued to use their inhaler, and none reported symptomatic improvement. Chest Radiograph Abnormalities The majority of radiographs had at least 1 abnormality (n = 110, 68.8%). Upper or lower zone preponderance were uncommon (n = 12, 10.9% and n = 13, 11.8%, respectively) compared with mid-zone abnormality. The most frequent abnormality was ring or tramlining pattern (n = 90, 56.3%). Two abnormalities (airspace shadowing and volume loss) were discriminatory for both CLD phenotypes (Table 2) and spirometric abnormality (Supplementary Table 3). Air space shadowing (n = 10, 6.3%) and loss of volume (n = 3, 1.9%) were associated with reduced FEV1 (P = .0032 and P = .010, respectively). Other radiographic findings were not significantly associated with differences in FEV1 or FVC z-score. Clinical Associations of Lung Function Potential associates of abnormal spirometry were investigated (Supplementary Data, Table 2). Only 1 strong association emerged: individuals reporting cough for more than 1 month were 2.9 times more likely to have abnormal spirometry (95% CI, 1.21–7.10). Microbiological Findings Sputum was obtained from 32 of 60 participants with cough, with the remainder unable to expectorate. There were 6 positive mycobacterial cultures; 2 M tuberculosis and 4 nontuberculous mycobacteria.

Clinical Associations of Lung Function Potential associates of abnormal spirometry were investigated (Supplementary Data, Table 2). Only 1 strong association emerged: individuals reporting cough for more than 1 month were 2.9 times more likely to have abnormal spirometry (95% CI, 1.21–7.10). Microbiological Findings Sputum was obtained from 32 of 60 participants with cough, with the remainder unable to expectorate. There were 6 positive mycobacterial cultures; 2 M tuberculosis and 4 nontuberculous mycobacteria. DISCUSSION This study demonstrates a high burden of symptoms in children aged 8 to 16 with vertically acquired HIV, consistent with a similar study from Zimbabwe [14]. Over half of our participants were coughing, wheezy, or breathless. Within our cohort, there are 2 definable, common, and independent phenotypes: children who cough (CLD cough), and those who have hypoxia at rest or desaturate with submaximal exercise (CLD hypoxia). Neither phenotype was associated with antiretroviral treatment. For the CLD-cough phenotype, cough, wheeze, and functional breathlessness were commonly associated with each other and also with radiological abnormalities of airspace shadowing and volume loss where parenchymal lung disease was likely. Previous treatment for TB was not a significant risk factor for this phenotype, and symptoms were mostly chronic (individuals with symptoms of acute infection were excluded). Abnormal spirometry was associated with this phenotype, but there was no preponderance of obstructive or restrictive types.

g disease was likely. Previous treatment for TB was not a significant risk factor for this phenotype, and symptoms were mostly chronic (individuals with symptoms of acute infection were excluded). Abnormal spirometry was associated with this phenotype, but there was no preponderance of obstructive or restrictive types. The CLD-hypoxia phenotype was predictably associated with tachypnoea. There was a suggestion that very low CD4 counts (<100) predicted hypoxia. Low numbers of individuals in this group limited our power to detect a difference. Although not independently associated, there was a higher than expected rate of finger clubbing in these individuals.

e was predictably associated with tachypnoea. There was a suggestion that very low CD4 counts (<100) predicted hypoxia. Low numbers of individuals in this group limited our power to detect a difference. Although not independently associated, there was a higher than expected rate of finger clubbing in these individuals. Chronic lung disease in these children is likely to be multifactorial and therefore difficult to clearly define [15]. Frequent bacterial, mycobacterial, and viral respiratory infections were reported in this population (18.8% of our cohort had received treatment for chest infection in the preceding year), and these can also contribute to bronchiectasis. Consistent with underlying bronchiectasis, there was a high rate of finger clubbing, reduced lung function, and radiological abnormalities consistent with bronchiectasis. These features are insensitive and nonspecific for its diagnosis in isolation [16]. A direct effect of HIV and chronic inflammation of the airways might cause reduced lung capacities and chronic chest x-ray findings, including lymphadenopathy. Findings that the pulmonary microbiota can be altered in adult HIV, notably for Tropheryma whipplei bacteria [17], raise the possibility that these changes may reflect or drive long-term disease in the airways, including chronic inflammation. High-resolution computerized tomography findings from similar patients in Zimbabwe that are suggestive of airways disease [18] can represent the final common pathway of many diseases, including postinfective change, although this is uncommon outside of allogeneic transplantation [19]. In our study, the clinical syndrome of CLD cough including nonreversible spirometry findings would be consistent with such pathology [20].

f airways disease [18] can represent the final common pathway of many diseases, including postinfective change, although this is uncommon outside of allogeneic transplantation [19]. In our study, the clinical syndrome of CLD cough including nonreversible spirometry findings would be consistent with such pathology [20]. Toro et al [21] demonstrated a high burden of pulmonary lymphoid hyperplasia and lymphoid interstitial pneumonitis (LIP) in early life associated with a wide variety of radiological changes, of which reticular infiltrates are most typical [22]. Early Western cohorts including the P2C2 study noted high rates of LIP and reported chest radiograph with LIP suggestive changes [23], but this condition has been almost eliminated with effective ART provision [24]. Our participants were considerably older than the usual age of LIP presentation [25], and they started ART later. In this case, the 2013 WHO guidelines to start all HIV- positive children under 5 years on ART may improve rates of CLD in the future. A Zimbabwean study has shown a high frequency of cardiac abnormalities and cor pulmonale in adolescents with vertically transmitted HIV infection [14]. This raises the possibility that CLD hypoxia might represent pulmonary vascular disease or interstitial lung disease with secondary cardiac involvement.

Toro et al [21] demonstrated a high burden of pulmonary lymphoid hyperplasia and lymphoid interstitial pneumonitis (LIP) in early life associated with a wide variety of radiological changes, of which reticular infiltrates are most typical [22]. Early Western cohorts including the P2C2 study noted high rates of LIP and reported chest radiograph with LIP suggestive changes [23], but this condition has been almost eliminated with effective ART provision [24]. Our participants were considerably older than the usual age of LIP presentation [25], and they started ART later. In this case, the 2013 WHO guidelines to start all HIV- positive children under 5 years on ART may improve rates of CLD in the future. A Zimbabwean study has shown a high frequency of cardiac abnormalities and cor pulmonale in adolescents with vertically transmitted HIV infection [14]. This raises the possibility that CLD hypoxia might represent pulmonary vascular disease or interstitial lung disease with secondary cardiac involvement. The degree of impairment of lung function is marked when measured against both internationally used and locally derived reference ranges. Adult HIV patients in the United States have higher rates of asthma than the general population [26], but the generalizability to our age group and geography is uncertain. The International Study of Asthma and Allergies in Childhood (ISAAC) study [27] did not cover Malawi, but prevalence of wheeze was 15.9% in English-speaking African countries. In our study, bronchodilator reversibility was minimal, and rates of wheeze were similar to regional rates in the general population, suggesting that asthma was unlikely to be a predominant pathology. The FEV1 improvement after inhaled bronchodilator was disappointing, and on average it was indistinguishable from zero. Therefore, it is possible that even those with >12% increase in FEV1 may represent bias related to regression to the mean. No participants found salbutamol helpful at 4 weeks: effective treatment options are urgently needed.

er inhaled bronchodilator was disappointing, and on average it was indistinguishable from zero. Therefore, it is possible that even those with >12% increase in FEV1 may represent bias related to regression to the mean. No participants found salbutamol helpful at 4 weeks: effective treatment options are urgently needed. Within our cohort, there is no strong evidence for clinically significant decline in lung function with age. However, this could be confounded by age of ART initiation, and a longitudinal study to specifically examine this is in progress. Declining FEV1 is reported in CLDs such as cystic fibrosis and chronic obstructive pulmonary disease. In other cohorts (chronic coughers with bronchiectasis which presented in childhood), FEV1 declined with age, but this was apparent only after many years [28]. Some decline may be artefactual relating to growth and maturation delay, although the significant baseline abnormality suggests that earlier life events have already strongly affected the lung architecture. In any case, the lack of clinical predictors of lung function abnormality suggests that considerable lung abnormalities, through intercurrent disease or other effects on lung growth, may not be identified unless spirometry is performed. Rates of reported household biomass fuel use were typical for many sub-Saharan countries. This important public health problem may have contributed to reduced lung function in our population [29].

lities, through intercurrent disease or other effects on lung growth, may not be identified unless spirometry is performed. Rates of reported household biomass fuel use were typical for many sub-Saharan countries. This important public health problem may have contributed to reduced lung function in our population [29]. Our data are limited by (1) the cross-sectional nature of the study, (2) the lack of total lung volume and transfer factor measurements, and (3) the absence of noninfected controls. Reversibility studies might be more easily interpreted with either universal reversibility testing or a control arm, but this was not possible within our study. We did not have access to HRCT imaging, echocardiography, or post mortem tissue biopsies, which would define the pathologies more clearly, and TB screening was limited by suboptimal diagnostics.

easily interpreted with either universal reversibility testing or a control arm, but this was not possible within our study. We did not have access to HRCT imaging, echocardiography, or post mortem tissue biopsies, which would define the pathologies more clearly, and TB screening was limited by suboptimal diagnostics. Prospective studies should examine our definition of the 2 phenotypes in relation to pathophysiology in a cohort in which intensive investigation is possible, for example, with high-resolution computed tomography scanning, echocardiography, and, possibly, autopsy studies. If the phenotypes correlate with disease (we hypothesize cough with bronchiectasis or bronchiolitis obliterans and hypoxia with interstitial lung disease), this could be useful to clinicians where such investigations are not available. Longitudinal cohort studies should assess long-term change in symptoms and lung function in CLD, and they would facilitate therapeutic trials of immunomodulation (for example, prednisolone in obliterative bronchiolitis) or antimicrobials (azithromycin in bronchiectasis).

ans where such investigations are not available. Longitudinal cohort studies should assess long-term change in symptoms and lung function in CLD, and they would facilitate therapeutic trials of immunomodulation (for example, prednisolone in obliterative bronchiolitis) or antimicrobials (azithromycin in bronchiectasis). CONCLUSIONS Widespread evidence of pulmonary disease presented here adds to the case for treatment of all HIV-infected children with antiretrovirals irrespective of CD4 count. At the very least, as a WHO HIV Stage 3 criterion, there should be a strong emphasis on identifying children with CLD and establishing early ART in those individuals. For this purpose, simple clinical definitions of CLD cough (in the absence of TB) and CLD hypoxia could be useful to clinicians in healthcare settings with few resources. Supplementary Data Supplementary materials are available at the Journal of The Pediatric Infectious Diseases Society online (http://jpids.oxfordjournals.org). Acknowledgments We thank all of the participants, their parents and guardians, and clinic staff. We acknowledge the authors of CFQ-R: Alexandra L. Quittner, Anne Buu, Marc Watrous, and Melissa A. Davis. Author contributions. J. R., S. J. R., T. M., E. L. C., and R. A. F. contributed to conception and design; B. O., J. J. v. O., and S. A. provided clinical advice; and E. L. W. provided statistical support. All authors contributed to writing the article and approved the final version.

Acknowledgments We thank all of the participants, their parents and guardians, and clinic staff. We acknowledge the authors of CFQ-R: Alexandra L. Quittner, Anne Buu, Marc Watrous, and Melissa A. Davis. Author contributions. J. R., S. J. R., T. M., E. L. C., and R. A. F. contributed to conception and design; B. O., J. J. v. O., and S. A. provided clinical advice; and E. L. W. provided statistical support. All authors contributed to writing the article and approved the final version. Financial support. T. M. was funded by the Commonwealth scholarship, with research costs from a grant fom Helse Nord Northern Norway Regional Health Authority. E. L. C., R. A. F., and J. R. are supported by Wellcome Trust Fellowships (Senior Fellowship in Clinical Sciences WT091769, Career Development Fellowship WT095878 and Clinical PhD Fellowship 086756/B/08/Z, respectively). Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest.

Invasive fungal disease (IFD) affects primarily patients with a compromised immune system, classically children with a hematological malignancy, especially those with acute leukemia, hematopoietic stem cell transplant (HSCT) recipients, solid-organ transplant (SOT) recipients, those with primary or acquired immunodeficiency, and premature neonates [1–3]. There is also an emerging appreciation of other patient groups at increased risk of IFD, such as those in a pediatric intensive care unit (PICU) [4, 5], patients who suffer a traumatic injury, those who have undergone surgery, particularly abdominal surgery or corrective surgery for congenital heart disease [6, 7], and patients with an autoimmune and/or autoinflammatory condition treated with immunomodulatory agents [8, 9]. Because the clinical disease phenotype is a result of the interaction between a fungal pathogen and the individual host immune response, a variety of disease presentations, determined mainly by the nature of the immune impairment, can be seen [10]. Knowledge of the clinical signs and symptoms of IFD, additional predisposing risk factors, and variations in disease phenotypes across different at-risk patient groups is essential for enabling early recognition and diagnosis of IFD and, ultimately, improving disease outcomes.

f the immune impairment, can be seen [10]. Knowledge of the clinical signs and symptoms of IFD, additional predisposing risk factors, and variations in disease phenotypes across different at-risk patient groups is essential for enabling early recognition and diagnosis of IFD and, ultimately, improving disease outcomes. PATIENTS WITH A HEMATOLOGICAL MALIGNANCY AND TRANSPLANT RECIPIENTS In children with a hematological malignancy or those who have undergone HSCT, persistent febrile neutropenia despite broad-spectrum antibiotic treatment is often the first and only clinical sign to alert the clinician to suspect an IFD [11, 12].

f the immune impairment, can be seen [10]. Knowledge of the clinical signs and symptoms of IFD, additional predisposing risk factors, and variations in disease phenotypes across different at-risk patient groups is essential for enabling early recognition and diagnosis of IFD and, ultimately, improving disease outcomes. PATIENTS WITH A HEMATOLOGICAL MALIGNANCY AND TRANSPLANT RECIPIENTS In children with a hematological malignancy or those who have undergone HSCT, persistent febrile neutropenia despite broad-spectrum antibiotic treatment is often the first and only clinical sign to alert the clinician to suspect an IFD [11, 12]. Risk Factors An initial risk profile can be derived on the basis of the underlying malignancy or transplant type and the specific characteristics of chemotherapy. For instance, patients with acute myeloid leukemia, high-risk (including relapsed) acute lymphoblastic leukemia, recipients of an allogeneic HSCT (particularly from a matched unrelated donor), and those who suffer from chronic or severe acute graft-versus-host disease are at particularly high risk of infection [1–3, 13–17]. Within the first 30 days after HSCT [14, 18, 19], a period of significant immunosuppression and profound neutropenia, patients are at the greatest risk of IFD. However, a second period of risk exists after neutrophil engraftment that coincides with acute or chronic graft-versus-host disease and requires ongoing vigilance well beyond neutrophil recovery [3, 14, 20]. The risk of developing IFD after autologous HSCT is considerably lower than that after allogeneic HSCT, and underlying disease can affect the incidence of IFD [1, 21, 22].

graftment that coincides with acute or chronic graft-versus-host disease and requires ongoing vigilance well beyond neutrophil recovery [3, 14, 20]. The risk of developing IFD after autologous HSCT is considerably lower than that after allogeneic HSCT, and underlying disease can affect the incidence of IFD [1, 21, 22]. IFD in children with a malignancy or after HSCT rarely occurs in the presence of an isolated predisposing host factor [17, 23]. Typically, multiple risk factors are present, such as prolonged neutropenia (absolute neutrophil count, ≤500/μL for ≥10 days), high-dose corticosteroid use (≥0.3 mg/kg per day of prednisone or equivalent), additional immunosuppressive therapy or chemotherapy, presence of a central venous catheter (CVC), use of parenteral nutrition, mucositis, concomitant bacterial infection, or preceding broad-spectrum antibiotic use [3, 5, 16, 23–26]. The depth and duration of neutropenia are associated with chemotherapy intensity, and the highest risk for IFD is reported during induction therapy [27, 28]. For instance, during induction chemotherapy to treat acute myeloid leukemia, the risk of invasive candidiasis (IC) is greater than that during subsequent chemotherapy courses (10% vs 6%, respectively) [28]. Data regarding the effect of age as an additional risk factor for IFD in children with a malignancy are conflicting. In 2 studies, an age of >10 years was associated with a higher incidence of IFD [11, 28], whereas a subsequent study found no significant differences between younger and older children [29].

ively) [28]. Data regarding the effect of age as an additional risk factor for IFD in children with a malignancy are conflicting. In 2 studies, an age of >10 years was associated with a higher incidence of IFD [11, 28], whereas a subsequent study found no significant differences between younger and older children [29]. Data concerning risk factors for IFD in pediatric SOT recipients have been more limited. A study that included 1854 pediatric heart transplantation recipients identified previous surgery and mechanical ventilation during transplantation as independent risk factors for IFD in a multivariate analysis [30]. For children who had undergone lung transplantation, colonization before transplantation, transplant rejection, cytomegalovirus mismatch, tacrolimus treatment, and older age increased the risk of developing IFD [31]. In a recently published US study of 397 children who were undergoing liver transplantation, the only significant risk factor for IC identified with multivariate analysis was admission to an ICU before transplantation [32]. Children who undergo a renal transplant are considered to be at low risk for IFD; candidemia related to intravascular catheter use is the most common presentation [33, 34].

transplantation, the only significant risk factor for IC identified with multivariate analysis was admission to an ICU before transplantation [32]. Children who undergo a renal transplant are considered to be at low risk for IFD; candidemia related to intravascular catheter use is the most common presentation [33, 34]. Clinical Presentation Invasive aspergillosis (IA) and IC are the most common IFDs in patients with a hematological malignancy and in HSCT or SOT recipients [1, 11, 12, 35]. Non-Aspergillus molds, such as Mucorales, are being seen with increased frequency in patients who have received an Aspergillus-active antifungal agent before their IFD is diagnosed [3, 36]. As a result of effective prophylaxis with trimethoprim-sulfamethoxazole, Pneumocystis jirovecii pneumonia (PCP) is seen rarely in these patients. However, a diagnosis of PCP should still be considered in patients who receive second-line PCP prophylaxis agents that are likely less effective than trimethoprim-sulfamethoxazole [37].

of effective prophylaxis with trimethoprim-sulfamethoxazole, Pneumocystis jirovecii pneumonia (PCP) is seen rarely in these patients. However, a diagnosis of PCP should still be considered in patients who receive second-line PCP prophylaxis agents that are likely less effective than trimethoprim-sulfamethoxazole [37]. Candidiasis IC is identified most commonly after the isolation of yeasts on blood culture [38, 39]; Candida spp now represent the third most common cause of nosocomial bloodstream infection in children [40, 41]. Candida spp need to be strongly considered when a clinical diagnosis of sepsis or septic shock in a neutropenic pediatric patient has been made, especially in the presence of an intravascular catheter and recent exposure to broad-spectrum antibiotics. However, although the isolation of Candida spp on blood culture is highly specific for IC, the sensitivity of such cultures is low, and disseminated infection can be present in the absence of a positive blood culture result [42–44]. Indeed, approximately half of the patients with IC will not have a positive blood culture result, and it might be more useful to consider IC as 3 distinct clinical entities, that is, candidemia in the absence of deep-seated infection, candidemia with disseminated infection, and deep-seated infection in the absence of candidemia [44, 45]. Dissemination can occur to almost any site, including the lungs, liver, spleen, kidneys, brain, eyes, and heart, although the symptoms of disseminated disease are frequently minimal and might become apparent only on immune reconstitution [45–47]. However, the identification of dissemination is important, because prolonged treatment might be required [45], and it is an independent risk factor for death in children with IC [48].

t, although the symptoms of disseminated disease are frequently minimal and might become apparent only on immune reconstitution [45–47]. However, the identification of dissemination is important, because prolonged treatment might be required [45], and it is an independent risk factor for death in children with IC [48]. Candida meningitis and meningoencephalitis occur more frequently in children than in adults with candidemia (11.4% vs 0.8%, respectively; P < .001) [49] and can present in the absence of candidemia [43]. In keeping with other manifestations of IC, patients are often asymptomatic; 5 (42%) of 12 children with Candida meningitis in 1 study displayed no symptoms other than fever [43]. When symptoms were present, the most common presenting feature was a reduced level of consciousness (50%), followed by seizures (33%), headache (25%), nuchal rigidity (25%), and cranial nerve palsies (17%). Skin lesions were also identified in one-third of the patients, which reflects the primarily hematogenous route of infection. Cerebrospinal fluid findings might be unremarkable, and the absence of cerebrospinal fluid abnormalities does not exclude central nervous system (CNS) infection [43].

ranial nerve palsies (17%). Skin lesions were also identified in one-third of the patients, which reflects the primarily hematogenous route of infection. Cerebrospinal fluid findings might be unremarkable, and the absence of cerebrospinal fluid abnormalities does not exclude central nervous system (CNS) infection [43]. Ocular involvement during IC is a rare but potentially sight-threatening complication. The most frequent manifestations are chorioretinitis and endophthalmitis [50]. Findings on examination can be unilateral or bilateral, and lesions typically appear as fluffy yellow-white retinal or vitreal balls with associated hemorrhage or vitreous haze [51]. Current practice guidelines from the Infectious Diseases Society of America recommend dilated ophthalmic examination within the first week of candidemia diagnosis in all nonneutropenic patients and within 1 week of neutrophil recovery in all neutropenic patients [45]. Examination findings are often minimal before neutrophil recovery, which explains the recommendation for delayed examination in neutropenic patients [45]. Information on visual outcomes in these patients has been limited; however, in 1 study, 3 of 4 children with endophthalmitis experienced subsequent complications that included retinal detachment and globe rupture [50]. Hepatosplenic candidiasis is rare in children [52], and only sparse information on its clinical presentation is available. Nausea and vomiting, right or left upper-quadrant pain, hepatosplenomegaly, or transaminitis can alert a clinician to the possibility of hepatosplenic infection [53].

Ocular involvement during IC is a rare but potentially sight-threatening complication. The most frequent manifestations are chorioretinitis and endophthalmitis [50]. Findings on examination can be unilateral or bilateral, and lesions typically appear as fluffy yellow-white retinal or vitreal balls with associated hemorrhage or vitreous haze [51]. Current practice guidelines from the Infectious Diseases Society of America recommend dilated ophthalmic examination within the first week of candidemia diagnosis in all nonneutropenic patients and within 1 week of neutrophil recovery in all neutropenic patients [45]. Examination findings are often minimal before neutrophil recovery, which explains the recommendation for delayed examination in neutropenic patients [45]. Information on visual outcomes in these patients has been limited; however, in 1 study, 3 of 4 children with endophthalmitis experienced subsequent complications that included retinal detachment and globe rupture [50]. Hepatosplenic candidiasis is rare in children [52], and only sparse information on its clinical presentation is available. Nausea and vomiting, right or left upper-quadrant pain, hepatosplenomegaly, or transaminitis can alert a clinician to the possibility of hepatosplenic infection [53]. Other rare sites of dissemination in children with IC include the heart and skeleton. Candida osteomyelitis in children typically affects the femoral metaphysis with complicating septic arthritis in the neighboring joint. The humerus, vertebrae, and ribs are other potential sites of infection. Local symptoms are usually present and include pain, tenderness, overlying erythema and edema, and limitation of movement; a majority of patients are febrile at presentation [54, 55]. Infective endocarditis secondary to Candida sp infection can affect children with an underlying hematological malignancy, particularly during periods of immunosuppression, but is seen more commonly in children with preexisting heart disease. Presentation is typically with fever and a new heart murmur. The classical signs of endocarditis, such as Osler’s nodes, Janeway lesions, and Roth spots, are seen rarely in children [56–58].

lignancy, particularly during periods of immunosuppression, but is seen more commonly in children with preexisting heart disease. Presentation is typically with fever and a new heart murmur. The classical signs of endocarditis, such as Osler’s nodes, Janeway lesions, and Roth spots, are seen rarely in children [56–58]. Aspergillosis The primary sites of IA are the lungs and the sinuses, yet only approximately half of the children with pulmonary IA display clinical signs and symptoms of respiratory infection [3, 36, 59–61]. When present, the most commonly reported symptoms are cough, dyspnea, and chest or pleuritic pain [15, 36, 59, 61], and tachypnea and oxygen requirement are the only reported clinical signs [60, 61]. Clinical symptoms of fungal rhinosinusitis can include fever, rhinorrhea, nasal congestion, facial pain or numbness, and headache [60–63]. However, symptoms can be nonspecific, and symptomatic disease might be a late presentation [62]. In a recent study by Cohn et al [60], who used a screening protocol that included direct nasal endoscopy performed at the bedside by an otorhinolaryngologist in addition to computed tomography of the chest and abdominal ultrasound in children with persistent febrile neutropenia despite the administration of broad-spectrum antibiotics, sinonasal disease was confirmed in 13 (42%) of 31 patients. Of the patients identified, 8 (62%) of 13 were asymptomatic, which suggests that fungal rhinosinusitis might be underappreciated in the pediatric oncology population if diagnostic modalities are withheld until specific signs and symptoms occur.

um antibiotics, sinonasal disease was confirmed in 13 (42%) of 31 patients. Of the patients identified, 8 (62%) of 13 were asymptomatic, which suggests that fungal rhinosinusitis might be underappreciated in the pediatric oncology population if diagnostic modalities are withheld until specific signs and symptoms occur. After pulmonary and sinus disease, the most common site of Aspergillus infection is the brain [61, 64]; between 6% and 15% of pediatric patients show evidence of CNS infection [17, 36, 59, 61, 65], and CNS symptoms are reported in up to half of all children with disseminated IA [66]. Multiple brain abscesses are the most common radiographic finding, followed by vasculitis and meningoencephalitis, although other more unusual clinical presentations, including intracerebral hemorrhage and hemorrhagic infarcts, have been reported also, which reflects the angioinvasive properties of Aspergillus hyphae [66]. Symptoms of brain abscess, such as headache and vomiting, are not typically seen in these patients. Instead, symptoms of disorientation, somnolence, general malaise, focal seizures, hemiparesis, and cranial nerve palsies are associated more frequently with CNS aspergillosis and might be the primary presenting feature of IA [61, 64, 66].

abscess, such as headache and vomiting, are not typically seen in these patients. Instead, symptoms of disorientation, somnolence, general malaise, focal seizures, hemiparesis, and cranial nerve palsies are associated more frequently with CNS aspergillosis and might be the primary presenting feature of IA [61, 64, 66]. Cutaneous involvement is far more common in children than in adults [17, 61], affecting between 8% and 41% of children with IA in various pediatric studies [17, 23, 36, 59, 61, 67]. This involvement can be a result of either local infection at the site of trauma (such as intravenous cannulas or CVC sites) or hematogenous dissemination. The clinical characteristics of cutaneous lesions vary from ulcers at intravenous sites to macules, papules, and nodular necrotic lesions with or without surrounding erythema and cellulitis [36, 61, 67]. Lesions can appear as purpuric nodules in the extremities as a result of hematogenous dissemination [64] and can progress to form necrotic eschars [67]. Isolated cutaneous Aspergillus infection has been associated with a more favorable outcome than those of other manifestations of IA [23, 24, 67, 68]. However, it is important to recognize that cutaneous lesions can represent the first sign of disseminated disease; 2 (33%) of 6 patients with clinically localized disease in a study by Abbasi et al [61] were found to have evidence of disseminated disease at autopsy, and in a more recent study by Burgos et al [17], 9 (47.4%) of 19 patients with cutaneous infection also had infection at other sites. When skin lesions are present, they can provide a useful source of diagnostic specimens; almost one-third of positive culture results in a 10-year retrospective study of invasive mold infections in pediatric oncology patients were isolated from the skin [36].

with cutaneous infection also had infection at other sites. When skin lesions are present, they can provide a useful source of diagnostic specimens; almost one-third of positive culture results in a 10-year retrospective study of invasive mold infections in pediatric oncology patients were isolated from the skin [36]. Cardiac involvement is also rare but has been reported consistently in studies of pediatric IA in neutropenic patients [17, 36, 61]. Clinical presentations include pericardial effusion, intracardiac thrombus, and endocarditis [17, 36, 61] Mucormycosis Similar to aspergillosis, the 2 primary sites of infection for mucormycosis are the pulmonary parenchyma and the sinuses. Among pediatric patients with a malignancy or those who were undergoing HSCT, 1 study found that the main clinical sites of mucormycosis were the lungs (25.6%), skin and soft tissues (12.8%), the paranasal sinuses/sinoorbital region (13.8%), and the rhinocerebral region (9.1%). Disseminated disease was present in 46.5% of these patients, which is higher than with IA [69]. The presenting symptoms of mucormycosis cannot be differentiated easily from those of IA and IFD caused by other molds, although signs and symptoms of hemorrhages and infarction are observed more frequently.

l region (9.1%). Disseminated disease was present in 46.5% of these patients, which is higher than with IA [69]. The presenting symptoms of mucormycosis cannot be differentiated easily from those of IA and IFD caused by other molds, although signs and symptoms of hemorrhages and infarction are observed more frequently. PATIENTS ADMITTED TO A PICU Although PICU admission is itself a risk factor for IFD [70], the majority of children who develop IFD in a PICU have other underlying risk factors that precede their PICU admission [71]. These factors include underlying malignancy, immunocompromise, a gastrointestinal disorder (particularly patients with short-gut syndrome), trauma, and surgery (particularly abdominal surgery, neurosurgery, or corrective surgery for congenital heart disease) [5, 71, 72]. In addition, PICU patients often require CVC or urinary catheter placement and frequently receive broad-spectrum antibiotic treatment, parenteral nutrition, and systemic steroid or other immunosuppressive treatment, which further increases their susceptibility to IFD [4, 5, 70, 71, 73].

ital heart disease) [5, 71, 72]. In addition, PICU patients often require CVC or urinary catheter placement and frequently receive broad-spectrum antibiotic treatment, parenteral nutrition, and systemic steroid or other immunosuppressive treatment, which further increases their susceptibility to IFD [4, 5, 70, 71, 73]. Risk Factors PICU patients are at risk of both IA and IC; however, IC is much more prevalent and has been better characterized [70]. Candida spp are now the third most common cause of bloodstream infection and the most frequent cause of IFD in PICU patients [74]. Predictive scoring systems for IC in adult ICU patients exist [75, 76], and attempts were made recently to develop pediatric predictive scoring systems to help identify PICU patients with likely IC [5, 7, 73]. Zaoutis et al [73] were the first to attempt such a system and found that the presence of a CVC or a malignancy or the use of vancomycin or agents with activity against anaerobic organisms for >3 days in the preceding 2 weeks were significant independent risk factors for IC in PICU patients [36]. The authors developed a prediction model by combining the aforementioned factors (>10% risk) and observed a predicted probability of candidemia that ranged from 10.7% to 46% [73]. However, an attempt to validate the prediction model in a multicenter study proved unsuccessful [77]. A separate predictive IC probability model for PICU patients, the ERICAP scoring system, was developed by Jordan et al [5]. It assigns points for the following clinical factors, identified in a multivariate analysis as significantly increasing the likelihood of IC in PICU patients and demonstrating high specificity for IC when present in combination: a pre-PICU hospital stay of ≥15 days; fever; thrombocytopenia; and use of parenteral nutrition [5]. Motta et al [7] also proposed a predictive scoring system for candidemia in children after surgery for congenital heart disease. They found the combination of a RACHS-1 (Risk Adjustment for Congenital Heart Surgery) (a scoring system that groups cardiac procedures into 1 of 6 categories on the basis of risk of death) score of ≥3, thrombocytopenia, and use of acid-suppression therapy resulted in a 58% predictive probability of candidemia. However, both scoring systems remain to be validated.

Adjustment for Congenital Heart Surgery) (a scoring system that groups cardiac procedures into 1 of 6 categories on the basis of risk of death) score of ≥3, thrombocytopenia, and use of acid-suppression therapy resulted in a 58% predictive probability of candidemia. However, both scoring systems remain to be validated. Another study found significant species-specific differences in risk factors; in particular, IC attributed to Candida albicans was associated significantly with chronic metabolic disease, gastrointestinal surgery, fever at PICU admission, and parenteral nutrition, whereas Candida parapsilosis–specific risk factors were previous yeast colonization, tracheostomy, parenteral nutrition, thrombocytopenia at PICU admission, and previous bacterial infection [5, 72]. Species-specific (C albicans versus non-albicans Candida spp) risk factors for IC in PICU patients were identified also in a study by Hegazi et al [78]; they found that the risk factors for acquiring non-albicans IC were an age of >1 year and isolation of a Candida species from a CVC or endotracheal tube. Further investigation to validate and improve the proposed clinical prediction models are of utmost importance to enable clinicians to better identify children in the PICU who are at the highest risk for IC and could benefit from targeted prophylactic or preemptive antifungal treatment.

from a CVC or endotracheal tube. Further investigation to validate and improve the proposed clinical prediction models are of utmost importance to enable clinicians to better identify children in the PICU who are at the highest risk for IC and could benefit from targeted prophylactic or preemptive antifungal treatment. Clinical Presentation Identifying IFD in PICU patients can be exceedingly difficult because symptoms frequently are indistinguishable from sepsis secondary to bacterial infection, and fever refractory to antibiotic treatment is the most common presenting feature [5, 72]. It is unfortunate that no study has addressed the clinical presentation of IFD in pediatric PICU patients, aside from the specific populations highlighted in this review. However, the presence of thrombocytopenia often raises the concern of IC in PICU patients. Recent studies associated pronounced and prolonged thrombocytopenia with candidemia in PICU patients after corrective surgery for congenital heart disease [5, 7] and in premature neonates [79–81]. Further investigation and validation of thrombocytopenia as a heralding sign of IC is warranted. PREMATURE NEONATES Neonates possess a number of endogenous and exogenous risk factors that predispose them to IFD, caused mainly by Candida spp [82, 83]. Few of these factors are intrinsic but instead have been associated with the immaturity of the immune system in these patients [83–85].

Clinical Presentation Identifying IFD in PICU patients can be exceedingly difficult because symptoms frequently are indistinguishable from sepsis secondary to bacterial infection, and fever refractory to antibiotic treatment is the most common presenting feature [5, 72]. It is unfortunate that no study has addressed the clinical presentation of IFD in pediatric PICU patients, aside from the specific populations highlighted in this review. However, the presence of thrombocytopenia often raises the concern of IC in PICU patients. Recent studies associated pronounced and prolonged thrombocytopenia with candidemia in PICU patients after corrective surgery for congenital heart disease [5, 7] and in premature neonates [79–81]. Further investigation and validation of thrombocytopenia as a heralding sign of IC is warranted. PREMATURE NEONATES Neonates possess a number of endogenous and exogenous risk factors that predispose them to IFD, caused mainly by Candida spp [82, 83]. Few of these factors are intrinsic but instead have been associated with the immaturity of the immune system in these patients [83–85]. Risk Factors Immaturity of the premature neonate’s epidermis and intestinal mucosal barriers enable Candida spp to translocate from the skin or gastrointestinal tract into the bloodstream [86]. Birth weight is correlated inversely with the incidence of IC; incidences range from 4% to 16% in extremely-low-birth-weight infants and 2% to 5% in very-low-birth-weight infants [87–90]. Apart from their immunosuppressed status, premature infants are often exposed to several risk factors for IC inherent in the provision of prolonged intensive care. In particular, these risk factors include parenteral nutrition, mechanical ventilation, central venous access, proton pump–inhibiting agents, postnatal corticosteroid use, and broad-spectrum antibiotic exposure (particularly to third-generation cephalosporins and carbapenems). Intestinal pathology and abdominal surgery, both common among neonatal intensive care unit patients, have been identified as risk factors also [87, 88, 91–96].

pump–inhibiting agents, postnatal corticosteroid use, and broad-spectrum antibiotic exposure (particularly to third-generation cephalosporins and carbapenems). Intestinal pathology and abdominal surgery, both common among neonatal intensive care unit patients, have been identified as risk factors also [87, 88, 91–96]. Colonization with Candida spp before the onset of IC is common among neonates; colonization rates range from 18% to 26% [84, 97–99]. Sources of colonization vary by Candida spp. For instance, C parapsilosis colonization occurs via horizontal transmission, typically >7 days after neonatal intensive care unit admission, whereas C albicans colonization occurs via vertical transmission in the perinatal period [100]. A recent study conducted by Barton et al [101] found that chorioamnionitis and vaginal delivery were strongly associated with the development of early-onset candidiasis (at ≤ 7 days of life).

l intensive care unit admission, whereas C albicans colonization occurs via vertical transmission in the perinatal period [100]. A recent study conducted by Barton et al [101] found that chorioamnionitis and vaginal delivery were strongly associated with the development of early-onset candidiasis (at ≤ 7 days of life). Although Candida spp are the predominant source of IFD in premature neonates, other fungal pathogens are sometimes opportunistic in this patient population. Malassezia spp are a group of lipid-dependent yeasts that frequently colonize the skin and gastrointestinal tract but are an infrequent cause of neonatal fungemia [102]. Infection with Malassezia furfur has been associated with the use of lipid infusions via a CVC in neonates [103]. IFD of the skin and soft tissues caused by molds such as Aspergillus spp and Mucorales have been reported infrequently. These infections have been described in relation to mild local trauma and skin contamination from the use of wooden tongue depressors, arm boards, and/or adhesive tapes [104–106].

C in neonates [103]. IFD of the skin and soft tissues caused by molds such as Aspergillus spp and Mucorales have been reported infrequently. These infections have been described in relation to mild local trauma and skin contamination from the use of wooden tongue depressors, arm boards, and/or adhesive tapes [104–106]. Clinical Presentation The most common presentation of IFD in premature neonates is a generalized sepsis that is indistinguishable from late-onset bacterial sepsis [107]. In a majority of premature infants with an IFD, infection presents around the third week of life and is caused predominantly by Candida spp. Almost 25% of candidemia infections in premature neonates are associated with a meningoencephalitis, even when no overt neurological symptoms are present [108]. Dissemination to the kidney (5%), eye (3%), and heart (5%) is seen, particularly in neonates with persistent candidemia [109, 110]. Isolated infections in the CNS [111], kidneys [112], heart [113], and bones and joints [114] in the presence of indwelling devices have been reported. Candida infection of the kidneys in neonates can be complicated by the development of a fungal bezoar (fungal ball) and lead to urinary tract obstruction [115]. Neonates with IC develop thrombocytopenia more frequently than those with bacteremia and have both a lower platelet nadir and a longer duration of thrombocytopenia [79–81]. In combination with the aforementioned existing risk factors, candidemia should be suspected in a neonate with clinical signs of sepsis and new thrombocytopenia. Hyperglycemia is also a common feature of neonatal fungal sepsis and acts as a clinical predictor of IC; the odds of IC increase as the blood glucose level rises [87].

ation with the aforementioned existing risk factors, candidemia should be suspected in a neonate with clinical signs of sepsis and new thrombocytopenia. Hyperglycemia is also a common feature of neonatal fungal sepsis and acts as a clinical predictor of IC; the odds of IC increase as the blood glucose level rises [87]. Infections caused by Aspergillus spp and Mucorales in neonates are often localized to the skin and soft tissues and affect the most premature and extremely low birth weight neonates, who have an impaired and immature skin barrier function [104–106]. Clear differences in the common sites of mucormycosis can be seen between premature neonates and older children with a malignancy. Gastrointestinal (54%) and cutaneous (36%) diseases are the predominant phenotypes in neonates, whereas sinopulmonary and rhinocerebral patterns of disease are noted mainly in older children with a malignancy [116]. PRIMARY AND ACQUIRED IMMUNODEFICIENCY IFD is highly unusual in the absence of impaired immunity but can represent the primary presenting feature of an underlying immunodeficiency. Therefore, the identification of IFD in an otherwise healthy child should prompt further investigation into possible immunodeficiency, and investigations should be targeted toward the most probable defective arm of host defense.

mmunity but can represent the primary presenting feature of an underlying immunodeficiency. Therefore, the identification of IFD in an otherwise healthy child should prompt further investigation into possible immunodeficiency, and investigations should be targeted toward the most probable defective arm of host defense. Risk Factors The main risk factor for IFD in children with immunodeficiency is the underlying immunodeficiency itself, and specific deficiencies in the host defense place the patient at risk for specific fungal pathogens [117]. Classical examples of this include IA with chronic granulomatous disease (CGD) and PCP in patients with severe combined immunodeficiency and acquired immunodeficiency secondary to human immunodeficiency virus (HIV) [118–123]. Deficiencies in T-cell immunity are the main predisposing factor for PCP, and patients at increased risk of infection include those with severe combined immunodeficiency, HIV, CD40 ligand deficiency, nuclear factor κB (NF-κB) essential modulator (NEMO) deficiency, hyperimmunoglobulin E syndrome (hyper-IgE) (Job syndrome), and X-linked hyperimmunoglobulin M syndrome [124, 125]. Prophylaxis with cotrimoxazole is highly effective, and breakthrough infection during prophylaxis should prompt consideration of noncompliance or antimicrobial resistance [37]. The majority of cryptococcosis cases occur in children with defective cell-mediated immunity, caused mainly by HIV infection or a primary immunodeficiency such as hyperimmunoglobulin M syndrome, hyper-IgE syndrome, and GATA2 deficiency [126, 127].

Deficiencies in T-cell immunity are the main predisposing factor for PCP, and patients at increased risk of infection include those with severe combined immunodeficiency, HIV, CD40 ligand deficiency, nuclear factor κB (NF-κB) essential modulator (NEMO) deficiency, hyperimmunoglobulin E syndrome (hyper-IgE) (Job syndrome), and X-linked hyperimmunoglobulin M syndrome [124, 125]. Prophylaxis with cotrimoxazole is highly effective, and breakthrough infection during prophylaxis should prompt consideration of noncompliance or antimicrobial resistance [37]. The majority of cryptococcosis cases occur in children with defective cell-mediated immunity, caused mainly by HIV infection or a primary immunodeficiency such as hyperimmunoglobulin M syndrome, hyper-IgE syndrome, and GATA2 deficiency [126, 127]. Disorders of host phagocyte function, such as CGD, place patients at increased risk for invasive mold infections such as those caused by Aspergillus spp and Mucorales. Indeed, patients with CGD remain at the highest lifetime risk of IA, despite effective antifungal prophylaxis [128–131]. Infections caused by Mucorales are rare and typically are seen in the setting of immunosuppressive treatment for inflammatory complications of CGD [132]. After infancy, IC is relatively uncommon in patients with CGD. However, the presence of additional risk factors, such as prolonged antibiotic treatment and the use of CVCs, places patients with CGD at increased risk for candidemia [131, 133].

g of immunosuppressive treatment for inflammatory complications of CGD [132]. After infancy, IC is relatively uncommon in patients with CGD. However, the presence of additional risk factors, such as prolonged antibiotic treatment and the use of CVCs, places patients with CGD at increased risk for candidemia [131, 133]. Although primary immunodeficiencies with impairment of interleukin 17 (IL-17) immunity traditionally present with chronic mucocutaneous candidiasis and have not been considered to confer increased risk of IFD [134], there are case reports of IC in such patients, including Candida endocarditis in a child with hyper-IgE syndrome [135] and meningoencephalitis due to Candida in children and adults with previously unrecognized CARD9 deficiency [136–138]. Therefore, consideration should be given to the possibility of an underlying IL-17 immunodeficiency, such as STAT3 or CARD9 deficiency, in a previously healthy child who presents with disseminated candidiasis. Clinical Presentation Classical PCP presents with hypoxia in excess of the degree of respiratory distress in a young infant (typically 3–6 months old). In contrast to most causes of pneumonia in infants, pyrexia and preceding coryzal symptoms frequently are absent. Instead, these children typically have a history of progressive dyspnea and dry cough with low-grade pyrexia, tachypnea, and hypoxia found on examination but an absence of adventitious sounds on auscultation [124, 139, 140]. Respiratory distress often progresses rapidly and necessitates significant respiratory support [124, 141].

ese children typically have a history of progressive dyspnea and dry cough with low-grade pyrexia, tachypnea, and hypoxia found on examination but an absence of adventitious sounds on auscultation [124, 139, 140]. Respiratory distress often progresses rapidly and necessitates significant respiratory support [124, 141]. Cryptococcosis most commonly manifests as meningoencephalitis, disseminated disease, and pneumonia. Pulmonary cryptococcosis without dissemination is a recognized but unusual clinical presentation in immunocompromised children [142–144]. The most common presenting clinical symptoms and signs in a review that included 53 pediatric patients were headache (79%), fever (77%), vomiting (70%), and neck pain and/or nuchal stiffness (49%). Hydrocephalus was reported for 6 patients [145]. It is notable that 26% of the children in this Brazilian study had no predisposing condition, and only 25% of the children had an underlying diagnosis of acquired immunodeficiency syndrome. The average age of the children was 7.7 years (range, 0–16 years), and only 5.6% were <2 years of age [145]. Other studies have found a comparable age distribution, with cryptococcosis occurring more frequently in middle childhood (6–12 years) and rarely appearing during the first 2 years of life [146, 147]. The most recent series of pediatric cryptococcosis came from a national survey of Colombian children <16 years of age [148]. Twenty-four percent of the children were HIV positive, and their mean age was 8.4 years, which is comparable with that in previously published studies [145–147]. In the Colombian series, neurocryptococcosis (87.8%) was most common, followed by disseminated disease (12.2%). Among the 5 patients with disseminated disease, 2 had skin involvement. Clinical signs and symptoms and their frequencies were similar to those reported from a study by Severo et al [145]. Results of several studies have suggested that disseminated cryptococcal disease is rare [145, 147, 148], although disseminated cryptococcosis was diagnosed in 47.8% of 23 pediatric patients in a case series in China. The affected organs in those 11 children included the lungs (n = 11), CNS (n = 7), lymph nodes (n = 10), liver (n = 9), and spleen (n = 7) [144]. None of the children had an identified immunocompromising condition, but 7 of them were malnourished. The differences in underlying disorders and geographical aspects might explain the variation observed in the clinical entities of cryptococcal disease.

7), lymph nodes (n = 10), liver (n = 9), and spleen (n = 7) [144]. None of the children had an identified immunocompromising condition, but 7 of them were malnourished. The differences in underlying disorders and geographical aspects might explain the variation observed in the clinical entities of cryptococcal disease. Failure to thrive was the most common (71%) presenting feature of IFD in children with CGD registered in the French National Database for Primary Immunodeficiency over a 25-year period [2]. The clinical presentation of IA in patients with CGD can be highly variable but is often indolent with minimal symptoms [118, 149]. Fever is reported in 61% in those presenting with IA [119]. However, a review of a French cohort of patients with CGD found that 37% reported neither fever nor respiratory symptoms at the time of IFD diagnosis [150], which is comparable with the one-third of patients with IA who presented to the National Institutes of Health who were asymptomatic at the time of diagnosis [151]. When present, symptoms associated with invasive pulmonary aspergillosis can include chest discomfort, cough (usually nonproductive), and progressive dyspnea, but hemoptysis is rare [119]. The second most common site of IA in patients with CGD is the bones, often with multifocal lesions; the thoracic vertebrae and ribs are affected most commonly [152, 153]. Clinical features have not been well described, but the most common manifestations seem to be localized pain and tenderness, often without fever. Vertebral invasion is associated with signs of spinal cord invasion in 45% of cases. Localized brain abscesses caused by Aspergillus spp are considered rare [133, 154, 155]. Clinical features vary from mild fever and headaches to seizures and localizing signs that mimic space-occupying lesions [119]. Other less common sites of Aspergillus infection include skin, lymph nodes, liver, and spleen [119, 133]. The clinical manifestations of skin infections are diverse, from erythematous plaques and papules to pustules and purulent ulcers, localized mainly to the extremities. Hepatic and splenic abscesses caused by infection with an Aspergillus sp typically are seen during disseminated infection rather than occurring in isolation [119].

l manifestations of skin infections are diverse, from erythematous plaques and papules to pustules and purulent ulcers, localized mainly to the extremities. Hepatic and splenic abscesses caused by infection with an Aspergillus sp typically are seen during disseminated infection rather than occurring in isolation [119]. Candida spp are the most common cause of fungal meningitis, fungemia, and fungal lymphadenitis in patients with CGD [119]. Young infants with CGD (ranging from 8 weeks to 4 months old) seem to be more prone to developing IFD caused by a Candida sp. Clinical signs are those of a septic infant (fever and irritability) sometimes associated with local signs of organ involvement, such as lymphadenopathy and hepatosplenomegaly, or signs of CNS involvement.

ts with CGD (ranging from 8 weeks to 4 months old) seem to be more prone to developing IFD caused by a Candida sp. Clinical signs are those of a septic infant (fever and irritability) sometimes associated with local signs of organ involvement, such as lymphadenopathy and hepatosplenomegaly, or signs of CNS involvement. SUMMARY A wide variety of vulnerable pediatric patients are at risk of developing IFD as a consequence of either an intrinsic impairment of the immune system, an acquired disorder, intensive management that interferes with normal immune function, or direct immunosuppressive therapies. Because the clinical manifestations of IFD are a result of the interaction between a fungus and the host immune system, a variety of disease phenotypes can be recognized. Nevertheless, clinical signs and symptoms are often nonspecific and develop late during disease progression. A high a priori clinical suspicion is needed and should be based on the severity and characteristics of the immune dysfunction and the presence of additional risk factors. Several features separate pediatric IFD from those that occur in adults, and these pediatric-specific features should be taken into account when developing clinical guidelines for and definitions of IFD. The European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) are currently preparing a second update of the consensus definitions of IFD [156] in which pediatric-specific features that aid in disease recognition are taken into account. This important step forward will increase the applicability of the consensus definitions in pediatric populations and facilitate the identification of IFD for clinical, epidemiological, and research purposes.

tions of IFD [156] in which pediatric-specific features that aid in disease recognition are taken into account. This important step forward will increase the applicability of the consensus definitions in pediatric populations and facilitate the identification of IFD for clinical, epidemiological, and research purposes. Notes Financial support. Prof Warris and Dr King are supported by the Wellcome Trust Strategic Award (grant 097377) and the Medical Research Council Centre for Medical Mycology (grant MR/N006364/1) at the University of Aberdeen. Supplement sponsorship. This article appears as part of the supplement “State of the Art Diagnosis of Pediatric Invasive Fungal Disease: Recommendations From the Joint European Organization for the Treatment of Cancer/Mycoses Study Group (EORTC/MSG) Pediatric Committee,” sponsored by Astellas. Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

A resurgence of pertussis across age groups has occurred in several countries in recent years [1]. Middle- and high-income countries that use an acellular pertussis vaccine for the primary vaccination series have been particularly affected [2, 3], and infants and adolescents have experienced the greatest increase [4]. Factors that may contribute to the increased risk of pertussis include rapidly waning immunity from those vaccinated with acellular vaccines [1, 5, 6], asymptomatic transmission from individuals vaccinated with acellular vaccines [7], genetic adaption of Bordetella pertussis [8], vaccination delay or refusal [9], improved surveillance and laboratory capabilities [2], and overall increased awareness of the continuing circulation of B pertussis [1]. Some countries experiencing epidemic pertussis, including the United States, United Kingdom, and Argentina, now recommend pertussis immunization in pregnancy and vaccination of close contacts [10, 11] to protect the youngest infants from pertussis before they can be vaccinated themselves [12]. Recent data from maternal vaccination trials demonstrate the ability of antibodies to be transferred from mothers to their infants in pregnancy and their persistence in infants [13].

and vaccination of close contacts [10, 11] to protect the youngest infants from pertussis before they can be vaccinated themselves [12]. Recent data from maternal vaccination trials demonstrate the ability of antibodies to be transferred from mothers to their infants in pregnancy and their persistence in infants [13]. Global estimates of pertussis show the highest childhood burden in Southeast Asia [14]. In this region, maternal pertussis vaccination during pregnancy may be a way to protect infants, similar to the approach using tetanus toxoid vaccine. However, globally only 1 population-based estimate of pertussis in infants from birth has been conducted (Senegal) [15], and surveillance and laboratory capabilities in Asia are lacking [16, 17]. The World Health Organization (WHO) recently recommended that countries using whole cell pertussis vaccines continue to do so in light of recent data indicating that acellular pertussis vaccines are less effective than whole cell pertussis vaccines [18]. Population-based data are needed, especially in low-income settings, to provide a more accurate estimate of the burden of pertussis in infants to inform childhood and maternal immunization policies [19, 20]. We report on a prospective cohort study following infants weekly in their homes to monitor for pertussis disease from birth to age 6 months. The objective was to provide a population-based estimate of laboratory-confirmed pertussis incidence in infants less than 6 months of age in the Sarlahi District, Nepal.

Global estimates of pertussis show the highest childhood burden in Southeast Asia [14]. In this region, maternal pertussis vaccination during pregnancy may be a way to protect infants, similar to the approach using tetanus toxoid vaccine. However, globally only 1 population-based estimate of pertussis in infants from birth has been conducted (Senegal) [15], and surveillance and laboratory capabilities in Asia are lacking [16, 17]. The World Health Organization (WHO) recently recommended that countries using whole cell pertussis vaccines continue to do so in light of recent data indicating that acellular pertussis vaccines are less effective than whole cell pertussis vaccines [18]. Population-based data are needed, especially in low-income settings, to provide a more accurate estimate of the burden of pertussis in infants to inform childhood and maternal immunization policies [19, 20]. We report on a prospective cohort study following infants weekly in their homes to monitor for pertussis disease from birth to age 6 months. The objective was to provide a population-based estimate of laboratory-confirmed pertussis incidence in infants less than 6 months of age in the Sarlahi District, Nepal. METHODS Settings and Population The study was nested within 2 consecutive randomized controlled trials of maternal influenza vaccination during pregnancy set in the Sarlahi District, located in the central Terai (low-lying plains) region of Nepal [21]. At the start of the trial, prevalent pregnancies were identified through a census of all households in the catchment area. For the duration of the trial, field workers visited all households in the communities, every 5 weeks, where married women (15–40 years) resided, for surveillance of incident pregnancies. Once a pregnancy was identified, women provided consent and were enrolled. From April 25, 2011 through September 9, 2013, women between 17 and 34 weeks gestation were randomized and vaccinated with either an influenza vaccine or placebo. The study was a population-based prospective cohort of infants followed from birth through 6 months postpartum. Approval for the study was obtained from the Institutional Review Boards at the Johns Hopkins Bloomberg School of Public Health, Cincinnati Children’s Medical Center, the Institute of Medicine at Tribhuvan University, Kathmandu, and the Nepal Health Research Council. The trials are registered at Clinicaltrials.gov (NCT01034254).

or the study was obtained from the Institutional Review Boards at the Johns Hopkins Bloomberg School of Public Health, Cincinnati Children’s Medical Center, the Institute of Medicine at Tribhuvan University, Kathmandu, and the Nepal Health Research Council. The trials are registered at Clinicaltrials.gov (NCT01034254). Data Collection At baseline, information was collected on household structure, socioeconomic status, and demographics. At enrollment, date of last menstrual period and pregnancy history data were collected. As soon as possible after delivery, the mother and infant were visited to collect detailed birth information including infant weight and breastfeeding status. From birth through 6 months, postpartum infants were visited weekly by a field worker, who recorded any infant respiratory symptoms in the past 7 days. If an infant had any of the following symptoms, a mid-nasal nylon flocked swab was collected: fever, cough, wheeze, difficulty breathing, or ear infection. Starting on August 17, 2012, new symptoms, more specific for pertussis, were added to the weekly morbidity visit: apnea, cyanosis, cough with vomit, or whoop/whooping cough. The swabs were stored for up to 1 week at room temperature in PrimeStore Molecular Transport Medium (Longhorn Diagnostics LLC, Bethesda, MD). In addition to these signs, mothers were asked which, if any, infant vaccinations were received in the past 7 days, including pertussis vaccination [22]. Mid-nasal swabs were also collected on a weekly basis from mothers from enrollment through 6 months postpartum who reported fever plus one additional morbidity (cough, sore throat, nasal congestion, or myalgia). All nasal swabs collected from infants were tested for B pertussis, Bordetella parapertussis, and Bordetella bronchispetica. Only the nasal swabs of mothers whose infants tested positive for any of these pathogens were tested for the same pathogens.

ditional morbidity (cough, sore throat, nasal congestion, or myalgia). All nasal swabs collected from infants were tested for B pertussis, Bordetella parapertussis, and Bordetella bronchispetica. Only the nasal swabs of mothers whose infants tested positive for any of these pathogens were tested for the same pathogens. Laboratory Assays Real-time polymerase chain reaction (PCR) testing was conducted at the University of Washington’s Molecular Virology Laboratory according to previously published methods [23]. Two-target PCR was used to assess the presence of 3 Bordetella species: B pertussis, B parapertussis, and B bronchiseptica. The amplified targets were chromosomal repeated insertion sequence IS481 (IS) and the polymorphic pertussis toxin ptxA promoter region (PT). After amplification, the melting points of the amplicons were measured in an iCycler (Bio-Rad). A sample was interpreted as positive when the target(s) had a melting temperature within the species-specific acceptable range and a computed tomography ≤42. A sample was negative if none of the targets tested positive or a single positive target was not reproducible. Maternal nasal swabs were tested for those mothers whose infants tested positive for any Bordetella species Polymerase chain reaction was also performed for several viral infections (influenza, rhinovirus [RV], respiratory syncytial virus [RSV], bocavirus [BoV], human metapneumovirus, coronavirus, adenovirus, and parainfluenza [1–4]) as previously described [21].

After amplification, the melting points of the amplicons were measured in an iCycler (Bio-Rad). A sample was interpreted as positive when the target(s) had a melting temperature within the species-specific acceptable range and a computed tomography ≤42. A sample was negative if none of the targets tested positive or a single positive target was not reproducible. Maternal nasal swabs were tested for those mothers whose infants tested positive for any Bordetella species Polymerase chain reaction was also performed for several viral infections (influenza, rhinovirus [RV], respiratory syncytial virus [RSV], bocavirus [BoV], human metapneumovirus, coronavirus, adenovirus, and parainfluenza [1–4]) as previously described [21]. Analytic Dataset Of 3693 women enrolled, 3646 infants were live born to 3621 women (Supplementary Figure 1). Infants were included in this analysis if they were followed for any length of the follow-up period (0 to 180 days); median total follow-up was 146 days per infant (Supplementary Figure 2). The final dataset consists of 3483 infants, contributing 1280 infant-years of observation, with at least 1 follow-up visit during the first 6 months. This includes infants from the entire trial period, both before and after more pertussis-specific additions to the weekly symptom questionnaire.

(Supplementary Figure 2). The final dataset consists of 3483 infants, contributing 1280 infant-years of observation, with at least 1 follow-up visit during the first 6 months. This includes infants from the entire trial period, both before and after more pertussis-specific additions to the weekly symptom questionnaire. At baseline, data on household structure were gathered. At enrollment, women reported their literacy status (binary) and pregnancy history. The field workers identified their ethnicity into 2 broad groups (Pahadi, a group originating from the hills; or Madeshi, a group originating from north India) from names and observation. Women were categorized as nulliparous or multiparous. Responses to 25 questions about household construction, water and sanitation, and household assets were used to develop an index to measure the socioeconomic status of households. Binary variables for each of the 25 questions and a mean SES score were calculated for each household.

orized as nulliparous or multiparous. Responses to 25 questions about household construction, water and sanitation, and household assets were used to develop an index to measure the socioeconomic status of households. Binary variables for each of the 25 questions and a mean SES score were calculated for each household. Gestational age was measured using a woman’s report of date of last menstrual period during pregnancy surveillance. Birth weight was collected as soon as possible after birth using a digital scale (Tanita model BD-585, precision to nearest 10 grams). Birth weights collected >72 hours after birth were excluded from the analysis. Small for gestational age (SGA) was calculated using the sex-specific 10th percentile cutoff described by Alexander et al [24] and the INTERGROWTH-21 standards [25]. Women were asked within how many hours of birth breastfeeding was initiated and binary breastfeeding categories were created (≤1 hour versus >1 hour postdelivery). Statistical Analysis Incidence was calculated as the number of pertussis cases per 1000 infant-years at risk. Poisson exact 95% confidence intervals (CIs) were constructed. Characteristics of infant pertussis cases were compared with nonpertussis cases using bivariate Poisson regression. Characteristics of all pertussis respiratory episodes were compared with nonpertussis respiratory episodes; t tests were used for continuous predictors and Fisher’s exact tests were used for categorical associations due to the low number of pertussis episodes. All statistical analyses were conducted in Stata/SE 14.1.

. Characteristics of all pertussis respiratory episodes were compared with nonpertussis respiratory episodes; t tests were used for continuous predictors and Fisher’s exact tests were used for categorical associations due to the low number of pertussis episodes. All statistical analyses were conducted in Stata/SE 14.1. RESULTS A total of 3483 infants had 4283 episodes of respiratory illness between May 18, 2011 and April 30, 2014. Thirty-nine percent (n = 1350) of infants experienced no respiratory episodes. The incidence of respiratory illness was 3.6 episodes per infant-year (95% CI, 3.5–3.7). Mean episode duration was 4.7 days (95% CI, 4.6–4.9). A total of 3930 (92%) episodes were matched to 1 or more pertussis-tested nasal swabs from 2026 infants (Supplementary Figure 1). Seventeen cases of B pertussis were identified from 19 nasal swabs (nasal swabs were positive on 2 consecutive weeks for 2 infants). The incidence of PCR-confirmed B pertussis was 13.3 cases per 1000-infant years (95% CI, 7.7–21.3). Five cases of B parapertussis were detected with an incidence of 3.9 cases per 1000 infant-years (95% CI, 1.3–9.1). No cases of B bronchiseptica were identified.

swabs were positive on 2 consecutive weeks for 2 infants). The incidence of PCR-confirmed B pertussis was 13.3 cases per 1000-infant years (95% CI, 7.7–21.3). Five cases of B parapertussis were detected with an incidence of 3.9 cases per 1000 infant-years (95% CI, 1.3–9.1). No cases of B bronchiseptica were identified. Bordetella Pertussis The average pertussis episode duration was 8 days (range, 2–33) (Table 1). Mean age of onset of symptoms was 83 days (range, 19–137) (median, 80; interquartile range, 63–109). The most common symptoms were cough, difficulty breathing, and cough with vomit. None of the additional symptoms related to pertussis that were added in year 2 (cyanosis, apnea, cough with vomit, and whoop) resulted in collection of nasal swabs based solely on these additional symptoms. Pertussis episodes were statistically significantly more likely to include difficulty breathing, cough with vomit, and whoop compared with other respiratory illness. Six infants had at least 1 pertussis vaccination before pertussis disease onset (three <2 weeks and three >2 weeks before pertussis illness) with a mean of 18 days from vaccination to illness compared with 49 days for nonpertussis episodes (P = .03). Five infants received their first pertussis vaccination postpertussis disease onset, whereas 6 infants received no pertussis vaccination in the first 180 days. Three fourths of pertussis episodes were coinfected with at least 1 virus, with RV and BoV the most common. Cases of pertussis were more likely to be infected with BoV than respiratory cases due to causes other than pertussis. The majority of cases occurred between February 2013 and January 2014 (Figure 1).

t 180 days. Three fourths of pertussis episodes were coinfected with at least 1 virus, with RV and BoV the most common. Cases of pertussis were more likely to be infected with BoV than respiratory cases due to causes other than pertussis. The majority of cases occurred between February 2013 and January 2014 (Figure 1). Table 1. Comparison of Pertussis Episodes to Nonpertussis Episodes Nonpertussis Episodes Pertussis Episodes (n = 3913) (n = 17) P Valuea Characteristic Proportion Mean Proportion Mean Symptomsb Cough 62% 71% .62 Difficulty breathing 40% 65% .05 Cough with vomit 12% 50% .00 Wheeze 45% 47% .99 Fever 53% 47% .64 Whoop 6% 33% .01 Apnea 4% 17% .08 Cyanosis 1% 8% .09 Ear Infection 5% 6% .59 Episode duration (days) 5 8 .07 Age at episode start (days) 91 83 .54 Coinfections RV 50% 53% .99 BoV 5% 24% .01 PIV3 4% 12% .17 RSV 9% 6% .99 Influenza 5% 6% .55 MPV 5% 6% .57 CoV 8% 6% .99 PIV1 2% 0% .99 PIV2 1% 0% .99 PIV4 2% 0% .99 AdV 2% 0% .99 Vaccination Received 1st pertussis vaccination 38% 35% 0.99 Days since vaccination 49 18 0.03 Abbreviations: AdV, adenovirus; BoV, bocavirus; CoV, coronavirus; MPV, human metapneumovirus; PIV, parainfluenza; RSV, respiratory syncytial virus; RV, rhinovirus. a t tests were used for continuous predictors and Fisher’s exact tests were used for categorical predictors; statistical significance of P < .05 indicated in bold. bCough with vomit, apnea, whoop, and cyanosis were only captured in year 2; denominator for these symptoms was 2034 episodes. Figure 1. Timing of respiratory episodes.

a t tests were used for continuous predictors and Fisher’s exact tests were used for categorical predictors; statistical significance of P < .05 indicated in bold. bCough with vomit, apnea, whoop, and cyanosis were only captured in year 2; denominator for these symptoms was 2034 episodes. Figure 1. Timing of respiratory episodes. No statistically significant differences between risk factors for pertussis and nonpertussis cases (Table 2) were documented. Given the low number of pertussis cases, the lack of a statistical association is not evidence of nonassociation. No deaths occurred in infants who had pertussis. Of the 8 mothers of B pertussis-positive infants who had a nasal swab collected (14 nasal swabs total) during their own follow-up, none were positive for any pertussis species. Table 2. Poisson Regression for Risk Factors for Pertussis in Infants

No statistically significant differences between risk factors for pertussis and nonpertussis cases (Table 2) were documented. Given the low number of pertussis cases, the lack of a statistical association is not evidence of nonassociation. No deaths occurred in infants who had pertussis. Of the 8 mothers of B pertussis-positive infants who had a nasal swab collected (14 nasal swabs total) during their own follow-up, none were positive for any pertussis species. Table 2. Poisson Regression for Risk Factors for Pertussis in Infants Risk Factor Nonpertussis Infants Pertussis Infants Unadjusted (n = 3466) (n = 17) IRR 95% CI P Value Male Sex 53% 59% 1.3 0.5 − 3.4 .61 Preterm (<37 weeks) 12% 24% 2.2 0.7 − 6.6 .18 Low birth weight (<2500 grams) 25% 36% 1.7 0.5 − 5.9 .38 Small for gestational age (IG) 37% 50% 1.7 0.5 − 5.8 .41 Small for gestational age (A) 48% 55% 1.3 0.4 − 4.3 .65 Breastfed in 1st hour 35% 38% 1.1 0.4 − 3.0 .86 Primiparous 42% 59% 2.0 0.8 − 5.2 .16 Pahadi ethnicity 58% 69% 1.6 0.6 − 4.6 .37 Literate 61% 60% 1.6 0.6 − 4.6 .37 Household size (mean) 5 4 0.9 0.7 − 1.1 .39 SES score (mean) 0.39 0.35 0.2 0.0 − 8.6 .42 Age (days) at first pertussis vaccination (mean) 85 96 1.0 1.0 − 1.0 .17 1st pertussis vaccination received by 6 months 56% 65% 1.5 0.5 − 3.9 .47 Abbreviations: A, Alexander standards; CI, confidence interval; IG, INTERGROWTH-21st standards; IRR, incidence rate ratios; SES, socioeconomic status.

0.35 0.2 0.0 − 8.6 .42 Age (days) at first pertussis vaccination (mean) 85 96 1.0 1.0 − 1.0 .17 1st pertussis vaccination received by 6 months 56% 65% 1.5 0.5 − 3.9 .47 Abbreviations: A, Alexander standards; CI, confidence interval; IG, INTERGROWTH-21st standards; IRR, incidence rate ratios; SES, socioeconomic status. Bordetella Parapertussis The 5 B parapertussis cases were primarily male whose mothers were primiparous, literate, and Pahadi ethnicity (Supplementary Table 1). No mothers of infants who had B parapertussis had a nasal swab collected during follow-up. The average B parapertussis episode duration was 4 days (Supplementary Table 2). Mean age of onset of symptoms was 58 days with a range of 7–95 days. The most common symptoms were cough and wheeze. Rhinovirus and RSV were the only coinfections observed. All B parapertussis cases occurred between September 2011 and February 2012 (Figure 1).

tussis episode duration was 4 days (Supplementary Table 2). Mean age of onset of symptoms was 58 days with a range of 7–95 days. The most common symptoms were cough and wheeze. Rhinovirus and RSV were the only coinfections observed. All B parapertussis cases occurred between September 2011 and February 2012 (Figure 1). DISCUSSION A low incidence of pertussis and generally mild clinical presentation were found in infants <6 months in Nepal. To our knowledge, this represents one of the first population-based active surveillance of PCR-confirmed pertussis among young infants in Asia. Acellular pertussis vaccine trials conducted in the 1990s found the average pertussis incidence in the whole cell vaccine groups ranged from 1 to 37 cases per 1000 infant-years [26]. Our finding of 13 B pertussis cases per 1000 infant-years was on the lower end of this range. In the United States in 2014, the estimated pertussis incidence in infants less than 6 months was 2 cases per 1000 infant-years [27], much lower than observed in our study; however, this passive surveillance system likely vastly underestimates pertussis incidence. Thus, there is a need for active surveillance data such as ours. Furthermore, given our highly sensitive case detection method, many of our pertussis cases would likely not have been detected in the previous acellular pertussis vaccine trials. More stringent respiratory symptom criteria would have lowered our incidence estimate even further. The low incidence was found in a population where pentavalent vaccine (Pentavac: Diphtheria, Tetanus, Pertussis [Whole Cell], Hepatitis-B and Haemophilus Type b Conjugate Vaccine; Serum Institute of India Pvt. Ltd), scheduled for administration at 6, 10, and 14 weeks, is received with significant delays (7% of infants received all 3 recommended pertussis vaccines by 6 months) [22]. These data support the WHO’s recommendation that countries using whole cell pertussis vaccine continue to do so given that the majority of outbreaks have been concentrated in countries using the acellular pertussis vaccine [2]. Recent studies suggest that protection from acellular pertussis vaccine is not as strong or long lasting as that conferred by the whole cell pertussis vaccine [6, 28].

pertussis vaccine continue to do so given that the majority of outbreaks have been concentrated in countries using the acellular pertussis vaccine [2]. Recent studies suggest that protection from acellular pertussis vaccine is not as strong or long lasting as that conferred by the whole cell pertussis vaccine [6, 28]. Another contributing factor to the low pertussis incidence observed could be that surveillance was conducted during a period of low pertussis transmission. Pertussis is a cyclical disease, thought to peak every 2 to 4 years, and we may have captured the burden at a low circulation period [6]. We observed over 70% of our B pertussis cases over a 1-year period. This increase from earlier observation periods could indicate a temporary rise in pertussis consistent with its cyclical pattern or a true increase in the baseline burden. Previous research on pertussis seasonality has in different places and time periods demonstrated various periods of peak transmission or no discernable patterns [29, 30]. Although our data do not support a seasonal pattern, the numbers observed are too low to be conclusive.

rn or a true increase in the baseline burden. Previous research on pertussis seasonality has in different places and time periods demonstrated various periods of peak transmission or no discernable patterns [29, 30]. Although our data do not support a seasonal pattern, the numbers observed are too low to be conclusive. Pertussis symptom duration and severity were mild compared with the classic pertussis case presentation. Only 3 of the 17 cases fulfilled the WHO criteria, which requires a minimum of 2 weeks of cough, whoop, or posttussive vomiting [31]. Studies on pertussis in infants have generally been clinic-based, hospital-based, or in an outbreak, which therefore required a certain severity of illness for parents to recognize a need for medical attention [29, 30, 32]. These study designs and passive surveillance efforts therefore may have missed milder pertussis cases [33]. Our study, which required only 1 respiratory symptom for a nasal swab to be collected, had increased sensitivity to detect a range of pertussis case presentations. An alternative explanation for the mild cases seen could be an increase in the proportion of mild compared with severe pertussis cases in Nepal.

cases [33]. Our study, which required only 1 respiratory symptom for a nasal swab to be collected, had increased sensitivity to detect a range of pertussis case presentations. An alternative explanation for the mild cases seen could be an increase in the proportion of mild compared with severe pertussis cases in Nepal. Although cough, difficulty breathing, and cough with vomit were the most common symptoms, no symptom was present in all B pertussis cases. During an epidemic period in Washington state, among infants <1 year, who had a minimum of 14 days cough plus an additional symptom, 82% had posttussive emesis, 29% had apnea, 26% had whoop, and 42% had cyanosis [32]. A study of US neonates with pertussis showed the symptom prevalence to be 97% for cough, 91% for cyanosis, 58% for apnea, and 3% for fever [34]. Our study found lower or equal symptom prevalence with the exception of fever. Fever prevalence was higher in our study, similar to that found in Peru [29]. Although not statistically significant, infants with pertussis were more likely to have been born preterm, low birth weight, and SGA, and their mothers were more likely to be primiparous. These findings are similar to previous studies showing no difference in pertussis cases by sex [29, 35, 36] or crowding [35] but showing differences by birth weight [36]. Coinfections were common, consistent with findings from other hospital-based studies [33]. Codetection of B pertussis and B parapertussis with respiratory viruses may be due to asymptomatic pertussis carriage.

fference in pertussis cases by sex [29, 35, 36] or crowding [35] but showing differences by birth weight [36]. Coinfections were common, consistent with findings from other hospital-based studies [33]. Codetection of B pertussis and B parapertussis with respiratory viruses may be due to asymptomatic pertussis carriage. The incidence of B parapertussis of 4 cases per 1000 person-years was comparable to that of 2 per 1000 person-years found in the Italian acellular pertussis vaccine trial in 1992–1993 [37]. The duration of illness was shorter for B parapertussis with a maximum duration of 6 days compared with a maximum of 33 days for B pertussis. A milder presentation is consistent with clinical knowledge of B parapertussis infection [37, 38]. Bordetella parapertussis cases occurred only during a 5-month period. Limitations There were several study design limitations. We cannot be certain whether the reported symptoms were caused by pertussis, another organism, or whether symptoms were related to 2 or more etiologic agents. We were unable to perform multivariate regression modeling for characteristics associated with pertussis disease and pertussis cases due to the small number of cases we detected.

ain whether the reported symptoms were caused by pertussis, another organism, or whether symptoms were related to 2 or more etiologic agents. We were unable to perform multivariate regression modeling for characteristics associated with pertussis disease and pertussis cases due to the small number of cases we detected. Infant respiratory symptoms were reported by parents, who may have missed signs that might have been observed by a healthcare worker. However, the criteria for collection of the nasal swab were broad and did not require sophisticated clinical skills. However, apnea and cyanosis may have been difficult for parents to identify. Although the criteria for specimen collection changed in year 2, no infant experienced a pertussis-specific symptom in isolation without also having one of the originally specified respiratory symptoms. These data support our assumption that we were unlikely to have missed pertussis cases in year 1 with our less sensitive respiratory symptom criteria.

n collection changed in year 2, no infant experienced a pertussis-specific symptom in isolation without also having one of the originally specified respiratory symptoms. These data support our assumption that we were unlikely to have missed pertussis cases in year 1 with our less sensitive respiratory symptom criteria. Nasal swabs were collected in the mid-nasal region for influenza virus detection, which may have lowered the sensitivity of pertussis detection. In a field site, the acceptability of an additional nasopharyngeal swab would likely have increased the participant refusal rate. This would have decreased the generalizability of our results to the entire population. Although nasopharyngeal swabs or nasopharyngeal aspirates are the recommended specimen collection method [39], the nasopharyngeal region was established as the collection area of choice when the diagnostic measure was culture, which has low sensitivity. Recent data demonstrated the comparability of using mid-nasal versus nasopharyngeal swabs in PCR pertussis detection [40].

s are the recommended specimen collection method [39], the nasopharyngeal region was established as the collection area of choice when the diagnostic measure was culture, which has low sensitivity. Recent data demonstrated the comparability of using mid-nasal versus nasopharyngeal swabs in PCR pertussis detection [40]. Strengths Strengths of the study included being a population-based, prospective study, with very low refusal rates. Risk factors, clinical symptoms, and coinfections were prospectively identified without the potential bias that may occur when these data are collected retrospectively or in clinical settings. The community-based design allows generalizability of these results to the entire population and not just those seeking care at a health facility or in an outbreak situation. The Sarlahi District is located in the Terai region where the majority of Nepalese reside, and it has similar demographics to the entire population of Nepal [41]. Sarlahi’s location near sea level and on the border with India supports the generalizability of these results to many populations living on the Indian subcontinent. The weekly active surveillance with sensitive criteria for pertussis testing was able to detect mild and atypical pertussis cases, which may have been missed by previous traditional surveillance. The multitarget PCR method allowed highly sensitive and specific detection of 2 additional Bordetella species beyond the primary B pertussis target.

llance with sensitive criteria for pertussis testing was able to detect mild and atypical pertussis cases, which may have been missed by previous traditional surveillance. The multitarget PCR method allowed highly sensitive and specific detection of 2 additional Bordetella species beyond the primary B pertussis target. CONCLUSIONS We observed a low incidence of pertussis in infants in a whole cell vaccine environment. Pertussis cases were generally milder than expected compared with traditional pertussis clinical definitions. These data support clinicians considering pertussis in their differential diagnosis of infants with mild respiratory symptoms. Policymakers in Nepal will need to weigh the benefit of an additional prenatal pertussis vaccine or a switch to acellular primary pertussis vaccine with the low burden of pertussis in infants less than 6 months. Our study demonstrated that mid-nasal swabs were able to detect pertussis using a sensitive multitarget PCR. The less invasive mid-nasal nasal swab is an attractive alternative for pertussis nasal swab collection, and further research is needed to compare this collection site with nasopharyngeal swabs. In the future, this method may enhance population-based surveillance efforts. Supplementary Data Supplementary materials are available at Journal of The Pediatric Infectious Diseases Society online. Notes Disclaimer. Neither of the funders had any role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; or preparation, review, or approval of the manuscript.

Supplementary Data Supplementary materials are available at Journal of The Pediatric Infectious Diseases Society online. Notes Disclaimer. Neither of the funders had any role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; or preparation, review, or approval of the manuscript. Financial support. This work was supported by grants from the Thrasher Research Fund (10470) and the Bill and Melinda Gates Foundation (50274). Potential conflicts of interest. J. A. E. has been a consultant for Pfizer, a member of a Data Safety Monitoring Board for GlaxoSmithKline (GSK) influenza antiviral studies, and her institution has received research support for clinical studies from GSK, Gilead, Chimerix, and Roche. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Supplementary Material Supplementary_Figure_1 Click here for additional data file. Supplementary_Figure_2 Click here for additional data file. eTable_1 Click here for additional data file. eTable_2 Click here for additional data file.

Measles is primarily a highly transmissible childhood viral disease caused by a single-stranded RNA paramyxovirus (genus Morbillivirus). Vaccination with currently used measles vaccines results in long-lasting immunity; seroconversion rates are 85% when children are vaccinated at 9 months of age and approximately 95% after 1 dose of vaccine among children aged 12 months or older [1, 2]. World Health Organization guidance recommends that all countries include a second routine dose of measles vaccine, even in countries with low national coverage of the first dose [3]. Countries in which measles is endemic, such as the Democratic Republic of the Congo (DRC), should routinely vaccinate children at 9 months and then at 12 to 15 months of age to protect those who might not have developed immunity after their first dose [4]. In 2015, the DRC reported 79% national coverage for the first dose of measles vaccination [3], and measles ranked 15th among the top causes of death in children less than 5 years of age in the DRC in 2016 [5].

n at 12 to 15 months of age to protect those who might not have developed immunity after their first dose [4]. In 2015, the DRC reported 79% national coverage for the first dose of measles vaccination [3], and measles ranked 15th among the top causes of death in children less than 5 years of age in the DRC in 2016 [5]. In addition to its high burden in DRC children, measles has more severe effects on child health as a result of widespread immunosuppression that stems from malnutrition. The DRC’s 2013–2014 Demographic and Health Survey (DHS) found that 23% of children younger than 5 years were acutely malnourished (wasted), and 43% were chronically malnourished (stunted) [6]. Malnutrition has been linked to dysfunction of cell-mediated immunity [7] and, in poor regions, increased risk of morbidity and death in those with measles. In 1998, Belamarich [8] estimated a 5% to 25% measles mortality rate in developing areas; for comparison, a 1% rate was noted in the United States during the measles epidemic in 1989–1990. In addition, measles infection greatly increases the risk of corneal ulceration and subsequent blindness among African children, particularly in those who are undernourished [1, 9].

les mortality rate in developing areas; for comparison, a 1% rate was noted in the United States during the measles epidemic in 1989–1990. In addition, measles infection greatly increases the risk of corneal ulceration and subsequent blindness among African children, particularly in those who are undernourished [1, 9]. Even in healthy children, measles virus infection is associated with transient but severe immunosuppression [10, 11]. This infection-associated lymphopenia is short lived (approximately 1 week in duration), and only 1% to 5% of total peripheral lymphocytes are infected [10, 12, 13]. However, it has long been known that measles virus can grow in the spleen, lymph nodes, and tonsils [14], and studies finding high percentages of measles virus–infected cells in lymphoid tissue [12] led de Vries et al [11] to reconsider lymphocyte depletion as a possible mechanism for virus-induced immunosuppression. They suggest that the marked increase of measles virus–specific and bystander lymphocytes in response to infection resolves measles’ acute global lymphopenia yet masks subsets of immune-mediated lymphocyte depletion. In addition, in a large population study in England, Wales, the United States, and Denmark, in which mortality rates in the pre–measles vaccine era were compared with those in the vaccine era, the authors noted that the increased number of deaths from nonmeasles infectious diseases, hypothesized to be a consequence of prolonged immunosuppression that resulted from a depletion of memory lymphocytes, after high population levels of measles was not transient but, rather, lasted 2 to 3 years [15].

e vaccine era, the authors noted that the increased number of deaths from nonmeasles infectious diseases, hypothesized to be a consequence of prolonged immunosuppression that resulted from a depletion of memory lymphocytes, after high population levels of measles was not transient but, rather, lasted 2 to 3 years [15]. In a resource-poor area such as the DRC, limited health care services, poor nutrition, and high levels of infectious disease make host immune function of crucial public health importance. Therefore, identifying risk factors (such as previous measles infection) for adverse disease outcomes (such as fever, cough, or diarrhea) that result from postmeasles immunosuppression can result in valuable recommendations for DRC public health policy and practice. METHODS Data Source and Study Population The 2013–2014 DHS was conducted between November 2013 and February 2014 and is a nationally representative survey based on a stratified 2-stage cluster design; the first stage consisted of enumeration-area formation, and the second stage involved sampling households from each enumeration area [16–18]. In the first stage, a stratified sample of geographic locations, or clusters (n = 540), was selected with proportional probability according to size. Complete listings of households within each cluster were created and selected with equal probability (n = 9000). For this study, 18827 women aged 15 to 49 years and 8656 men aged 15 to 59 years in 50% of the selected households were interviewed.

s (n = 540), was selected with proportional probability according to size. Complete listings of households within each cluster were created and selected with equal probability (n = 9000). For this study, 18827 women aged 15 to 49 years and 8656 men aged 15 to 59 years in 50% of the selected households were interviewed. The DHS survey collected biomarker data on children if they were 6 to 59 months of age and in a household in which a male interview was conducted. Collected data included weight, anemia status, health outcomes, vaccination history, and vaccine-preventable disease serology. The University of California, Los Angeles (UCLA)–DRC laboratory at the National Laboratory for Biomedical Research in Kinshasa processed dried blood spots (DBSs) collected from participants after parental consent and assessed them for immunity to vaccine-preventable diseases. Survey data were recorded on paper questionnaires and transferred to an electronic format using the Census and Survey Processing System (US Census Bureau, ICF Macro). Data were entered twice, and the resulting 2 data sets compared and verified.

tal consent and assessed them for immunity to vaccine-preventable diseases. Survey data were recorded on paper questionnaires and transferred to an electronic format using the Census and Survey Processing System (US Census Bureau, ICF Macro). Data were entered twice, and the resulting 2 data sets compared and verified. Laboratory Analysis DBS samples were extracted using a modified extraction protocol [19] and processed at the UCLA-DRC laboratory at the National Laboratory for Biomedical Research in Kinshasa. The DBS extraction and assay protocol and multiplex technology have been described elsewhere [20]. The Dynex Multiplier chemiluminescent automated immunoassay platform with a research-use-only SmartPLEX assay for measles, mumps, rubella, varicella-zoster virus, and tetanus (Dynex Technologies, Chantilly, Virginia) was used to test samples for immunoglobulin G antibody response. An assay score (AS) was calculated for each assay as the ratio to a five-donor, pooled positive control included in each run. The limit of quantitation (LOQ) was calculated as an AS of 0.03, and this value (as a supplement to maternal report of measles) was used to indicate previous infection among children for whom a history of measles was reported. We used the LOQ in conjunction with maternal report to avoid missing cases of infants who might have had measles but failed to mount a robust immune response because of maternal antibody interference. As an internal validation, we compared measles antibody titers of children with a maternal measles report and those in children with no report, and we also conducted this analysis while limiting reports to those from mothers with a higher level of education to examine potential differences in reporting according to maternal education.

alidation, we compared measles antibody titers of children with a maternal measles report and those in children with no report, and we also conducted this analysis while limiting reports to those from mothers with a higher level of education to examine potential differences in reporting according to maternal education. Study Design We assessed the association of past measles disease with an increased prevalence of infectious disease markers among 2350 children aged 9 to 59 months. Mothers selected for the DHS household interview reported whether their child had ever had measles and his or her approximate age (in months) at the time of illness. Of these children, those whose AS was less than the minimum antibody level of 0.03 were considered to be uninfected. This minimum antibody level was used to indicate measles infection among those children for whom measles had already been reported, whereas antibody found among those for whom no disease was reported was considered vaccine induced. A child was considered vaccinated if, during the interview, his or her mother presented the interviewer with a vaccination card (provided by a healthcare worker) that contained the date that the child was vaccinated against measles [17]. Unvaccinated children were those reported as such in the DHS survey. Because of the cross-sectional nature of this study, we assessed the prevalence of maternally reported acute infectious disease markers (fever, cough, and diarrhea) during the 2-week reporting period before interview, as defined in the DHS survey. The final sample size was determined by variables available for each child.

ause of the cross-sectional nature of this study, we assessed the prevalence of maternally reported acute infectious disease markers (fever, cough, and diarrhea) during the 2-week reporting period before interview, as defined in the DHS survey. The final sample size was determined by variables available for each child. Statistical Analysis DHS surveys oversample or undersample different geographic areas, and the inclusion of individual-, stratum-, and cluster-level weights is required for unbiased estimates and confidence intervals (CIs) [18]. We used methods in SAS 9.4 (SAS Institute, Cary, North Carolina) that accounted for complex survey design [21], and to estimate variance correctly, all final analyses were single level.

inclusion of individual-, stratum-, and cluster-level weights is required for unbiased estimates and confidence intervals (CIs) [18]. We used methods in SAS 9.4 (SAS Institute, Cary, North Carolina) that accounted for complex survey design [21], and to estimate variance correctly, all final analyses were single level. In addition to a descriptive analysis according to measles status, we performed a second descriptive analysis according to the distribution of fever, cough, diarrhea, and fever/cough/diarrhea (all 3 reported within the previous 2 weeks) outcomes to assess the imbalance of fever, cough, and diarrhea outcomes across demographic and socioeconomic variables. A logistic regression model that accounted for survey design was used to examine the association between measles and episodes of fever, cough, or diarrhea. The model was run again to examine the association of measles with the occurrence of all 3 outcomes within the same 2-week period and then to examine the association of outcomes with time (months) since measles. To account for potential miscategorization of time since measles infection, we categorized this analysis in 2 ways, by dividing at <12, <24, and 24 to 58 months (model 1) and by dividing by approximately 9-month intervals up until 36 months and limiting the recall period to 3 years or less (model 2). To examine trends beyond the expected window for short-term immunosuppression caused by measles, children for whom measles infection was reported to have occurred less than 2 months before the interview were removed from the time-since-measles analysis. Because of the small cell sizes in this analysis, we also show the frequency data for all outcomes of interest within each month category.

ssion caused by measles, children for whom measles infection was reported to have occurred less than 2 months before the interview were removed from the time-since-measles analysis. Because of the small cell sizes in this analysis, we also show the frequency data for all outcomes of interest within each month category. Sensitivity Analysis To examine potential effects of misclassification on odds ratio estimates, we included a sensitivity analysis in which we selected a simple random sample of measles cases to be considered misclassified and included these selected cases in the nonmeasles group. In their study, Doshi et al [22] found that 48.6% of measles-like illness reports made using the DRC’s case-based surveillance system in 2010–2012 were laboratory confirmed to be positive. We therefore ran 4 models, first considering only 29% of cases to be true positive and the remaining 71% to be false positive (and thus categorized as nonmeasles), and then we did the same for 39%, 49%, and 59% of the 193 measles cases in the full sample.

ce system in 2010–2012 were laboratory confirmed to be positive. We therefore ran 4 models, first considering only 29% of cases to be true positive and the remaining 71% to be false positive (and thus categorized as nonmeasles), and then we did the same for 39%, 49%, and 59% of the 193 measles cases in the full sample. Covariate Selection Covariate selection was based on a priori confounders (age, sex) and other potential confounders identified in the literature. Because areas with a high level of malnutrition tend to manifest greater severity in measles cases, we controlled for chronic malnutrition by categorizing children as stunted or normally or overnourished, as calculated in the DHS and according to National Center for Health Statistics/Centers for Disease Control and Prevention/World Health Organization international reference standards [17]. Malaria status was included because it is a common cause of febrile illness [23] in this region, and vaccination, breastfeeding, maternal education (7 or more years), and low parity were included because they have been found to be protective against child death [24–26]. Hobcraft et al [26] also found that 3 or more births in a 2- to 6-year period potentially increases the mortality rate in children younger than 3 years, and we applied this criterion to define high versus low parity. To control for poverty, we created a binary variable from the DHS categorical wealth index variable (in quintiles) by categorizing the 2 lowest categories (“poorest” and “poorer”) as “poor” and the 3 highest categories (“middle,” “wealthy,” and “wealthiest”) as “middle income/wealthy.” We previously found that measles vaccination and wealth index have an interactive effect on markers of infectious disease outcomes and thus included this interaction in our analysis; likewise, measles vaccination and DRC province have an interactive effect that we incorporated into our final model for fever, cough, and diarrhea outcomes. In addition, because the DHS wealth index variable depends on items more frequently found in urban than in rural residences, we included an interaction variable (wealth index × residence) in the final model [27].

ve an interactive effect that we incorporated into our final model for fever, cough, and diarrhea outcomes. In addition, because the DHS wealth index variable depends on items more frequently found in urban than in rural residences, we included an interaction variable (wealth index × residence) in the final model [27]. Although measles is known to have seasonal variation in nonindustrialized nations [28], the inclusion of seasonality was found to be minimally informative in this analysis and was not included in the final model because the data were collected within a short 4-month period and weather patterns across the DRC differ. Although previous work on long-term immune effects of measles examined death as an outcome [15], data on all-cause deaths between measles and nonmeasles cases were not available. Because the DHS primarily provides information on living children, we examined clinical markers of infection as our outcome of interest, which, to our knowledge, has not yet been examined in this context. Analyses were completed using SAS 9.4. Ethical approval was obtained by the UCLA Fielding School of Public Health, the Kinshasa School of Public Health, and the Centers for Disease Control and Prevention. Because the children were younger than the standard age of assent, the parent or guardian of each participating child provided consent on that child’s behalf.

cal approval was obtained by the UCLA Fielding School of Public Health, the Kinshasa School of Public Health, and the Centers for Disease Control and Prevention. Because the children were younger than the standard age of assent, the parent or guardian of each participating child provided consent on that child’s behalf. RESULTS Internal Validation of Measles Categorization Among the children with a maternal report of measles, 193 (77%) of 252 had a measles antibody level of at least 0.03 AS. As an internal validation, we examined the correlation of measles antibody titer with maternal report of measles infection. Median measles antibody titer levels were higher among children with a maternal report of measles infection (median AS value, 0.507 [95% CI, 0.223–0.791]) than among those with no report (median AS value, 0.176 [95% CI, 0.152–0.199]) (Supplementary Table 1). To explore the potential effect of maternal education on measles reporting, we conducted this analysis limited to mothers with 7 or more years of education and again found that children with maternal report of measles infection had higher measles antibody titer levels (median AS value, 0.742 [95% CI, 0.009–1.476]) than those with no report of measles infection (median AS value, 0.203 [95% CI, 0.160–0.242]), although the CI for the group with report of measles infection among more highly educated mothers (n = 39) is notably wider than that from the group that included all observations (n = 705).

alue, 0.742 [95% CI, 0.009–1.476]) than those with no report of measles infection (median AS value, 0.203 [95% CI, 0.160–0.242]), although the CI for the group with report of measles infection among more highly educated mothers (n = 39) is notably wider than that from the group that included all observations (n = 705). Descriptive Analyses We observed differences (Table 1) between history of measles disease and vaccination status (P = .0036, Wald χ2), age (P < .0001, Wald χ2), breastfeeding status (P < .0001, Wald χ2), years of maternal education (P = .0086, Wald χ2), DRC province (P = .0006, Wald χ2), and fever outcome (P = .0485, Wald χ2). Examination of demographic and socioeconomic variables according to fever, cough, and diarrhea outcomes (Supplementary Table 2) revealed differences according to vaccination status for diarrhea outcome (P = .0131, Wald χ2), age (P = .0025, .0032, <.0001, and .0003 for fever, cough, diarrhea, and fever/cough/diarrhea, respectively, Wald χ2), and breastfeeding status for all outcomes except cough. Other differences included cough outcome according to wealth index and residence (P = .0114, Wald χ2) and fever outcome according to malaria result and measles history (P = .0018, Wald χ2). Controlled for covariates, logistic regression analyses (Table 2) revealed that children with a history of measles disease had greater odds of experiencing a fever episode within the 2 weeks before interview (odds ratio [OR], 1.80 [95% CI, 1.25–2.60]). Measles vaccination was associated with decreased odds of fever, diarrhea, and fever/cough/diarrhea, and a positive malaria test result was associated with increased odds of fever. The sensitivity analysis that examined changes in outcome according to potential percentage of measles cases misclassified (Supplementary Table 3) revealed a continued association with previous measles infection and increased odds of fever if as many as half of the measles reports had been misclassified. The trend in association continued for higher levels of misclassification but was not statistically significant.

sles cases misclassified (Supplementary Table 3) revealed a continued association with previous measles infection and increased odds of fever if as many as half of the measles reports had been misclassified. The trend in association continued for higher levels of misclassification but was not statistically significant. Table 1. Descriptive Data of Children Aged 9 to 59 Months With and Those Without a History of Measles Infection All Children Children With Measles Infection History Variable n % of Total n % of Category Pa Vaccinated against measles .0036 No 1608 68 157 10 Yes 742 32 36 5 Age (mo) <.0001 9–11 264 11 11 4 12–23 725 31 36 5 24–35 547 23 42 8 36–47 434 18 57 13 48–59 381 16 47 12 Breastfeeding <.0001 Never 52 2 4 8 Past 1395 59 151 11 Current 964 41 38 4 Maternal education .0086 <7 years 1605 68 154 10 ≥7 years 745 32 39 5 DRC province .0006 Kinshasa 149 6 8 5 Bandundu 318 14 20 6 Bas-Congo 83 4 0 0 Equateur 366 16 42 11 Kasai-Occidental 234 10 6 3 Kasai-Oriental 287 12 35 12 Katanga 282 12 23 8 Maniema 87 4 5 6 Nord-Kivu 245 10 19 8 Orientale 165 7 31 19 Sud-Kivu 133 6 4 3 Sex .5963 Male 1134 48 97 9 Female 1216 52 96 8 Wealth indexb .3213 Poor 1149 49 104 9 Middle income/wealthy 1201 51 89 7 Residence .2117 Urban 704 30 48 7 Rural 1646 70 145 9 Chronically malnourishedc .2924 Yes 1122 48 99 9 No 1228 52 94 8 Children <5 years old in household .5027 <3 children 1602 68 126 8 ≥3 children 747 32 67 9 Malaria (blood smear) result .4685 Negative 1825 78 145 8 Positive 525 22 48 9 Fever .0485

ealthy 1201 51 89 7 Residence .2117 Urban 704 30 48 7 Rural 1646 70 145 9 Chronically malnourishedc .2924 Yes 1122 48 99 9 No 1228 52 94 8 Children <5 years old in household .5027 <3 children 1602 68 126 8 ≥3 children 747 32 67 9 Malaria (blood smear) result .4685 Negative 1825 78 145 8 Positive 525 22 48 9 Fever .0485 Yes 830 35 84 10 No 1519 65 109 7 Cough .5885 Yes 832 35 73 9 No 1518 65 120 8 Diarrhea .7523 Yes 534 23 42 8 No 1816 77 151 8 Fever/cough/diarrhea .3323 Yes 200 9 21 11 No 2150 91 172 8 Total 2350 — 193 8 aWald χ2 test for independence of measles status and row variables. Values in bold type indicate statistical significance. bWealth index is the Demographic and Health Survey composite measure of a household’s cumulative living standard. On the basis of household ownership of previously selected assets and using principal components analysis, households were placed within 1 of 5 quintiles. For the dichotomized variable, we combined the 2 lowest categories into the “poor” category and the 3 wealthiest into the “middle income/wealthy” category. cCalculated according to the National Center for Health Statistics/Centers for Disease Control and Prevention/World Health Organization international reference standard for height and age, dichotomized as −2.0 to less than or equal to −3.0 standard deviations (SDs) below the mean for chronically malnourished children and normal to ≥3.0 SDs above the mean for normally and overnourished children.

r Disease Control and Prevention/World Health Organization international reference standard for height and age, dichotomized as −2.0 to less than or equal to −3.0 standard deviations (SDs) below the mean for chronically malnourished children and normal to ≥3.0 SDs above the mean for normally and overnourished children. Table 2. Association of Measles Disease History With Acute Infectious Disease Episodes of Fever, Cough, Diarrhea, and Fever/Cough/Diarrhea in the 2 Weeks Before Interview Among Children Aged 9 to 59 Months Variable OR (95% CI) fora: Feverb Cough Diarrhea Fever/Cough/Diarrheac Measlesd 1.80 (1.25–2.60) 1.24 (0.82–1.86) 1.24 (0.80–1.93) 1.74 (0.96–3.15) Selected covariates Received measles vaccination 0.53 (0.35–0.82) 0.76 (0.51–1.13) 0.25 (0.17–0.37) 0.51 (0.30–0.88) Malaria positive 1.54 (1.16–2.03) 0.89 (0.66–1.19) 1.03 (0.78–1.35) 0.94 (0.61–1.45) Abbreviations: CI, confidence interval; OR, odds ratio. aValues in bold type indicate statistical significance. bControlled for the following additional covariates: measles vaccination (vx), wealth index (WI), vx × WI interaction, breastfeeding, maternal education, parity, age, sex, malaria-positive status, rural versus urban residence, residence × WI interaction, (old) DRC province, vx × DRC province interaction, and chronic malnutrition (according to National Center for Health Statistics/Centers for Disease Control and Prevention/World Health Organization international reference standards for height and age standard deviations).

sus urban residence, residence × WI interaction, (old) DRC province, vx × DRC province interaction, and chronic malnutrition (according to National Center for Health Statistics/Centers for Disease Control and Prevention/World Health Organization international reference standards for height and age standard deviations). cBecause of the reduced number of outcomes for fever/cough/diarrhea, the vx × DRC province interaction variable was removed from the model. dWe used 2350 observations in the regression model for all outcomes of fever, cough, diarrhea, and fever/cough/diarrhea.

sus urban residence, residence × WI interaction, (old) DRC province, vx × DRC province interaction, and chronic malnutrition (according to National Center for Health Statistics/Centers for Disease Control and Prevention/World Health Organization international reference standards for height and age standard deviations). cBecause of the reduced number of outcomes for fever/cough/diarrhea, the vx × DRC province interaction variable was removed from the model. dWe used 2350 observations in the regression model for all outcomes of fever, cough, diarrhea, and fever/cough/diarrhea. Time-Since-Measles Analysis Of 177 children for whom the date of measles infection was given and who met inclusion criteria, the median time since disease was 14.8 months (standard error, 1.08 months; range, 0–57 months). We performed logistic regression analyses to examine associations with acute fever, cough, or diarrhea episodes and a categorical “time (months) since measles” variable (Table 3). Trends of increased odds for fever outcome among children with measles disease were found in all 3 time categories (reference category, no history of measles) but were statistically significant in only the 13–24 month category (OR, 2.08 [95% CI, 0.96–4.51]) for model 1 and for the 10- to 18-month (OR, 1.93 [95% CI, 1.03–3.62]) and 19- to 27-month (OR, 3.01 [95% CI, 1.18–7.67]) age categories for model 2. Although not all estimates were statistically significant, the fever outcome showed consistently elevated trends. In Supplementary Table 4, model 1 data show that approximately 50% of the children with measles disease history in the previous 2 years also had a report of fever within the previous 2 weeks, compared with 35% of the children with no measles disease history and a report of recent fever. Model 2, which categorized time in 8- to 9-month intervals, shows a similar trend for children with history of measles disease in the previous 3 years.

previous 2 years also had a report of fever within the previous 2 weeks, compared with 35% of the children with no measles disease history and a report of recent fever. Model 2, which categorized time in 8- to 9-month intervals, shows a similar trend for children with history of measles disease in the previous 3 years. Table 3. Association of Time in Months Since Measles Disease With Acute Infectious Disease Episode of Fever, Cough, or Diarrhea in the Previous Two Weeks Among Children 9–59 Months of Age. Time Since Measles (months)b Fever Cough Diarrhea OR and 95% CIa OR and 95% CI OR and 95% CI Model 1 (2–57 months) 2–12 (n = 62) 2.08 (0.96–4.51) 1.53 (0.70–3.33) 1.02 (0.50–2.11) 13–24 (n = 54) 2.14 (1.12–4.08) 0.94 (0.41–2.17) 1.44 (0.75–2.78) 25–57 (n = 60) 1.54 (0.82–2.87) 1.37 (0.76–2.46) 1.58 (0.74–3.37) Model 2 (2–36 months) 2–9 (n = 54) 1.79 (0.80–3.99) 1.71 (0.74–3.97) 1.14 (0.50–2.63) 10–18 (n = 46) 1.93 (1.03–3.62) 1.02 (0.50–2.10) 1.36 (0.68–2.71) 19–27 (n = 24) 3.01 (1.18–7.67) 1.54 (0.63–3.75) 1.08 (0.35–3.32) 28–36 (n = 30) 1.94 (0.80–4.71) 1.59 (0.68–3.73) 2.58 (0.93–7.13) aControlling for the following additional covariates: measles vaccination (vx), wealth index (WI), vx*WI interaction, breastfeeding, maternal education, parity, age, sex, malaria positive status, rural versus urban residence, residence*WI interaction, (old) province, vx*province interaction, and chronic malnutrition (according to NCHS/CDC/WHO international references standard for height/age SD). bReference = No history of measles (n=2,157).

aControlling for the following additional covariates: measles vaccination (vx), wealth index (WI), vx*WI interaction, breastfeeding, maternal education, parity, age, sex, malaria positive status, rural versus urban residence, residence*WI interaction, (old) province, vx*province interaction, and chronic malnutrition (according to NCHS/CDC/WHO international references standard for height/age SD). bReference = No history of measles (n=2,157). DISCUSSION The results of our analysis suggest that previous measles infection, as reported via maternal recall and meeting serologic criteria, was associated with increased odds of fever outcomes among children aged 9 to 59 months, which supports the hypothesis of immune-amnesia leading to a prolonged period of increased risk for death as a result of non-measles infectious diseases. The association of infectious disease markers with time in months since measles infection also suggests some support for this hypothesis. Although most estimates were not statistically significant, this lack of statistical significance might have been a result of the small number of children with a history of measles in each category, and we found a trend of prolonged association with fever beyond the weeks after measles infection. The lack of association between measles and cough might have been a result of environmental or indoor pollution having a greater effect on respiratory illness than previous history of measles disease since environmental pollution is known to cause respiratory symptoms in children [29], and indoor air-pollution exposure is a public health concern in many areas of sub-Saharan Africa [30].We also found no association between past measles infection and recent diarrhea. A possible reason is that measles virus might not target all intestinal lymphoid tissue. Although findings of de Swart and coworkers [12] indicated that lymphoid tissue in the small and large intestinal submucosa of the macaque is a target for measles virus, no evidence of infected cells in the epithelial layer was found. If prolonged measles-induced immunosuppression were to affect primarily cells of the submucosa and leave lymphoid cells in the intestinal epithelium unaffected, this may suggest that these intact cells could provide some compensation for immunomodulation caused by submucosal lymphoid cell depletion.

al layer was found. If prolonged measles-induced immunosuppression were to affect primarily cells of the submucosa and leave lymphoid cells in the intestinal epithelium unaffected, this may suggest that these intact cells could provide some compensation for immunomodulation caused by submucosal lymphoid cell depletion. We found that measles vaccination had a protective association against fever, diarrhea, and fever/cough/diarrhea. Measles vaccination may exert beneficial nonspecific effects that are thought to affect resistance to infectious diseases other than the targeted disease [31], and both observational and randomized trials from low-income countries have found an association between measles vaccination and reduced overall death and morbidity that cannot be explained by the prevention of measles alone [32–39]. A potential reason for why a protective association between measles vaccine between all 3 symptoms, but not cough alone, was found is that, as described above, other risk factors may mask a potential underlying association between measles vaccination and cough that becomes statistically significant when a child experiences all 3 outcomes. Alternatively, children who experience all 3 symptoms might be more likely to have a cough of infectious origin than those children who had only cough. Malaria symptoms and treatment are commonly associated with fever [23], which agrees with the associations found in our analyses.

en a child experiences all 3 outcomes. Alternatively, children who experience all 3 symptoms might be more likely to have a cough of infectious origin than those children who had only cough. Malaria symptoms and treatment are commonly associated with fever [23], which agrees with the associations found in our analyses. Additional research is warranted to determine how much of the association of protection against fever, diarrhea, and fever/cough/diarrhea is a result of measles vaccination itself apart from the prevention of measles disease. The nearly null association with cough could be because of the high prevalence of asthma and other chronic respiratory disorders that are significant causes of morbidity and death in sub-Saharan Africa [40], but that we were unable to assess it in this study.

ination itself apart from the prevention of measles disease. The nearly null association with cough could be because of the high prevalence of asthma and other chronic respiratory disorders that are significant causes of morbidity and death in sub-Saharan Africa [40], but that we were unable to assess it in this study. To our knowledge, ours is the first study to have examined the association of previous measles infection with a prolonged increase in the prevalence of acute infectious disease clinical markers. The strengths of this study include its large, nationally representative sample, increased confidence in maternal measles report because of serologic test results that revealed differences in median serum antibody levels between children with and those without a report of measles infection, and collection of the date of measles infection to assess its association with episodes of fever, cough, or diarrhea over time. There were several limitations to this study. (1) Measles reports were not laboratory confirmed. Although measles diagnosis based on clinical signs has been shown to be unreliable in more highly vaccinated countries, it is still thought to be a sensitive method for detecting measles infection [41, 43], and the high prevalence of measles in the DRC [42] might increase the positive predictive value (PPV) of a clinical measles diagnosis compared to that in countries with lower measles prevalence; previous work found the PPV of clinical measles diagnosis to be improved in higher-prevalence locations [43]. (2) Although clinical signs of measles, particularly Koplik spots [43], can provide a higher PPV in higher-prevalence areas, measles reports were obtained from mothers rather than trained healthcare workers and so might have been less reliable. However, previous work has indicated that maternal recall of symptoms associated with childhood deaths, including death caused by measles, in sub-Saharan Africa were reported with a high degree of accuracy, and recollections of signs and symptoms within 1 month versus within 6 months of a child’s death were found to be similar [44]. Moreover, despite the lack of healthcare practitioner reports of disease, we substantiated maternal reports of measles infection with serology and therefore lessened the possibility of exposure misclassification.

signs and symptoms within 1 month versus within 6 months of a child’s death were found to be similar [44]. Moreover, despite the lack of healthcare practitioner reports of disease, we substantiated maternal reports of measles infection with serology and therefore lessened the possibility of exposure misclassification. If misclassification of measles did occur, it would likely be nondifferential, because inaccurate maternal memory or assessment of measles disease would likely be relatively equal across demographic groups, which would bias estimates toward the null. An exception to this tendency toward non-differential misclassification might be found among reports from more highly educated mothers. This group did demonstrate a statistically significant difference in reported measles cases compared to those from less educated mothers, but this difference could be a result of mothers in the highly educated group (in urban settings) being more likely to have vaccinated their children against measles [45]. Such differential misclassification could bias the estimate toward or away from the null [46], but we attempted to account for this limitation with a sensitivity analysis, which revealed a continued association with past measles disease and fever even if nearly half of the measles cases had been misclassified. (3) Elevated levels of malnutrition in regions such as the DRC can drive measles-related mortality rates as high as 6% [47]. Survivor bias might have occurred in our study, because the most severely affected children might have died and thus would not have been included in the analyses; however, survivor bias would likely influence estimates toward the null. (4) The cross-sectional nature of the survey might have missed acute infectious disease episodes in children who truly experienced an overall increased incidence caused by previous measles infection but who did not experience disease in the 2-week reporting window for the DHS survey.

nfluence estimates toward the null. (4) The cross-sectional nature of the survey might have missed acute infectious disease episodes in children who truly experienced an overall increased incidence caused by previous measles infection but who did not experience disease in the 2-week reporting window for the DHS survey. In conclusion, our findings provide support for the hypothesis of measles-induced prolonged immunosuppression suggested by others and underscore the need for continued evaluation and improvement of the measles vaccination program in the DRC. Elevating vaccination coverage in the least-reached areas is an important component of strengthening the national vaccination program to control and eliminate measles [45]. In addition, more studies in wealthy countries could further clarify the immune-amnesia hypothesis. An increase in the number of infectious disease episodes over the long term in children who have a history of measles infection puts added strain on both the local health system and on the wellness of other children with whom the infected child comes in contact. The profound effect of measles on the lives and health of children in sub-Saharan Africa warrants concerted efforts toward the mitigation and elimination of this disease, and better defining the risks that measles poses to child health can drive and refine vaccination program policy. Supplementary Data Supplementary materials are available at Journal of the Pediatric Infectious Diseases Society online. piy099_suppl_Supplementary_Table_1 Click here for additional data file.

In conclusion, our findings provide support for the hypothesis of measles-induced prolonged immunosuppression suggested by others and underscore the need for continued evaluation and improvement of the measles vaccination program in the DRC. Elevating vaccination coverage in the least-reached areas is an important component of strengthening the national vaccination program to control and eliminate measles [45]. In addition, more studies in wealthy countries could further clarify the immune-amnesia hypothesis. An increase in the number of infectious disease episodes over the long term in children who have a history of measles infection puts added strain on both the local health system and on the wellness of other children with whom the infected child comes in contact. The profound effect of measles on the lives and health of children in sub-Saharan Africa warrants concerted efforts toward the mitigation and elimination of this disease, and better defining the risks that measles poses to child health can drive and refine vaccination program policy. Supplementary Data Supplementary materials are available at Journal of the Pediatric Infectious Diseases Society online. piy099_suppl_Supplementary_Table_1 Click here for additional data file. piy099_suppl_Supplementary_Table_2 Click here for additional data file. piy099_suppl_Supplementary_Table_3 Click here for additional data file. piy099_suppl_Supplementary_Table_4 Click here for additional data file. Notes

Supplementary Data Supplementary materials are available at Journal of the Pediatric Infectious Diseases Society online. piy099_suppl_Supplementary_Table_1 Click here for additional data file. piy099_suppl_Supplementary_Table_2 Click here for additional data file. piy099_suppl_Supplementary_Table_3 Click here for additional data file. piy099_suppl_Supplementary_Table_4 Click here for additional data file. Notes Acknowledgments. We thank Jeff Gornbein and Philip Eckhoff for their thoughtful feedback on statistical analyses and interpretation and Steve Meshnik for quality control assistance. Disclaimer. The views expressed here are the authors’ alone and do not reflect the official policy or position of the Department of the Army, Department of Defense, or the US government. Financial support. This work was supported by the Faucett Catalyst Fund and the Estimating Population Immunity to Poliovirus in the Democratic Republic of the Congo grant by the Bill and Melinda Gates Foundation (OPP1066684). Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

Financial support. This work was supported by the Faucett Catalyst Fund and the Estimating Population Immunity to Poliovirus in the Democratic Republic of the Congo grant by the Bill and Melinda Gates Foundation (OPP1066684). Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Previous presentation:This work was presented at Fondation Merieux’s “Communication of vaccine benefit beyond the infection prevented” conference, Les Pensières Center for Global Health, Veyrier-du-Lac, France (4–6 December 2017); at the Society for Epidemiologic Research 2017 annual meeting in Seattle, Washington (20–23 June 2017); and at the 2016 annual American Society of Tropical Medicine and Hygiene conference, Atlanta, Georgia (13–17 November 2016).

% for SCR and ≥70% for SPR [21]. The EMA Committee for Medicinal Products for Human Use criteria for adults aged 18–60 years are that the point estimates of the following 3 thresholds are met: SCR ≥ 40%, SPR ≥ 70%, SCF ≥ 2.5 [20]. The primary cohort for analysis of safety/reactogenicity was the total vaccinated cohort. RESULTS Study Population Seventeen centers participated in this study. The data of children at 1 study center were excluded due to concerns regarding protocol compliance, and all results presented here exclude that center. Sensitivity analyses were performed, and adding back these data resulted in no meaningful changes in the results. A total of 390 children were enrolled, and 374 children were vaccinated (Supplementary Figure 1). Baseline characteristics of the 374 participants by treatment group are seen in Supplementary Table 1. The according-to-protocol cohort for immunogenicity was comprised of 299 children (Supplementary Figure 1). One hundred forty-one children in the Flu-0.25 group, 146 children in the Flu-0.50 group, and 37 children in the Vaxi-0.25 group were unprimed and received 2 doses of vaccine. Table 1. Comparison of Full- and Half-Dose Trivalent Seasonal Influenza Vaccine in Children Aged 6–<36 Months

children (Supplementary Figure 1). One hundred forty-one children in the Flu-0.25 group, 146 children in the Flu-0.50 group, and 37 children in the Vaxi-0.25 group were unprimed and received 2 doses of vaccine. Table 1. Comparison of Full- and Half-Dose Trivalent Seasonal Influenza Vaccine in Children Aged 6–<36 Months Treatment Group Vaccine Strain Age Strata Flu-0.50a Flu-0.25b Adj. GMT Ratioc (95% CI)d A/Brisbane All 109.3 87.2 1.25 (0.90–1.75) 6–23 mo 78.8 56.9 1.38 (0.94–2.04) 24–35 mo 263.4 237.6 1.11 (0.62–1.98) A/Uruguay All 116.9 104.8 1.11 (0.83–1.49) 6–23 mo 100.2 73.0 1.37 (0.97–1.95) 24–35 mo 218.7 277.3 0.79 (0.48–1.30) B/Florida All 161.5 126.9 1.27 (0.93–1.74) 6–23 mo 128.2 91.6 1.40 (0.94–2.02) 24–35 mo 252.2 215.0 1.17 (0.70–1.96) Abbreviations: Adj., adjusted; CI, confidence interval; Flu-0.25, 0.25-mL dose of thimerosal free (TF)–TIV; Flu-0.50, 0.50-mL dose of TF-TIV; GMT, geometric mean titer; TIV, trivalent seasonal influenza vaccine. aFlu-0.50: n = 132 for all; n = 91 for 6–23 months; and n = 41 for 24–35 months. bFlu-0.25: n = 131 for all; n = 90 for 6–23 months; and n = 41 for 24–35 months. cAdj. GMT ratio: Geometric mean antibody titer adjusted for baseline titer, FLU-0.50/FLU-0.25. d95% CI: lower limit–upper limit for adjusted GMTs (Ancova model: adjustment for prior flu vaccination, baseline titer – pooled variance).

aFlu-0.50: n = 132 for all; n = 91 for 6–23 months; and n = 41 for 24–35 months. bFlu-0.25: n = 131 for all; n = 90 for 6–23 months; and n = 41 for 24–35 months. cAdj. GMT ratio: Geometric mean antibody titer adjusted for baseline titer, FLU-0.50/FLU-0.25. d95% CI: lower limit–upper limit for adjusted GMTs (Ancova model: adjustment for prior flu vaccination, baseline titer – pooled variance). Immune Responses Higher GMTs were observed for all 3 influenza strains (H1N1, H3N2, and B) in the 0.50-mL dose of TF-TIV compared with the 0.25-mL dose (Table 1), but these GMTs were not statistically significantly different. The CBER criterion for SPR (lower limit of 95% CI ≥ 70%) was met only for the B strain in all 3 treatment groups (Table 2). The point estimate for SPR for the H1N1 strain was <70% for both Flu-0.25 and Flu-0.50 groups, but >70% for Vaxi-0.25 (83.3%, 67.2%–93.6%). The CBER criterion for SCR (lower limit of 95% CI ≥ 40%) was met for all strains in all 3 treatment groups (Table 2). The EMA adult immunogenicity criterion for HI response (SCR > 40% and SCF > 2.5) was met for all virus strains included and at both doses for all vaccine groups (Table 2). Table 2. Summary of Immunogenicity Results Pre- and Postvaccination (According-to-Protocol Cohort for Immunogenicity)

An estimated 5%–15% of the world's population experiences an influenza virus infection each year [1], with an estimated 90 million cases occurring in children [2]. Significant complications of influenza are most likely to occur in persons with underlying medical conditions, the elderly, and children, especially those aged <5 years [3, 4]. Children aged <3 years have the highest attack rates [5, 6], and otherwise healthy children aged <1 year have influenza-related hospitalization rates similar to high-risk adults [3]. Children are also efficient disseminators of influenza infection in households [7]. Although annual vaccination of infants and young children is recommended in many jurisdictions [8, 9], a limited number of studies have been conducted in this population, particularly in children aged <24 months. The estimated efficacy of trivalent influenza vaccine (TIV) in these young children varies from no protection to as high as 70% [10, 11]. Live attenuated influenza vaccine has higher efficacy in young children than TIV [12, 13] and is available for children aged >2 years in Canada, the United States, and, more recently, Europe. More data on the immunogenicity, efficacy, and safety of TIV in young children are needed.

rotection to as high as 70% [10, 11]. Live attenuated influenza vaccine has higher efficacy in young children than TIV [12, 13] and is available for children aged >2 years in Canada, the United States, and, more recently, Europe. More data on the immunogenicity, efficacy, and safety of TIV in young children are needed. Various strategies have been used to improve influenza vaccine immune responses in young children, including use of adjuvants [14], administration via the intradermal instead of the intramuscular route [15], and use of different antigen doses, such as giving the adult dose [16] and doubling the adult dose [17]. Children in their first years of life do not benefit from the immunologic priming that results from multiple lifetime exposures to influenza infection or immunization, and consequently 2 influenza vaccine doses are recommended in the first year that younger children receive the vaccine [8]. Generally infants and toddlers are given half of the adult dose of influenza vaccines, a practice begun to avoid the reactogenicity associated with whole virus vaccines [18] that were evaluated >30 years ago. The dose of influenza antigen is known to play an important role in influenza vaccine immunogenicity, but little data are available on the relative safety and immunogenicity of a full (adult) dose (0.50 mL) compared with a half dose (0.25 mL) of TIV in children aged <3 years. In this study, the immunogenicity and safety of a preservative-free, prefilled syringe formulation of TIV (thimerosal-free TIV; TF-TIV) provided as the full adult dose of 0.50 mL compared with the usual children's dose of 0.25 mL were assessed in young children.

h a half dose (0.25 mL) of TIV in children aged <3 years. In this study, the immunogenicity and safety of a preservative-free, prefilled syringe formulation of TIV (thimerosal-free TIV; TF-TIV) provided as the full adult dose of 0.50 mL compared with the usual children's dose of 0.25 mL were assessed in young children. METHODS Study Design This was a randomized, observer-blind, multicenter study conducted in 17 centers in Canada between November 2008 and August 2009 in healthy children aged 6–35 months at the time of vaccination. Exclusion criteria included use of any investigational or nonregistered product within 30 days preceding administration of the study vaccine or planned use during the study period; a history of hypersensitivity or allergy to any vaccine or component of the vaccine, such as egg or chicken protein; immunodeficiency; acute disease at the time of enrollment; history of Guillain–Barré syndrome within 6 weeks of receipt of prior TIV; receipt of a nonstudy influenza vaccine during the 2008–09 influenza immunization campaign; receipt of any immunoglobulins or blood products within 3 months of study enrollment or planned administration during the study period. Children were not to have received analgesics/antipyretics within 12 hours before scheduled receipt of test vaccine, but if this had occurred, vaccination could be rescheduled at a later time. Participants were randomized using a 4:4:1 blocking scheme to 1 of 3 treatment groups by an Internet-based, central randomization system that balanced the distribution of enrolled children by center, prior influenza immunization status, and age (6–23 months and 24–35 months).

ation could be rescheduled at a later time. Participants were randomized using a 4:4:1 blocking scheme to 1 of 3 treatment groups by an Internet-based, central randomization system that balanced the distribution of enrolled children by center, prior influenza immunization status, and age (6–23 months and 24–35 months). The 3 treatment groups were a TF-TIV 0.25 mL (Flu-0.25) group, a TF-TIV 0.5 mL (Flu-0.50) group, and a group treated with 0.25 mL of the active comparator Vaxigrip (Sanofi-Pasteur) (Vaxi-0.25) (Supplementary Figure 1). All vaccines were trivalent, inactivated, split virion influenza vaccines containing hemagglutinin (HA) from each of the 3 recommended influenza A and B strains for the 2008–09 season: A/Brisbane/59/2007 (H1N1), A/Uruguay/716/2007 (an A/Brisbane/10/2007 [H3N2]–like virus), and B/Florida/4/2006 [19]. The study vaccine is a TF-TIV. Thimerosal-free TIV provided in single dose vials (a thimerosal-containing multidose TIV is currently available as FluLaval in the United States, and FluVIral elsewhere). The TF-TIV was administered as either a 0.25 mL dose of vaccine with 7.5 µg HA of each influenza strain or a 0.50 mL dose of vaccine with 15 µg HA per strain. The active comparator was administered as a 0.25 mL dose of vaccine with 7.5 µg of HA of each influenza strain. Thimerosal-free TIV 0.5 mL and 0.25 mL were in single-dose presentation, and Vaxigrip was provided as a multidose vial that contained thimerosal (per World Health Organization recommendations [20]). Figure 1. Solicited local (I) and general (I) symptoms occurring within 4 days of vaccination. Treatment groups were: Flu-0.25, 0.25-mL dose of thimerosal-free (TF) trivalent seasonal influenza vaccine (TIV); Flu-0.50, 0.50-mL dose of TF-TIV; and Vaxi-0.25, 0.25-mL dose of Vaxigrip. Data is presented as the percentage of participants reporting the symptom, with the error bars indicating the 95% confidence level.

Treatment groups were: Flu-0.25, 0.25-mL dose of thimerosal-free (TF) trivalent seasonal influenza vaccine (TIV); Flu-0.50, 0.50-mL dose of TF-TIV; and Vaxi-0.25, 0.25-mL dose of Vaxigrip. Data is presented as the percentage of participants reporting the symptom, with the error bars indicating the 95% confidence level. Children received either 1 injection on day 0 (“primed” participants; ie, children who had a prior 2-dose priming influenza immunization) or 2 injections, with the first on day 0 and the second 28–35 days later (day 28) (“unprimed” participants; ie, children who had not previously received a complete 2-dose priming influenza immunization). Primed and unprimed children were allocated in approximately equal proportions to all treatment groups. The injection site was the deltoid region of the nondominant arm for children aged 12 months or above or the anterolateral thigh for children aged <12 months at study entry. Unblinded study personnel administered the vaccine and then had no further contact with study participants. All protocols and study documentation were approved by the relevant and properly constituted local ethical review bodies following the International Conference on Harmonization principles of Good Clinical Practice, the Declaration of Helsinki, and the Tri-Council Policy Statement: Ethical Conduct for Research Involving Humans [19]. Written informed consent was obtained from the parent or legally acceptable representative.

l review bodies following the International Conference on Harmonization principles of Good Clinical Practice, the Declaration of Helsinki, and the Tri-Council Policy Statement: Ethical Conduct for Research Involving Humans [19]. Written informed consent was obtained from the parent or legally acceptable representative. Study Assessments Blood samples for measurement of hemagglutinin inhibition (HI) antibody responses were collected on day 0 and 28 days following completion of the vaccination schedule. All serological testing was performed in a GlaxoSmithKline Biologicals laboratory using standardized procedures with controls. Diary cards were used to record postvaccination local symptoms (pain, redness, swelling) and systemic solicited symptoms (drowsiness, irritability, loss of appetite, and fever, defined as a temperature of ≥38.0°C) on days 0–3 postvaccine. Unsolicited symptoms were collected until day 28 and medically-attended adverse events (AE), new onset of chronic illness, and serious AEs were collected through the 6-month safety follow-up period.

Diary cards were used to record postvaccination local symptoms (pain, redness, swelling) and systemic solicited symptoms (drowsiness, irritability, loss of appetite, and fever, defined as a temperature of ≥38.0°C) on days 0–3 postvaccine. Unsolicited symptoms were collected until day 28 and medically-attended adverse events (AE), new onset of chronic illness, and serious AEs were collected through the 6-month safety follow-up period. Statistical Analysis The sample size and power determination were based on prior studies in this age group showing log10 HI titer standard deviation values ranging from 0.56–0.86. A target sample size of 450 subjects with 200 subjects in each of the TF-TIV groups and 50 subjects in the active comparator group was calculated to be required for the study. Assuming a 5% drop-out rate, this sample would allow detection of differences of 1.7–1.8-fold in geometric mean titers (GMTs) and approximately 80% power to detect differences of 4.4-, 2.7-, 2.0-, and 1.6-fold in the incidence rates of common reactogenicity events occurring at background rates of 2%, 5%, 10%, and 20% in the Flu-0.25 group.

t rate, this sample would allow detection of differences of 1.7–1.8-fold in geometric mean titers (GMTs) and approximately 80% power to detect differences of 4.4-, 2.7-, 2.0-, and 1.6-fold in the incidence rates of common reactogenicity events occurring at background rates of 2%, 5%, 10%, and 20% in the Flu-0.25 group. Immunogenicity was assessed in the according-to-protocol cohort, for each group and strain and by age stratification (aged 6–23 months and aged 24–35 months). The primary immunogenicity outcome was the GMTs at 28 days following the final influenza vaccine (1 or 2 doses depending on previous vaccination). Secondarily, seroconversion rates (SCRs), seroconversion factors (SCFs), and seroprotection rates (SPRs), and their 95% confidence intervals (CIs) 28 days following the completion of the vaccine regimen were also determined. The SCR was defined as the percentage of vaccinees who had either a prevaccination titer <1:10 and a postvaccination titer of ≥1:40 or a prevaccination titer ≥1:10 and at least a 4-fold increase in postvaccination titer at approximately 28 days following the last dose of the vaccine. The SPR was defined as the percentage of vaccinees with a serum HI titer ≥1:40 (protection titer deemed likely to correlate with a reduction in disease risk, based on adult data) 28 days following the last dose of the vaccine. The SCF was defined as the fold increase in serum HI GMTs at approximately 28 days following the last dose of the vaccine compared with prevaccination (day 0). These immunogenicity parameters and their 95% CIs were compared with the United States Food and Drug Administration Center for Biologics Evaluation and Research (CBER) criteria and European Medicines Agency (EMA) criteria for young adults because no criteria exist for children. The CBER criteria for adults aged <65 years and the pediatric population are that the lower limits of the 95% CIs are ≥40% for SCR and ≥70% for SPR [21]. The EMA Committee for Medicinal Products for Human Use criteria for adults aged 18–60 years are that the point estimates of the following 3 thresholds are met: SCR ≥ 40%, SPR ≥ 70%, SCF ≥ 2.5 [20]. The primary cohort for analysis of safety/reactogenicity was the total vaccinated cohort.

3 treatment groups (Table 2). The EMA adult immunogenicity criterion for HI response (SCR > 40% and SCF > 2.5) was met for all virus strains included and at both doses for all vaccine groups (Table 2). Table 2. Summary of Immunogenicity Results Pre- and Postvaccination (According-to-Protocol Cohort for Immunogenicity) Treatment Group Strain Flu-0.25 Flu-0.50 Vaxi-0.25 A/Brisbane GMT (95% CI) PRE 8.3 (6.9–10.1) 8.5 (7.0–10.3) 8.1 (5.9–11.0) POST 56.3 (39.5–80.2) 70.7 (50.7–98.6) 120.9 (73.4–199.0) SPR, % (95% CI) PRE 13.0 (7.7–20.0) 15.9 (10.1–23.3) 13.9 (4.7–29.5) POST 53.4 (44.5–62.2) 63.6 (54.8–71.8) 83.3 (67.2–93.6)a SCR, % (95% CI) POST 51.1 (42.3–60.0)a,b 62.1 (53.3–70.4)a,b 80.6 (64.0–91.8)a,b SCF (95% CI) POST 6.8 (5.2–8.9)a 8.3 (6.6–10.6)a 14.9 (9.6–23.3)a A/Uruguay GMT (95% CI) PRE 7.0 (5.9–8.3) 9.2 (7.4–11.4) 7.3 (4.9–11.1) POST 64.5 (48.2–86.4) 89.5 (67.4–119.0) 97.8 (59.0–162.4) SPR, % (95% CI) PRE 7.6 (3.7–13.6) 17.4 (11.4–25.0) 8.3 (1.8–22.5) POST 62.6 (53.7–70.9) 75.0 (66.7–82.1)a 83.3 (67.2–93.6)a SCR, % (95% CI) POST 61.8 (52.9–70.2)a,b 74.2 (65.9–81.5)a,b 77.8 (60.8–89.9)a,b SCF (95% CI) POST 9.2 (7.3–11.7)a 9.7 (8.0–11.9)a 13.3 (8.9–19.9)a B/Florida GMT (95% CI) PRE 7.9 (6.7–9.4) 7.9 (6.6–9.4) 10.8 (6.9–16.9) POST 128.7 (100.3–165.1) 163.7 (130.1–206.0) 190.3 (119.0–304.3) SPR, % (95% CI) PRE 13.0 (7.7–20.0) 15.2 (9.5–22.4) 19.4 (8.2–36.0) POST 84.7 (77.4–90.4)a,b 92.4 (86.5–96.3)a,b 91.7 (77.5–98.2) a,b SCR, % (95% CI) POST 80.9 (73.1–87.3)a,b 86.4 (79.3–91.7)a,b 86.1 (70.5–95.3)a,b SCF (95% CI) POST 16.2 (12.8–20.5)a 20.7 (16.3–26.2)a 17.6 (10.4–29.9)a Abbreviations: CI, confidence interval; Flu-0.25, 0.25-mL dose of thimerosal-free (TF) trivalent seasonal influenza vaccine (TIV); Flu-0.50, 0.50-mL dose of TF-TIV; GMT, geometric mean titer; POST, Postvaccination (day 28 for primed children, day 56 for unprimed children); PRE, Prevaccination dose 1 (day 0); SCF, seroconversion factor; SCR, seroconversion rate; SPR, seroprotection rate; Vaxi-0.25, 0.25-mL dose of Vaxigrip.

influenza vaccine (TIV); Flu-0.50, 0.50-mL dose of TF-TIV; GMT, geometric mean titer; POST, Postvaccination (day 28 for primed children, day 56 for unprimed children); PRE, Prevaccination dose 1 (day 0); SCF, seroconversion factor; SCR, seroconversion rate; SPR, seroprotection rate; Vaxi-0.25, 0.25-mL dose of Vaxigrip. aCommittee for Medicinal Products for Human Use criteria met or exceeded (SPR>70%, SCR>40%, SCF>2.5). bUnited States Food and Drug Administration Center for Biologics Evaluation and Research criteria met or exceeded (lower limit of the 95% CI for SPR≥70%, SCR≥40%). Analysis of Immune Response by Age Stratification Immunogenicity results for the 3 vaccine groups stratified by age (6–23 months and 24–35 months) are seen in Table 3 and Supplementary Table 2. Only the Vaxi-0.25 group met all EMA criteria. The EMA SCF criteria were met by both the Flu-0.50 and Flu-0.25 groups for all strains, but only the B/Florida strain met criteria for SPR. The SCR EMA criteria were not met by the lower dose FLU-0.25 group. Table 3. Summary of Immunogenicity Results 28 Days Postvaccination for Age Stratification (6–23 Months vs 24–35 Months)

SCF criteria were met by both the Flu-0.50 and Flu-0.25 groups for all strains, but only the B/Florida strain met criteria for SPR. The SCR EMA criteria were not met by the lower dose FLU-0.25 group. Table 3. Summary of Immunogenicity Results 28 Days Postvaccination for Age Stratification (6–23 Months vs 24–35 Months) Treatment Groupa Age stratum Strain Flu-0.25 Flu-0.50 Vaxi-0.25 6–23 months A/Brisbane GMT (95% CI) 30.0 (20.5–43.8) 39.8 (27.6–57.5) 100.2 (59.8–168.0) SPR, % ( 95% CI) 40.0 (29.8–50.9) 50.5 (39.9–61.2) 84.6 (65.1–95.6)b SCR, % ( 95% CI) 40.0 (29.8–50.9) 49.5 (38.8–60.1)b 84.6 (65.1–95.6)b,c SCF (95% CI) 4.5 (3.3–6.1)b 5.7 (4.3–7.6)b 14.4 (8.7–23.7)b A/Uruguay GMT (95% CI) 36.7 (28.2–47.9) 57.2 (42.1–77.8) 77.8 (44.3–136.6) SPR, % ( 95% CI) 51.1 (40.3–61.8) 67.0 (56.4–76.5) 76.9 (56.4–76.5)b SCR, % ( 95% CI) 51.1 (40.3–61.8)b,c 65.9 (55.3–75.5)b,c 73.1 (52.2–88.4)b,c SCF (95% CI) 6.9 (5.3–8.8)b 8.3 (6.5–10.8)b 12.2 (7.6–19.8)b B/Florida GMT (95% CI) 93.9 (72.5–121.7) 134.2 (101.7–177.1) 160.0 (96.5–265.2) SPR, % ( 95% CI) 82.2 (72.7–89.5)b,c 90.1 (82.1–95.4)b,c 92.3 (74.9–99.1)b,c SCR, % ( 95% CI) 76.7 (66.6–84.9)b,c 84.6 (75.5–91.3)b,c 84.6 (65.1–95.6)b,c SCF (95% CI) 12.7 (9.5–17.0)b 17.5 (13.1–23.3)b 15.6 (8.2–29.5)b 24–35 months A/Brisbane GMT (95% CI) 224.3 (124.5–404.0) 252.4 (148.4–429.4) 196.8 (51.3–755.0) SPR, % ( 95% CI) 82.9 (67.9–92.8)b 92.7 (80.1–98.5)b,c 80.0 (44.4–97.5)b SCR, % ( 95% CI) 75.6 (59.7–87.6)b,c 90.2 (76.9–97.3)b,c 70.0 (34.8–93.3)b SCF (95% CI) 16.4 (10.2–26.3)b 19.1 (13.6–26.9)b 16.6 (5.5–50.1)b A/Uruguay GMT (95% CI) 222.5 (124.1–398.7) 242.1 (145.8–401.8) 177.5 (53.9–584.9) SPR, % ( 95% CI) 87.8 (73.8–95.9)b,c 92.7 (80.1–98.5)b,c 100 (69.2–100)b SCR, % ( 95% CI) 85.4 (70.8–94.4)b,c 92.7 (80.1–98.5)b,c 90.0 (55.5–99.7)b SCF (95% CI) 17.7 (11.1–28.2)b 13.8 (10.4–18.1)b 16.6 (7.1–38.6)b B/Florida GMT (95% CI) 256.7 (153.8–428.6) 254.6 (171.9–377.0) 298.6 (89.7–993.4) SPR, % ( 95% CI) 90.2 (76.9–97.3)b,c 97.6 (87.1–99.9)b,c 90.0 (55.5–99.7)b SCR, % ( 95% CI) 90.2 (76.9–97.3)b,c 90.2 (76.9–97.3)b,c 90.0 (55.5–99.7)b,c SCF (95% CI) 27.5 (18.8–40.1)b 30.2 (20.1–45.2)b 24.3 (8.1–73.0)b Abbreviations: CI, confidence interval; Flu-0.25, 0.25-mL dose of thimerosal-free (TF) trivalent seasonal influenza vaccine (TIV); Flu-0.50, 0.50-mL dose of TF-TIV; GMT, geometric mean titer; SCF, seroconversion factor; SCR, seroconversion rate; SPR, seroprotection rate; Vaxi-0.25, 0.25-mL dose of Vaxigrip.

(8.1–73.0)b Abbreviations: CI, confidence interval; Flu-0.25, 0.25-mL dose of thimerosal-free (TF) trivalent seasonal influenza vaccine (TIV); Flu-0.50, 0.50-mL dose of TF-TIV; GMT, geometric mean titer; SCF, seroconversion factor; SCR, seroconversion rate; SPR, seroprotection rate; Vaxi-0.25, 0.25-mL dose of Vaxigrip. aFlu-0.25: 6–23 months, n = 90; 24–35 months, n = 41; Flu-0.50: 6–23 months, n = 91; 24–35 months, n = 41; Vaxi-0.25: 6–23 months, n = 26; 24–35 months, n = 10. bCHMP criteria met or exceeded (SPR>70%, SCR>40%, SCF>2.5). cUnited States Food and Drug Administration Center for Biologics Evaluation and Research criteria met or exceeded (lower limit of the 95% CI for SPR≥70%, SCR≥40). Immune responses were significantly higher in children aged 24–35 months than in those aged 6–23 months. The EMA criteria for SCR, SPR, and SCF were met for all strains in all treatment groups. The CBER criteria for SCR and SPR were met for all strains in the Flu-0.50 group. All CBER criteria were met by the Flu-025 group except for the SPR of the H1N1 component. The SCR was met for all strains and the SPR met for H3N2 and B strains. The SCR for the B and H3N2 strains met the CBER criteria in the Vaxi-0.25 group.

CBER criteria for SCR and SPR were met for all strains in the Flu-0.50 group. All CBER criteria were met by the Flu-025 group except for the SPR of the H1N1 component. The SCR was met for all strains and the SPR met for H3N2 and B strains. The SCR for the B and H3N2 strains met the CBER criteria in the Vaxi-0.25 group. Safety and Reactogenicity There was no difference in reactogenicity following dose 2 compared with dose 1. The incidence of any symptom (solicited and unsolicited) following dose 1 was 62.8% (103 of 164; 95% CI, 54.9–70.2) in Flu-0.25, 71.3% (119 of 167; 95% CI, 63.8–78.0) in Flu-0.50, and 65.1% (28 of 43; 95% CI, 49.1–79.0) in Vaxi-0.25 compared with 61.0% (83 of 136, 95% CI, 52.3–69.3) in Flu-0.25, 56.0% (79 of 141; 95% CI, 47.4–64.4) in Flu-0.50, and 67.6% (25 of 37; 95% CI, 50.2–82.0) in Vaxi-0.25 following dose 2 (Figure 1 and Supplementary Table 3). Injection-site pain was the most common local solicited symptom. Only 1 child in the Flu-0.25 group and 1 child in the Flu-0.50 group were reported to have grade 3 pain, and there were no reports of redness or swelling >50 mm in any of the treatment groups. The most common general solicited symptom was irritability. Most symptoms lasted 1–2.5 days postvaccination, and no symptom persisted >4 days.

the Flu-0.25 group and 1 child in the Flu-0.50 group were reported to have grade 3 pain, and there were no reports of redness or swelling >50 mm in any of the treatment groups. The most common general solicited symptom was irritability. Most symptoms lasted 1–2.5 days postvaccination, and no symptom persisted >4 days. Unsolicited adverse events occurred in 65.9% (108 of 164) of the Flu-0.25, 67.1% (112 of 167) of the Flu-0.50, and 55.8% (24 of 43) of the Vaxi-0.25 group. Medically attended events (MAEs) were reported for 52 children (31.7%) in the Flu-0.25 group, 40 children (24.0%) in the Flu-0.50 group, and 9 (20.9%) children in the Vaxi-0.25 group. During the 6-month extended safety follow-up, 46.3% (76 of 164) of children in the Flu-0.25 group experienced unsolicited AEs compared with 38.9% (65 of 167) in the Flu-0.50 group and 32.6% (14 of 43) in the Vaxi-0.25 group. Unsolicited MAEs were reported for 44.5% (73 of 164) of children in the Flu-0.25 group compared with 34.1% (57 of 167) in the Flu-0.50 group and 32.6% (14 of 43) in the Vaxi-0.25 group. There were 2 SAEs reported in the active phase of the study: 1 case of pneumonia in the Flu-0.25 group (resolved) and 1 case of bronchial hyper-reactivity in the Flu-0.50 group (in resolving stage). Two additional SAEs were reported in the extended safety follow-up period: 1 case of lobar pneumonia (Flu-0.25 group) and 1 case of viral pharyngitis (Flu-0.50 group). Both SAEs were reported to be resolved, and neither was deemed to be related to vaccination.

yper-reactivity in the Flu-0.50 group (in resolving stage). Two additional SAEs were reported in the extended safety follow-up period: 1 case of lobar pneumonia (Flu-0.25 group) and 1 case of viral pharyngitis (Flu-0.50 group). Both SAEs were reported to be resolved, and neither was deemed to be related to vaccination. DISCUSSION This study evaluated the use of 2 dose options of a TF-TIV in children aged <3 years. A recent similar study in 252 children aged 6–23 months found superior immunogenicity of a full dose of a TIV for 2 of 3 components in children aged 6–11 months [16]. Although we enrolled 374 children, the sample size was insufficient to demonstrate any superiority of the 0.5 mL dose over the 0.25 mL dose in children aged <3 years. However, these data suggest that increased influenza antigen content is associated with moderate improvement in immunogenicity with no increase in reactogenicity in this age group.

ildren, the sample size was insufficient to demonstrate any superiority of the 0.5 mL dose over the 0.25 mL dose in children aged <3 years. However, these data suggest that increased influenza antigen content is associated with moderate improvement in immunogenicity with no increase in reactogenicity in this age group. Young children often respond with lower HA antibody titers than older children [16, 21, 22]. In this study we observed higher titers in children aged 24–35 months than in those aged 6–23 months. However, in both children aged 6–23 months and children aged 24–35 month, the Committee for Medicinal Products for Human Use (CHMP) adult criterion were met (SCF > 2.5) by both doses of TIV for all virus strains. The GMTs induced by the TF-TIV at the 0.50 mL dose were higher than those of the 0.25 mL dose for all virus strains, particularly in children aged 6–23 months, which is consistent with at least 1 study using an adjuvanted influenza vaccine [17]. This greater immunogenic response with a full-dose strategy is encouraging because children aged <2 years have higher rates of illness and hospitalization than children aged ≥2 years [3, 23]. Although a single protective titer of HI antibodies that can be generalized to all influenza strains is elusive and pediatric data are lacking, the adult experience repeatedly suggests that higher HI titers are associated with lower risk of influenza [24]. Canada's immunization recommendation body recently recommended that all children receive the 0.5-mL dose of influenza vaccine [25] given the moderate improvement in immunogenicity with this dose and the likelihood that this would simplify the administration schedule. Other strategies to improve immunogenicity in the youngest children, particularly the use of adjuvants, are being studied. Given the small amount of data comparing these 2 doses, further study is warranted.

ovement in immunogenicity with this dose and the likelihood that this would simplify the administration schedule. Other strategies to improve immunogenicity in the youngest children, particularly the use of adjuvants, are being studied. Given the small amount of data comparing these 2 doses, further study is warranted. The 0.25-mL dose of the active comparator was apparently more immunogenic than the TF-TIV in many comparisons. In particular, the Vaxi-0.25 group met or exceeded the CHMP criteria for SPR for the H1N1 strain (A/Brisbane) in all children and both age categories unlike the TF-TIV at both doses. Of note, the only study to show superior immunogenicity of the 0.5-mL formulation used the Vaxigrip vaccine [16]. Although both vaccines are trivalent, inactivated, split virion influenza vaccines, the manufacturing processes used to produce the TF-TIV and Vaxigrip vaccines have some differences in terms of splitting agents, inactivation procedures, and excipients in the final formulation. Thimerosal-free TIV has undergone a detergent treatment to disrupt intact influenza virus particles, but complete clearance of intact virus might also diminish immunogenicity. An additional difference between the 2 vaccines used in this study is the method of delivery. Thimerosal-free TIV was supplied as prefilled syringes that provided a dose of 0.25 mL or 0.50 mL, whereas the active comparator, Vaxigrip, was supplied as a multidose vial with preservative.

ight also diminish immunogenicity. An additional difference between the 2 vaccines used in this study is the method of delivery. Thimerosal-free TIV was supplied as prefilled syringes that provided a dose of 0.25 mL or 0.50 mL, whereas the active comparator, Vaxigrip, was supplied as a multidose vial with preservative. The second primary objective of this study was to describe the safety of 2 doses of the TF-TIV in terms of solicited local and general symptoms (days 0–3), unsolicited AEs 28 days following vaccination, and unsolicited MAEs and SAEs throughout the study. The similar safety profile of the 2 TF-TIV vaccination groups suggests that doubling the volume and total antigen dose of TF-TIV did not alter meaningfully the reactogenicity to the vaccine. Fever was less frequent in the TF-TIV 0.50-mL group compared with the active comparator group, and the only occurrences of grade 3 fever (temperature >39.0°C) were in the TF-TIV 0.25-mL group. Previous studies with a virosomal-adjuvanted influenza vaccine and an unadjuvanted TIV also showed that increased antigen content did not correspondingly increase reactogenicity in young children [16, 17]. Children in our study were followed for 6 months after the final vaccine dose, and no reactogenicity signal was observed.

s studies with a virosomal-adjuvanted influenza vaccine and an unadjuvanted TIV also showed that increased antigen content did not correspondingly increase reactogenicity in young children [16, 17]. Children in our study were followed for 6 months after the final vaccine dose, and no reactogenicity signal was observed. Interestingly, the EMA and CBER criteria in terms of SCR and SPR were met for the B/Florida strain in all treatment groups. Influenza B strains are derived from 2 separate lineages, B/Victoria or B/Yamagata, with strains from only 1 lineage included in the TIV recommended for a particular season. Difficulties in obtaining good influenza B responses in children have been reported, particularly following changes in the lineage of the B strain from 1 vaccination season to the next [22, 26]. Influenza epidemics relating to the B strain have occurred with higher morbidity rates than normal in children [27]; in the 2008 influenza season in the southern hemisphere, this was the predominant strain in Australia and most Asian countries [28]. The main limitation of this study was that it was not powered to make statistical comparisons; immunogenicity was assessed primarily on point estimates and 95% CIs around postimmunization GMTs. Further study of the adult dose in children aged 6–35 months is clearly needed.

Interestingly, the EMA and CBER criteria in terms of SCR and SPR were met for the B/Florida strain in all treatment groups. Influenza B strains are derived from 2 separate lineages, B/Victoria or B/Yamagata, with strains from only 1 lineage included in the TIV recommended for a particular season. Difficulties in obtaining good influenza B responses in children have been reported, particularly following changes in the lineage of the B strain from 1 vaccination season to the next [22, 26]. Influenza epidemics relating to the B strain have occurred with higher morbidity rates than normal in children [27]; in the 2008 influenza season in the southern hemisphere, this was the predominant strain in Australia and most Asian countries [28]. The main limitation of this study was that it was not powered to make statistical comparisons; immunogenicity was assessed primarily on point estimates and 95% CIs around postimmunization GMTs. Further study of the adult dose in children aged 6–35 months is clearly needed. In summary, the TF-TIV met the CHMP adult immunogenicity criterion for all 3 virus strains and at both doses. There was a trend toward greater immunogenicity in recipients of the 0.50-mL dose compared with recipients of the 0.25-mL dose, particularly in children aged 6–23 months. The reactogenicity profiles of the 3 vaccine regimens were comparable.

CHMP adult immunogenicity criterion for all 3 virus strains and at both doses. There was a trend toward greater immunogenicity in recipients of the 0.50-mL dose compared with recipients of the 0.25-mL dose, particularly in children aged 6–23 months. The reactogenicity profiles of the 3 vaccine regimens were comparable. Supplementary Data Supplementary materials are available at the Journal of the Pediatric Infectious Diseases Society online (http://jpids.oxfordjournals.org). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

ordjournals.org). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author. Acknowledgments The authors are indebted to the participating study volunteers, study doctors and trial nurses, and laboratory technicians at the study sites and to the sponsor's project staff for their support and contributions throughout the study, in particular Drs Kathryn Bigsby, Pierre Bonin, William Bowler, Georges Caouette, Amarjit Cheema, David Connor, Allen Greenspoon, Sam Henein, Pierre-Alain Houle, Marc LeBel, Bhanu Muram, Michael O'Mahony and Rizwan Somani, as investigators; Arshad Amanullah for preparation of the study report; Sandra Fenstermacher for global study management; Dorothy Slavin, Clinical Safety Representative; and Richard Ippersiel, Regulatory Affairs representative. We thank Dr Claire Marie Seymour (XPE Pharma & Science) for writing assistance and Dr Geraldine Verplancke (Keyrus Biopharma) who provided support with coordinating the circulation of the manuscript to all coauthors, collecting comments received from coauthors, and ensuring that the recommendations of the International Committee of Medical Journal Editors (ICMJE) were fulfilled. GlaxoSmithKline, the funding source for this study, was involved in all stages of the study conduct and analysis. All authors participated in the implementation of the study including substantial contributions to conception and design, the gathering of the data, or analysis and interpretation of the data. All authors had full access to the data. All authors were involved in the drafting of the article or revising it critically for important intellectual content and final approval of the manuscript.

substantial contributions to conception and design, the gathering of the data, or analysis and interpretation of the data. All authors had full access to the data. All authors were involved in the drafting of the article or revising it critically for important intellectual content and final approval of the manuscript. Financial support. This work was supported by GlaxoSmithKline Biologicals. Potential conflicts of interest. J. M. L.'s institution has received research funding from GlaxoSmithKline, Sanofi Pasteur, and Novartis to conduct influenza vaccine studies. O. G. V. has received funding from GSK to conduct the present clinical trial. H. A. G. received compensation for travel expenses to investigator meeting and honoraria for participation in meetings related to the study from GSK. J. H. has received funding from GSK to conduct the present clinical trial and has served as a consultant to GSK and received compensation for travel expenses to investigator meetings. V. C. and V. K. J. are employees of GlaxoSmithKline and report ownership of GlaxoSmithKline stock options as part of a compensation package. L. F. is a former employee of GlaxoSmithKline and reports ownership of GlaxoSmithKline stock options as part of a compensation package. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

Neisseria meningitidis is an important cause of invasive bacterial infection worldwide [1]. Disease manifestations include bacteremia and sepsis, meningitis, septic arthritis, and pericarditis. Death and long-term sequelae including hearing loss, neurological disabilities, and limb loss in survivors are not uncommon after invasive meningococcal disease [2]. Although there are 12 immunologically distinct serogroups of N meningitidis, 6 serogroups (A, B, C, W-135, X, Y) cause most disease in humans [1, 2]. Polysaccharide vaccines against serogroups A, C, W-135, and Y have been available for decades, but, like other polysaccharide vaccines, they are limited in the durability of the protective responses, fail to reduce nasopharyngeal carriage, do not provide opportunities for boosting with subsequent doses, and, except for serogroup A, are not immunogenic in children under 2 years of age [3]. The development of conjugate vaccines, where the polysaccharide antigens are covalently linked to proteins, has led to more immunogenic vaccines for use in children for the prevention of several infectious diseases, including invasive meningococcal disease. Meningococcal C (MenC) conjugate vaccines have been approved in Europe, Canada, and elsewhere for use at 2 months of age and older beginning in 1999 [4]. Addition of MenC conjugate vaccines to the routine pediatric vaccination schedule has resulted in dramatic decreases in invasive MenC disease in many countries including Australia, the United Kingdom, the Netherlands, and Canada [5–9]. Two quadrivalent meningococcal conjugate vaccines using diphtheria toxoid or diphtheria toxoid cross-reacting material (CRM) as the conjugate protein (MenACWY-DT and MenACWY-CRM, respectively) have been developed and approved for use in individuals 9 months (MenACWY-DT) or 2 years (MenACWY-CRM) to 55 years of age, and they are now recommended for universal preadolescent and adolescent immunization in the United States and some provinces in Canada [10, 11]. The MenB capsular polysaccharide is poorly immunogenic because of antigenic similarities to human neural tissue, which has precluded development of MenB conjugate vaccines [12].

d they are now recommended for universal preadolescent and adolescent immunization in the United States and some provinces in Canada [10, 11]. The MenB capsular polysaccharide is poorly immunogenic because of antigenic similarities to human neural tissue, which has precluded development of MenB conjugate vaccines [12]. Therefore, broadly protective MenB vaccines are being developed using universally expressed N meningitidis surface proteins [13, 14]. Recently, a novel quadrivalent MenACWY conjugate vaccine using tetanus toxoid as the carrier protein (MenACWY-TT) has been developed and has undergone clinical trials in toddlers, adolescents, and adults [15–22]. The purpose of this study was to evaluate the safety and immunogenicity of MenACWY-TT compared with a marketed MenACWY-DT. Because the percentage of O-acetylation of the MenA polysaccharide may be important in its immunogenicity [23], 2 representative lots of MenACWY-TT covering the manufacturing range of O-acetylation of the MenA polysaccharide were compared.

the safety and immunogenicity of MenACWY-TT compared with a marketed MenACWY-DT. Because the percentage of O-acetylation of the MenA polysaccharide may be important in its immunogenicity [23], 2 representative lots of MenACWY-TT covering the manufacturing range of O-acetylation of the MenA polysaccharide were compared. METHODS Study Design This was a randomized, observer-blinded, multicentered, phase 2 clinical trial conducted in 33 centers in Canada and the United States between August 2010 and March 2011. Healthy individuals between 10 and 25 years of age were randomly allocated in a 1:1:1 ratio to 1 of 2 lots of MenACWY-TT or to MenACWY-DT. The study was conducted according to the International Conference on Harmonisation principles of Good Clinical Practice, the Declaration of Helsinki, and the Tri-Council Policy Statement: Ethical Conduct for Research Involving Humans. The protocol received ethics approval at each participating center; written informed consent was obtained from all participants or their parent or legal guardian (ClinicalTrials.Gov NCT01165242).

Practice, the Declaration of Helsinki, and the Tri-Council Policy Statement: Ethical Conduct for Research Involving Humans. The protocol received ethics approval at each participating center; written informed consent was obtained from all participants or their parent or legal guardian (ClinicalTrials.Gov NCT01165242). Study Participants Healthy males and females from 10 to 25 years of age, inclusive, were eligible to participate in the study. Individuals were excluded from participation for the following reasons: if they had used any other investigational drug or vaccine within the previous 30 days; if they were receiving immunosuppressive medication or had an immunocompromising condition; if they had previously received a meningococcal vaccine or had a history of meningococcal infection; if they had received tetanus toxoid containing vaccine within the previous month; if they had an allergy to any of the vaccine components; if they had received immunoglobulin or blood products within the 3 previous months or planned administration during the study period; if they were female and pregnant, planning to become pregnant, lactating, or unwilling to use effective contraceptive during the study; if they had a bleeding disorder; if they had a serious chronic infection or congenital defect; if they had any neurological disorder; or if they had an acute illness at the time of enrollment.

were female and pregnant, planning to become pregnant, lactating, or unwilling to use effective contraceptive during the study; if they had a bleeding disorder; if they had a serious chronic infection or congenital defect; if they had any neurological disorder; or if they had an acute illness at the time of enrollment. Vaccines The MenACWY-TT investigational vaccine (GlaxoSmithKline, Belgium) contained in each 0.5 mL dose 5 µg of each polysaccharide antigen (serogroup A, C, W-135, Y) conjugated to a total of approximately 44 µg tetanus toxoid. Two lots of MenACWY-TT were included in the study: lot A had 68% and lot B had 92% O-acetylation of the MenA polysaccharide, respectively. The MenACWY-DT control vaccine (Sanofi Pasteur, Swiftwater, PA) contained in each 0.5 mL dose 4 µg of each of the 4 meningococcal serogroup polysaccharides (A, C, W-135, Y) conjugated to a total of approximately 48 µg of diphtheria toxoid.

ad 68% and lot B had 92% O-acetylation of the MenA polysaccharide, respectively. The MenACWY-DT control vaccine (Sanofi Pasteur, Swiftwater, PA) contained in each 0.5 mL dose 4 µg of each of the 4 meningococcal serogroup polysaccharides (A, C, W-135, Y) conjugated to a total of approximately 48 µg of diphtheria toxoid. Study Objectives The primary study objective was to demonstrate the noninferiority of MenACWY-TT (lot A) when compared with MenACWY-DT in terms of the percentage of participants with a serum bactericidal antibody response against each serogroup 1 month postvaccination. Vaccine response was defined as a serum bactericidal titer of at least 1:8 in participants initially seronegative (titer <1:4) and as a 4-fold increase in titer in participants initially seropositive (titer ≥1:4). Secondary objectives included evaluation of the immune response by comparing geometric mean antibody titers (GMT) of the 3 vaccines; comparison of MenACWY-TT lots A and B; and evaluation of local and general solicited symptoms, unsolicited adverse events (AEs), new onsets of chronic illness, and serious AEs.

ter ≥1:4). Secondary objectives included evaluation of the immune response by comparing geometric mean antibody titers (GMT) of the 3 vaccines; comparison of MenACWY-TT lots A and B; and evaluation of local and general solicited symptoms, unsolicited adverse events (AEs), new onsets of chronic illness, and serious AEs. Study Procedures After obtaining informed consent, a medical history was taken, a physical examination was performed, and 10 mL of blood was obtained by venipuncture for baseline serology. After a urine pregnancy test (in females of childbearing potential), participants were randomly allocated to receive a single dose of 0.5 mL of MenACWY-DT or 1 of the 2 lots of MenACWY-TT intramuscularly in the deltoid muscle of the nondominant arm. Randomization was performed at GlaxoSmithKline using MATEX, a program developed for use in the Statistical Analysis System software (version 9.2; SAS Institute Inc., Cary, NC). The randomization algorithm used a minimization procedure accounting for center and age strata (10 through 17 years and 18 through 25 years). The 2 vials of the 2 lots of MenACWY-TT differed in appearance and required reconstitution before injection; MenACWY-DT was supplied in a ready-to-administer liquid. Therefore, to maintain blinding, vaccine preparation and injection was performed by study personnel not otherwise involved in study evaluation procedures (observer blind). A second visit was scheduled 1 month postvaccination for repeat blood sampling for antibody determination.

supplied in a ready-to-administer liquid. Therefore, to maintain blinding, vaccine preparation and injection was performed by study personnel not otherwise involved in study evaluation procedures (observer blind). A second visit was scheduled 1 month postvaccination for repeat blood sampling for antibody determination. Safety Assessments Participants were monitored for 30 minutes postvaccination for any immediate AEs. Solicited injection-site reactions and systemic AEs were recorded by the participants or their parents on a diary card on the day of and for 3 days after vaccination. Solicited injection-site reactions included pain, redness, and swelling; solicited systemic AEs included fever, headache, fatigue, and gastrointestinal symptoms (abdominal pain, nausea, vomiting, and diarrhea). Injection-site redness and swelling were measured, and the greatest diameter was recorded; intensity was graded as 1 (>0 to ≤20 mm), 2 (>20 to ≤50 mm), or 3 (>50 mm). Oral temperature was recorded and graded as 1 (≥37.5○C to ≤ 38.5○C), 2 (>38.5○C to ≤ 39.5○C), or 3 (>39.5○C). Intensity of pain was graded as 1 (mild, not interfering with normal activities), 2 (moderate, painful when limb moved and interfering with normal activities), or 3 (severe, significant pain at rest and preventing normal activities). Systemic AEs were graded in relation to interference with normal activities similar to injection-site pain. Unsolicited AEs were recorded by participants or their parents on the day of and for 30 days postvaccination and graded relative to interference with normal activities. Solicited and unsolicited AEs as well as concomitant medications, new onsets of chronic illness, and serious AEs were collected by the investigator from participants or their parents at the 1-month visit. A telephone contact took place 6 months postvaccination for collection of any new onset chronic illnesses, concomitant medications, and serious AEs.

well as concomitant medications, new onsets of chronic illness, and serious AEs were collected by the investigator from participants or their parents at the 1-month visit. A telephone contact took place 6 months postvaccination for collection of any new onset chronic illnesses, concomitant medications, and serious AEs. Immunogenicity Assessments Blood samples were obtained before and 30 days after vaccination. Serum aliquots were shipped frozen to Glaxo-SmithKline Vaccine's laboratories in Rixensart, Belgium, and Laval, Canada, and were assayed by technicians blinded to vaccine allocation. Serogroup-specific, functional antimeningococcal antibodies were determined by a serum bactericidal assay using human complement (hSBA) based on the Centers for Disease Control and Prevention protocol [24]. The cutoff for the assay was a dilution of 1:4; titers were expressed as the reciprocal of the dilution resulting in 50% bacterial killing.

onal antimeningococcal antibodies were determined by a serum bactericidal assay using human complement (hSBA) based on the Centers for Disease Control and Prevention protocol [24]. The cutoff for the assay was a dilution of 1:4; titers were expressed as the reciprocal of the dilution resulting in 50% bacterial killing. Statistical Considerations The primary analysis of immunogenicity used the according-to-protocol (ATP) cohort for immunogenicity, defined as all evaluable participants (those meeting all eligibility criteria, compliance with protocol procedures, and with no elimination criteria during the study) for whom assay results were available for antibodies against at least 1 serogroup for the blood taken 1 month postvaccination (defined as 21–48 days postvaccination). The primary analysis for safety used the total vaccinated cohort (TVC), which included all vaccinated subjects for whom data were available. For each treatment group and for each antibody at each time point, the GMT and 95% confidence interval (CI) were calculated. The proportion and 95% CIs of participants with hSBA titers against prespecified cutoffs (≥1:4, ≥1:8) and the proportion and 95% CIs of participants with hSBA vaccine responses were also calculated. The primary objective of noninferiority of MenACWY-TT (lot A) compared with MenACWY-DT with respect to the serogroup A, C, W-135, Y vaccine responses were evaluated through computation of the 95% CIs for the difference in the percentage of participants with an hSBA vaccine response (MenACWY-TT lot A minus MenACWY-DT) 1 month postvaccination. Statistical noninferiority of MenACWY-TT was defined as a lower limit of the 95% CI greater than or equal to the predefined clinical limit of −10%. Exploratory immunological analyses included (1) computation of the 95% CIs of the difference in the percentage of participants with hSBA titers ≥1:4 and ≥1:8 1 month after vaccination (MenACWY-TT lot A minus MenACWY-DT, MenACWY-TT lot B minus MenACWY-DT, and MenACWY-TT lot A minus MenACWY-TT lot B) and (2) computation of the 95% CIs of the difference in the proportion of participants with hSBA vaccine response 1 month after vaccination (MenACWY-TT lot A minus MenACWY-DT and MenACWY-TT lot B minus MenACWY-DT). In the exploratory analyses, 2 vaccine groups were said to be significantly different for percentages if the 95% CI for the difference in rates did not contain the value 0.

ion of participants with hSBA vaccine response 1 month after vaccination (MenACWY-TT lot A minus MenACWY-DT and MenACWY-TT lot B minus MenACWY-DT). In the exploratory analyses, 2 vaccine groups were said to be significantly different for percentages if the 95% CI for the difference in rates did not contain the value 0. Computation of the 95% CIs of the hSBA GMT ratios (ACWY-A over ACWY-DT, ACWY-B over ACWY-DT, and ACWY-A over ACWY-B) were also performed using an analysis of covariance (ANCOVA) model on the logarithm10 transformation of the titers including the vaccine group as fixed effect and using the prevaccination (ie, the month 0 blood sampling) logarithm10 transformation of the titers and the age strata as covariates. Two groups were considered significantly different for GMTs if the 95% CI for the GMT ratio between the 2 groups did not contain the value 1. Results of the exploratory analyses should be interpreted with caution because no adjustment for multiplicity of comparisons was made. Safety analyses included the percentage and 95% CI of participants with at least 1 injection-site AE, at least 1 systemic AE, and with any AE on the day of and 3 days postvaccination. The analysis also included the percentage and 95% CI of participants reporting individual solicited injection-site and systemic AEs (any grade, ≥grade 2, and grade 3) and relationship to vaccination, unsolicited AEs, new onset chronic illness, and serious AEs.

, and with any AE on the day of and 3 days postvaccination. The analysis also included the percentage and 95% CI of participants reporting individual solicited injection-site and systemic AEs (any grade, ≥grade 2, and grade 3) and relationship to vaccination, unsolicited AEs, new onset chronic illness, and serious AEs. A sample size of 900 participants (300 per group) was calculated to provide 92% power to meet the primary objective; a sample size of 335 per group was selected to account for up to 10% of participants being excluded from the ATP cohort for immunogenicity. Statistical analyses were done using SAS software (version 9.2). RESULTS Demographics and Participant Disposition A total of 1016 participants were enrolled and 1011 participants were vaccinated (MenACWY-TT lot A 337, MenACWY-TT lot B 336, MenACWY-DT 338) (Figure 1). A total of 60 participants were excluded from the ATP for immunogenicity cohort: 20 were from MenACWY-TT lot A, 16 were from MenACWY-TT lot B, and 24 were from MenACWY-DT. Reasons for exclusion were similar amongst the 3 groups and were mostly due to participant ineligibility, noncompliance with blood sample schedule, and missing essential serological data. A total of 977 (96.6%) vaccinated participants completed the study and were equally distributed amongst the vaccine groups; the most frequent reasons for noncompletion were loss to follow-up and consent withdrawal. Figure 1. Participant disposition.

ce with blood sample schedule, and missing essential serological data. A total of 977 (96.6%) vaccinated participants completed the study and were equally distributed amongst the vaccine groups; the most frequent reasons for noncompletion were loss to follow-up and consent withdrawal. Figure 1. Participant disposition. The 3 vaccine groups were similar for demographic characteristics (Table 1). The mean age of participants was 16.3 years (range, 10–25 years) and varied from 16.2 to 16.4 years amongst the 3 vaccine groups. A total of 58.9% of participants were between 10 and 17 years of age, and 41.1% were between 18 and 25 years of age. Females comprised 51.4% of participants, and participants ranged from 50.3% to 52.1% amongst the vaccine groups. The majority of participants (73.6%–76.0%) were of white-Caucasian/European heritage. Table 1. Summary of Demographic Characteristics (Total Vaccinated Cohort) Characteristics Parameter or Category MenACWY-TT lot A (N = 337) MenACWY-TT lot B (N = 336) MenACWY-DT (N = 338) Age Mean (SD) 16.4 (5.16) 16.3 (5.16) 16.2 (4.97) Gender Female, n (%) 175 (51.9) 169 (50.3) 176 (52.1) Male, n (%) 162 (48.1) 167 (49.7) 162 (47.9) Race African heritage/African American, n (%) 38 (11.3) 29 (8.6) 40 (11.8) Asian-Central/South Asian heritage, n (%) 17 (5.0) 17 (5.1) 17 (5.0) White-Caucasian/European heritage n (%) 248 (73.6) 249 (74.1) 257 (76.0) Other n (%) 34 (10.1) 41 (12.2) 24 (7.1) Abbreviations: N, total number of participants; n (%), number (percentage) of participants in a given category; SD, standard deviation.

) Asian-Central/South Asian heritage, n (%) 17 (5.0) 17 (5.1) 17 (5.0) White-Caucasian/European heritage n (%) 248 (73.6) 249 (74.1) 257 (76.0) Other n (%) 34 (10.1) 41 (12.2) 24 (7.1) Abbreviations: N, total number of participants; n (%), number (percentage) of participants in a given category; SD, standard deviation. Safety Diary cards recording AEs were returned by most participants (97.6% of MenACWY-TT lot A, 97.9% of MenACWY-TT lot B, and 96.2% and 96.4% of MenACWY-DT for solicited injection-site and systemic AEs, respectively). At least 1 symptom was reported during the 4-day safety follow-up period in 70.0% of MenACWY-TT lot A recipients, 66.1% of MenACWY-TT lot B recipients, and 70.1% of MenACWY-DT recipients. Injection-site reactions were reported by 58.8%, 56.3%, and 59.5% of recipients, respectively, and systemic AEs were reported by 44.8%, 39.3%, and 42.3% of recipients, respectively. Injection-site pain was the most frequently reported solicited AE, and headache and fatigue were the most common systemic AEs reported (Table 2). Grade 3 (severe) injection-site and systemic AEs were uncommon. Rates of injection-site and systemic AEs and grade 3 AEs were similar amongst the 3 vaccine groups. Table 2. Percentage of Participants Experiencing Solicited Local and General Symptoms in the MenACWY-TT and MenACWY-DT Groups During 4-Day Period After Vaccination (Total Vaccinated Cohort)

nd systemic AEs were uncommon. Rates of injection-site and systemic AEs and grade 3 AEs were similar amongst the 3 vaccine groups. Table 2. Percentage of Participants Experiencing Solicited Local and General Symptoms in the MenACWY-TT and MenACWY-DT Groups During 4-Day Period After Vaccination (Total Vaccinated Cohort) Symptom Type MenACWY-TT lot A (N = 329) MenACWY-TT lot B (N = 329) MenACWY-DT (N = 325) n % (95% CI) n % (95% CI) n % (95% CI) Local Pain All 169 51.4 (45.8, 56.9) 167 50.8 (45.2, 56.3) 180 55.4 (49.8, 60.9) Grade 3 8 2.4 (1.1, 4.7) 5 1.5 (0.5, 3.5) 2 0.6 (0.1, 2.2) Redness (mm) All 85 25.8 (21.2, 30.9) 60 18.2 (14.2, 22.8) 66 20.3 (16.1, 25.1) Grade 3 3 0.9 (0.2, 2.6) 2 0.6 (0.1, 2.2) 6 1.8 (0.7, 4.0) Swelling (mm) All 63 19.1 (15.0, 23.8) 40 12.2 (8.8, 16.2) 44 13.5 (10.0, 17.7) Grade 3 3 0.9 (0.2, 2.6) 3 0.9 (0.2, 2.6) 3 0.9 (0.2, 2.7) General Fatigue All 96 29.2 (24.3, 34.4) 94 28.6 (23.8, 33.8) 89 27.3 (22.5, 32.5) Grade 3 9 2.7 (1.3, 5.1) 7 2.1 (0.9, 4.3) 5 1.5 (0.5, 3.5) Gastrointestinala All 43 13.1 (9.6, 17.2) 43 13.1 (9.6, 17.2) 44 13.5 (10.0, 17.7) Grade 3 4 1.2 (0.3, 3.1) 3 0.9 (0.2, 2.6) 4 1.2 (0.3, 3.1) Headache All 86 26.1 (21.5, 31.2) 87 26.4 (21.8, 31.6) 83 25.5 (20.8, 30.6) Grade 3 5 1.5 (0.5, 3.5) 2 0.6 (0.1, 2.2) 6 1.8 (0.7, 4.0) Fever All 17 5.2 (3.0, 8.1) 14 4.3 (2.3, 7.0) 16 4.9 (2.8, 7.8) Grade 3b 1 0.3 (0.0, 1.7) 0 0 (0.0, 1.1) 0 0 (0.0, 1.1) Abbreviations: CI, confidence interval; N, total number of participants, with the documented dose; n/%, number/percentage of participants reporting the symptom at least once.

.1, 2.2) 6 1.8 (0.7, 4.0) Fever All 17 5.2 (3.0, 8.1) 14 4.3 (2.3, 7.0) 16 4.9 (2.8, 7.8) Grade 3b 1 0.3 (0.0, 1.7) 0 0 (0.0, 1.1) 0 0 (0.0, 1.1) Abbreviations: CI, confidence interval; N, total number of participants, with the documented dose; n/%, number/percentage of participants reporting the symptom at least once. aGastrointestinal symptoms included nausea, vomiting, diarrhea, and/or abdominal pain. bTemperature >39.5°C. At the 6-month follow-up, at least 1 new onset chronic illness was reported by 3 participants in the MenACWY-TT lot A group, including hypersensitivity, insulin resistance, asthma, and bronchial hyperreactivity; none were reported in the other 2 groups. Serious AEs were reported by 1 participant in the MenACWY-TT lot A group (asthma), 5 participants in the MenACWY-TT lot B group (8 serious AEs: tooth infection; appendicitis; asthma, influenza and pneumonia; and pneumonia and hypoxia), and 2 participants in the MenACWY-DT group (jaw fracture and postprocedural hematoma); none of the serious AEs were considered by the investigators to be vaccine-related.

articipants in the MenACWY-TT lot B group (8 serious AEs: tooth infection; appendicitis; asthma, influenza and pneumonia; and pneumonia and hypoxia), and 2 participants in the MenACWY-DT group (jaw fracture and postprocedural hematoma); none of the serious AEs were considered by the investigators to be vaccine-related. Immunogenicity At baseline, 79 (25.5%) of 310 participants in the MenACWY-TT lot A group, 84 (27.2%) of 309 in the MenACWY-TT lot B group, and 89 (29.1%) of 306 participants in the MenACWY-DT group were seropositive for MenA antibody. For MenC antibody, 175 (60.8%) of 288 in the MenACWY-TT lot A group, 188 (65.7%) of 286 in the MenACWY lot B group, and 197 (68.2%) of 289 in the MenACWY-DT group were seropositive. For MenW-135 antibody, 99 (33.8%) of 293 in the MenACWY-TT lot A group, 98 (33.8%) of 290 in the MenACWY-TT lot B group, and 102 (34.1%) of 299 in the MenACWY-DT group were seropositive. For MenY antibody, 215 (72.6%) of 296 in the MenACWY-TT lot A group, 224 (73.2%) of 306 in the MenACWY-TT lot B group, and 234 (77.0%) of 304 in the MenACWY-DT group were seropositive. The proportion of participants in each vaccine group with a vaccine response against the 4 serogroups is depicted in Table 3. Vaccine response rates were consistently higher in participants who were initially seronegative at baseline (data not shown). The noninferiority of MenACWY-TT (lot A) compared with MenACWY-DT was demonstrated in terms of the percentage of participants with a vaccine response as measured by hSBA against serogroups A, C, W-135, and Y 1 month after vaccination. The difference in vaccine response rate (MenACWY-TT lot A minus MenACWY-DT) was 6.01% (95% CI −1.45 to 13.44) for MenA, 0.95% (−6.10 to 8.00) for MenC, 6.95% (−0.76 to 14.59) for MenW-135, and 12.21% (4.17–20.10) for MenY, all above the noninferiority threshold for the lower limit of the 95% CI of −10%. In the exploratory analyses, a statistically significantly higher vaccine response rate was observed for MenACWY-TT lot A compared with MenACWY-DT for hSBA Men Y and for MenACWY-TT lot B compared with MenACWY-DT for hSBA MenW-135 and MenY. No statistically significant differences were detected for vaccine response between any of the serogroups between MenACWY-TT lots A and B. Table 3. Geometric Mean Antibody Titers and Percentage of Participants With a Vaccine Response at 1 Month After Vaccination in the MenACWY-TT and MenACWY-DT Groups (ATP Cohort for Immunogenicity)

ly significant differences were detected for vaccine response between any of the serogroups between MenACWY-TT lots A and B. Table 3. Geometric Mean Antibody Titers and Percentage of Participants With a Vaccine Response at 1 Month After Vaccination in the MenACWY-TT and MenACWY-DT Groups (ATP Cohort for Immunogenicity) Antibody GMT (95% CI) Adjusted GMT Group Ratio MenACWY-TT lot A or B/MenACWY-DT Adjusted GMT Group Ratio MenACWY-TT lot A/MenACWY-TT lot B Vaccine Response Group N Pre N Post (95% CI) (95% CI) N %a (95% CI) MenA MenACWY-TT lot A 310 3.6 (3.1, 4.0) 315 54.2 (43.5, 67.4) 1.34 (0.97, 1.84) 1.08 (0.79, 1.47) 310 70.3 (64.9, 75.4) MenACWY-TT lot B 309 3.6 (3.2, 4.1) 309 49.6 (39.6, 62.1) 1.24 (0.90, 1.73) – 300 71.3 (65.9, 76.4) MenACWY-DT 306 3.6 (3.2, 4.1) 305 41.3 (32.3, 52.9) – – 297 64.3 (58.6, 69.8) MenC MenACWY-TT lot A 288 15.6 (12.3, 19.9) 307 687.1 (510.5, 924.9) 1.35 (0.92, 1.99) 0.92 (0.62, 1.38) 281 77.2 (71.9, 82.0) MenACWY-TT lot B 286 16.0 (12.8, 20.0) 304 755.8 (557.3, 1025.0) 1.47 (1.00, 2.17) – 274 82.5 (77.5, 86.8) MenACWY-DT 289 18.0 (14.4, 22.6) 296 543.3 (411.2, 718.0) – – 274 76.3 (70.8, 81.2) MenW-135 MenACWY-TT lot A 293 7.7 (6.1, 9.7) 298 174.5 (138.6, 219.6) 1.60 (1.15, 2.23)b 1.04 (0.76, 1.43) 279 71.0 (65.3, 76.2) MenACWY-TT lot B 290 7.6 (6.0, 9.6) 292 161.6 (128.3, 203.5) 1.55 (1.11, 2.15) – 270 72.6 (66.9, 77.8) MenACWY-DT 299 7.4 (5.9, 9.2) 297 101.7 (77.9, 132.7) – – 289 64.0 (58.2, 69.6) MenY MenACWY-TT lot A 296 45.7 (35.9, 58.2) 313 349.1 (298.1, 408.8) 1.54 (1.21, 1.97) 0.91 (0.73, 1.14) 293 51.2 (45.3, 57.1) MenACWY-TT lot B 306 49.8 (39.1, 63.4) 307 387.4 (329.7, 455.1) 1.66 (1.30, 2.13) – 294 51.0 (45.2, 56.9) MenACWY-DT 304 55.3 (43.7, 69.9) 305 253.8 (204.9, 314.5) – – 295 39.0 (33.4, 44.8) Abbreviations: Adjusted GMT, geometric mean antibody titer adjusted for age strata and baseline titer; ATP, according to protocol; CI, confidence interval; GMT, geometric mean antibody titer (reciprocal dilution) calculated on all participants; N, number of subjects with results available; post, postvaccination at month 1; pre, prevaccination at month 0.

ic mean antibody titer adjusted for age strata and baseline titer; ATP, according to protocol; CI, confidence interval; GMT, geometric mean antibody titer (reciprocal dilution) calculated on all participants; N, number of subjects with results available; post, postvaccination at month 1; pre, prevaccination at month 0. aPercentage of participants with a vaccine response. bBolded text indicates statistically significant group difference in GMT based on exploratory analysis.

ic mean antibody titer adjusted for age strata and baseline titer; ATP, according to protocol; CI, confidence interval; GMT, geometric mean antibody titer (reciprocal dilution) calculated on all participants; N, number of subjects with results available; post, postvaccination at month 1; pre, prevaccination at month 0. aPercentage of participants with a vaccine response. bBolded text indicates statistically significant group difference in GMT based on exploratory analysis. Prevaccination, GMTs against the 4 serogroups were similar amongst the 3 vaccine groups (Table 3). Postvaccination, all groups demonstrated increased GMTs against all 4 serogroups. Differences in GMTs between groups were explored using GMT ratios; GMTs for serogroups W-135 and Y were statistically higher in recipients of either MenACWY-TT lot A or MenACWY-TT lot B compared with MenACWY-DT; no differences were observed between MenACWY-TT (lots A and B). The proportion of participants postvaccination with hSBA titers ≥1:4 for the 4 serogroups ranged between 73.1% and 80.3% for serogroup A, between 96.1% and 98.3% for serogroup C, between 83.2% and 91.3% for serogroup W-135, and between 94.1% and 98.4% for serogroup Y (Figure 2). The percentage of participants with antibody titers ≥1:4 was significantly higher at day 28 for recipients of MenACWY-TT lot A for serogroups A, W-135, and Y compared with MenACWY-DT, and statistically higher for recipients of MenACWY-TT lot B for serogroups W-135 and Y compared with MenACWY-DT. There were no differences between MenACWY-TT lots A and B. A similar pattern was observed for the proportion of participants with postvaccination titers ≥1:8; most participants who achieved protective titers of ≥1:4 also exceeded the ≥1:8 threshold (Figure 2). Figure 2. Percentage of participants with hSBA titers equal to or above the cutoff values of 1:4 (left) and 1:8 (right) against (A) MenA, (B) MenC, (C) MenW-135, (D) MenY prevaccination (open bars), and postvaccination (closed bars) in the according-to-protocol cohort for immunogenicity. Error bars represent the 95% confidence intervals.

e of participants with hSBA titers equal to or above the cutoff values of 1:4 (left) and 1:8 (right) against (A) MenA, (B) MenC, (C) MenW-135, (D) MenY prevaccination (open bars), and postvaccination (closed bars) in the according-to-protocol cohort for immunogenicity. Error bars represent the 95% confidence intervals. DISCUSSION In this study, MenACWY-TT met the primary immunogenicity noninferiority vaccine response criteria relative to a marketed MenACWY-DT vaccine for all 4 serogroups in adolescents and young adults. In the secondary immunogenicity analyses, both lots of MenACWY-TT elicited significantly higher GMTs against serogroups W-135 and Y and proportions of participants achieving titers ≥1:4 against serogroups W-135 and Y, as well as the proportion achieving titers ≥1:4 for lot A against serogroup A. Taken in aggregate, the data suggest that the immunogenicity of both lots of MenACWY-TT was at least as high as that of the licensed comparator vaccine for all 4 serogroups.

participants achieving titers ≥1:4 against serogroups W-135 and Y, as well as the proportion achieving titers ≥1:4 for lot A against serogroup A. Taken in aggregate, the data suggest that the immunogenicity of both lots of MenACWY-TT was at least as high as that of the licensed comparator vaccine for all 4 serogroups. Injection-site and systemic AEs were similar between recipients of both lots of MenACWY-TT and MenACWY-DT. Pain was the most common injection-site reaction, and headache and fatigue were the most commonly reported systemic AEs. Although new onset chronic illness was reported only by recipients of MenACWY-TT, the events did not represent a specific disease or syndrome; all 3 participants were in lot A group, likely representing chance occurrence. Serious AEs were reported by 6 MenACWY-TT participants (1 in lot A and 5 in lot B, likely representing chance clustering) and 2 MenACWY-DT recipients, reflecting the 2:1 MenACWY-TT/MenACWY-DT allocation.

esent a specific disease or syndrome; all 3 participants were in lot A group, likely representing chance occurrence. Serious AEs were reported by 6 MenACWY-TT participants (1 in lot A and 5 in lot B, likely representing chance clustering) and 2 MenACWY-DT recipients, reflecting the 2:1 MenACWY-TT/MenACWY-DT allocation. The immunogenicity results of this study support the findings of a previous study of 784 adolescents and young adults 11–25 years of age who received a single dose of MenACWY-TT or MenACWY-DT where similarity of the MenACWY-TT vaccine was demonstrated in exploratory analyses as well as significantly higher GMTs and proportions of participants achieving hSBA titers ≥1:4 for some or all serogroups [20]. The current study also demonstrated that the 2 lots of MenACWY-TT with differing levels of O-acetylation did not differ in either reactogenicity or immunogenicity. The MenA capsular polysaccharide is approximately 70%–95% O-acetylated at carbon 3 [23, 25, 26]. The 2 lots of MenACWY-TT vaccine spanned the range of O-acetylation and performed similarly in this study. O-acetylation is an important factor in the immunogenicity of MenA polysaccharide conjugates; deacetylation during the conjugation process has been previously reported to markedly affect the immunogenicity of MenA vaccines in bactericidal assays [23]; however, this was not confirmed in our study.

milarly in this study. O-acetylation is an important factor in the immunogenicity of MenA polysaccharide conjugates; deacetylation during the conjugation process has been previously reported to markedly affect the immunogenicity of MenA vaccines in bactericidal assays [23]; however, this was not confirmed in our study. At the time of this clinical trial, 2 other quadrivalent meningococcal conjugate vaccines were marketed worldwide. Menactra (Sanofi Pasteur, Swiftwater, PA), which uses diphtheria toxoid as the carrier protein, was the first quadrivalent meningococcal conjugate vaccine to market, and it was used as the comparator vaccine in this study. Menveo (Novartis Vaccines and Diagnostics, Cambridge, MA), which uses CRM197 as the carrier protein, was second to market, and it demonstrated noninferiority to Menactra in a prelicensure clinical trial conducted in adolescents [27]. Similar to this study with the novel MenACWY-TT vaccine, Menveo showed increased GMTs compared with Menactra [27]. MenACWY-TT has also been shown to be noninferior to quadrivalent meningococcal polysaccharide vaccine in children aged 2 to 10 years [21], Asian adolescents [18], and European adolescents and young adults [15]. Four different formulations of the novel MenACWY-TT vaccine compared with age-appropriate, marketed meningococcal vaccines (either MenC conjugate vaccine or quadrivalent meningococcal conjugate vaccine) were studied in children 1–5 years of age [16], and noninferiority of MenACWY-TT compared with MenC was demonstrated in children 12–23 months of age [22]. There was no detrimental effect on reactogenicity or immunogenicity when MenACWY-TT was coadministered with a combined diphtheria, tetanus, acellular pertussis, inactivated poliovirus, Haemophilus influenzae type b conjugate, hepatitis B vaccine in toddlers [17], with combined measles-mumps-rubella-varicella vaccine in toddlers [22], or combined hepatitis A-hepatitis B vaccine in adolescents [19]. In this study, MenACWY‐TT vaccine was well tolerated and elicited an immune response that was noninferior to that of a marketed MenACWY‐DT vaccine. Taken collectively, these studies provide the data needed to support the use of MenACWY-TT in various national meningococcal vaccination programs.

cine in adolescents [19]. In this study, MenACWY‐TT vaccine was well tolerated and elicited an immune response that was noninferior to that of a marketed MenACWY‐DT vaccine. Taken collectively, these studies provide the data needed to support the use of MenACWY-TT in various national meningococcal vaccination programs. In the United States, quadrivalent meningococcal conjugate vaccines are licensed and recommended for all adolescents, as well as for children 9 months to 2 years (2-dose regimen) of age and individuals 2–55 years of age (single dose) with conditions that put them at increased risk for invasive meningococcal disease [28–32]. In Canada, some provinces recommend an adolescent booster dose of quadrivalent meningococcal conjugate vaccines after infant MenC conjugate vaccination [33]. Most European countries have implemented MenC conjugate vaccination programs because of the predominance of serogroup C disease [34]. MenACWY-TT has now received market authorization in the European Union for administration as a single dose to individuals 12 months of age or older, expanding the options for invasive meningococcal disease control.

implemented MenC conjugate vaccination programs because of the predominance of serogroup C disease [34]. MenACWY-TT has now received market authorization in the European Union for administration as a single dose to individuals 12 months of age or older, expanding the options for invasive meningococcal disease control. Acknowledgments We thank the individuals and their parents or legal guardians who participated in the study, and we thank all of the other investigators involved in conducting the study (T. Araki, D. Connor, B. Lasko, D. Shu, A. McIntosh, W. Andrews, R. Broker, W. Ellison, R. Hines, I. Rarick, J. Borders, B. Fox, N. Segall, and L. Wadsworth). We also thank the nurses and research assistants at all enrollment sites for their careful attention to detail. Finally, we thank F. Del Buono (GlaxoSmithKline Vaccines) for protocol development, GlaxoSmithKline Vaccines study manager A. Lee, and B. van Heertum (XPE Pharma & Science on behalf of GlaxoSmithKline Vaccines) for manuscript coordination. Financial support. This work was supported by GlaxoSmithKline Biologicals SA. Potential conflicts of interest S. A. H., J. M. L., and S. A. M. have served on ad hoc advisory boards for the GlaxoSmithKline group of companies and other vaccine manufacturers and governments. J. B. D. received compensation for lectures from the GlaxoSmithKline group of companies. C. I. B., L. R. F., Y. B., V. B., and J. M. M. are employees of the GlaxoSmithKline group of companies. C. I. B., L. R. F., Y. B., and J. M. M. declare stock ownership in the GlaxoSmithKline group of companies.

ers and governments. J. B. D. received compensation for lectures from the GlaxoSmithKline group of companies. C. I. B., L. R. F., Y. B., V. B., and J. M. M. are employees of the GlaxoSmithKline group of companies. C. I. B., L. R. F., Y. B., and J. M. M. declare stock ownership in the GlaxoSmithKline group of companies. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

INTRODUCTION The interpretation of positive sexually transmitted infection (STI) diagnostic test results from young children poses particular challenges. Nucleic acid amplification tests for STIs are very sensitive and do not discriminate between living and dead material. Therefore, false positives are always a concern. Although such false positives will always have potentially serious consequences, they are of particular forensic and medico-legal significance in young children where a positive STI diagnostic test may be regarded as a strong indicator that sexual contact has occurred. Clinical guidelines regarding the interpretation of STI diagnoses in young children are imprecise and do not contain numerical information regarding positive predictive values of such diagnoses to indicate sexual contact. In the United States, guidelines from the Centers for Disease Control state that confirmed diagnoses of gonorrhoea, syphilis, human immunodeficiency virus, or chlamydia in a nonneonatal prepubertal child “suggests sexual abuse”, and that the significance “varies by pathogen” [1]. In the United Kingdom, guidelines state only that “results need to be interpreted based on the limitations of the tests used” [2]. In Australia, guidelines are generated at a state or territory level and, in general, specify that STI diagnosis in a young child is insufficient to conclude that sexual abuse has occurred [3, 4]. In the Northern Territory (NT), a positive STI result in a young child prompts a mandatory notification to NT Government child protection authorities and an investigation.

e or territory level and, in general, specify that STI diagnosis in a young child is insufficient to conclude that sexual abuse has occurred [3, 4]. In the Northern Territory (NT), a positive STI result in a young child prompts a mandatory notification to NT Government child protection authorities and an investigation. In the NT, the challenges regarding interpretation of positive STI tests in young children are amplified. Approximately 30% of the NT population are Indigenous Australians, with a large proportion living in remote communities. Sexually transmitted infections are highly prevalent in the NT Indigenous population [5]. In 2006, the NT Government commissioned a report titled “Ampe Akelyernemane Meke Mekarle” (Little Children are Sacred). The authors of this document worked with Indigenous communities to collate evidence of sexual abuse of children and make policy recommendations. The impetus for this report was the frequency of anecdotal evidence for abuse [6]. This document was in large part the catalyst for the socially and politically contentious Australian Commonwealth Government “Northern Territory National Emergency Response” and the subsequent “Stronger Futures in the NT” program.

tions. The impetus for this report was the frequency of anecdotal evidence for abuse [6]. This document was in large part the catalyst for the socially and politically contentious Australian Commonwealth Government “Northern Territory National Emergency Response” and the subsequent “Stronger Futures in the NT” program. In this context of heightened community awareness regarding sexual abuse, frontline service providers in the NT may on occasion test children for STIs in the absence of specific disclosure of abuse. In this scenario, a positive test may be virtually the only evidence that sexual contact has occurred, and the pretest probability of sexual contact is potentially low. Accordingly, the postpositive test probability of sexual contact may be very sensitive to the frequency of STI positive tests in the absence of sexual contact. Two broad categories of postulated mechanisms for false-positive STI diagnoses are (1) those that result from the direct transfer of STI agent environmental contaminants into diagnostic samples and (2) those that result from infection or contamination of the urogenital tract in the absence of sexual contact. These categories are amenable to fundamentally different interventions for reducing the impact of false positives. In this study, we address the first category.

mental contaminants into diagnostic samples and (2) those that result from infection or contamination of the urogenital tract in the absence of sexual contact. These categories are amenable to fundamentally different interventions for reducing the impact of false positives. In this study, we address the first category. The results of recent studies have revealed environmental contamination with nucleic acid from STI agents in clinical settings [7-9]. These studies incorporated experiments that were designed to indicate the potential for contaminants to be transferred to diagnostic specimens. However, the experiments were small scale or made use of artificially contaminated surfaces. Here, we report a multisite study (1) to determine the extent of STI nucleic acid contamination of toilets and bathrooms in different types of clinical facilities throughout the NT and (2) to directly test the potential of contaminating material to be transferred to sterile urine specimens from the environment by means of finger contact.

ultisite study (1) to determine the extent of STI nucleic acid contamination of toilets and bathrooms in different types of clinical facilities throughout the NT and (2) to directly test the potential of contaminating material to be transferred to sterile urine specimens from the environment by means of finger contact. METHODS Sampling Sites, Locations, and Schedule Ten clinics located in the NT were included in the study: the 2 Sexual Assault Referral Centre (SARC) clinical facilities in Darwin and Alice Springs, 2 sexual health clinics in Darwin and Alice Springs, 2 primary health clinics in other regional urban centres, and 4 primary health clinics in 4 widely separated remote Indigenous communities. The remote community locations included 2 in Arnhem Land in the far north of the NT and 2 in the south of the NT in central Australia. SARC is an NT Government medical and forensic specialist service responsible for the management of child and adult victims of sexual assault. At each clinic visit, sampling was performed in 1 male and 1 female toilet or bathroom regularly used by clients to supply urine specimens for diagnostic purposes. Each of the 10 clinics was visited for sampling 7 times, at intervals of 1–2 months. The clinical and managerial staff at each clinic were made aware of impending visits by the investigators, but they agreed not to alter their normal toilet and bathroom cleaning procedures.

pply urine specimens for diagnostic purposes. Each of the 10 clinics was visited for sampling 7 times, at intervals of 1–2 months. The clinical and managerial staff at each clinic were made aware of impending visits by the investigators, but they agreed not to alter their normal toilet and bathroom cleaning procedures. Assessment of Surface Contamination At each clinic visit, 10 surfaces within each of the male and female toilet or bathrooms were sampled by swabbing. These surfaces were as follows: door handle and lock on the side of the door facing into the room or cubicle being assessed; the inner surface of the door to the room or cubicle being tested; the left and right walls of the room or cubicle; the floor close to toilet pedestal; toilet flush buttons; toilet cistern or wall behind toilet; upper surface of toilet seat; toilet bowl and rim; shelf or wash basin edge closest to toilet; and wash basin taps. The surfaces were sampled by swabbing a ∼20 × 20 cm area where possible. For small areas such as taps, the entire surface was swabbed. Either sterile cotton swabs wetted with sterile water or APTIMA Vaginal Swab Specimen Collection kits (Gen-Probe) were used, depending on the analysis procedure (see below).

basin taps. The surfaces were sampled by swabbing a ∼20 × 20 cm area where possible. For small areas such as taps, the entire surface was swabbed. Either sterile cotton swabs wetted with sterile water or APTIMA Vaginal Swab Specimen Collection kits (Gen-Probe) were used, depending on the analysis procedure (see below). Simulation of Contamination Procedure of Sterile Urine Surrogate We endeavoured to replicate collection of urine samples by a patient or guardian. This procedure was performed in the same toilet or bathrooms as the swab sampling. The essential elements of the procedure were contact between the experimenter's gloved hands and all the surfaces that were subjected to swab sampling, and also with the insides of lids of urine collection jars, followed by transfer of 20 mL of a urine surrogate solution from a test tube into 2 urine collection jars (approximately 10 mL into each). To maximize the probability of contamination, all 10 surfaces were touched by the experimenter before each transfer of 20 mL of urine surrogate, and the transfer was performed 5 times at each visit to toilet or bathroom, without changing gloves. In this way, each instance of touching of all the clinic and bathroom surfaces resulted in two 10 mL urine surrogate samples. The two 10 mL aliquots arising from each transfer event were combined and then split into two 10 mL aliquots, because it was posited that these would yield duplicate samples that would assist subsequent statistical analysis. The detailed procedure is shown in Figure 1. The urine surrogate used was described by Martino et al [10] and contained the following: 0.65 g/L CaCl2; 0.65 g/L MgCl2; 4.6 g/L NaCl; 2.3 g/L Na2SO4; 0.65 g/L Na3-citrate; 0.02 g/L Na2-oxalate; 2.8 g/L KH2PO4; 1.6 g/L KCl; 1.0 g/L NH4Cl; 25.0 g/L urea; 1.1 g/L creatinine; and 5% Luria Bertani broth (v/v). The pH was adjusted to 5.8 with HCl, and the solution was sterilized by filtration through a 0.22 µm filter. The urine surrogate was stored at 4°C and used within 1 month. Figure 1. The method for simulation of collection of urine specimens using a urine surrogate solution.

creatinine; and 5% Luria Bertani broth (v/v). The pH was adjusted to 5.8 with HCl, and the solution was sterilized by filtration through a 0.22 µm filter. The urine surrogate was stored at 4°C and used within 1 month. Figure 1. The method for simulation of collection of urine specimens using a urine surrogate solution. Because gloves were not changed between the 5 replicates of the urine collection simulation performed in each analysis of a toilet or bathroom, it is reasonable to assume that if a glove becomes sufficiently contaminated to transfer material to a urine surrogate specimen, then there is an increased probability that subsequent specimens arising from that set of 5 transfers may be contaminated. To circumvent this potential confounder, all results from urine surrogate samples from a particular visit to toilet or bathroom that were generated subsequent to a sample that gave a positive result were omitted from analysis (eg, if a positive result was obtained from at least 1 of the duplicate samples from the 2nd of 5 replicate urine surrogate transfer, then the results arising from the 3rd, 4th, and 5th transfers would be discarded).

hroom that were generated subsequent to a sample that gave a positive result were omitted from analysis (eg, if a positive result was obtained from at least 1 of the duplicate samples from the 2nd of 5 replicate urine surrogate transfer, then the results arising from the 3rd, 4th, and 5th transfers would be discarded). Analysis of Samples Samples from each clinic were transported to, and analyzed for Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis nucleic acid by, the diagnostic service provider used for STI diagnosis services by that clinic. At the time of the study, the SARC and sexual health clinics used Royal Darwin Hospital Pathology, and the regional and remote clinics used Western Diagnostics for STI diagnostic services. Both of these organizations are accredited by the Australian National Association of Testing Authorities. Royal Darwin Hospital Pathology used the Versant CT/GC DNA 1 0 Assay (kPCR) (Siemens), and Western Diagnostics used the APTIMA Combo 2 Assay system (Gen-Probe). The reports were provided in the same format and with the same information as is routinely provided to the clinics for genuine diagnostic samples. Role of the Funding Source The study sponsors had no role in the study design, the collection, analysis or interpretation of data, the preparation of the manuscript, or the decision to submit the manuscript for publication.

Analysis of Samples Samples from each clinic were transported to, and analyzed for Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis nucleic acid by, the diagnostic service provider used for STI diagnosis services by that clinic. At the time of the study, the SARC and sexual health clinics used Royal Darwin Hospital Pathology, and the regional and remote clinics used Western Diagnostics for STI diagnostic services. Both of these organizations are accredited by the Australian National Association of Testing Authorities. Royal Darwin Hospital Pathology used the Versant CT/GC DNA 1 0 Assay (kPCR) (Siemens), and Western Diagnostics used the APTIMA Combo 2 Assay system (Gen-Probe). The reports were provided in the same format and with the same information as is routinely provided to the clinics for genuine diagnostic samples. Role of the Funding Source The study sponsors had no role in the study design, the collection, analysis or interpretation of data, the preparation of the manuscript, or the decision to submit the manuscript for publication. RESULTS Each of the 10 sites was visited 7 times, and a total of 1400 swab samples and 1400 urine surrogate samples were collected. All of the swab samples and 1398 of the urine surrogate samples were analyzed for C trachomatis and N gonorrhoeae. The 2 remaining urine surrogate samples were lost as a result of leakage during transit to the diagnostic service provider. One thousand three hundred and sixty of the swab samples and 1358 of the urine surrogate samples were analyzed for T vaginalis. Forty swab samples and 40 urine surrogate samples were inadvertently not analyzed for T vaginalis. An additional 7 test results were not received from service providers. In total, 4156 results were obtained from swabs and 4151 results were obtained from urine surrogate samples.

te samples were analyzed for T vaginalis. Forty swab samples and 40 urine surrogate samples were inadvertently not analyzed for T vaginalis. An additional 7 test results were not received from service providers. In total, 4156 results were obtained from swabs and 4151 results were obtained from urine surrogate samples. The results from the swab samples are shown in Table 1. Sexually transmitted infection agents were detected in all classes of clinics apart from the SARC clinics, which yielded no positive results. The clinics in the remote Indigenous communities were more contaminated than the sexual health and regional clinics. T vaginalis was the most common contaminant in the regional and remote Indigenous communities, whereas N gonorrhoeae was most common in the sexual health clinics. Table 1. Surface Contamination of Each Measured STI Agent At Each Clinic in Studya

nities were more contaminated than the sexual health and regional clinics. T vaginalis was the most common contaminant in the regional and remote Indigenous communities, whereas N gonorrhoeae was most common in the sexual health clinics. Table 1. Surface Contamination of Each Measured STI Agent At Each Clinic in Studya SARC Sexual Health Regional Remote Indigenous A B A B A B A B C D Chlamydia trachomatis 0 (0 of 139) 0 (0 of 140) 0 (0 of 140) 0.7 (1 of 140) 5.0 (7 of 140) 0 (0 of 140) 3.6 (5 of 140) 6.4 (9 of 140) 7.1 (10 of 140) 9.4 (13 of 139) Neisseria gonorrhoeae 0 (0 of 140) 0 (0 of 140) 6.4 (9 of 140) 5.0 (7 of 140) 0 (0 of 140) 0 (0 of 140) 0 (0 of 140) 2.1 (3 of 140) 25.0 (35 of 140) 36.7 (51 of 139) Trichomonas vaginalis 0 (0 of 140) 0 (0 of 140) 0.7 (1 of 140) 0.7 (1 of 140) 7.1 (10 of 140) 14.3 (20 of 140) 26.1 (31 of 119) 44.2 (53 of 120) 37.9 (53 of 140) 32.1 (45 of 140) Positive swabs (any STI agent) 0 (0 of 140) 0 (0 of 140) 7.1 (10 of 140) 6.4 (9 of 140) 10 (14 of 140) 14.3 (20 of 140) 24.3 (34 of 140) 40.7 (57 of 140) 50.7 (71 of 140) 57.1 (80 of 140) Abbreviations: SARC, Sexual Assault Referral Centre; STI, sexually transmitted infection. aClinics are divided into geographical categories. Data are expressed as a percentage. The fraction of the number of positive tests over total number of tests is stated in parentheses.

SARC Sexual Health Regional Remote Indigenous A B A B A B A B C D Chlamydia trachomatis 0 (0 of 139) 0 (0 of 140) 0 (0 of 140) 0.7 (1 of 140) 5.0 (7 of 140) 0 (0 of 140) 3.6 (5 of 140) 6.4 (9 of 140) 7.1 (10 of 140) 9.4 (13 of 139) Neisseria gonorrhoeae 0 (0 of 140) 0 (0 of 140) 6.4 (9 of 140) 5.0 (7 of 140) 0 (0 of 140) 0 (0 of 140) 0 (0 of 140) 2.1 (3 of 140) 25.0 (35 of 140) 36.7 (51 of 139) Trichomonas vaginalis 0 (0 of 140) 0 (0 of 140) 0.7 (1 of 140) 0.7 (1 of 140) 7.1 (10 of 140) 14.3 (20 of 140) 26.1 (31 of 119) 44.2 (53 of 120) 37.9 (53 of 140) 32.1 (45 of 140) Positive swabs (any STI agent) 0 (0 of 140) 0 (0 of 140) 7.1 (10 of 140) 6.4 (9 of 140) 10 (14 of 140) 14.3 (20 of 140) 24.3 (34 of 140) 40.7 (57 of 140) 50.7 (71 of 140) 57.1 (80 of 140) Abbreviations: SARC, Sexual Assault Referral Centre; STI, sexually transmitted infection. aClinics are divided into geographical categories. Data are expressed as a percentage. The fraction of the number of positive tests over total number of tests is stated in parentheses. The frequencies of contamination on the different surfaces are shown in Table 2. Contamination was found on all classes of surface. A comparison of the contamination of female and male toilets and bathrooms is shown in Table 3. The pooled results from the STI agents were not significantly different. However, the female toilet or bathrooms were more heavily contaminated with C trachomatis and T vaginalis, whereas the male toilet or bathrooms were more heavily contaminated with N gonorrhoeae. Table 2. Frequencies, Expressed As Percentages, of Contamination of Different Locations Within the Toilet or Bathrooms Included in the Study

male toilet or bathrooms were more heavily contaminated with C trachomatis and T vaginalis, whereas the male toilet or bathrooms were more heavily contaminated with N gonorrhoeae. Table 2. Frequencies, Expressed As Percentages, of Contamination of Different Locations Within the Toilet or Bathrooms Included in the Study DHL CD LRW TF TFB TC TS TB WBE WBT Chlamydia trachomatis 1.4 (2 of 140) 1.4 (2 of 140) 0.7 (1 of 140) 10.8 (15 of 139) 0.7 (1 of 140) 0 (0 of 140) 5.0 (7 of 139) 7.9 (11 of 140) 2.9 (4 of 140) 1.4 (2 of 140) Neisseria gonorrhoeae 7.9 (11 of 140) 4.3 (6 of 140) 11.4 (16 of 140) 17.1 (24 of 140) 7.9 (11 of 140) 3.6 (5 of 140) 7.2 (10 of 139) 7.1 (10 of 140) 7.1 (10 of 140) 1.4 (2 of 140) Trichomonas vaginalis 11.0 (15 of 136) 5.1 (7 of 136) 15.4 (21 of 136) 27.9 (38 of 136) 14.0 (19 of 136) 5.9 (8 of 136) 25.0 (34 of 136) 27.9 (38 of 136) 14.8 (20/135) 103 (14 of 136) Abbreviations: CD, cubicle door; DHL, door handle and lock; LRW, left and right walls; TB, toilet bowl; TC, toilet cistern; TF, toilet floor; TFB, toilet flush button; TS, toilet seat; WBE, wash basin edge; WBT, wash basin taps. Table 3. Frequencies, Expressed As Percentages, of Surface Contamination in Female and Male Bathroom and Toilets Female Male P Valuea Chlamydia trachomatis 4.6 (32 of 699) 1.9 (13 of 699) .004 Neisseria gonorrhoeae 3.6 (25 of 700) 11.4 (80 of 699) <.0001 Trichomonas vaginalis 20.9 (142 of 680) 10.6 (72 of 679) <.0001 aPearson's χ2 test.

Table 3. Frequencies, Expressed As Percentages, of Surface Contamination in Female and Male Bathroom and Toilets Female Male P Valuea Chlamydia trachomatis 4.6 (32 of 699) 1.9 (13 of 699) .004 Neisseria gonorrhoeae 3.6 (25 of 700) 11.4 (80 of 699) <.0001 Trichomonas vaginalis 20.9 (142 of 680) 10.6 (72 of 679) <.0001 aPearson's χ2 test. The results of the analyses of the urine surrogate samples are shown in Table 4. The frequency of urine surrogate contamination was much less than the frequency of swab contamination. Consistent with the swab results, no contaminated urine surrogate samples were obtained from the SARC clinics. Additional details regarding the contaminated urine surrogate samples are provided in Table S1. It is noteworthy that our attempt to generate duplicate samples by splitting then recombining the 20 mL samples or surrogate urine was unsuccessful, with instances where 1 of each pair of samples was positive (Table S1). As a result, each 10 mL sample was regarded as independent in subsequent analyses. The pooled results in the lowermost row of Table 4 were provided because a critical parameter is the probability of a sample being contaminated with any of the STIs for which it is tested, and it is usual practice to test urine specimens for more than 1 STI. Table 4. Numbers and Frequencies, Expressed As Percentages of Urine Surrogate Contaminationa

most row of Table 4 were provided because a critical parameter is the probability of a sample being contaminated with any of the STIs for which it is tested, and it is usual practice to test urine specimens for more than 1 STI. Table 4. Numbers and Frequencies, Expressed As Percentages of Urine Surrogate Contaminationa SARC Sexual Health Regional Remote Indigenous Total (95% CI) A B A B A B A B C D Chlamydia trachomatis 0 (0 of 140) 0 (0 of 138) 0 (0 of 139) 0 (0 of 140) 0.7 (1 of 137) 0 (0 of 139) 0 (0 of 140) 0.7 (1 of 131) 0 (0 of 140) 0 (0 of 140) 0.14 (0.02–0.57) (2 of 1384) Neisseria gonorrhoeae 0 (0 of 140) 0 (0 of 139) 1.4 (2 of 131) 0 (0 of 140) 0 (0 of 140) 0 (0 of 139) 0 (0 of 140) 0 (0 of 140) 0 (0 of 140) 0 (0 of 140) 0.14 (0.02–0.57) (2 of 1389) Trichomonas vaginalis 0 (0 of 140) 0 (0 of 139) 0 (0 of 139) 0 (0 of 140) 0 (0 of 140) 0 (0 of 139) 0 (0 of 120) 2.1 (3 of 101) 0 (0 of 140) 0 (0 of 140) 0.22 (0.06–0.71) (3 of 1338) Positive for any STI agent (95% CI) 0 (0–1.3) (0 of 279) 0.72 (0.1–2.6) (2 of 279) 0.36 (0–2.0) (1 of 279) 0.72 (0.2–1.8) (4 of 558) Abbreviations: CI, confidence interval; SARC, Sexual Assault Referral Centre; STI, sexually transmitted infection. a The 95% CI figures were calculated using the Exact Binomial method.

SARC Sexual Health Regional Remote Indigenous Total (95% CI) A B A B A B A B C D Chlamydia trachomatis 0 (0 of 140) 0 (0 of 138) 0 (0 of 139) 0 (0 of 140) 0.7 (1 of 137) 0 (0 of 139) 0 (0 of 140) 0.7 (1 of 131) 0 (0 of 140) 0 (0 of 140) 0.14 (0.02–0.57) (2 of 1384) Neisseria gonorrhoeae 0 (0 of 140) 0 (0 of 139) 1.4 (2 of 131) 0 (0 of 140) 0 (0 of 140) 0 (0 of 139) 0 (0 of 140) 0 (0 of 140) 0 (0 of 140) 0 (0 of 140) 0.14 (0.02–0.57) (2 of 1389) Trichomonas vaginalis 0 (0 of 140) 0 (0 of 139) 0 (0 of 139) 0 (0 of 140) 0 (0 of 140) 0 (0 of 139) 0 (0 of 120) 2.1 (3 of 101) 0 (0 of 140) 0 (0 of 140) 0.22 (0.06–0.71) (3 of 1338) Positive for any STI agent (95% CI) 0 (0–1.3) (0 of 279) 0.72 (0.1–2.6) (2 of 279) 0.36 (0–2.0) (1 of 279) 0.72 (0.2–1.8) (4 of 558) Abbreviations: CI, confidence interval; SARC, Sexual Assault Referral Centre; STI, sexually transmitted infection. a The 95% CI figures were calculated using the Exact Binomial method. The frequencies obtained are our best estimate but are subject to sampling error. Even though the samples are not entirely independent, we contend that confidence intervals (CIs) calculated assuming independence will provide useful guidance regarding extremely conservative limits for the frequencies of false positives by this mechanism. Upper boundaries for the 95% CIs for the proportions of urine surrogate samples that caused positive tests, calculated using the Exact Method, are 2.6%, 2.0%, and 1.8% for the sexual health, regional, and remote clinics, respectively. The remote clinic figure (2.0%) is probably the most useful because it is derived from clinics with the highest proportion of contaminated urine samples and the largest number of samples. Another useful indicator may be the upper boundary of the frequencies of C trachomatis + N gonorrhoeae positive urine surrogate samples, if T vaginalis is regarded as a less strong indicator of sexual contact than C trachomatis or N gonorrhoeae. The upper boundary of the 95% CI for the proportion of remote clinic urine surrogate samples that are positive for either of these 2 agents (1 of 558) is 1.2%.

s + N gonorrhoeae positive urine surrogate samples, if T vaginalis is regarded as a less strong indicator of sexual contact than C trachomatis or N gonorrhoeae. The upper boundary of the 95% CI for the proportion of remote clinic urine surrogate samples that are positive for either of these 2 agents (1 of 558) is 1.2%. DISCUSSION To our knowledge, this is the largest study to address contamination of surfaces within clinic bathroom and toilet facilities with nucleic acid from STI agents, and it is the only study to encompass multiple visits to multiple clinics. Overall, the degree of environmental contamination in our study is slightly lower than that found in other studies. Meader et al [9] detected C trachomatis from 41 of 104 (39%) swabs (range, 0%–88%) from surfaces such as toilets, curtains, and draining boards within an English genitourinary clinic. Lewis et al [8] sampled similar surfaces at a similar clinic and found that 20 of 154 (13%) of surfaces tested positive for either C trachomatis or N gonorrhoeae. In our study, the highest proportion of C trachomatis-positive swabs was 9.4%, which is much lower than the proportions we sometimes saw for N gonorrhoeae and T vaginalis. The lower contamination rates with C trachomatis in the NT remote clinics compared with sexual health clinics in England may reflect that these remote clinics are general healthcare and not sexual health clinics. However, rates of contamination in NT sexual health clinics were substantially lower still. Whereas the proportion of surface contamination by the individual STI agents is largely consistent with published prevalence of the STI agents in the NT [5], the rate of surface contamination with C trachomatis was lower than expected, possibly due to differences in shedding and environmental stability of the different STI agents. The STI environmental survival and nucleic acid environmental persistence may be an interesting area of study in the hot and often humid NT. Gender distributions of the STI agents were consistent with the higher prevalence of trichomoniasis in women than in men in the NT [5], but there is little difference between the prevalence of C trachomatis and N gonorrhoeae in the NT (5). Although the high frequency of contamination of the floor and the toilet bowl was unsurprising, it is noteworthy that surfaces such as the door handle, toilet flush button, and wash basin taps, which are in elevated positions and frequently touched by hands, also showed appreciable contamination.

rrhoeae in the NT (5). Although the high frequency of contamination of the floor and the toilet bowl was unsurprising, it is noteworthy that surfaces such as the door handle, toilet flush button, and wash basin taps, which are in elevated positions and frequently touched by hands, also showed appreciable contamination. This study is also the only one to include a large-scale assessment of the potential of environmental contaminants to be transferred to urine specimens obtained within a variety of facilities. The procedure was designed to provide a greater probability of contamination than would be expected in reality. By incorporating extensive contact between gloved hands and the built environment and deliberate contact with between gloved hands and the inside of the urine jar lids with every tested specimen, we have mimicked what may be regarded as a worst-case scenario for specimen contamination. The rationale was that the results would provide a conservative (ie, high) upper boundary for the probability of sample contamination. This result, in combination with the use of the Exact Method for calculating 95% CI for the proportions, has yielded upper boundaries that are conservative in the extreme.

ontamination. The rationale was that the results would provide a conservative (ie, high) upper boundary for the probability of sample contamination. This result, in combination with the use of the Exact Method for calculating 95% CI for the proportions, has yielded upper boundaries that are conservative in the extreme. We feel justified in stating that the probability that a urine sample from the 4 remote clinics in this study will be contaminated with C trachomatis, N gonorrhoeae, or T vaginalis of toilet or bathroom environmental origin cannot reasonably exceed 2.0%, and it is probably much less. Likewise, the probability of contamination with C trachomatis or N gonorrhoea cannot reasonably exceed 1.2% and is also probably much less. If false positives do occur, even at a low rate, this result has considerable implications when the pretest probability of a true positive is low. For example, using the point estimate urine surrogate contamination rate of 0.7% for the remote clinics (Table 4), and assuming the combined STI testing has a sensitivity of 1, the positive likelihood ratio for the test is 143. Hypothetically, if the pretest probability of a true STI is 0.01, the posttest probability of a true STI is just 0.59. This hypothesis emphasizes the potential peril of broad community-based testing of children for STIs as a screen for young children who have been subjected to sexual contact. Our study provides no evidence to suggest that broad screening is advisable.

ue STI is 0.01, the posttest probability of a true STI is just 0.59. This hypothesis emphasizes the potential peril of broad community-based testing of children for STIs as a screen for young children who have been subjected to sexual contact. Our study provides no evidence to suggest that broad screening is advisable. This study may be used to estimate the relationship between the extent of toilet or bathroom contamination, as measured by swab sampling, and the probability that a urine specimen will be contaminated. In previous studies, researchers had attempted such estimations to a limited extent. Meader et al [9] demonstrated that a hand that had touched an artificially heavily contaminated wet surface could potentially transfer C trachomatis nucleic acid to a swab. Lewis et al [8] tested the outer surfaces of the caps of 46 sample containers and found that none were contaminated. Chan et al [7] exposed to the air 60 samples of distilled water and 10 unused swabs while the toilet in a sexual health clinic was flushed, and they found none of the samples to be positive for C trachomatis. It is difficult to argue that these experiments provided useful numerical data. Using data from the remote clinics, the ratio between the proportion of swabs positive and the proportion of urine specimens positive is 0.017 (95% CI, .006–.045). Thus, urine surrogate specimens were ∼60.3 (95% CI, 22–167) times less likely to be positive compared with environmental swabs from the same toilet or bathroom. Continued surveillance using standardized procedures would result in refinement of this relationship. This finding may provide a widely applicable means to extrapolate from surface contamination to a “worst case” urine sample contamination probability.

compared with environmental swabs from the same toilet or bathroom. Continued surveillance using standardized procedures would result in refinement of this relationship. This finding may provide a widely applicable means to extrapolate from surface contamination to a “worst case” urine sample contamination probability. A limitation of this study is that gloved hands instead of bare skin were used in the simulation of urine specimen collection. This process was done in the interests of the health and safety of the experimenters. However, there are recent reports that gloves are effective at transferring microorganisms, suggesting that our experiment does provide information relevant to the potential of bare skin to transfer environmental contaminants [11, 12]. Another limitation was that different diagnostic service providers using different STI diagnosis systems were used for different clinics. This result has the potential to confound comparison between clinics. However, the desirability of using the diagnostic service suppliers used for genuine samples from the clinics in this study outweighed other considerations. Large differences between clinics using the same providers were observed, making it very unlikely that differences between clinics were a function of the diagnostic service provider.

bility of using the diagnostic service suppliers used for genuine samples from the clinics in this study outweighed other considerations. Large differences between clinics using the same providers were observed, making it very unlikely that differences between clinics were a function of the diagnostic service provider. We can suggest a number of ways to reduce the probability of contamination of urine samples. First, cleaning procedures affect environmental contamination. The SARC clinics are treated daily with bleach to remove environmental nucleic acid, and we found no evidence of environmental or urine surrogate contamination in these clinics. Second, patients and caregivers can be instructed to minimize touching of environmental surfaces when obtaining samples. Gloves could be worn to circumvent the effects of preexisting STI agent contamination on the fingers. Third, if it were particularly important to reduce the risk of the contribution of this mode of contamination to false positives, such as when there was an indication to test a child for STIs, trained clinic staff should obtain the sample and duplicate samples could be taken, with different gloves worn for each collection. This process would also eliminate any consequences of preexisting contamination of a parent's or guardian's fingers.

all 4 groups, as shown by calculated GMC ratios in Table 4. Table 4. Geometric Mean Concentrations (GMCs) for Varicella-Zoster Virus (VZV) Antibodies, and Baseline-Adjusted GMCs and GMC Ratios for Antibodies to Hepatitis A Virus and PCV7 Pneumococcal Serotypes at Day 42 (According-to-Protocol Cohort for Immunogenicity) Antibody MMRII MMR-RIT-1 MMR-RIT-2 MMR-RIT-3 N GMC (95% CI) N GMC (95% CI) N GMC (95% CI) N GMC (95% CI) VZV 246 256 (240; 272) 245 246 (229; 263) 238 235 (217; 254) 240 256 (240; 272) MMRII MMR-RIT-1 MMR-RIT-2 MMR-RIT-3 N Adjusted GMC N Adjusted GMC Adjusted GMC ratioa (95% CI) N Adjusted GMC Adjusted GMC ratioa (95% CI) N Adjusted GMC Adjusted GMC ratioa (95% CI) Hepatitis A virus 124 42.0 117 33.9 0.81 (0.64; 1.02) 112 39.2 0.93 (0.74; 1.18) 111 39.5 0.94 (0.74; 1.19) S.PNEU-4 116 3.68 122 3.69 1.00 (0.82; 1.24) 125 3.78 1.03 (0.84; 1.26) 124 3.26 0.89 (0.72; 1.09) S.PNEU-6B 111 6.50 117 5.86 0.90 (0.75; 1.09) 122 5.87 0.90 (0.75; 1.09) 123 5.81 0.89 (0.74; 1.07) S.PNEU-9V 120 7.32 121 6.65 0.91 (0.76; 1.08) 125 7.23 0.99 (0.83; 1.17) 127 5.80 0.79 (0.64; 0.94) S.PNEU-14 118 7.87 127 8.91 1.13 (0.95; 1.35) 127 8.29 1.05 (0.88; 1.26) 126 7.89 1.00 (0.84; 1.20) S.PNEU-18C 119 6.58 123 6.29 0.96 (0.79; 1.15) 126 6.62 1.01 (0.84; 1.21) 126 5.98 0.91 (0.76; 1.09) S.PNEU-19F 115 2.39 122 2.41 1.01 (0.83; 1.22) 126 2.52 1.05 (0.87; 1.27) 126 2.33 0.97 (0.80; 1.18) S.PNEU-23F 113 10.21 121 9.56 0.94 (0.76; 1.16) 127 9.69 0.95 (0.77; 1.17) 126 8.37 0.82 (0.67; 1.01) Abbreviations: Adjusted GMC, geometric mean antibody concentration adjusted for baseline antibody concentration; MMR, measles-mumps-rubella; N, Number of subjects with both pre- and post-vaccination results available; 95% CI, 95% confidence interval for the adjusted GMC ratio (ANCOVA model: adjustment for baseline concentration - pooled variance with more than 2 groups); S.PNEU, Streptococcus pneumoniae.

sitives, such as when there was an indication to test a child for STIs, trained clinic staff should obtain the sample and duplicate samples could be taken, with different gloves worn for each collection. This process would also eliminate any consequences of preexisting contamination of a parent's or guardian's fingers. In conclusion, we have performed a multisite investigation of the potential of environmental contaminants in NT clinic bathroom or toilets to be transferred to urine specimens and deduced very conservative upper boundaries for the probabilities of such events. Although the impetus for this investigation was child protection related, the results are of general relevance to STI diagnosis. Acknowledgments We thank Barbara Kelly (Northern Territory Government Sexual Assault Referral Centre [Darwin]), and Michael Leung (Western Diagnostics, Western Australia); and Pathwest, (West Australia Government) for facilitating the early stages of this project. We also thank Nathan Ryder (Northern Territory Government Centre for Disease Control) and Terry Donald (Women's and Children's Hospital, Adelaide, South Australia) for critical review of the manuscript prior to submission. Finally, we thank the management and staff of the participating clinics for their participation and assistance. The authors thank Mark Chatfield (Menzies School of Health Research) for statistics advice.

men's and Children's Hospital, Adelaide, South Australia) for critical review of the manuscript prior to submission. Finally, we thank the management and staff of the participating clinics for their participation and assistance. The authors thank Mark Chatfield (Menzies School of Health Research) for statistics advice. Financial support. This work was supported by the National Health and Medical Research Council (project grant 1004123; and postdoctoral training fellowship 508829 to S. Y. C. T.); and the Northern Territory (Australia) Research and Innovation Board (Research and Innovation Grant “Urine surrogates in the quality control of Chlamydia diagnosis”). Potential conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. The standard license has been updated to Open Access since the original version.

INTRODUCTION Despite the successful introduction of routine immunization with combined live attenuated measles-mumps-rubella (MMR) vaccines in the 1970s [1], outbreaks and generally increased prevalence of mumps and measles are still noted among the vaccinated and unvaccinated populations, respectively, across the United States [2–4]. Maintenance of high vaccine coverage rates remains an essential component of efforts to control these diseases. A 2-dose MMR vaccination schedule is recommended in the United States. Children receive dose-1 at 12–15 months concomitantly with other recommended vaccines, including hepatitis A vaccine (HAV), varicella vaccine (VAR), and pneumococcal conjugate vaccine (PCV) [5–8]. MMR dose-2 is administered at age 4–6 years to induce immune responses in those who fail to respond to the initial dose. Two-dose catch-up schedules, with a minimum 4-week interval between MMR doses, are recommended for children and adolescents who miss the first dose [9]. As Merck's M-M-RTMII (MMRII), a human serum albumin-free vaccine, is currently the only MMR vaccine licensed in the United States, any interruption in its availability would pose a critical public health risk. Therefore, GlaxoSmithKline Vaccines is currently evaluating its trivalent MMR vaccine PriorixTM (MMR-RIT) for use in the United States. MMR-RIT is routinely given in over 100 countries from the second year of life onwards [10]. The formulation of MMR-RIT used in this study does not contain human serum albumin, thereby minimizing any theoretical risk of microbial contamination as compared to previous formulations [11]. This formulation is also consistent with the recommendation from the European Medicines Agency to eliminate the use of blood-derived products of human origin [12, 13].

tudy does not contain human serum albumin, thereby minimizing any theoretical risk of microbial contamination as compared to previous formulations [11]. This formulation is also consistent with the recommendation from the European Medicines Agency to eliminate the use of blood-derived products of human origin [12, 13]. This Phase-2 exploratory study assessed immunologic responses to 3 lots of MMR-RIT (containing a range of mumps virus titers) and to MMRII used as a first dose in 12–15-month-old children in the United States. The study was used as a preliminary evaluation of the minimum effective mumps virus titer for the candidate vaccine to allow planning of a Phase-3 study, and was used also to generate preliminary data on the safety and immunogenicity of co-administration of MMR-RIT with routine childhood vaccines: VAR, HAV, and 7-valent PCV (PCV7).

used as a preliminary evaluation of the minimum effective mumps virus titer for the candidate vaccine to allow planning of a Phase-3 study, and was used also to generate preliminary data on the safety and immunogenicity of co-administration of MMR-RIT with routine childhood vaccines: VAR, HAV, and 7-valent PCV (PCV7). METHODS This randomized, observer-blind, Phase-2 study was conducted at 51 centers in the Unites States in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. The study was approved by a national, regional, or investigational center institutional review board or independent ethics committee. Written informed consent was obtained from parents/guardians before enrollment. The study consisted of an active phase (immediate post-vaccination interval; Days 0–42), an extended safety follow-up phase (Days 43–180), and an antibody persistence phase ending approximately 2 years post-vaccination. Only the results of a planned analysis conducted for immunogenicity and safety data from the active phase are reported here.

n active phase (immediate post-vaccination interval; Days 0–42), an extended safety follow-up phase (Days 43–180), and an antibody persistence phase ending approximately 2 years post-vaccination. Only the results of a planned analysis conducted for immunogenicity and safety data from the active phase are reported here. Eligible healthy 12- to 15-month-old male and female subjects had not been previously immunized against (and had no previous history of) measles, mumps, rubella, varicella, and hepatitis A, and had received 3 doses of PCV7 within the first year of life (third dose administered ≥30 days before enrollment). Other key exclusion criteria included: exposure to measles, mumps, rubella, or varicella ≤30 days before study start; previous (≤30 days before study start) or planned administration of investigational products during the study; administration of other vaccines (except influenza and Haemophilus influenzae type b) ≤30 days before study vaccination until Day 42; chronic immunosuppressants/immune-modifying drugs, polyclonal immunoglobulins, or blood products received ≤6 months before study vaccination; immunosuppressive or immunodeficient conditions; contraindication to vaccination; a history of neurologic disorders or seizures; acute disease at enrollment; and severe chronic illness or major congenital defects.

odifying drugs, polyclonal immunoglobulins, or blood products received ≤6 months before study vaccination; immunosuppressive or immunodeficient conditions; contraindication to vaccination; a history of neurologic disorders or seizures; acute disease at enrollment; and severe chronic illness or major congenital defects. Subjects visited the study site at Days 0, 42, 180, 365, and 730. At Day 0 (Visit 1), subjects were randomized using a blocking scheme (3:3:3:[1:1:1] ratio) to 1 of 4 parallel treatment groups: 3 groups received a single dose of 1 of 3 MMR-RIT lots (containing either high [MMR-RIT-1], medium [MMR-RIT-2], or low [MMR-RIT-3] RIT 4385 mumps strain titers); the fourth group received a single dose of 1 of 3 commercial lots of MMRII (Merck & Co Inc. [14]) (Table 1). The randomization list was generated at GlaxoSmithKline Biologicals using SAS® software. Treatment allocation was performed at the investigator site via a central internet-based randomization system. Subjects concomitantly received a single dose each of HAV (HavrixTM, GlaxoSmithKline Vaccines [15]), VAR (VarivaxTM, Merck & Co. Inc. [16]), and PCV7 (PrevnarTM, Wyeth [17]) at Day 0. Immune response against measles, mumps, and rubella viruses was assessed at Day 42 (Visit 2). MMR-RIT or MMRII were administered subcutaneously into the upper right arm, VAR subcutaneously into the upper left arm, and HAV and PCV7 intramuscularly into the left and right thighs, respectively. A second HAV dose was administered at Day 180 (Visit 3). Vaccine recipients, parents/guardians, and those responsible for evaluation of study endpoints were blinded to study treatment. Vaccine reception, storage, preparation, reconstitution, and administration were performed by study personnel who did not participate in outcome evaluation. Table 1. Formulation of 3 Lots of Candidate MMR-RIT (Measles, Mumps, Rubella) Vaccine and Commercially Available Comparator Vaccine (MMRII, Merck & Co., Inc.)

ent. Vaccine reception, storage, preparation, reconstitution, and administration were performed by study personnel who did not participate in outcome evaluation. Table 1. Formulation of 3 Lots of Candidate MMR-RIT (Measles, Mumps, Rubella) Vaccine and Commercially Available Comparator Vaccine (MMRII, Merck & Co., Inc.) Vaccine Lot Number(s) Log10 CCID50 Measlesa Mumpsb Rubellac MMRII 1291X 4.0 4.8 4.2 1255X 3.9 4.8 4.0 1362X 3.8 4.8 4.1 MMR-RIT-1 AMJRB721A 3.8 4.8 (high) 3.9 MMR-RIT-2 DMJRA002A 4.1 4.1 (medium) 3.9 MMR-RIT-3 DMJRA003A 4.0 3.7 (low) 4.1 Abbreviation: CCID50, Median cell culture infective dose. Note: All CCID50 values for vaccine components of MMRII and MMR-RIT were determined by GlaxoSmithKline. aMeasles was Schwarz strain for GlaxoSmithKline vaccines and Moraten Edmonston-Enders strain for MMRII. bMumps was RIT 4385 strain for GlaxoSmithKline vaccines and Jeryl Lynn for MMRII. cRubella strain was the same for MMRII and each MMR-RIT (ie, Wistar RA 27/3).

Note: All CCID50 values for vaccine components of MMRII and MMR-RIT were determined by GlaxoSmithKline. aMeasles was Schwarz strain for GlaxoSmithKline vaccines and Moraten Edmonston-Enders strain for MMRII. bMumps was RIT 4385 strain for GlaxoSmithKline vaccines and Jeryl Lynn for MMRII. cRubella strain was the same for MMRII and each MMR-RIT (ie, Wistar RA 27/3). Immunogenicity Blood for antibody determination was obtained from subjects on Days 0 (pre-immunization), 42, 365, and 730. Analysis of blood obtained at Days 365 and 730 for evaluation of antibody persistence is ongoing and will be reported separately. Sera were stored at -20°C until assayed in a blinded manner at a central laboratory (GlaxoSmithKline Biologicals, Rixensart, Belgium). Mumps virus antibody response was determined using an in-house plaque-reduction assay (GlaxoSmithKline Biologicals [18, 19]) via neutralization of wild-type virus (Mu90LO1) in the presence of complement and anti-human globulin. Immunoglobulin (Ig) G antibodies to measles, rubella, and varicella-zoster virus (VZV) were measured with commercial enzyme-linked immunosorbent assay (ELISA) (Enzygnost, Dade Behring, Marburg GmbH, Germany); antibodies to hepatitis A virus were determined in a randomized subset of 50% of subjects. Antibodies to PCV7 pneumococcal serotypes were measured in the remaining 50% with an in-house ELISA (GlaxoSmithKline Biologicals [20]). Assay seronegativity cut-off values for antibodies to vaccine viral antigens were: measles <150 mIU/mL, mumps <24 ED50, rubella <4 IU/mL, VZV <25 mIU/mL, hepatitis A <15 mIU/mL, and Streptococcus pneumoniae <0.05 µg/mL. The seronegativity cut-offs evaluated in this study were determined empirically as part of the assay validation protocol and were accepted by the US Food and Drug Administration (FDA). Post-vaccination seroresponses for MMR vaccine viral antigens in initially seronegative subjects were defined as antibody concentrations/titers of: measles ≥200 mIU/mL [21]; mumps ≥51 ED50 (no known correlate of protection threshold) and rubella ≥10 IU/mL [22]. The seroresponse thresholds were accepted by the FDA as those defining active immunization offering clinical benefit.

ns in initially seronegative subjects were defined as antibody concentrations/titers of: measles ≥200 mIU/mL [21]; mumps ≥51 ED50 (no known correlate of protection threshold) and rubella ≥10 IU/mL [22]. The seroresponse thresholds were accepted by the FDA as those defining active immunization offering clinical benefit. VAR response was defined as a post-vaccination antibody concentration ≥75 mIU/mL in initially seronegative subjects. HAV response was defined as a post-vaccination antibody concentration ≥15 mIU/mL in initially seronegative subjects, or a ≥2-fold increase in the pre-vaccination antibody concentration in initially seropositive subjects. Reactogenicity and Safety Reactogenicity and safety were assessed at each visit and via subject diary cards completed by parents/guardians. Solicited injection site symptoms (pain, redness, swelling for study vaccines only) were recorded from Days 0–3. Solicited general symptoms (fever, rash, parotid/salivary gland swelling, febrile convulsions, irritability/fussiness, drowsiness, and loss of appetite), and unsolicited symptoms were recorded from Days 0–42. Serious adverse events (SAEs) were recorded throughout the study. Fever was assessed daily with a tympanic thermometer or rectally if the tympanic reading indicated fever (≥38.0°C). For each reported symptom, parents/guardians were asked what medical attention (if any) the subject had received.

recorded from Days 0–42. Serious adverse events (SAEs) were recorded throughout the study. Fever was assessed daily with a tympanic thermometer or rectally if the tympanic reading indicated fever (≥38.0°C). For each reported symptom, parents/guardians were asked what medical attention (if any) the subject had received. Statistics This was a “hypothesis-generating” exploratory study conducted to provide estimations of response rates, which will be used to develop statistical criteria for a formal Phase-3 trial to support licensure of the candidate vaccine on the basis of non-inferior immunogenicity compared to the licensed comparator. All analyses in this study were descriptive, and no formal statistical comparison was prespecified. Enrollment of 1200 subjects (300/group) was planned to ensure ≥240 evaluable subjects/group. Subjects in the MMRII group were randomized across 3 commercial MMRII lots; no lot-by-lot analysis was done and results were pooled. The primary analysis of immunogenicity was conducted on the according-to-protocol (ATP) cohort for immunogenicity, which included eligible subjects who had received the study vaccine via the correct administration route and complied with study procedures, and who were below the assay cut-off for at least 1 MMR vaccine antigen at baseline, with pre-vaccination and post-vaccination serology results available. Safety analysis was performed on the total vaccinated cohort (TVC), which included all vaccinated subjects.

administration route and complied with study procedures, and who were below the assay cut-off for at least 1 MMR vaccine antigen at baseline, with pre-vaccination and post-vaccination serology results available. Safety analysis was performed on the total vaccinated cohort (TVC), which included all vaccinated subjects. The primary endpoint was seroresponse rates for antibodies to measles, mumps, and rubella viruses at Day 42; the proportions of subjects with antibody concentration/titer at or above specified assay cut-offs were calculated with exact 95% confidence intervals (CIs) both pre- and post-vaccination. Secondary endpoints included pre- and post-vaccination (Day 42) antibody concentration/titers, summarized by geometric mean concentrations/titers (GMC/Ts) with 95% CI. Exploratory analyses included standardized asymptotic 2-sided 95% CIs calculated for group differences (MMR-RIT group minus MMRII) in Day-42 seroresponse rates for antibodies to MMR viruses. In addition, 95% CIs for GMC ratios (MMR-RIT:MMRII) for antibodies to hepatitis A virus and PCV7 pneumococcal serotypes were obtained using an analysis of covariance model on the logarithm10-transformed Day-42 concentrations. For the safety analysis, the number and percentage of subjects reporting a symptom were calculated with exact 95% CIs. Symptoms were categorized according to intensity and relationship to study vaccine. All data processing and analyses were performed using SAS® version 9.2 (SAS Institute Inc., Cary, NC). Proc StatXact 8.1 derived exact 95% CIs for a proportion within a group as well as standardized asymptotic 95% CI for the group difference in proportions.

ized according to intensity and relationship to study vaccine. All data processing and analyses were performed using SAS® version 9.2 (SAS Institute Inc., Cary, NC). Proc StatXact 8.1 derived exact 95% CIs for a proportion within a group as well as standardized asymptotic 95% CI for the group difference in proportions. RESULTS Subject Disposition and Baseline Demography The first subject was enrolled on June 3, 2009, and the last visit of the active (43-day) phase was completed on July 21, 2010. Of 1259 enrolled subjects, 1224 were randomized and 4 did not receive study vaccine. The TVC consisted of 1220 subjects: MMR-RIT-1 (n = 304), MMR-RIT-2 (n = 304), MMR-RIT-3 (n = 304), and MMRII (n = 308). Of these, 1117 completed Day 42 and 103 were withdrawn (Figure 1). Figure 1. Disposition of subjects in the total vaccinated cohort (TVC) (enrolled = 1259 subjects, randomized = 1224 subjects, vaccinated = 1220 subjects). Abbreviations: MMR, measles-mumps-rubella; SAE, serious adverse event; ATP, according-to-protocol.

Of these, 1117 completed Day 42 and 103 were withdrawn (Figure 1). Figure 1. Disposition of subjects in the total vaccinated cohort (TVC) (enrolled = 1259 subjects, randomized = 1224 subjects, vaccinated = 1220 subjects). Abbreviations: MMR, measles-mumps-rubella; SAE, serious adverse event; ATP, according-to-protocol. Overall, of 1220 subjects in the TVC, 1026 subjects were included in the ATP-immunogenicity cohort (194 subjects were excluded): MMR-RIT-1 (n = 261); MMR-RIT-2 (n = 254); MMR-RIT-3 (n = 251), and MMRII (n = 260). Demographic characteristics of the 4 treatment groups were comparable between the TVC and ATP-immunogenicity cohorts. Mean (standard deviation) age in the TVC was 12.3 (0.71) months, 75.8% of subjects were white, and 51.1% were male. In the ATP-immunogenicity cohort, 100%, 86.2%, and 99.8%, of subjects were seronegative for measles, mumps and rubella antibody, respectively, before study vaccination; overall baseline seronegativity rates were comparable across the 4 groups (Table 2). Table 2. Demographic and Baseline Data for the Total Vaccinated Cohort (TVC) and According-to-Protocol (ATP) Cohort for Immunogenicity

e antibody concentration; MMR, measles-mumps-rubella; N, Number of subjects with both pre- and post-vaccination results available; 95% CI, 95% confidence interval for the adjusted GMC ratio (ANCOVA model: adjustment for baseline concentration - pooled variance with more than 2 groups); S.PNEU, Streptococcus pneumoniae. aRatio of MMR-RIT lot: MMRII. Safety and Reactogenicity Overall incidence of solicited and unsolicited symptoms in the TVC (Days-0–42) was 80.9% (95% CI: 76.0; 85.2) for MMR-RIT-1, 75.7% (70.4; 80.4) for MMR-RIT-2, 74.0% (68.7; 78.9) for MMR-RIT-3, and 75.3% (70.1; 80.0) for MMRII. The most frequently observed solicited symptom (Days 0–3) at MMR-RIT and MMRII injection sites was pain, reported in ≥24.5% of subjects in each group (Table 5), although Grade-3 pain (subject cried when the limb was moved/spontaneously painful) was reported in ≤1.5%. Two subjects (1 each in the MMR-RIT-2 and MMRII groups) had Grade-3 injection site swelling (diameter >20 mm). Table 5. Incidence of Solicited Injection Site (Days 0; 3) and General Symptoms During the 43-Day Post-vaccination Period (Total Vaccinated Cohort)

s were seronegative for measles, mumps and rubella antibody, respectively, before study vaccination; overall baseline seronegativity rates were comparable across the 4 groups (Table 2). Table 2. Demographic and Baseline Data for the Total Vaccinated Cohort (TVC) and According-to-Protocol (ATP) Cohort for Immunogenicity MMR-RIT-1 MMR-RIT-2 MMR-RIT-3 MMRII Total TVC N = 304 ATP N = 261 TVC N = 304 ATP N = 254 TVC N = 304 ATP N = 251 TVC N = 308 ATP N = 260 TVC N = 1220 ATP N = 1026 Age (mo), mean (SD) 12.4 (0.75) 12.4 (0.69) 12.4 (0.73) 12.4 (0.73) 12.2 (0.56) 12.2 (0.57) 12.4 (0.75) 12.4 (0.73) 12.3 (0.71) 12.3 (0.69) Gender Female, n (%) 156 (51.3) 134 (51.3) 144 (47.4) 120 (47.2) 157 (51.6) 128 (51.0) 139 (45.1) 118 (45.4) 596 (48.9) 500 (48.7) Male, n (%) 148 (48.7) 127 (48.7) 160 (52.6) 134 (52.8) 147 (48.4) 123 (49.0) 169 (54.9) 142 (54.6) 624 (51.1) 526 (51.3) Pre-vaccination status of ATP cohort for immunogenicity, no. of seronegative subjects (%)a Antibody (cut-off point) MMR-RIT-1 N = 261b MMR-RIT-2 N = 254b MMR-RIT-3 N = 251b MMRII N = 260b Total N = 1026b Measles (<150 mIU/mL) 259/259 (100) 253/253 (100) 249/249 (100) 258/258 (100) 1019/1019 (100) Mumps (ED50 <24) 216/252 (85.7) 219/248 (88.3) 213/246 (86.6) 213/243 (86.6) 861/999 (86.2) Rubella (<4 IU/mL) 259/259 (100) 251/252 (99.6) 248/249 (99.6) 258/258 (100) 1016/1018 (99.8) Abbreviations: MMR, measles-mumps-rubella; N, total number of subjects; SD, standard deviation.

49 (100) 258/258 (100) 1019/1019 (100) Mumps (ED50 <24) 216/252 (85.7) 219/248 (88.3) 213/246 (86.6) 213/243 (86.6) 861/999 (86.2) Rubella (<4 IU/mL) 259/259 (100) 251/252 (99.6) 248/249 (99.6) 258/258 (100) 1016/1018 (99.8) Abbreviations: MMR, measles-mumps-rubella; N, total number of subjects; SD, standard deviation. aCalculated for subjects in TVC for which pre-vaccination status was known (ie, % = n/[n + seropositive] × 100, where n = no. of seronegative subjects). bNumber of subjects regardless of the status of baseline detection of antibody (ie, sum of seronegative, seropositive, and unknown).

49 (100) 258/258 (100) 1019/1019 (100) Mumps (ED50 <24) 216/252 (85.7) 219/248 (88.3) 213/246 (86.6) 213/243 (86.6) 861/999 (86.2) Rubella (<4 IU/mL) 259/259 (100) 251/252 (99.6) 248/249 (99.6) 258/258 (100) 1016/1018 (99.8) Abbreviations: MMR, measles-mumps-rubella; N, total number of subjects; SD, standard deviation. aCalculated for subjects in TVC for which pre-vaccination status was known (ie, % = n/[n + seropositive] × 100, where n = no. of seronegative subjects). bNumber of subjects regardless of the status of baseline detection of antibody (ie, sum of seronegative, seropositive, and unknown). Immunogenicity Seroresponse to MMR Vaccines Measles virus seroresponse rates were 98.3–99.2% for MMR-RIT groups and 99.6% for the MMRII group; GMCs of measles virus antibodies were >2500 mIU/mL in all 4 groups. Day-42 seroresponse rates for mumps virus antibodies were 89.7% for MMR-RIT-3 (low mumps titer), 90.6% for MMR-RIT-2 (medium mumps titer), 90.7% for MMR-RIT-1 (high mumps titer), and 91.1% for MMRII. Mumps virus antibody GMTs were at least 10-fold greater than the assay cut-off for seronegativity in all 4 groups. Day-42 rubella virus seroresponse rates were 97.5–98.8% for MMR-RIT groups and 100% for the MMRII group. Observed rubella virus antibody GMCs for MMR-RIT groups (68.2–77.7 IU/mL) and MMRII (89.4 IU/mL) were above the assay seronegativity cut-off of 10 IU/mL (Table 3). Table 3. Seroresponse Rates and Geometric Mean Concentrations/Titers (GMC/Ts) for Antibodies to Measles, Mumps, and Rubella Viruses at Day 42 in Initially Seronegative Subjects (According-to-Protocol Cohort for Immunogenicity)

mL) and MMRII (89.4 IU/mL) were above the assay seronegativity cut-off of 10 IU/mL (Table 3). Table 3. Seroresponse Rates and Geometric Mean Concentrations/Titers (GMC/Ts) for Antibodies to Measles, Mumps, and Rubella Viruses at Day 42 in Initially Seronegative Subjects (According-to-Protocol Cohort for Immunogenicity) Measles (≥200 mIU/mL) Mumps (≥51 ED50) Rubella (≥10 IU/mL) N Seroresponse GMC (95% CI) N Seroresponse GMC (95% CI) N Seroresponse GMC (95% CI) n (%) (95% CI) % Diff. vs. MMRII (95% CI) n (%) (95% CI) % Diff. vs. MMRII (95% CI) n (%) (95% CI) % Diff. vs. MMRII (95% CI) MMR-RIT-1 247 245 (99.2) (97.1; 99.9) −0.41 (−2.55; 1.50) 2799 (2545; 3078) 193 175 (90.7) (85.7; 94.4) −0.47 (−6.42; 5.46) 242 (205; 287) 247 244 (98.8) (96.5; 99.7) −1.21 (−3.51; 0.32) 72.2 (65.6; 79.6) MMR-RIT-2 240 236 (98.3) (95.8; 99.5) −1.27 (−3.85; 0.74) 2878 (2607; 3178) 202 183 (90.6) (85.7; 94.2) −0.55 (−6.41; 5.35) 265 (222; 317) 238 235 (98.7) (96.4; 99.7) −1.26 (−3.64; 0.27) 77.7 (70.4; 85.7) MMR-RIT-3 240 236 (98.3) (95.8; 99.5) −1.27 (−3.85; 0.74) 2593 (2350; 2861) 195 175 (89.7) (84.6; 93.6) −1.40 (−7.47; 4.62) 253 (213; 301) 239 233 (97.5) (94.6; 99.1) −2.51 (−5.37; −0.97) 68.2 (61.8; 75.3) MMRII 249 248 (99.6) (97.8; 100) Reference 2950 (2698; 3224) 192 175 (91.1) (86.2; 94.8) Reference 268 (224; 320) 249 249 (100) (98.5; 100) Reference 89.4 (81.4; 98.2) Abbreviations: MMR, measles-mumps-rubella; N, number of subjects with available results; n (%), number/percentage of subjects who seroconverted; 95% CI, 95% confidence interval; GMC/T, geometric mean concentrations/titers for measles, mumps, and rubella virus antibodies.

49 (100) (98.5; 100) Reference 89.4 (81.4; 98.2) Abbreviations: MMR, measles-mumps-rubella; N, number of subjects with available results; n (%), number/percentage of subjects who seroconverted; 95% CI, 95% confidence interval; GMC/T, geometric mean concentrations/titers for measles, mumps, and rubella virus antibodies. Co-administration of VAR, HAV, and PCV7 Day-42 seroresponse rates for antibodies to VZV among initially seronegative subjects when VAR was co-administered with MMR-RIT or MMRII were 95.8–98.0%. GMCs for VZV antibodies were >200 mIU/mL in all 4 groups. Day-42 response rates for antibodies to hepatitis A virus were 83.0–89.3% among initially seronegative vaccinees across the 4 groups. Day-42 GMCs adjusted for baseline serostatus for antibodies to hepatitis A virus or pneumococcal serotypes appeared to be similar for all 4 groups, as shown by calculated GMC ratios in Table 4. Table 4. Geometric Mean Concentrations (GMCs) for Varicella-Zoster Virus (VZV) Antibodies, and Baseline-Adjusted GMCs and GMC Ratios for Antibodies to Hepatitis A Virus and PCV7 Pneumococcal Serotypes at Day 42 (According-to-Protocol Cohort for Immunogenicity)

limb was moved/spontaneously painful) was reported in ≤1.5%. Two subjects (1 each in the MMR-RIT-2 and MMRII groups) had Grade-3 injection site swelling (diameter >20 mm). Table 5. Incidence of Solicited Injection Site (Days 0; 3) and General Symptoms During the 43-Day Post-vaccination Period (Total Vaccinated Cohort) Symptom MMR-RIT-1 (N = 283) MMR-RIT-2 (N = 275) MMR-RIT-3 (N = 283) MMRII (N = 277) n % (95% CI) n % (95% CI) n % (95% CI) n % (95% CI) Days 0; 3 Pain 70 24.8 (19.9; 30.3) 70 25.5 (20.5; 31.1) 79 28.0 (22.9; 33.6) 67 24.5 (19.5; 30.0) Redness 45 16.0 (11.9; 20.8) 47 17.2 (12.9; 22.1) 41 14.5 (10.6; 19.2) 47 17.2 (12.9; 22.1) Swelling 20 7.1 (4.4; 10.7) 26 9.5 (6.3; 13.6) 19 6.7 (4.1; 10.3) 15 5.5 (3.1; 8.9) Days 0; 14 Irritability/fussiness 180 63.6 (57.7; 69.2) 141 51.3 (45.2; 57.3) 150 53.0 (47.0; 58.9) 153 55.2 (49.2; 61.2) Drowsiness 133 47.0 (41.1; 53.0) 106 38.5 (32.8; 44.6) 113 39.9 (34.2; 45.9) 109 39.4 (33.6; 45.4) Loss of appetite 111 39.2 (33.5; 45.2) 77 28.0 (22.8; 33.7) 110 38.9 (33.2; 44.8) 94 33.9 (28.4; 39.8) Fever (rectal temp. ≥38.0°C) 65 23.0 (18.2; 28.3) 79 28.7 (23.5; 34.5) 64 22.6 (17.9; 27.9) 56 20.2 (15.6; 25.4) Fever (rectal temp. >39.5°C) 10 3.5 (1.7; 6.4) 7 2.5 (1.0; 5.2) 9 3.2 (1.5; 6.0) 8 2.9 (1.3; 5.6) Days 0; 42 Fever (rectal temp. ≥38.0°C) 103 36.4 (30.8; 42.3) 104 37.8 (32.1; 43.8) 104 36.7 (31.1; 42.7) 85 30.7 (25.3; 36.5) Fever (rectal temp. >39.5°C) 20 7.1 (4.4; 10.7) 14 5.1 (2.8; 8.4) 18 6.4 (3.8; 9.9) 13 4.7 (2.5; 7.9) Localized/generalized rash 72 25.4 (20.5; 30.9) 74 26.9 (21.8; 32.6) 60 21.2 (16.6; 26.4) 69 24.9 (19.9; 30.4) Parotid gland swelling 3 1.1 (0.2; 3.1) 3 1.1 (0.2; 3.2) 5 1.8 (0.6; 4.1) 2 0.7 (0.1; 2.6) Abbreviations: MMR, measles-mumps-rubella; N, number of subjects having received the documented dose; n/%, number/percentage of subjects reporting a specified symptom; 95% CI, exact 95% confidence interval.

) 69 24.9 (19.9; 30.4) Parotid gland swelling 3 1.1 (0.2; 3.1) 3 1.1 (0.2; 3.2) 5 1.8 (0.6; 4.1) 2 0.7 (0.1; 2.6) Abbreviations: MMR, measles-mumps-rubella; N, number of subjects having received the documented dose; n/%, number/percentage of subjects reporting a specified symptom; 95% CI, exact 95% confidence interval. During Days 0–14, irritability or fussiness (overall incidence, 51.3–63.6%) and drowsiness (38.5–47.0%) were the most frequently reported solicited general symptoms (Table 5). Incidence of fever (rectal temperature ≥38.0°C) during Days 0–14 was 20.2–28.7% across the 4 groups; fever >39.5°C occurred in ≤3.5% subjects in any group. Incidence of fever requiring medical attention reported during Days 0–14 in each group was as follows: 6.0% (95% CI: 3.5; 9.4) for MMR-RIT-1, 8.0% (5.1; 11.9) for MMR-RIT-2, 8.5% (5.5; 12.4) for MMR-RIT-3, and 6.9% (4.2; 10.5) for MMRII. Overall incidence of fever during Days 0–42 was 36.4–37.8% for MMR-RIT groups and 30.7% for MMRII recipients (Table 5). For all groups, prevalence of fever peaked 5–12 days after study vaccination (Figure 2); in an exploratory post hoc analysis, the observed incidence of fever between Days 5 and 12 was 14.8% (95% CI: 10.9; 19.5) for MMR-RIT-1, 23.3% (18.4; 28.7) for MMR-RIT-2, 17.3% (13.1; 22.2) for MMR-RIT-3, and 14.8% (10.8; 19.5) for MMRII. Figure 2. Prevalence of any fever from Day 0 to Day 42 after vaccination (total vaccinated cohort). Abbreviation: MMR, measles-mumps-rubella.

of fever between Days 5 and 12 was 14.8% (95% CI: 10.9; 19.5) for MMR-RIT-1, 23.3% (18.4; 28.7) for MMR-RIT-2, 17.3% (13.1; 22.2) for MMR-RIT-3, and 14.8% (10.8; 19.5) for MMRII. Figure 2. Prevalence of any fever from Day 0 to Day 42 after vaccination (total vaccinated cohort). Abbreviation: MMR, measles-mumps-rubella. For Days 0–42, incidence of rash of any type varied between 21.2% and 26.9% across the 4 groups. Measles or rubella-like rashes were reported in MMR-RIT-1 (n = 6), MMR-RIT-2 (n = 7), MMR-RIT-3 (n = 5), and MMRII (n = 9) recipients; varicella-like rash was reported for MMR-RIT-2 (n = 4) and MMRII (n = 2) subjects. Overall incidence of parotid-gland swelling was low (Table 5). There were 2 cases of febrile convulsion during Days 0–42. One MMR-RIT-2 recipient experienced a simple febrile convulsion at Day 29. This was not considered vaccine-related by the investigator since the peak prevalence of vaccine-related fever, and hence febrile convulsions, occurs in the second week following vaccination with measles-containing vaccines [23]. One MMRII recipient had a complex febrile seizure on Day 0, which led to hospitalization and the mother's withdrawal of the subject from the study; this event, classified as an SAE, was considered to be related to the study vaccine.

For Days 0–42, incidence of rash of any type varied between 21.2% and 26.9% across the 4 groups. Measles or rubella-like rashes were reported in MMR-RIT-1 (n = 6), MMR-RIT-2 (n = 7), MMR-RIT-3 (n = 5), and MMRII (n = 9) recipients; varicella-like rash was reported for MMR-RIT-2 (n = 4) and MMRII (n = 2) subjects. Overall incidence of parotid-gland swelling was low (Table 5). There were 2 cases of febrile convulsion during Days 0–42. One MMR-RIT-2 recipient experienced a simple febrile convulsion at Day 29. This was not considered vaccine-related by the investigator since the peak prevalence of vaccine-related fever, and hence febrile convulsions, occurs in the second week following vaccination with measles-containing vaccines [23]. One MMRII recipient had a complex febrile seizure on Day 0, which led to hospitalization and the mother's withdrawal of the subject from the study; this event, classified as an SAE, was considered to be related to the study vaccine. Of 15 SAEs reported in 11 subjects (Days 0–42), 2 SAEs were considered to be related to study treatment: febrile convulsion in a 12-month-old female MMRII recipient (described above), and idiopathic thrombocytopenic purpura (onset at Day 20) in a 13-month-old female MMR-RIT-2 recipient who was hospitalized for treatment and discharged after 3 days. Both subjects with vaccine-related SAEs were withdrawn from the study. All SAEs resolved without sequelae.

courses of ribavirin and IVIG. Treatment with ribavirin alone (n = 2) was initiated 2 and 4 days after diagnosis, respectively. Of the 7 patients who received ribavirin, 6 (86%) received inhaled ribavirin at a dose of 2 g 3 times daily for a 5-day course, and 1 (14%) received an 11-day course of intravenous ribavirin. Mortality Attributed to hMPV Three patients (5%) died of respiratory failure related to hMPV pneumonia. Two of these deaths occurred in HSCT recipients who were diagnosed with hMPV prior to transplant (Figures 1 and 2B: Patients 2 and 3). One HSCT recipient had not engrafted at time of death, while the other engrafted the day prior to death. Both were treated with ribavirin and IVIG. The third death occurred in a patient with ALL and secondary acute myelogenous leukemia on salvage chemotherapy who did not receive hMPV-specific treatment (Figure 2B: Patient 5). HMPV was considered to be a contributing factor to her death, though the primary cause of death was thought to be progression of ALL. The median time to death was 37 days (range, 37–64 days).

-old female MMRII recipient (described above), and idiopathic thrombocytopenic purpura (onset at Day 20) in a 13-month-old female MMR-RIT-2 recipient who was hospitalized for treatment and discharged after 3 days. Both subjects with vaccine-related SAEs were withdrawn from the study. All SAEs resolved without sequelae. Concomitant Medication Rates of concomitant medication use (Days 0–42) were the following: MMR-RIT-1 (76.6%), MMR-RIT-2 (72.0%), MMR-RIT-3 (74.0%), and MMRII (70.1%). Rates of antipyretic medication use were the following: MMR-RIT-1 (65.1%), MMR-RIT-2 (59.2%), MMR-RIT-3 (64.1%), and MMRII (57.5%). DISCUSSION This Phase-2 multicenter exploratory study assessed immune responses to the first dose of MMR-RIT with 3 differing mumps virus titers and to commercially available MMRII when concomitantly administered with HAV, VAR, and PCV7 in healthy children in the United States, aged 12–15 months. The results indicate that a single dose of any 1 of the 3 MMR-RIT lots elicited an acceptable immune response with respect to seroresponse rates to MMR viruses, 42 days post-vaccination. Previous randomized comparative studies have shown that MMR-RIT administered as a primary vaccination to children in the second year of life produced similar seroconversion rates for antibodies to MMR vaccine viruses compared to those seen with MMRII [24–28].

seroresponse rates to MMR viruses, 42 days post-vaccination. Previous randomized comparative studies have shown that MMR-RIT administered as a primary vaccination to children in the second year of life produced similar seroconversion rates for antibodies to MMR vaccine viruses compared to those seen with MMRII [24–28]. The current formulation of MMR-RIT used in this study, when co-administered to young children with other recommended vaccines (HAV, VAR, and PCV7), elicited measles and rubella virus antibody concentrations meeting the predefined threshold for seroresponse in ≥97.5% MMR-RIT recipients, and in ≥99.6% MMRII recipients when co-administered with the same vaccines.

this study, when co-administered to young children with other recommended vaccines (HAV, VAR, and PCV7), elicited measles and rubella virus antibody concentrations meeting the predefined threshold for seroresponse in ≥97.5% MMR-RIT recipients, and in ≥99.6% MMRII recipients when co-administered with the same vaccines. Day-42 mumps virus antibody titers after vaccination with all 3 MMR-RIT lots met the seroresponse threshold in 90.7%, 90.6%, and 89.7% recipients, respectively, and no obvious dose–response relationship was observed. Of note, 13.8% of subjects were seropositive for mumps at baseline. This high baseline seropositivity rate is likely attributed to complement enhancement of the mumps PRN assay rather than prior exposure to mumps or persistence of maternal antibodies. Complement-enhanced mumps PRN assays have been shown to have higher seroresponse rates than unenhanced assays [29], and this may also be reflected in the baseline seropositivity. Rubella seroresponse rates across all 4 groups were high (97.5–100%) and GMCs for antibodies to rubella virus following all 3 MMR-RIT lots were substantially higher than the cut-off level of ≥10 IU/mL and would provide effective immunization. Long-term follow-up in this study will allow the evaluation of antibody persistence until approximately 2 years post-vaccination.

high (97.5–100%) and GMCs for antibodies to rubella virus following all 3 MMR-RIT lots were substantially higher than the cut-off level of ≥10 IU/mL and would provide effective immunization. Long-term follow-up in this study will allow the evaluation of antibody persistence until approximately 2 years post-vaccination. The immune response to vaccines routinely co-administered with MMR dose-1 in the United States (HAV, VAR, and PCV7) was also assessed. Observed seroresponse rates for antibodies to VZV were consistently high (≥95.8%) across all 4 treatment groups, and hepatitis A virus antibody response rates to HAV dose-1 were ≥83.0% in each group. Furthermore, Day-42 baseline-adjusted GMCs for antibodies to hepatitis A virus and Streptococcus pneumoniae serotypes appeared comparable when these vaccines were administered with MMR-RIT or MMRII in this exploratory analysis of between-group GMC ratios.

antibody response rates to HAV dose-1 were ≥83.0% in each group. Furthermore, Day-42 baseline-adjusted GMCs for antibodies to hepatitis A virus and Streptococcus pneumoniae serotypes appeared comparable when these vaccines were administered with MMR-RIT or MMRII in this exploratory analysis of between-group GMC ratios. MMR-RIT had an acceptable reactogenicity profile when co-administered with HAV, VAR, and PCV7. Injection site symptoms at the MMR injection site occurred in all 4 treatment groups within 4 days of vaccination, although the incidence of severe symptoms was low. Consistent with previous reports [25–28], vaccination with MMR-RIT or MMRII was associated with fever (rectal temperature ≥38.0°C) during the first 2 weeks, which peaked during days 5–12 after vaccine administration. MMR-RIT, which contains the Jeryl Lynn–derived RIT 4385 strain, has demonstrated a good reactogenicity profile in previous clinical trials [25–28, 30]. Accordingly, in this study, a low incidence of other solicited general symptoms, including measles/rubella- or varicella-like rash, parotid gland swelling, and febrile convulsions, was reported among both MMR-RIT and MMRII recipients during the follow-up period.

secondary acute myelogenous leukemia on salvage chemotherapy who did not receive hMPV-specific treatment (Figure 2B: Patient 5). HMPV was considered to be a contributing factor to her death, though the primary cause of death was thought to be progression of ALL. The median time to death was 37 days (range, 37–64 days). CONCLUSIONS Human metapneumovirus infections in our immunocompromised patient population was associated with high rates of LRTI at presentation, and an attributable mortality of 5%, rates substantially higher than that in the general population where hMPV is most commonly a self-limited URTI. In our study, 12 (23%) of immunocompromised children with hMPV were classified as having severe disease, and neutropenia was identified as a significant risk factor for disease severity. This rate is comparable or even higher than previous rates reported in seriously immunocompromised adult patients [13].

genicity profile in previous clinical trials [25–28, 30]. Accordingly, in this study, a low incidence of other solicited general symptoms, including measles/rubella- or varicella-like rash, parotid gland swelling, and febrile convulsions, was reported among both MMR-RIT and MMRII recipients during the follow-up period. Strengths of this study include use of a computer-generated randomization list and blinding of study vaccines, thereby addressing potential biases in study conduct, and comparing the immune responses to the study vaccine with the US-licensed standard of care (MMRII). Furthermore, the MMR-RIT formulation used in this study was free of human serum albumin, thus eliminating the theoretical risk of microbial contamination associated with human serum albumin–containing vaccines [14]. Additionally, omitting a human blood–derived component from the MMR-RIT formulation might make it more socially acceptable. Lastly, concomitant administration of routine vaccines (HAV, VAR, and PCV7) did not affect the immunogenicity of MMR-RIT and vice versa. Limitations of the study include the relatively small study population that is consistent with a Phase-2 planning study. Larger Phase-3 clinical trials are required in future to substantiate the immunogenicity and safety profile of MMR-RIT. Furthermore, extended follow-up studies are important to evaluate the long-term protection offered by MMR-RIT.

e the relatively small study population that is consistent with a Phase-2 planning study. Larger Phase-3 clinical trials are required in future to substantiate the immunogenicity and safety profile of MMR-RIT. Furthermore, extended follow-up studies are important to evaluate the long-term protection offered by MMR-RIT. CONCLUSIONS This Phase-2 study demonstrated an acceptable immune response to 3 candidate MMR-RIT lots containing differing mumps virus titers with respect to seroresponse rates to all 3 MMR virus components. There was no obvious dose–response relationship for the 3 mumps virus titers evaluated; based on the current results, all 3 lots would provide effective immunization, with an acceptable reactogenicity profile. MMR-RIT can be given concomitantly with HAV, VAR, and PCV7 without interfering with the immune response to these co-administered vaccines, as was shown for MMRII. Confirmatory Phase-3 studies to support licensure of MMR-RIT on the basis of immunogenic noninferiority to the licensed comparator are warranted. Trademark Statement MMRII is a registered trademark of Merck & Co. Inc., Whitehouse Station, NJ, United States. Priorix, Havrix, and Varivax are registered trademarks of the GlaxoSmithKline group of companies. Prevnar is a trademark of Wyeth LLC, New York, NY. Enzygnost is a registered trademark of Dade Behring, Marburg GmbH, Germany. SAS is a registered trademark of SAS Institute Inc., Cary, NC.

Trademark Statement MMRII is a registered trademark of Merck & Co. Inc., Whitehouse Station, NJ, United States. Priorix, Havrix, and Varivax are registered trademarks of the GlaxoSmithKline group of companies. Prevnar is a trademark of Wyeth LLC, New York, NY. Enzygnost is a registered trademark of Dade Behring, Marburg GmbH, Germany. SAS is a registered trademark of SAS Institute Inc., Cary, NC. Author Contributions C.D.C. and B.I. conceived the study, designed the trial, and obtained research funding. All authors substantially contributed to the conception, the design, the acquisition of data, and the analysis and interpretation of data. M.M., C.D., M.L., C.H., S.G., A.C., S.C.T., R.J., and A.Q. recruited patients. M.P. provided statistical advice on study design and analyzed the data. All authors contributed substantially to the drafting of the article. They revised it critically for important intellectual content and approved the version to be published. Acknowledgments The authors would like to thank the parents, children, and investigators who participated in this clinical trial. We also gratefully acknowledge the work of the nurses and other staff members involved. The authors also thank Dr. Sarah Hopwood (Scinopsis) for medical writing and Véronique Duquenne, Shruti MP, and Ashmita Ravishankar (GlaxoSmithKline group of companies) for editorial support and publication coordination.

ical trial. We also gratefully acknowledge the work of the nurses and other staff members involved. The authors also thank Dr. Sarah Hopwood (Scinopsis) for medical writing and Véronique Duquenne, Shruti MP, and Ashmita Ravishankar (GlaxoSmithKline group of companies) for editorial support and publication coordination. Financial support. This work was supported by GlaxoSmithKline Biologicals SA. As study sponsor, GlaxoSmithKline Biologicals SA was involved in all stages of study conduct, including data analysis and took charge of all costs associated with the study, including the development and publication of the manuscript. Potential conflicts of interest. C.D.C, B.L.I., and O.N., former and current employees of the sponsor, may own stock or stock options in the company. M.P. is an employee of GlaxoSmithKline group of companies. M.A.M. received travel support for a meeting for the study and payment for lectures, including service on speakers bureau (Merck). C.J.H. declares that his institution receives grants to conduct the trials for which he is the investigator; he has also received honoraria for lectures, including service on speakers bureau (Merck and Sanofi). S.G. received support for travel, consulting fees, payment for lectures, and fees for participation in review. M.L. has in the past, and is currently being paid by the sponsor as a principal investigator for vaccine research. C.D, A.C, S.C.T., R.JF, A.Q.D.R., and G.B have indicated they have no financial relationship or conflict of interest relevant to this article to disclose.

or lectures, and fees for participation in review. M.L. has in the past, and is currently being paid by the sponsor as a principal investigator for vaccine research. C.D, A.C, S.C.T., R.JF, A.Q.D.R., and G.B have indicated they have no financial relationship or conflict of interest relevant to this article to disclose. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

Annual influenza attack rates are highest in young children, and their rates of complicated influenza that require medical care and hospitalization parallel those in persons older than 65 years [1]. In the United States from 1993 to 2008, the estimated rates of influenza-associated hospitalizations were 91.5 per 100 000 for children in the first 12 months of life and 21.9 per 100 000 for children 1 to 4 years of age [1]. Influenza is also associated with outpatient and emergency department visits, diagnoses of otitis media, and prescriptions for antibiotics. Although influenza vaccines are accepted worldwide as the most effective method for preventing influenza [2], there is a paucity of data on the efficacy of inactivated influenza vaccines in young children, especially those younger than 2 years. Indeed, recent systematic reviews of methodologically rigorous studies or randomized controlled trials [3-7] found few data demonstrating the immunogenicity or efficacy of the influenza vaccine in preventing laboratory-confirmed influenza in young children. More research on the influenza vaccine in this age group is needed [4].

ecent systematic reviews of methodologically rigorous studies or randomized controlled trials [3-7] found few data demonstrating the immunogenicity or efficacy of the influenza vaccine in preventing laboratory-confirmed influenza in young children. More research on the influenza vaccine in this age group is needed [4]. The emergence of 2 lineages of influenza B virus since the 1980s, with a subsequent mismatch of circulating and vaccine strains, led to the development of quadrivalent influenza vaccines (QIVs) containing both the Yamagata and the Victoria lineages. QIVs, both live attenuated and inactivated, were approved in the United States beginning in the 2013–2014 season. QIVs containing both B virus lineages are estimated to reduce the numbers of illnesses, hospitalizations, and deaths caused by influenza [5]. Studies of the immunogenicity [6–8] and effectiveness [9] of various QIVs in children from 6 months to 17 years of age have met regulatory immunogenicity criteria for seasonal influenza vaccines; 1 of the approved QIVs showed 73% efficacy in preventing moderate-to-severe influenza in children aged 3 to 8 years [9]. In an open-label study in 6- to 35-month-olds conducted in the 2010–2011 season, a QIV was immunogenic and had a safety profile consistent with that of other inactivated influenza vaccines, and in a concurrent randomized controlled study in 3- to 17-year-olds, no interference with immune responses to the QIV occurred compared to a trivalent influenza vaccine (TIV) containing a B Victoria component [6].

V was immunogenic and had a safety profile consistent with that of other inactivated influenza vaccines, and in a concurrent randomized controlled study in 3- to 17-year-olds, no interference with immune responses to the QIV occurred compared to a trivalent influenza vaccine (TIV) containing a B Victoria component [6]. In this Phase 3 randomized controlled study of children 6 through 35 months of age conducted in the 2012–2013 season, we assessed the immunogenicity, reactogenicity, and safety of a QIV containing influenza B strains from both lineages versus those of a TIV containing the same H1N1 and H3N2 and a B Yamagata component. In the 2012–2013 season, the recommended B Yamagata antigen had changed (to B/Hubei-Wujiagang/158/09) from that of the 2010–2011 season, providing an opportunity to assess the safety, reactogenicity, and immune responses to a vaccine containing influenza antigens from another combination of influenza strains. METHODS This study was a randomized (1:1) controlled double-blind comparison of QIV and TIV in 6- to 35-month-old children to determine safety and immunogenicity (Figure 1). The study was initiated on November 1, 2012, and the day 180 visit concluded on June 19, 2013. Figure 1. Participant flow. Abbreviations: QIV, quadrivalent influenza vaccine; TIV-YB, trivalent influenza vaccine containing Yamagata lineage of the B strain.

-old children to determine safety and immunogenicity (Figure 1). The study was initiated on November 1, 2012, and the day 180 visit concluded on June 19, 2013. Figure 1. Participant flow. Abbreviations: QIV, quadrivalent influenza vaccine; TIV-YB, trivalent influenza vaccine containing Yamagata lineage of the B strain. The study (registered under ClinicalTrials.gov identifier NCT01711736) was undertaken in compliance with Good Clinical Practice guidelines, the Declaration of Helsinki, and national regulatory requirements and was approved by a local, regional, or national institutional review board at each study site. Participants Eligible children were in stable health and between 6 and 35 months of age at the time of the first vaccination. Children were excluded if they were febrile (temperature, ≥38.0°C) or acutely ill at the time of enrollment, known to be immunocompromised, known to be allergic to any of the vaccine components, had a history of Guillain-Barré syndrome within 6 weeks of receipt of a previous influenza vaccine, had a coagulation disorder, had received influenza vaccine in the previous 6 months, had received immunoglobulins or blood products within 3 months, or had received an investigational product within 30 days before the first study vaccine. A parent/guardian provided written informed consent for each participant. The study was conducted at 8 sites in 3 countries (Canada, Dominican Republic, and Honduras).

ad received immunoglobulins or blood products within 3 months, or had received an investigational product within 30 days before the first study vaccine. A parent/guardian provided written informed consent for each participant. The study was conducted at 8 sites in 3 countries (Canada, Dominican Republic, and Honduras). Vaccines All vaccines were provided in a single-dose thimerosal-free formulation and administered by the intramuscular route. The QIV contained 15 µg of hemagglutinin (HA) from 4 strains recommended for the 2012–2013 season [10]: A/California/7/2009(H1N1)pdm09, A/Victoria/361/2011(H3N2), B/Brisbane/60/2008 (Victoria lineage), and B/Hubei-Wujiagang/158/2009 (Yamagata lineage). The TIV contained the same H1N1, H3N2, and B Yamagata components but no B Victoria lineage component. The vaccines were manufactured by GlaxoSmithKline Vaccines and provided in prefilled 0.5-mL syringes. The QIV was produced in Sainte-Foy, Quebec, Canada, according to the process used to manufacture FluLaval™, and the licensed TIV was produced in Dresden, Germany, according to the process used to manufacture Fluarix™. The vaccines, which were both opalescent off-white to grayish suspensions, were labeled with the treatment number but no identifying information.

Canada, according to the process used to manufacture FluLaval™, and the licensed TIV was produced in Dresden, Germany, according to the process used to manufacture Fluarix™. The vaccines, which were both opalescent off-white to grayish suspensions, were labeled with the treatment number but no identifying information. Study Procedures After the consent process, the study staff determined treatment allocation using an internet-based central randomization system. The randomization sequence was generated by using MATEX, a software program developed for use in SAS (SAS Institute, Cary, NC) that uses a minimization algorithm to balance treatment allocation between age groups (6–17 months and 18–35 months), between prestudy influenza vaccine priming statuses (primed vs unprimed), and among study centers.

nce was generated by using MATEX, a software program developed for use in SAS (SAS Institute, Cary, NC) that uses a minimization algorithm to balance treatment allocation between age groups (6–17 months and 18–35 months), between prestudy influenza vaccine priming statuses (primed vs unprimed), and among study centers. Before the first vaccination, a brief history-directed physical examination was performed, and blood was drawn. Children who had previously received ≥2 doses of an influenza vaccine at least 1 month apart or who had received at least 1 dose before the previous season were considered vaccine primed. Vaccine-primed children received one 0.5-mL dose of study vaccine on day 0 and had blood collected on day 28 (visit 2). Vaccine-unprimed participants received a second 0.5-mL dose of vaccine on day 28 and had blood collected on day 56. Receipt of previous influenza vaccines was determined by parental/guardian history. Vaccines were administered intramuscularly in the anterolateral region of the left thigh (for children aged 6–11 months) or the deltoid region of the nondominant arm (for those aged ≥12 months) using a 25-mm (1-inch) 22- to 25-gauge needle, as determined by the vaccinator on the basis of participant age and muscle mass. The children were observed at the study site for 30 minutes after administration of the study vaccine. Routine childhood vaccines were permitted to be given concurrently.

hose aged ≥12 months) using a 25-mm (1-inch) 22- to 25-gauge needle, as determined by the vaccinator on the basis of participant age and muscle mass. The children were observed at the study site for 30 minutes after administration of the study vaccine. Routine childhood vaccines were permitted to be given concurrently. Parents/guardians were instructed on the use of a diary card for recording any solicited injection-site or general adverse events (AEs) for 7 days and any unsolicited AEs for 28 days and to bring this diary with them at the next study visit. Parents were instructed to contact the investigator immediately if the child displayed any symptoms they perceived as serious. Antipyretics taken between 6 hours before and 12 hours after vaccination were recorded. On day 180, parents/guardians were asked if changes in the child's health had occurred since the last vaccination to identify unsolicited AEs. Participant flow is seen in Figure 1. Outcomes Immunogenicity Antibody titers against the vaccine strains were measured in serum samples by hemagglutination-inhibition (HI) assays performed at the GlaxoSmithKline Vaccines laboratory in Dresden, Germany, using standardized procedures [11]. The primary objective was to assess the immunogenicity of the QIV 28 days after the completion of dosing (day 28 for vaccine-primed children and day 56 for vaccine-unprimed children) on the basis of the Center for Biologics Evaluation and Research's (CBER's) seroconversion rate (SCR) criterion (lower limit [LL] of the 95% confidence interval [CI] for each of the 4 strains) [12].

QIV 28 days after the completion of dosing (day 28 for vaccine-primed children and day 56 for vaccine-unprimed children) on the basis of the Center for Biologics Evaluation and Research's (CBER's) seroconversion rate (SCR) criterion (lower limit [LL] of the 95% confidence interval [CI] for each of the 4 strains) [12]. A secondary objective was to determine the immunogenic superiority of the B/Victoria strain in the QIV, compared to that in the TIV, 28 days after the final vaccination in terms of the geometric mean titer (GMT) ratio (QIV/TIV) (success criterion, LL of the 95% CI, >1.5), adjusted by the baseline antibody titer (analysis of covariance model) and the SCR difference (QIV – TIV) (success criterion, LL of the 95% CI, >10%). Immunogenicity was also described for each group, age stratum, and priming status, with 95% CIs for the following parameters: the GMT of HI and seroprotection rate (SPR) on day 0 and 28 days after the final vaccination and the SCR and mean geometric increase (MGI) 28 days after the final vaccination with 95% CIs. Reactogenicity The frequencies of solicited injection-site AEs (pain, redness, and swelling) and solicited general AEs (drowsiness, fever, irritability/fussiness, loss of appetite) were described (primary safety objective). Fever was defined as a temperature of ≥38.0°C as measured by any method. Intensity scales were used for the description of each symptom (Supplementary Table 1). The relative risk of fever in the 4 days after vaccination (QIV group/TIV group) was explored.

ss, loss of appetite) were described (primary safety objective). Fever was defined as a temperature of ≥38.0°C as measured by any method. Intensity scales were used for the description of each symptom (Supplementary Table 1). The relative risk of fever in the 4 days after vaccination (QIV group/TIV group) was explored. Unsolicited AEs were recorded for 28 days after each dose. Safety was assessed further by consideration of serious AEs (SAEs), medically attended AEs (MAEs), and potentially immune-mediated diseases (pIMDs) during the entire study period (up to day 180). MAEs were defined as events for which a child was hospitalized, visited the emergency department, or had a visit with a physician for any complaint. All injection-site reactions were considered vaccine-related events. The causality of all other AEs was assessed by the investigators. All AEs were classified according to the Medical Dictionary for Regulatory Activities (MedRA). Statistical Analysis We planned for a sample size of 255 evaluable participants in each group to obtain an overall power of 99.99% to demonstrate the primary objective of meeting the CBER SCR criterion simultaneously for all 4 strains. A target of 600 children (300 per group) was set to account for an attrition rate of 15%. The according-to-protocol (ATP) cohort for analysis of immunogenicity was defined as children who did not meet elimination or exclusion criteria during the study, those for whom we had assay results for at least 1 study vaccine antigen after vaccination, and those who complied with the time requirements of the study.

Statistical Analysis We planned for a sample size of 255 evaluable participants in each group to obtain an overall power of 99.99% to demonstrate the primary objective of meeting the CBER SCR criterion simultaneously for all 4 strains. A target of 600 children (300 per group) was set to account for an attrition rate of 15%. The according-to-protocol (ATP) cohort for analysis of immunogenicity was defined as children who did not meet elimination or exclusion criteria during the study, those for whom we had assay results for at least 1 study vaccine antigen after vaccination, and those who complied with the time requirements of the study. Reactogenicity and safety were assessed in the total vaccinated cohort (TVC), which included vaccinated participants for whom data were available. AEs, SAEs, pIMDs, and MAEs were analyzed descriptively by tabulating the percentage of subjects with at least 1 AE in each category and any AE after each vaccine dose and overall, with a 95% CI.

fety were assessed in the total vaccinated cohort (TVC), which included vaccinated participants for whom data were available. AEs, SAEs, pIMDs, and MAEs were analyzed descriptively by tabulating the percentage of subjects with at least 1 AE in each category and any AE after each vaccine dose and overall, with a 95% CI. RESULTS Participants Six hundred one children (QIV, n = 299; TIV, n = 302) were enrolled and randomly assigned (Figure 1). The ATP cohort for immunogenicity consisted of 571 children (95%). The mean ages of the participants at the first vaccination visit were 18.2 months (standard deviation [SD], 8.17 months) in the TVC and 18.1 months (SD, 8.34 months) in the QIV and TIV groups (Table 1). Overall, 53% (317 of 601) of the children were 6 to 17 months of age, and 47% (284 of 601) were 18 to 35 months of age. Girls comprised 50.1% of the participants (301 of 601). Table 1. Demographic Characteristics at Enrolment: Total Vaccinated Cohort

nd 18.1 months (SD, 8.34 months) in the QIV and TIV groups (Table 1). Overall, 53% (317 of 601) of the children were 6 to 17 months of age, and 47% (284 of 601) were 18 to 35 months of age. Girls comprised 50.1% of the participants (301 of 601). Table 1. Demographic Characteristics at Enrolment: Total Vaccinated Cohort Characteristic QIV (n = 299) TIV (n = 302) Total (n = 601) Mean age in months (SD; median; range) 18.2 (8.17; 17.0; 6–35) 18.1 (8.34; 16.5; 6–35) 18.1 (8.25; 17.0; 6–35) Male, n (%) 144 (48.2) 156 (51.7) 300 (49.9) Female, n (%) 155 (51.8) 146 (48.3) 301 (50.1) Hispanic/Latino ethnicity 231 (77.3) 233 (77.2) 464 (77.2) Not Hispanic/Latino ethnicity 68 (22.7) 69 (22.8) 137 (22.8) Heritage/race European heritage/Caucasian 47 (15.7) 52 (17.2) 99 (16.5) Asian 16 (5.4) 16 (5.3) 32 (5.3) African heritage/African American 4 (1.3) 2 (0.7) 6 (1.0) American Indian or Native Alaskan 0 0 0 Pacific Islander/Native Hawaiian 0 0 0 Other 232 (77.6) 232 (76.8) 464 (77.2) Median BMI (kg/m2), SD 17.0 (1.94) 17.1 (2.42) 17.0 (2.2) QIV, quadrivalent influenza vaccine; SD, standard deviation; TIV, trivalent influenza vaccine; BMI, body mass index, weight/ height. Of 601 children, only 39 (6.5%) had previously received an influenza vaccine in any previous season (QIV, n = 16; TIV, n = 23); thus, 573 (95%) of the 601 children were considered vaccine unprimed.

Characteristic QIV (n = 299) TIV (n = 302) Total (n = 601) Mean age in months (SD; median; range) 18.2 (8.17; 17.0; 6–35) 18.1 (8.34; 16.5; 6–35) 18.1 (8.25; 17.0; 6–35) Male, n (%) 144 (48.2) 156 (51.7) 300 (49.9) Female, n (%) 155 (51.8) 146 (48.3) 301 (50.1) Hispanic/Latino ethnicity 231 (77.3) 233 (77.2) 464 (77.2) Not Hispanic/Latino ethnicity 68 (22.7) 69 (22.8) 137 (22.8) Heritage/race European heritage/Caucasian 47 (15.7) 52 (17.2) 99 (16.5) Asian 16 (5.4) 16 (5.3) 32 (5.3) African heritage/African American 4 (1.3) 2 (0.7) 6 (1.0) American Indian or Native Alaskan 0 0 0 Pacific Islander/Native Hawaiian 0 0 0 Other 232 (77.6) 232 (76.8) 464 (77.2) Median BMI (kg/m2), SD 17.0 (1.94) 17.1 (2.42) 17.0 (2.2) QIV, quadrivalent influenza vaccine; SD, standard deviation; TIV, trivalent influenza vaccine; BMI, body mass index, weight/ height. Of 601 children, only 39 (6.5%) had previously received an influenza vaccine in any previous season (QIV, n = 16; TIV, n = 23); thus, 573 (95%) of the 601 children were considered vaccine unprimed. Immunogenicity The primary immunogenicity objective was met: the LL of the 2-sided 95% CI for the SCR in QIV recipients ranged from 66.6% to 81.3%, which was ≥ 40% against all 4 strains, and the SCR point estimates for each of the 4 strains ranged from 72.2% to 85.9% (Figure 2 and Table 2) . Table 2. Immunogenicity of QIV and TIV in Children Aged 6 to 35 months: ATP Immunogenicity Cohort

the 2-sided 95% CI for the SCR in QIV recipients ranged from 66.6% to 81.3%, which was ≥ 40% against all 4 strains, and the SCR point estimates for each of the 4 strains ranged from 72.2% to 85.9% (Figure 2 and Table 2) . Table 2. Immunogenicity of QIV and TIV in Children Aged 6 to 35 months: ATP Immunogenicity Cohort Vaccine Age (mo) Timing n GMT Value (95% CI) SCR* (% [95% CI]) SPR* (% [95% CI]) MGI Value (% [95% CI]) A/California/7/2009 (H1N1) QIV 6–35 Pre 284 9.6 (8.1–11.3) 16.2 (12.1–21.0) 6–35 Post 284 157.1 (132.8–185.9) 85.9 (81.3–89.7) 89.4 (85.3–92.8) 16.4 (14.3–18.7) 6–17 Pre 151 7.0 (5.9–8.3) 7.3 (3.7–12.7) 6–17 Post 151 103.2 (82.2–129.5) 80.8 (73.6–86.7) 82.8 (75.8–88.4) 14.8 (12.1–18.1) 18–35 Pre 133 13.7 (10.3–18.3) 26.3 (19.1–34.7) 18–35 Post 133 253.2 (201.7–317.7) 91.7 (85.7–95.8) 97.0 (92.5–99.2) 18.4 (15.6–21.8) TIV 6–35 Pre 287 9.8 (8.3–11.6) 16.4 (12.3–21.2) 6–35 Post 287 61.2 (49.2–76.2) 53.7 (47.7–59.5) 58.9 (53.0–64.6) 6.2 (5.3–7.3) 6–17 Pre 149 6.9 (5.8–8.2) 8.1 (4.2–13.6) 6–17 Post 149 28.6 (21.7–37.7) 38.3 (30.4–46.6) 39.6 (31.7–47.9) 4.1 (3.4–5.1) 17–35 Pre 138 14.4 (10.8–19.1) 25.4 (18.3–33.5) 17–35 Post 138 139.3 (104.1–186.4) 70.3 (61.9–77.8) 79.7 (72.0–86.1) 9.7 (7.8–12.0) A/Victoria/361/2011 (H3N2) QIV 6–35 Pre 284 17.4 (14.1–21.5) 32.7 (27.3–38.5) 6–35 Post 284 159.4 (129.4–196.3) 72.2 (66.6–77.3) 81.3 (76.3–85.7) 9.1 (8.0–10.5) 6–17 Pre 151 12.4 (9.5–16.1) 24.5 (17.9–32.2) 6–17 Post 151 108.3 (81.0–144.8) 72.2 (64.3–79.2) 76.2 (68.6–82.7) 8.7 (7.3–10.5) 18–35 Pre 133 25.6 (18.4–35.7) 42.1 (33.6–51.0) 18–35 Post 133 247.1 (185.9–328.6) 72.2 (63.7–79.6) 87.2 (80.3–92.4) 9.6 (7.9–11.8) TIV 6–35 Pre 287 13.8 (11.4–16.8) 25.8 (20.8–31.3) 6–35 Post 287 103.0 (83.7–126.7) 55.7 (49.8–61.6) 66.6 (60.8–72.0) 7.5 (6.4–8.7) 6–17 Pre 149 9.2 (7.3–11.6) 14.8 (9.5–21.5) 6–17 Post 149 53.5 (40.9–70.1) 42.3 (34.2–50.6) 50.3 (42.0–58.6) 5.8 (4.8–7.1) 18–35 Pre 138 21.4 (15.9–29.0) 37.7 (29.6–46.3) 18–35 Post 138 208.7 (158.4–274.9) 70.3 (61.9–77.8) 84.1 (76.9–89.7) 9.7 (7.7–12.2) B/Brisbane/60/2008 (Victoria) QIV 6–35 Pre 284 10.6 (9.1–12.4) 19.7 (15.3–24.8) 6–35 Post 284 111.4 (91.9–135.2) 73.9 (68.4–79.0) 76.1 (70.7–80.9) 10.5 (9.2–11.9) 6–17 Pre 151 6.9 (6.0–7.9) 6.6 (3.2–11.8) 6–17 Post 151 66.6 (53.0–83.6) 66.9 (58.8–74.3) 68.2 (60.1–75.5) 9.7 (7.9–11.8) 18–35 Pre 133 17.4 (13.4–22.7) 34.6 (26.6–43.3) 18–35 Post 133 200.1 (149.2–268.3) 82.0 (74.4–88.1) 85.0 (77.7–90.6) 11.5 (9.8–13.4) TIV 6–35 Pre 287 9.3 (8.0–10.7) 15.7 (11.7–20.4) 6–35 Post 287 15.6

7 Pre 151 6.9 (6.0–7.9) 6.6 (3.2–11.8) 6–17 Post 151 66.6 (53.0–83.6) 66.9 (58.8–74.3) 68.2 (60.1–75.5) 9.7 (7.9–11.8) 18–35 Pre 133 17.4 (13.4–22.7) 34.6 (26.6–43.3) 18–35 Post 133 200.1 (149.2–268.3) 82.0 (74.4–88.1) 85.0 (77.7–90.6) 11.5 (9.8–13.4) TIV 6–35 Pre 287 9.3 (8.0–10.7) 15.7 (11.7–20.4) 6–35 Post 287 15.6 (13.3–18.5) 9.8 (6.6–13.8) 25.8 (20.8–31.3) 1.7 (1.5–1.9) 6–17 Pre 149 5.6 (5.2–6.0) 1.3 (0.2–4.8) 6–17 Post 149 8.6 (7.4–9.9) 7.4 (3.7–12.8) 8.7 (4.7–14.5) 1.5 (1.3–1.8) 18–35 Pre 138 16.1 (12.4–21.0) 31.2 (23.6–39.6) 18–35 Post 138 29.9 (22.8–39.2) 12.3 (7.3–19.0) 44.2 (35.8–52.9) 1.9 (1.6–2.2) B/Hubei–Wujiagang/158/2009 (Yamagata) QIV 6–35 Pre 284 7.7 (6.9–8.7) 9.2 (6.1–13.1) 6–35 Post 284 114.2 (100.0–130.5) 78.9 (73.7–83.5) 85.2 (80.5–89.1) 14.8 (12.8–17.1) 6–17 Pre 151 7.5 (6.4–8.8) 8.6 (4.7–14.3) 6–17 Post 151 93.3 (78.2–111.3) 74.2 (66.4–80.9) 81.5 (74.3–87.3) 12.5 (10.2–15.3) 18–35 Pre 133 8.0 (6.8–9.4) 9.8 (5.3–16.1) 18–35 Post 133 143.8 (118.2–174.8) 84.2 (76.9–90.0) 89.5 (83.0–94.1) 18.0 (14.6–22.1) TIV 6–35 Pre 287 7.2 (6.5–8.0) 8.4 (5.4–12.2) 6–35 Post 287 107.2 (92.2–124.6) 77.4 (72.1–82.1) 79.8 (74.7–84.3) 14.8 (12.8–17.2) 6–17 Pre 149 6.9 (6.0–7.9) 6.7 (3.3–12.0) 6–17 Post 149 71.8 (59.0–87.5) 68.5 (60.3–75.8) 71.8 (63.9–78.9) 10.5 (8.6–12.7) 18–35 Pre 138 7.7 (6.5–9.0) 10.1 (5.7–16.4) 18–35 Post 138 165.3 (134.1–203.7) 87.0 (80.2–92.1) 88.4 (81.9–93.2) 21.5 (17.4–26.6) Abbreviations: Pre, prevaccination; Post, postvaccination.

.8–17.2) 6–17 Pre 149 6.9 (6.0–7.9) 6.7 (3.3–12.0) 6–17 Post 149 71.8 (59.0–87.5) 68.5 (60.3–75.8) 71.8 (63.9–78.9) 10.5 (8.6–12.7) 18–35 Pre 138 7.7 (6.5–9.0) 10.1 (5.7–16.4) 18–35 Post 138 165.3 (134.1–203.7) 87.0 (80.2–92.1) 88.4 (81.9–93.2) 21.5 (17.4–26.6) Abbreviations: Pre, prevaccination; Post, postvaccination. *Each of the four strains contained in the QIV met CBER licensure criteria for immunogenicity (LL of the 95% CI for SCR of at least 40% and a postvaccination SPR of at least 70%). Figure 2. SCRs for HI antibodies 28 days after the last vaccine dose (28 days after dose 1 [day 28] for primed subjects; 28 days after dose 2 [day 56] for unprimed subjects) in the ATP cohort for immunogenicity. The SCR was defined as an antibody titer of ≥40 1/DIL after vaccination for initially seronegative subjects and an antibody titer after vaccination of ≥4-fold the prevaccination antibody titer for initially seropositive subjects. % indicates the percentage of seroconverted subjects; error bars indicate 95% CIs. Abbreviations: Q-QIV, quadrivalent influenza vaccine; Q, manufactured in Quebec; TIV-YB, trivalent influenza vaccine containing Yamagata lineage of B strain; D, manufactured in Dresden.

antibody titer for initially seropositive subjects. % indicates the percentage of seroconverted subjects; error bars indicate 95% CIs. Abbreviations: Q-QIV, quadrivalent influenza vaccine; Q, manufactured in Quebec; TIV-YB, trivalent influenza vaccine containing Yamagata lineage of B strain; D, manufactured in Dresden. The immunogenic superiority of the B/Victoria strain present in the QIV (in terms of GMT and SCR) over that in the TIV was concluded, because the LL of the 2-sided 95% CI of the adjusted GMT ratio (QIV/TIV) (6.28 [95% CI, 5.32–7.41]) was greater than 1.5, and the LL of the 2-sided 95% CI for the difference in SCRs (QIV – TIV, 64.19% [95% CI, 57.65%–69.95%]) was greater than 10% (Figure 3). The adjusted GMT ratio (adjusted for HI antibody titers at baseline) was 6.28 (95% CI, 5.32–7.41). Figure 3. Immunogenic superiority of QIV versus TIV-YB for the B Victoria strain in the ATP cohort for immunogenicity. Adjusted GMT indicates GMT adjusted for baseline titer; SCRs, an antibody titer of ≥40 1/DIL after vaccination for initially seronegative subjects and an antibody titer after vaccination of ≥4-fold the prevaccination antibody titer for initially seropositive subjects. Criteria for superiority were that the LL of the 2-sided 95% CI of the GMT ratio (Q-QIV /D-TIV-YB) was >1.5 and the LL of the 2-sided 95% CI on the SCR difference (Q-QIV – D-TIV-YB) was >10%. Error bars indicate 95% CIs.

after vaccination of ≥4-fold the prevaccination antibody titer for initially seropositive subjects. Criteria for superiority were that the LL of the 2-sided 95% CI of the GMT ratio (Q-QIV /D-TIV-YB) was >1.5 and the LL of the 2-sided 95% CI on the SCR difference (Q-QIV – D-TIV-YB) was >10%. Error bars indicate 95% CIs. The GMTs, SCRs, SPRs, and MGIs for the QIV and TIV recipients before and after vaccination (day 28 after the last dose) and according to age stratum (6–17 months or 18–35 months of age) are shown in Table 2. Immune responses were lower in the younger age stratum (6–17 months) than in the 18- to 35-month-olds. The SPR point estimates for the 3 antigens contained in both vaccines ranged from 76.2% to 82.8% for the 6- to 17-month-old QIV recipients and from 87.2% to 97.0% for the 18- to 35-month-old QIV recipients. In contrast, SPRs in the TIV recipients ranged from 39.6% to 71.8% and 79.7% to 88.4% in the younger and older age strata, respectively. SCRs for the 3 antigens contained in both study vaccines ranged from 72.2% to 80.8% in the 6- to 17-month-old QIV recipients and– from 72.2% to 91.7% in 18- to 35-month-old QIV recipients,. In TIV recipients, the SCRs ranged from 38.3% to 68.5% and 70.3% to 87.0% in the younger and older age strata, respectively.

vely. SCRs for the 3 antigens contained in both study vaccines ranged from 72.2% to 80.8% in the 6- to 17-month-old QIV recipients and– from 72.2% to 91.7% in 18- to 35-month-old QIV recipients,. In TIV recipients, the SCRs ranged from 38.3% to 68.5% and 70.3% to 87.0% in the younger and older age strata, respectively. Reactogenicity The frequencies of injection-site and general solicited AEs overall during the 7 days after vaccination are shown in Figures 4 and 5. Injection-site pain was the most frequently reported solicited injection-site AE (QIV, 32.6%; TIV, 30.6%), with grade 3 pain in 2.4% and 1.0% of participants, respectively. Irritability/fussiness was the most frequently reported solicited general AE (40.7% and 41.6% of subjects in the QIV and TIV groups, respectively). Grade 3 irritability/fussiness was reported in 5.2% and 4.7% of the QIV and TIV recipients, respectively. Figure 4. Incidence and nature of local injection-site adverse events on days 0 to 6 after each vaccine dose in the TVC. Grade 3 pain was defined as a child crying when his or her limb was moved and/or spontaneously painful; grade 3, redness/swelling of >100 mm. Error bars indicate 95% CIs.

IV recipients, respectively. Figure 4. Incidence and nature of local injection-site adverse events on days 0 to 6 after each vaccine dose in the TVC. Grade 3 pain was defined as a child crying when his or her limb was moved and/or spontaneously painful; grade 3, redness/swelling of >100 mm. Error bars indicate 95% CIs. Figure 5. Incidence and nature of systemic adverse events on days 0 to 6 after each vaccine dose in the TVC. Grade 3 drowsiness was defined as drowsiness that prevented normal activity; grade 3 irritability/fussiness (irritability/fuss), crying that could not be comforted and/or prevented normal activity; grade 3 loss of appetite, child did not eat at all; grade 3 fever (or higher), temperature of ≥39.0°C (grade 4 fever also included here). Error bars indicate 95% CIs. A temperature of ≥38°C in the 7-day postvaccine period was reported for 14.5% (95% CI, 10.7%–19.1%) (QIV) and 14.5% (95% CI, 10.7%–19.1%) (TIV) of the recipients after the first dose and in 10.3% (95% CI, 7.0%–14.5%) (QIV) and 9.1% (95% CI, 5.9%–13.1%) (TIV) of the children after the second dose. A temperature of ≥39.0°C in the same time period was reported in 3.8% (95% CI, 1.9%–6.7%) and 3.0% (95% CI, 1.4%–5.7%) of the QIV and TIV recipients, respectively.

limited URTI. In our study, 12 (23%) of immunocompromised children with hMPV were classified as having severe disease, and neutropenia was identified as a significant risk factor for disease severity. This rate is comparable or even higher than previous rates reported in seriously immunocompromised adult patients [13]. Similar retrospective studies performed in pediatric cancer patients have looked at morbidity and mortality related to other respiratory viruses. Parainfluenza virus was associated with LRTI in 20–36% of patients with a mortality rate of 0–7% in a mixed population of pediatric cancer and transplant patients [14, 15]. Similarly, respiratory syncytial virus (RSV) has been associated with LRTI in 28% and mortality in 8% of a mixed group of pediatric and transplant patients [16]. More recently, pandemic H1N1 and seasonal influenza A were associated with LRTI rates of 20–42% and 4–27% and mortality rates of 10–12% and 2–4%, respectively, despite treatment of the majority of patients with antiviral drugs [17, 18]. An additional study performed in a larger group of patients described a lower rate of LRTI (16%) associated with pandemic H1N1 with only 1 severe case, with no related mortality [19]. Prospective studies of hMPV in adults with hematologic malignancies demonstrated that 9% of respiratory tract infections were attributed to hMPV, and that the majority of these were in HSCT recipients [13]. Among HSCT recipients, hMPV was associated with 19% mortality.

V) of the recipients after the first dose and in 10.3% (95% CI, 7.0%–14.5%) (QIV) and 9.1% (95% CI, 5.9%–13.1%) (TIV) of the children after the second dose. A temperature of ≥39.0°C in the same time period was reported in 3.8% (95% CI, 1.9%–6.7%) and 3.0% (95% CI, 1.4%–5.7%) of the QIV and TIV recipients, respectively. In an exploratory analysis, the relative risks (RRs) (QIV/TIV) of a temperature of ≥38.5°C were 1.40 (95% CI, 0.66–2.94; P = .43) after the first vaccine dose and 1.62 (95% CI, 0.76–3.46; P = .23) after the second vaccine dose. The RRs of a temperature of >39.0°C (102.2°F, grade 3) during the 4-day follow-up period were 1.54 (95% CI, 0.47–5.03; P = .54) after dose 1 and 2.54 (95% CI, 0.85–7.58; P = .11) after dose 2. The frequencies of fever were similar across the age strata (data not shown). An antipyretic agent was taken by 31.8% of QIV recipients and 32.8% of TIV recipients after the vaccine. Although the administration of routine childhood vaccines was permitted, only 2 of 601 subjects in the TVC received pneumococcal conjugate vaccine concomitantly. During the 28-day postvaccination period, at least 1 unsolicited AE was reported for 47.5% and 54.6% of participants in the QIV and TIV groups, respectively. Nasopharyngitis (26.1% and 29.8% of participants) and diarrhea (12.7% and 12.6% of participants) were the only 2 unsolicited AEs reported for more than 5% of the children in the QIV and TIV groups, respectively. At least 1 unsolicited grade 3 AE was reported for 3.0% and 1.7% of participants in the QIV and TIV groups, respectively.

is (26.1% and 29.8% of participants) and diarrhea (12.7% and 12.6% of participants) were the only 2 unsolicited AEs reported for more than 5% of the children in the QIV and TIV groups, respectively. At least 1 unsolicited grade 3 AE was reported for 3.0% and 1.7% of participants in the QIV and TIV groups, respectively. During the 180-day follow-up period, 25 nonfatal SAEs were reported for 17 participants (QIV, n = 9 [3%]; TIV, n = 8 [2.6%]). Only 1 SAE was considered by the investigators to be related to the study vaccine: a simple partial seizure associated with fever in an 18-month-old 6 hours after the first dose of QIV. The child recovered without sequelae, and the second dose of vaccine was given with no fever reported. All SAEs were resolved by the end of the study. At least 1 MAE was reported for 52.2% (n = 156) and 51.7% (n = 156) of the QIV and TIV recipients, respectively. The most common MAEs were the unsolicited AEs: nasopharyngitis, diarrhea, and pharyngitis. Two pIMDs were reported in the TIV group (MedRA codes “alopecia areata” and “colitis ulcerative”), and they resolved by day 180. No pIMDs were reported in the QIV group. No AEs led to participant discontinuation.

s, respectively. The most common MAEs were the unsolicited AEs: nasopharyngitis, diarrhea, and pharyngitis. Two pIMDs were reported in the TIV group (MedRA codes “alopecia areata” and “colitis ulcerative”), and they resolved by day 180. No pIMDs were reported in the QIV group. No AEs led to participant discontinuation. DISCUSSION In this randomized double-blind controlled trial of 6- to 35-month-old children, the QIV met regulatory criteria for immunogenicity and was superior to the TIV for the B/Victoria strain present in the QIV. QIVs are expected to improve protection against influenza B when 2 B lineages are cocirculating or there is mismatch between the B lineage in the TIV and the circulating strain. Although immunogenicity is an indirect measure of clinical protection against influenza-associated illness, it is notable that in a recent phase 3 efficacy trial in 3- to 8-year-old children, the efficacy of the QIV was confirmed. The vaccine effectiveness rate in this older age group against reverse-transcriptase polymerase chain reaction–confirmed influenza was 55.4% (95% CI, 39.2%–67.3%) in the TVC and against moderate-to severe-influenza was 73.1% (95% CI, 47.1%–86.3%) [9]. An efficacy trial in children aged 6 to 35 months (ClinicalTrials.gov identifier NCT01439360) is underway.

older age group against reverse-transcriptase polymerase chain reaction–confirmed influenza was 55.4% (95% CI, 39.2%–67.3%) in the TVC and against moderate-to severe-influenza was 73.1% (95% CI, 47.1%–86.3%) [9]. An efficacy trial in children aged 6 to 35 months (ClinicalTrials.gov identifier NCT01439360) is underway. Attaining robust immune responses to influenza vaccine in infants in the first years of life has been an ongoing challenge. In this study, immune responses in the younger age stratum (6–17 months) were lower than those of the 18- to 35-month-olds, and the SPRs for H3N2 and the B virus (Victoria lineage) did not meet CBER immunogenicity criteria. We note that in a predominately vaccine-unprimed population, such as in this study, SCRs may be a better measure of vaccine response. Strategies for overcoming the reduced immunogenicity of vaccine have included using 2 doses in the first year a child is vaccinated, using a 0.5-ml “adult dose” [13–15], and adjuvantation with oil-in-water emulsions [16, 17] or virosomes [18].

on, such as in this study, SCRs may be a better measure of vaccine response. Strategies for overcoming the reduced immunogenicity of vaccine have included using 2 doses in the first year a child is vaccinated, using a 0.5-ml “adult dose” [13–15], and adjuvantation with oil-in-water emulsions [16, 17] or virosomes [18]. The frequencies of both local and systemic AEs in the week after immunization were comparable in the QIV and TIV recipients, after dose 1 or 2, and across the age strata. Adverse events were somewhat less common than those observed in a previous open-label (unblinded) trial of this QIV product in the same age group except for fever, which was slightly higher [6]. Grade 3 events were also similar across groups and uncommon. There are few published data on the reactogenicity of inactivated nonadjuvanted influenza vaccines in children younger than 3 years to which our results can be compared. Most systematic reviews of randomized controlled trials of influenza vaccines in children have focused instead on immunogenicity and/or effectiveness. Two narrative reviews found few primary studies of inactivated split-virus vaccines that documented AEs in young children [19, 20].

to which our results can be compared. Most systematic reviews of randomized controlled trials of influenza vaccines in children have focused instead on immunogenicity and/or effectiveness. Two narrative reviews found few primary studies of inactivated split-virus vaccines that documented AEs in young children [19, 20]. Fever is probably the most concerning AE after influenza immunization in children under 5 years of age because of its association with febrile seizures. In the 2010–2011 and 2011–2012 seasons, the Centers for Disease Control and Prevention noted an increase in febrile seizures in the 48 hours after TIV receipt in 6- to 48-month-old children, at a frequency of less than 1 in 1000 children vaccinated [10]. No increase was seen in the subsequent season. In this study of 601 children from 6 to 35 months of age, an exploratory analysis of the RR of fever in QIV recipients compared to TIV recipients during a 4-day follow-up period did not detect any significant findings, but the CIs were wide. Similar to previous QIV study results, no excess reactogenicity seems to occur with the higher dose of influenza antigen (60 µg).

f age, an exploratory analysis of the RR of fever in QIV recipients compared to TIV recipients during a 4-day follow-up period did not detect any significant findings, but the CIs were wide. Similar to previous QIV study results, no excess reactogenicity seems to occur with the higher dose of influenza antigen (60 µg). A limitation of this study is that it was conducted in children in stable health, and the results may not be generalizable to children in this age group with chronic conditions, particularly immunodeficiency. Also, the study end points, antibody responses, are surrogate outcomes for clinical protection. Although efficacy studies of this QIV in children aged 3 to 8 years have shown clinical protection [9], clinical efficacy for this QIV was shown in 3- to 8-year-olds but not in 6- to 35-month-olds. Other limitations are that most children in this study had not previously received influenza vaccine; children in this age group with previous influenza vaccine exposure (vaccine-primed) may respond differently to a subsequent inactivated influenza vaccine. Although routine childhood immunizations were permitted in this study, the number of children who concurrently received vaccines was insufficient to evaluate immune responses and reactogenicity with concurrent administration.

posure (vaccine-primed) may respond differently to a subsequent inactivated influenza vaccine. Although routine childhood immunizations were permitted in this study, the number of children who concurrently received vaccines was insufficient to evaluate immune responses and reactogenicity with concurrent administration. In summary, in this randomized controlled trial in children aged 6 to 35 months, an inactivated QIV had an immunogenicity that was superior to that of a TIV for the added B strain and acceptable immunogenicity for the shared strains, with no notable difference in reactogenicity and safety compared to the TIV. Supplementary Data Supplementary materials are available at the Journal of the Pediatric Infectious Diseases Society online (http://jpids.oxfordjournals.org). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

ordjournals.org). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author. Acknowledgments We are indebted to the participating study volunteers and their parents, clinicians, nurses, and laboratory technicians at the study sites and to the sponsor's project staff for their support and contributions throughout the study. In particular, we thank M. Dionne, A. Greenspoon, P. Rheault, Y. Hernandez, and G. Tellier. We are grateful to all teams at GlaxoSmithKline Vaccines for their contributions to this study, especially S. Van de Voorde (Emtex, on behalf of GlaxoSmithKline Vaccines) and N. Hilgert for writing support of the clinical study report, T. Ryckaert (Harrison, on behalf of GlaxoSmithKline Vaccines) for clinical study management, C. Stalens from the clinical and serological laboratory teams, O. Gascard (Keyrus Biopharma, on behalf of GlaxoSmithKline Vaccines) for data management, P. Boutet for project management, W. Jiang from the clinical safety team, S. Ravault from the clinical regulatory affairs team, J. Herrera from the clinical research associate team, M. M. Castrejon-Alba from the clinical research and development team, and S. Wu from the project statistician team. Finally, we thank B. Dumont (Business and Decision Life Sciences, on behalf of GlaxoSmithKline Vaccines) for editorial assistance and manuscript coordination.

ra from the clinical research associate team, M. M. Castrejon-Alba from the clinical research and development team, and S. Wu from the project statistician team. Finally, we thank B. Dumont (Business and Decision Life Sciences, on behalf of GlaxoSmithKline Vaccines) for editorial assistance and manuscript coordination. Financial support. This work was supported by GlaxoSmithKline Biologicals SA. GlaxoSmithKline Biologicals SA was involved in all stages of study conduct and analysis and bore the costs associated with the development and publishing of the present manuscript. All the authors had full access to the data, and the corresponding author had final responsibility for submission of the publication. J.M.L. is supported by a Canadian Institutes of Health-GlaxoSmithKline Chair in Pediatric Vaccinology at Dalhousie University. All authors participated in the implementation of the study, including providing substantial contributions to conception and design, gathering of the data, or analysis and interpretation of the data. The corresponding author drafted the manuscript; all authors were involved in revising the manuscript critically for important intellectual content and in its final approval.

study, including providing substantial contributions to conception and design, gathering of the data, or analysis and interpretation of the data. The corresponding author drafted the manuscript; all authors were involved in revising the manuscript critically for important intellectual content and in its final approval. Potential conflicts of interest. V.K.J., V.C., L.W., A.L., P.L., and B.L.I. are employees of the GlaxoSmithKline group of companies. V.K.J., A.L., L.W., P.L., and B.L.I. report ownership of stock options and/or restricted shares. All the investigators or their institutions received payments from the GlaxoSmithKline group of companies for the conduct of the study. J.M.L. reports payments and grants to her institution as follows: payment from Astra Zeneca for an advisory board, grants from Sanofi Pasteur, and grants from Pfizer, outside the submitted work, and having served on independent committees that are advisory to provincial and national immunization making. S.A.H. reports payments and grants to his institution as follows: grants from Sanofi Pasteur, grants from Novartis, payments from Novartis, and payments from the GlaxoSmithKline group of companies, outside the submitted work. S.M. reports payments from the GlaxoSmithKline group of companies, grants from Sanofi Pasteur, and grants from Pfizer Canada, outside the submitted work. L.P.M., L.R., L.C., A.B., and N.A. have nothing to disclose.

ents from Novartis, and payments from the GlaxoSmithKline group of companies, outside the submitted work. S.M. reports payments from the GlaxoSmithKline group of companies, grants from Sanofi Pasteur, and grants from Pfizer Canada, outside the submitted work. L.P.M., L.R., L.C., A.B., and N.A. have nothing to disclose. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Trademarks. Fluarix™ and FluLaval™ are trademarks of the GlaxoSmithKline group of companies.

There is a growing body of evidence that respiratory virus infections play an important role in pulmonary morbidity and exacerbations in children with cystic fibrosis (CF) [1–14]. Previous studies using clinic- or hospital-based testing have likely underestimated the true impact of respiratory viruses on CF pulmonary exacerbations due to delays in sample collection relative to onset of symptoms and the lack of sensitive molecular testing methods. Investigations to further elucidate the impact of respiratory virus infections in CF will require timely diagnoses using molecular methods.

ue impact of respiratory viruses on CF pulmonary exacerbations due to delays in sample collection relative to onset of symptoms and the lack of sensitive molecular testing methods. Investigations to further elucidate the impact of respiratory virus infections in CF will require timely diagnoses using molecular methods. Nasal washes or invasive nasopharyngeal swabs collected by medical personnel have historically been considered “gold standard” samples for respiratory virus detection. In children, nasal swabs (NS) have been shown to have reasonable performance for polymerase chain reaction (PCR) detection of most respiratory viruses [15–18]. Community-based studies have used parent- or self-collected swabs for respiratory virus research [19–24]. Although previous studies using self-collection have involved transport medium and storage at 4°C, we have recently developed a simple, sensitive, and noninvasive method for self-collection of respiratory samples using foam swabs that does not require transport media or refrigeration [25]. Furthermore, we found that swabs collected with the use of saline spray were superior to swabs collected without the use of saline spray [25]. In the current study, we investigated the feasibility of home self-collection of NS in children with CF experiencing onset of new respiratory illness, with samples mailed to a central laboratory for respiratory virus detection by real-time PCR. Although the main objective was to assess feasibility, we also sought to study whether NS collected at home would compare favorably to swabs collected in clinic for detection of respiratory viruses, and whether foam NS collected with the use of saline spray would perform comparably to swabs collected without saline.

R. Although the main objective was to assess feasibility, we also sought to study whether NS collected at home would compare favorably to swabs collected in clinic for detection of respiratory viruses, and whether foam NS collected with the use of saline spray would perform comparably to swabs collected without saline. METHODS Human Subjects The study protocol was approved by the Seattle Children's Institutional Review Board. Children ages 6–18 years with a diagnosis of CF were eligible to enroll if they met the following criteria: (1) attended at least 2 CF clinic visits during the previous 12 months; (2) currently experiencing a new respiratory illness or willing to return for a clinic visit or collect home samples when experiencing a new respiratory illness; (3) willing to perform self-collection of NS (collected by the child or parent); and (4) written informed consent provided. Children were excluded if they had received antiviral medications during the 30 days before enrollment or if they were awaiting or had previously received a lung transplant. The period of study enrollment was February 2009 to January 2010, with up to 15 months of follow-up per patient.

ritten informed consent provided. Children were excluded if they had received antiviral medications during the 30 days before enrollment or if they were awaiting or had previously received a lung transplant. The period of study enrollment was February 2009 to January 2010, with up to 15 months of follow-up per patient. Sample Collection and Symptom Surveys If respiratory illness with onset of symptoms in the previous 7 days was present at a clinic visit, subjects were first asked to blow their nose to remove mucus that might inhibit PCR, and paired swabs were obtained as follows: a deep nasal (mid-turbinate) sample was collected by research staff, by first measuring from the opening of one naris to the nasal bridge, and then inserting a standard flexible nasopharyngeal flocked nylon swab (Copan Diagnostics Inc, Murrieta, CA; catalog No. 503CS01) until mild resistance was encountered, approximately one-half to two-thirds the length of the nose. This approach to using nasopharyngeal swabs to collect deep nasal samples was previously found to be well accepted by participants in a study of respiratory viruses in CF patients at our hospital [2]. Next, a polyurethane foam NS (Super Brush, LLC, Chicopee, MA; catalog No. 71-4541) was collected in the opposite naris after instillation of saline nasal spray. Nasal spray was used to closely replicate a standard nasal wash. In brief, 5 sprays of saline from a polyethylene metered bottle (0.1 mL/spray) were instilled into the naris, followed by NS insertion into the anterior naris as far as comfortably possible, and rotation of the swab while exhaling through the nose for 5–7 seconds. During the clinic visit, the research nurse instructed the patient regarding self-collection of foam NS, and either the patient or parent collected the swab while observed and directed by the research nurse, or the swab was collected by the research nurse as an opportunity to demonstrate the proper technique for collection to the patient and parent. Detailed written instructions regarding self-collection were reviewed at the clinic visit and were provided to the family for reference when swab collection at home was indicated (see Supplementary Material). Also provided were kits containing all supplies necessary for collection and mailing of foam NS to be obtained at home at onset of symptoms of a new respiratory illness.

re reviewed at the clinic visit and were provided to the family for reference when swab collection at home was indicated (see Supplementary Material). Also provided were kits containing all supplies necessary for collection and mailing of foam NS to be obtained at home at onset of symptoms of a new respiratory illness. Subsequent collections at home consisted of foam NS collected either by the participant or parent with (“saline”) and without (“dry”) the use of nasal saline spray to evaluate whether the spray was essential to the procedure, as previously described [25]. Dry swabs were collected by inserting a foam swab into the anterior naris as far as comfortably possible, followed by slow rotation of the swab for 5–7 seconds while exhaling through the nose. For home collections, participants were asked to collect the dry swab before the saline swab.

re, as previously described [25]. Dry swabs were collected by inserting a foam swab into the anterior naris as far as comfortably possible, followed by slow rotation of the swab for 5–7 seconds while exhaling through the nose. For home collections, participants were asked to collect the dry swab before the saline swab. During initial development and optimization of the self-collection procedure, we previously evaluated virus stability over time from foam NS (for influenza A and parainfluenza virus type 3), and we found no difference in viral recovery between room temperature and 4°C, transport medium, and dry tubes, at 1, 2, or 7 days [25]. Thus, for this study each foam swab was placed into an empty dry transport tube (no transport media added) and stored at room temperature. Home-collected NS were mailed to the University of Washington Molecular Virology Laboratory using US Postal Service Pre-paid Priority Mailers with appropriate packaging and labeling for Category B Infectious Substances. Deep NS collected in clinic were placed into lysis buffer and stored at 4°C until laboratory testing.

ome-collected NS were mailed to the University of Washington Molecular Virology Laboratory using US Postal Service Pre-paid Priority Mailers with appropriate packaging and labeling for Category B Infectious Substances. Deep NS collected in clinic were placed into lysis buffer and stored at 4°C until laboratory testing. Study participants received phone or e-mail reminders every 1–2 weeks to collect samples at home at onset of new respiratory illness; home sample collection was allowed during up to 2 illnesses per participant. Each participant (or parent) completed a standardized symptom survey in conjunction with each sample collection. Criteria for a new respiratory illness included (1) presence of at least 1 of the 15 symptoms listed on the standardized symptom survey and (2) symptom duration of a minimum of 24 hours and ≤ 7 days. Questionnaires related to tolerability of self-collection were also completed. Participants were instructed to mail home swabs and surveys within 1 day of collection.

d (1) presence of at least 1 of the 15 symptoms listed on the standardized symptom survey and (2) symptom duration of a minimum of 24 hours and ≤ 7 days. Questionnaires related to tolerability of self-collection were also completed. Participants were instructed to mail home swabs and surveys within 1 day of collection. Polymerase Chain Reaction Testing Swabs were processed in the laboratory as soon as possible after receipt. Specimens were tested for qualitative detection by a panel of 8 single or multiplexed real time reverse-transcription (RT)–PCR (for respiratory syncytial virus, influenza virus types A and B, parainfluenza virus types 1–4, human metapneumovirus, human coronaviruses [subtypes OC43, 229E, NL63, and HKU1] and rhinoviruses) and PCR (for adenovirus and bocavirus) using previously described methods [25–31]. Samples were considered positive if the PCR amplification plot crossed the threshold at less than 40 cycles (cycle threshold [CT] <40). All PCR methods were performed according to College of American Pathologist standards, and the laboratory passed proficiency testing in viral diagnostics.

ly described methods [25–31]. Samples were considered positive if the PCR amplification plot crossed the threshold at less than 40 cycles (cycle threshold [CT] <40). All PCR methods were performed according to College of American Pathologist standards, and the laboratory passed proficiency testing in viral diagnostics. Statistical Analysis Data were summarized using counts and proportions and means and standard deviations (SD). Linear regression analyses were used to compare differences in time from collection to laboratory processing, symptom duration, and number of symptoms between swab collections performed at home versus in clinic. A similar method was used to compare differences in RT-PCR CT values between swabs positive for rhinovirus alone that were collected at the same time and between swabs collected at home versus in clinic. Too few swabs were positive for other virus types to perform statistical analyses. Regression analyses included clustering on participant to account for repeated observations per participant, and 95% confidence interval (CI) estimates were calculated using robust variance estimates. All analyses were performed using STATA version 10.1 (StataCorp, College Station, TX).

to perform statistical analyses. Regression analyses included clustering on participant to account for repeated observations per participant, and 95% confidence interval (CI) estimates were calculated using robust variance estimates. All analyses were performed using STATA version 10.1 (StataCorp, College Station, TX). RESULTS Sample Collection A total of 35 children were enrolled, 28 of whom provided paired swab sets collected in clinic or at home during a new respiratory illness. Baseline characteristics were similar between the total study population and those who had samples collected (Table 1). Paired swabs were collected during new respiratory illnesses as follows: 18 sets (deep nasal vs foam NS with saline) collected at clinic visits and 43 sets (foam NS with and without saline) collected at home (7 participants with a single home collection and 18 with 2 home collections). Study samples thus included a total of 61 swab sets collected during new respiratory illnesses, representing a total of 122 swabs available for PCR testing. For the 43 home collections, 27 (63%) swab sets were collected by the parent, 14 (33%) by the participant, and 2 by another adult individual. The mean age of participants who performed self-collection was older than that of participants who had samples collected by someone else (15.7 vs 10.3 years, respectively). Table 1. Baseline Characteristics of the Study Population

collected by the parent, 14 (33%) by the participant, and 2 by another adult individual. The mean age of participants who performed self-collection was older than that of participants who had samples collected by someone else (15.7 vs 10.3 years, respectively). Table 1. Baseline Characteristics of the Study Population All Enrolled (N = 35) n (%) Samples Collected (N = 28) n (%) Sex Male 16 (45.7) 12 (42.9) Female 19 (54.3) 16 (57.1) Race/ethnicity Caucasian (not Hispanic) 34 (97.1) 28 (100) Hispanic 1 (2.9) – Genotype Homozygous dF508 23 (65.7) 18 (64.3) Heterozygous dF508 11 (31.4) 9 (32.1) Other 1 (2.9) 1 (3.6) Pancreatic status Sufficient 3 (8.6) 3 (10.7) Insufficient 32 (91.4) 25 (89.3) Mean (SD) Mean (SD) Age enrolled (years)a 11.7 (4.0) 11.3 (3.8) Sweat chloride (mEq/L)b 109.4 (19.2) 112 (19.2) Abbreviation: SD, standard deviation. aAge at enrollment ranged from 6.5 to 18.2 years among all enrolled participants and among the 28 participants with samples collected. bSweat chloride was not required if there were 2 identifiable mutations consistent with cystic fibrosis. Sweat chloride values were available for 25 enrollees, including 20 participants with samples collected.

aAge at enrollment ranged from 6.5 to 18.2 years among all enrolled participants and among the 28 participants with samples collected. bSweat chloride was not required if there were 2 identifiable mutations consistent with cystic fibrosis. Sweat chloride values were available for 25 enrollees, including 20 participants with samples collected. Respiratory Virus Detections and Symptoms Viral PCR results are presented for paired swab sets collected in clinic and at home (Table 2). Among the 122 swabs tested, 81 (66.4%) had 1 or more viruses detected, and 41 (33.6%) were negative for all viruses tested. The most prevalent finding was rhinovirus, which was detected in 59 swabs (48.4%). For swabs collected in clinic, overall percent agreement was observed for 13 of 18 pairs (72.2%), including 4 pairs with both swabs positive for the same virus and 9 pairs with both swabs negative. For NS collected at home, overall percent agreement was observed for 39 of 43 pairs (90.7%), including 31 pairs with both swabs positive for the same virus and 8 pairs with both swabs negative (2 swab pairs with a virus detected by dry swab alone, and 2 with an additional virus detected by dry swab but not by saline swab, were not counted as exact agreements). Table 2. Viral Polymerase Chain Reaction Results for Respiratory Swab Pairs Collected in Clinic and At Home

virus and 8 pairs with both swabs negative (2 swab pairs with a virus detected by dry swab alone, and 2 with an additional virus detected by dry swab but not by saline swab, were not counted as exact agreements). Table 2. Viral Polymerase Chain Reaction Results for Respiratory Swab Pairs Collected in Clinic and At Home Clinic Visitsa Home Collectionsb Total Deep nasal flocked swab (n = 18) Saline foam swab (n = 18) Dry foam swab (n = 43 ) Saline foam swab (n = 43) All swab types (n = 122) No viruses detected 10 (55.6) 13 (72.2) 8 (18.6) 10 (23.3) 41 (33.6) Any virus detected 8 (44.4) 5 (27.8) 35 (81.4) 33 (76.7) 81 (66.4) Rhinovirus 6 (33.3) 4 (22.2) 25 (58.1) 24 (55.8) 59 (48.4) Coronavirus 3 (7.0) 3 (7.0) 6 (4.9) Rhinovirus and coronavirus 3 (7.0) 2 (4.7) 5 (4.1) Respiratory syncytial virus 2 (4.7) 2 (4.7) 4 (3.3) Parainfluenza type 3 1 (5.6) 1 (5.6) 2 (1.6) Parainfluenza type 4 1 (2.3) 1 (2.3) 2 (1.6) Influenza A (2009 H1N1) 1 (5.6) 1 (2.3) 2 (1.6) Influenza A (2009 H1N1) and adenovirus 1 (2.3) 1 (0.8) aAmong swab pairs collected in clinic, the following paired results were observed: 4 pairs with the same virus detected by deep nasal swab and saline foam swab, 9 pairs with both swabs negative, 4 pairs with deep nasal flocked swab positive (3 rhinovirus, 1 influenza A) and saline foam swab negative, and 1 pair with saline foam swab positive (rhinovirus) and deep nasal swab negative.

s were observed: 4 pairs with the same virus detected by deep nasal swab and saline foam swab, 9 pairs with both swabs negative, 4 pairs with deep nasal flocked swab positive (3 rhinovirus, 1 influenza A) and saline foam swab negative, and 1 pair with saline foam swab positive (rhinovirus) and deep nasal swab negative. bAmong swab pairs collected at home, the following paired results were observed: 31 pairs with the same virus detected by dry foam swab and saline foam swab, 8 pairs with both swabs negative, 2 pairs with dry foam swab positive (rhinovirus) and saline foam swab negative, and 2 pairs with dry foam swab detecting an additional virus not detected by saline foam swab (influenza A by both swabs and adenovirus by dry swab only; rhinovirus by both swabs and coronavirus by dry swab only).

with both swabs negative, 2 pairs with dry foam swab positive (rhinovirus) and saline foam swab negative, and 2 pairs with dry foam swab detecting an additional virus not detected by saline foam swab (influenza A by both swabs and adenovirus by dry swab only; rhinovirus by both swabs and coronavirus by dry swab only). Among the 59 samples positive for rhinovirus alone, the amount of virus did not differ between swab pairs collected at the same time: mean CT value (95% CI) was 31.0 (27.1, 35.0) and 30.9 (25.2, 36.7) for 6 clinic-collected deep NS and 4 saline NS, respectively, and 28.0 (25.5, 30.5) and 26.3 (25.4, 28.1) for 25 home-collected dry and 24 home-collected saline NS, respectively. The difference in mean PCR CT values was –3.8 comparing home versus clinic collections (95% CI –6.8, –0.9; P = .014), indicating that CT values were significantly lower on average (ie, more virus present) for swabs collected at home. Comparing only wet self-collected NS, the difference in mean CT values for 24 home versus 4 clinic collections was −4.6, 95% CI (−8.1, −1.1; P = .013), indicating higher viral load on average for swabs collected at home. The time from collection to laboratory processing averaged 1.1 day (SD = 0.9) for swab sets collected in clinic, 5.4 days (SD = 3.6) for the first home collection, and 6.4 days (SD = 4.0) for the second home collection. There was no association between longer time from collection to laboratory processing and likelihood of negative results by viral PCR.

tory processing averaged 1.1 day (SD = 0.9) for swab sets collected in clinic, 5.4 days (SD = 3.6) for the first home collection, and 6.4 days (SD = 4.0) for the second home collection. There was no association between longer time from collection to laboratory processing and likelihood of negative results by viral PCR. Symptom surveys were summarized according to timing of sample collection (Table 3). The most common symptoms reported at clinic collections were increased nasal congestion, increased cough, and increased sputum production; increased nasal congestion, sore throat, and increased cough were the most common symptoms reported at home collections. The mean difference in number of symptoms was 1.1 comparing home versus clinic collections (95% CI −0.1, 2.4; P = .08), but this result was not statistically significant. The mean difference in days with increased symptoms was −2.3 comparing home versus clinic collections (95% CI −3.5, −1.2; P < .001), indicating significantly shorter duration of symptoms at the time of collection for swabs collected at home. Table 3. Symptoms Reported at the Time of Swab Collections

ically significant. The mean difference in days with increased symptoms was −2.3 comparing home versus clinic collections (95% CI −3.5, −1.2; P < .001), indicating significantly shorter duration of symptoms at the time of collection for swabs collected at home. Table 3. Symptoms Reported at the Time of Swab Collections Symptom Clinic Visits (18 surveys) n (%) Home Collection #1 (25 surveys) n (%) Home Collection #2 (18 surveys) n (%) Fever 1 (5.6) 5 (20.0) 5 (27.8) Chills/rigors – 3 (12.0) 3 (16.7) Decreased appetite 1 (5.6) 4 (16.0) 4 (22.2) Muscle aches 3 (16.7) 2 (8.0) 3 (16.7) Headache 1 (5.6) 7 (28.0) 10 (55.6) Increased nasal congestion 17 (94.4) 24 (96.0) 17 (94.4) Sore throat 3 (16.7) 14 (56.0) 11 (61.1) Increased cough 13 (72.2) 13 (52.0) 9 (50.0) Increased sputum production 9 (50.0) 9 (36.0) 7 (38.9) Change in sputum appearance 2 (11.1) 4 (16.0) 5 (27.8) Wheezing 1 (5.6) 1 (4.0) 5 (27.8) Shortness of breath 1 (5.6) 2 (8.0) 2 (11.1) Increased chest congestion 4 (22.2) 4 (16.0) 4 (22.2) Chest pain 2 (11.1) 0 (0.0) 0 (0.0) Increased fatigue 2 (11.1) 7 (28.0) 7 (38.9) Mean (SD) Mean (SD) Mean (SD) Total No. of symptoms reported 3.3 (2.0) 4.0 (1.8) 5.1 (2.6) No. of days with new or increased symptoms 5.3 (2.5) 2.4 (1.9) 3.9 (2.9) Abbreviation: SD, standard deviation.

congestion 4 (22.2) 4 (16.0) 4 (22.2) Chest pain 2 (11.1) 0 (0.0) 0 (0.0) Increased fatigue 2 (11.1) 7 (28.0) 7 (38.9) Mean (SD) Mean (SD) Mean (SD) Total No. of symptoms reported 3.3 (2.0) 4.0 (1.8) 5.1 (2.6) No. of days with new or increased symptoms 5.3 (2.5) 2.4 (1.9) 3.9 (2.9) Abbreviation: SD, standard deviation. Feasibility and Safety Tolerability surveys were completed by all 28 participants on 59 occasions (16 clinic visits and 43 home collections) and indicated that self-collection of anterior nasal foam swabs was acceptable and not difficult for participants. Questions were answered using a 5-point response scale (strongly agree, agree, neither, disagree, or strongly disagree). Using results from the first tolerability survey completed by each participant, we found that participants regarded that collection of anterior nasal foam swabs was comfortable (71% agree or strongly agree responses, 11% neither agree nor disagree, and 18% disagree responses). Among the 18% (5 subjects) who disagreed that the procedure was comfortable, specific comments included that the swab was “large and uncomfortable” and that self-collection was “hard to do” when not feeling well. Subjects thought that self-collection was simple (96% agree or strongly agree responses) and that instructions were clear and easy to follow. The majority of participants indicated willingness to participate in future studies using the self-collection procedure (85% agree or strongly agree responses). Restricting to responses obtained at the 16 clinic visits, participants indicated that the procedure for collection of anterior nasal foam swabs was preferred over collection of the deep nasal flocked swab (94% agree or strongly agree responses).

ng the self-collection procedure (85% agree or strongly agree responses). Restricting to responses obtained at the 16 clinic visits, participants indicated that the procedure for collection of anterior nasal foam swabs was preferred over collection of the deep nasal flocked swab (94% agree or strongly agree responses). Adverse events were minimal. One episode of mild and self-limited epistaxis occurred following a clinic swab collection. In 5 home collections, 3 participants reported cough, 1 reported sneezing, and 1 subject reported “blood smear on sample.”

ng the self-collection procedure (85% agree or strongly agree responses). Restricting to responses obtained at the 16 clinic visits, participants indicated that the procedure for collection of anterior nasal foam swabs was preferred over collection of the deep nasal flocked swab (94% agree or strongly agree responses). Adverse events were minimal. One episode of mild and self-limited epistaxis occurred following a clinic swab collection. In 5 home collections, 3 participants reported cough, 1 reported sneezing, and 1 subject reported “blood smear on sample.” DISCUSSION In this pilot study, we demonstrated that self- and parent-collection of foam NS at home and mailing to the laboratory for respiratory virus diagnosis was feasible for patients with CF. This method of home collection was simple, comfortable, and safe, and mailing time did not affect the likelihood of virus detection. Swabs collected at home yielded a higher proportion of positive virus detections compared with swabs collected in clinic (81% vs 47%), likely explained by our finding that home-collected NS were collected significantly closer to the onset of illness. Similarly, rates of virus positivity in the home-collected NS were higher when compared with other recent clinic- or hospital-based studies of respiratory virus surveillance using PCR in CF patients with respiratory symptoms or pulmonary exacerbations, with reported detection rates of 50%–60% [8, 12–14]. Likewise, we found that among PCR-positive swabs for rhinovirus, the amount of virus in swabs collected at home was significantly greater than for swabs collected in clinic.

s surveillance using PCR in CF patients with respiratory symptoms or pulmonary exacerbations, with reported detection rates of 50%–60% [8, 12–14]. Likewise, we found that among PCR-positive swabs for rhinovirus, the amount of virus in swabs collected at home was significantly greater than for swabs collected in clinic. In this study among children with CF, the self-collection method was feasible using foam swabs collected with saline or dry swabs with comparable rates of viral detection. A recent manuscript showed that self-collected foam NS following the use of nasal saline spray had increased sensitivity over dry swabs for detection of respiratory viruses [25]. These results were in a mostly adult population, whereas the mean age in the current study was 11.7 years. It is likely that children shed high quantities of respiratory virus, and larger studies are needed to evaluate whether the use of saline spray is essential to the self-collection procedure in children.

uses [25]. These results were in a mostly adult population, whereas the mean age in the current study was 11.7 years. It is likely that children shed high quantities of respiratory virus, and larger studies are needed to evaluate whether the use of saline spray is essential to the self-collection procedure in children. There are few other studies that have used self-sampling and mailing to study respiratory viral pathogens, although use of self-collected swabs mailed via the postal service in the United Kingdom has been reported to be a feasible method of enhancing community-based syndromic surveillance for influenza [20, 32]. In these studies, viral transport medium was required for shipping, and there were no significant differences in mean times from swabbing to laboratory analysis between positive and negative samples. Our approach allows for samples to be mailed in dry tubes, simplifying the procedure and eliminating the risk of spilling the transport diluent, as previously reported [32].

for shipping, and there were no significant differences in mean times from swabbing to laboratory analysis between positive and negative samples. Our approach allows for samples to be mailed in dry tubes, simplifying the procedure and eliminating the risk of spilling the transport diluent, as previously reported [32]. Because of the small sample size of our study and limited diversity of virus types identified, we are unable to make conclusions regarding the sensitivity of the various swab methods used for collection, especially for viruses other than rhinovirus. The high proportion of swabs positive for rhinovirus may simply reflect the high frequency of rhinoviruses detected in nonmedically attended acute respiratory illness. However, it is important to note that the home-collected swabs did detect a variety of important respiratory viruses, in addition to rhinovirus. Although it is has been documented that children with CF shed respiratory viruses even in the absence of symptoms [2, 8, 14], the increased detection of viruses in home-collected samples in the setting of new respiratory illness suggests that this method is useful to detect incident viral infections.

ion to rhinovirus. Although it is has been documented that children with CF shed respiratory viruses even in the absence of symptoms [2, 8, 14], the increased detection of viruses in home-collected samples in the setting of new respiratory illness suggests that this method is useful to detect incident viral infections. Because the majority of the virus detections were rhinovirus, and because the more than 100 rhinovirus serotypes do not amplify with the same efficiency using our RT-PCR assay, quantitative RT-PCR testing was not performed. Using RT-PCR CT values as a semiquantitative evaluation of viral load for rhinoviruses detected, we found a difference in viral load between swabs collected in clinic versus home. The nearly 3.8 CT difference between mean CT values likely represents at least a 10-fold greater viral load in home-collected compared with clinic-collected swabs.

as a semiquantitative evaluation of viral load for rhinoviruses detected, we found a difference in viral load between swabs collected in clinic versus home. The nearly 3.8 CT difference between mean CT values likely represents at least a 10-fold greater viral load in home-collected compared with clinic-collected swabs. In summary, these results provide a unique method for larger studies of respiratory virus shedding and transmission among children with CF. Studies involving home self-collection of respiratory samples will provide more accurate data on the incidence of respiratory virus infections among children with CF and will help to elucidate the role of viral infections in chronic bacterial colonization and pulmonary function decline in CF lung disease. Because CF patients are generally followed at quarterly clinic visits, home sample collection could provide critical interim data regarding respiratory virus infections that might otherwise go undocumented. Home collection of samples could also prove useful for clinical management of patients with CF, providing more prompt recognition of these infections and potentially decreasing unnecessary or prolonged antibiotic use. Thus, we recommend this approach for future studies of respiratory illness and pulmonary exacerbations in patients with CF.

mples could also prove useful for clinical management of patients with CF, providing more prompt recognition of these infections and potentially decreasing unnecessary or prolonged antibiotic use. Thus, we recommend this approach for future studies of respiratory illness and pulmonary exacerbations in patients with CF. Supplementary Data Supplementary materials are available at the Journal of the Pediatric Infectious Diseases Society online (http://jpids.oxfordjournals.org). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author. Acknowledgments We thank Janine Jijina, Libby Brockman, Alicia Stone-Zipse, and Linnea Brody for study coordination and assistance; and Nancy Wright, Reggie Sampoleo, and Rohit Shankar for laboratory expertise. Financial support. This work was supported by the Firland Foundation and the Center for Clinical and Translational Research/Institute of Translational Health Sciences at the University of Washington. A. P. C. also received support from the National Institutes of Health (L40 AI071572) and the Seattle Children's Center for Clinical and Translational Research and the Clinical and Translational Science Award program (Grant ULI RR025014). Potential conflicts of interest. All authors: No reported conflicts.

Financial support. This work was supported by the Firland Foundation and the Center for Clinical and Translational Research/Institute of Translational Health Sciences at the University of Washington. A. P. C. also received support from the National Institutes of Health (L40 AI071572) and the Seattle Children's Center for Clinical and Translational Research and the Clinical and Translational Science Award program (Grant ULI RR025014). Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

BACKGROUND Human metapneumovirus (hMPV) is a paramyxovirus first identified in 2001 as a pathogen associated with upper and lower respiratory tract infections in children [1]. Seroprevalence studies show that almost all children acquire infection by 5 years of age, and that frequent reinfections occur through life [2, 3]. The prevalence of hMPV infection among hospitalized children with acute respiratory infection or fever is similar to that of other respiratory viruses. Clinical disease most closely resembles that associated with respiratory syncytial virus infection (RSV), a related paramyxovirus [4]. A prospective cohort study of hMPV revealed a 9.4% seroprevalence in children hospitalized with respiratory tract infections, with 63% of patients requiring supplemental oxygen and 3% requiring intensive care unit (ICU) admission [5]. Lower respiratory tract illnesses (LRTI) caused by hMPV includes bronchiolitis, pneumonia, and croup. hMPV is also associated with acute otitis media, as well as more rarely conjunctivitis, gastroenteritis, and rash [6, 7]. In adults with hematologic malignancy, hMPV is associated with high rates of progression from upper to lower respiratory tract disease and substantial mortality [8, 9].

olitis, pneumonia, and croup. hMPV is also associated with acute otitis media, as well as more rarely conjunctivitis, gastroenteritis, and rash [6, 7]. In adults with hematologic malignancy, hMPV is associated with high rates of progression from upper to lower respiratory tract disease and substantial mortality [8, 9]. The treatment of hMPV is mainly supportive. Animal data support the use of bronchodilators and corticosteroids, but no controlled trials have been done to assess their efficacy in human populations [10]. Ribavirin is a nucleoside analogue shown in in-vitro studies to have activity against hMPV [11]. Ribavirin is approved for use in treatment of respiratory syncytial virus (RSV), and is often used in combination with intravenous immunoglobulin (IVIG) in severely immunocompromised individuals. Few prior descriptions of the clinical management and treatment of hMPV respiratory tract infections in a pediatric immunocompromised population, including children with leukemia and solid organ transplant recipients, are available. We describe hMPV respiratory tract infections in 55 pediatric immunocompromised patients at Seattle Children’s Hospital, reporting on their clinical presentation, management, and outcomes.

in a pediatric immunocompromised population, including children with leukemia and solid organ transplant recipients, are available. We describe hMPV respiratory tract infections in 55 pediatric immunocompromised patients at Seattle Children’s Hospital, reporting on their clinical presentation, management, and outcomes. METHODS Screening of all positive laboratory results for hMPV by both direct fluorescent antibody (DFA) and real-time reverse-transcriptase polymerase chain reaction (RT-qPCR) was performed to identify patients between the ages of newborn to 19 years who were diagnosed at Seattle Children’s Hospital in Seattle, WA during the years 2006–2010. Of 368 total patients with positive hMPV laboratory results, we performed retrospective chart review to identify a subset of 55 patients with immunocompromised conditions. We defined an immunocompromised condition as presence of hematologic malignancy, solid tumor, rheumatologic disease receiving immunosuppressive therapy, solid organ transplant, primary immunodeficiency, receipt of chronic immunosuppressive therapy, or receipt of a hematopoietic stem cell transplant (HSCT). Using electronic chart review, we abstracted sociodemographic variables, symptoms, laboratory and radiologic values, clinical course, and treatment outcomes for these patients.

transplant, primary immunodeficiency, receipt of chronic immunosuppressive therapy, or receipt of a hematopoietic stem cell transplant (HSCT). Using electronic chart review, we abstracted sociodemographic variables, symptoms, laboratory and radiologic values, clinical course, and treatment outcomes for these patients. Respiratory specimens were obtained by nasal washes for patients with suspected respiratory viral infections by attending physicians, or from bronchoalveolar lavage fluid (BAL) when this was performed. DFA was performed using virus-specific mouse monoclonal antibodies (Chemicon, Temecula, CA), and RT-qPCR was performed using previously published methods at the University of Washington Virology Laboratories [12]. Viral load values were obtained on a subset of patients whose samples were tested by RT-qPCR. Estimated viral load data were calculated from cycle threshold values on RT-qPCR analysis using stored standard curve data for hMPV. Upper respiratory tract infection (URTI) was defined as hMPV documented in an upper respiratory tract specimen in a patient with compatible symptoms in the absence of radiographic or clinical evidence of pneumonia. LRTI was defined as a new pulmonary infiltrate or presence of lower respiratory tract symptoms (wheezing or hypoxia) in association with a positive lower respiratory tract specimen or a positive upper respiratory tract specimen if the patient did not undergo BAL. Neutropenia was defined as absolute neutrophil count <1000 cells/mL. Lymphopenia was defined as lymphocyte count <300 cells/mL (not age-adjusted), and severe lymphopenia as lymphocyte count <100 cells/mL.

lower respiratory tract specimen or a positive upper respiratory tract specimen if the patient did not undergo BAL. Neutropenia was defined as absolute neutrophil count <1000 cells/mL. Lymphopenia was defined as lymphocyte count <300 cells/mL (not age-adjusted), and severe lymphopenia as lymphocyte count <100 cells/mL. Infections were defined as nosocomial if the patient was hospitalized within 7 days of diagnosis, and otherwise classified as community acquired. Severe disease was defined as requiring ICU stay and/or use of supplemental oxygen ≥FiO2 0.28. Mortality attributable to hMPV was defined as death due to respiratory failure from pneumonia during the hospitalization where hMPV was diagnosed, and hMPV was considered a contributor to lung injury. Data were entered into a password-protected Excel spreadsheet, and analyzed using Stata 11.0 (STATA Corp, College Station, TX). Comparison of clinical characteristics was performed using Fisher’s exact tests for categorical variables and t tests with unequal variance for continuous variables. This study was approved by the Seattle Children’s Hospital IRB.

tected Excel spreadsheet, and analyzed using Stata 11.0 (STATA Corp, College Station, TX). Comparison of clinical characteristics was performed using Fisher’s exact tests for categorical variables and t tests with unequal variance for continuous variables. This study was approved by the Seattle Children’s Hospital IRB. RESULTS Clinical Characteristics Overall, 55 hMPV-infected immunocompromised patients were identified from May 29, 2006 to March 26, 2010. The characteristics of these patients are listed in Table 1. The median age was 5 years (range, 5 months–19 years), and the majority had a hematologic malignancy as an underlying condition. Of these, most had acute lymphoblastic leukemia (ALL) (n = 21; 88%). The nine solid tumors included Wilms tumor (n = 3; 33%), osteosarcoma (n = 1; 11%), and ovarian cancer (n = 1; 11%). Nine (16%) patients were undergoing HSCT. Indications for transplant included aplastic anemia (n = 4; 44%) and severe combined immunodeficiency (SCID) (n = 2; 22%). Eight (15%) patients were solid organ transplant recipients, and all were receiving immunosuppressive therapy. Eight (15%) hMPV infections were acquired nosocomially, while 46 (85%) were community acquired. Of these 46 cases, 24 (52%) patients were hospitalized for evaluation of their hMPV infection. Table 1. Characteristics of Immunocompromised Children With Human Metapneumovirus (HMPV) Infection. Data Are Shown as Number (n [%]) Unless Otherwise Indicated

re acquired nosocomially, while 46 (85%) were community acquired. Of these 46 cases, 24 (52%) patients were hospitalized for evaluation of their hMPV infection. Table 1. Characteristics of Immunocompromised Children With Human Metapneumovirus (HMPV) Infection. Data Are Shown as Number (n [%]) Unless Otherwise Indicated Characteristics n = 5 Age in years, median (range) 5 (0.4–19) Female sex 26 (47) Underlying condition Hematologic malignancy 24 (44) ALL 21 (88) HSCT recipient 9 (16) Pre-transplant 3 (33) Post-transplant 6 (67) Solid organ transplant 8 (15) Heart 4 (50) Kidney 1 (13) Liver 3 (38) Solid tumors 9 (16) Primary immunodeficiency 3 (5) Other 3 (5) Chemotherapy recipienta (n = 29) 29 (100) Steroid use 12 (22) Season of infection Winter (Dec–Feb) 19 (35) Spring (March–May) 32 (58) Community acquiredb 46 (85) Nosocomial 8 (15) aIn patients with hematologic malignancy or solid tumors. bDefined as outpatients at the time of hMPV diagnosis. The overall characteristics of the hMPV illness episodes are listed in Table 2. The majority of patients presented with fever (n = 44; 80%) and/or cough (n = 42; 82%). Lymphopenia was present in 7 (13%) patients at diagnosis, and neutropenia was present in 19 (35%). Of the 35 patients who presented with URTI symptoms alone, only 2 (6%) progressed to LRTI. As compared to those presenting with fever or URTI, children presenting with LRTIs did not differ by age (P = .80) or presence of fever (P = .70), cough (P = 1.00), neutropenia (P = .21), or lymphopenia (P = .09). Table 2. Characteristics of Human Metapneumovirus Illness Episodes

alone, only 2 (6%) progressed to LRTI. As compared to those presenting with fever or URTI, children presenting with LRTIs did not differ by age (P = .80) or presence of fever (P = .70), cough (P = 1.00), neutropenia (P = .21), or lymphopenia (P = .09). Table 2. Characteristics of Human Metapneumovirus Illness Episodes Number of Infections 55 Severe diseasea 8 (15) Neutropenia (ANC < 1000) 19 (35) Lymphopenia (ALC < 300) [n = 54] 7 (13) Severe lymphopenia (ALC < 100) [n = 54] 5 (9) Clinical symptoms Cough [n = 51] 42 (86) Fever 44 (80) Stage at presentation Asymptomatic 1 (2) Fever 3 (5) Upper respiratory tract infection 35 (64) Lower respiratory tract infection 16 (29) Abnormal chest imaging [n = 36] 16 (44) Median initial viral load in log10 copies/mL (range) [n = 6] 6.96 (2.75–7.22) Diagnostic modality Direct fluorescent antibody 54 (98) Polymerase chain reaction 6 (11) Co-infections 12 (22) Adenovirus 3 (5) Cytomegalovirus 3 (5) Epstein Barr virus 1 (2) Influenza B 1 (2) Parainfluenza 1(2) Human herpes virus 81 (2) Rhinovirus 1 (2) Staphylococcus 3 (7) Other 3 (7) Hospitalization required [n = 46]a 24 (52) Treatment received Ribavirin and intravenous immunoglobulin 5 (9) Ribavirin alone 2 (4) Intravenous immunoglobulin alone 2 (4) Receipt of antibiotics 22 (40) Intensive care unit stay [n = 52] 6 (12) Receipt of supplemental oxygen [n = 54] 9 (17) Severe disease [n = 52]b 12 (23) Death attributed to human metapneumovirus infection 3 (5) aOf the 46 patients who were outpatient at the time of diagnosis.

2 (4) Intravenous immunoglobulin alone 2 (4) Receipt of antibiotics 22 (40) Intensive care unit stay [n = 52] 6 (12) Receipt of supplemental oxygen [n = 54] 9 (17) Severe disease [n = 52]b 12 (23) Death attributed to human metapneumovirus infection 3 (5) aOf the 46 patients who were outpatient at the time of diagnosis. bSevere disease is defined as intensive care unit stay and/or use of supplemental oxygen (FiO2 ≥ 0.28). Samples tested included nasal wash (n = 43; 91%), BAL (n = 1; 2%), or both (n = 3; 6%). Diagnosis of hMPV was made by DFA alone in 49 (89%) patients, by PCR alone in 1 patient (2%), and by DFA and PCR in 5 (9%) patients. Twelve (23%) patients met criteria for severe disease, 6 (50%) of whom were admitted to the intensive care unit (ICU). Those with severe disease were more likely to be neutropenic (P = .02), but otherwise did not differ by age (P = .27), HSCT recipient status (P = .19), or presence of lymphopenia (P = .09). ICU stay alone was not associated with age (P = .59) or presence of neutropenia (P = .38), or lymphopenia (P = .54), though HSCT recipients were more likely to require ICU stay (P < .01).

nic (P = .02), but otherwise did not differ by age (P = .27), HSCT recipient status (P = .19), or presence of lymphopenia (P = .09). ICU stay alone was not associated with age (P = .59) or presence of neutropenia (P = .38), or lymphopenia (P = .54), though HSCT recipients were more likely to require ICU stay (P < .01). Nine (16%) patients were HSCT recipients; indications for transplant included aplastic anemia (n = 4, 44%), SCID (n = 2, 22%), Ewing sarcoma (n = 1, 11%), acute lymphoblastic leukemia (ALL) (n = 1, 11%), and osteopetrosis (n = 1, 11%). In this subset, 4 (50%) had abnormal chest imaging, and 1 (11%) required supplemental oxygen. The chest radiograph and computed tomography imaging of 3 representative patients are illustrated in Figure 1 (Patients 1, 2, and 3). Three (33%) of these patients had a diagnosis of hMPV prior to transplant, including 2 children with SCID and 1 with aplastic anemia. In general, the policy at our institution is to delay transplant if patients have respiratory tract infections; however, in these 3 cases, it was felt that due to their underlying disease and lack of functioning immune system, these patients would not be able to clear their viral infection without HSCT. Two of these transplant recipients subsequently died due to hMPV infection. Figure 1. Chest imaging findings of 3 patients. (A) Patient 1 is an 11-month-old boy with severe combined immunodeficiency (SCID) who survived and cleared human metapneumovirus (hMPV) 511 days after diagnosis. His chest computed tomography (CT) scan shows right lower lobe consolidation. (B) Patient 2 is a 5-month-old girl with SCID who died of respiratory failure secondary to hMPV pneumonia. She has bilateral lower lobe consolidation on chest CT. (C) Patient 3 is a 2-year-old boy with aplastic anemia who died of respiratory failure secondary to hMPV pneumonia. His chest CT shows airspace consolidation with air bronchograms.

nth-old girl with SCID who died of respiratory failure secondary to hMPV pneumonia. She has bilateral lower lobe consolidation on chest CT. (C) Patient 3 is a 2-year-old boy with aplastic anemia who died of respiratory failure secondary to hMPV pneumonia. His chest CT shows airspace consolidation with air bronchograms. Eight (15%) patients were solid organ transplant recipients. The median time from transplant to hMPV infection was 12 months (range, 5–57 months). None required ICU stay or supplemental oxygen. Three (50%) of the 6 who had chest imaging performed had abnormalities on chest radiograph. None received treatment with ribavirin or IVIG, and though there was 1 death, it was not attributed to hMPV infection. As compared to other immunocompromised patients, solid organ transplant recipients were no more likely to have fever (P = .19), cough (P = .62), abnormal chest imaging (P = 1.00), or to be classified as LRTI disease on initial clinical presentation (P = .35).

there was 1 death, it was not attributed to hMPV infection. As compared to other immunocompromised patients, solid organ transplant recipients were no more likely to have fever (P = .19), cough (P = .62), abnormal chest imaging (P = 1.00), or to be classified as LRTI disease on initial clinical presentation (P = .35). Virologic Characteristics of hMPV Episodes Six patients had respiratory samples collected at multiple time points. The median number of samples collected was 7 (range, 2–44 samples), and the median duration of follow-up was 37 days (range, 4–562 days). Types of samples were primarily nasal wash samples, but also included two BAL, one sinus biopsy, and one lung biopsy sample in Patient 1, as well as 1 BAL sample in Patient 2. Median initial viral load was estimated as 6.96 log10 copies/mL (range, 2.75–7.22 log10 copies/mL). The viral load data for all 6 patients are illustrated in Figures 2A (Patient 1) and 2B (Patients 2–6). In the 3 patients who died, the median time between initial and final sample was 7 days (range, 2–20 days), and the median change in viral load was ­1.31 log10 copies/mL (range, −6.23 to 0.36 log10 copies/mL). For the 3 patients who survived, the median time to viral clearance was 43 days (range, 4–511 days). Figure 2. (A) Viral loads in Patient 1 (11-month-old with severe combined immunodeficiency) over time. IVIG, intravenous immunoglobulin; HSCT, hematopoietic stem cell transplant; hMPV, human metapneumovirus. (B) Viral loads in Patients 2 through 6 over time. ALL, acute lymphoblastic leukemia; mo, month; SCID, severe combined immunodeficiency syndrome; yo, year old.

h-old with severe combined immunodeficiency) over time. IVIG, intravenous immunoglobulin; HSCT, hematopoietic stem cell transplant; hMPV, human metapneumovirus. (B) Viral loads in Patients 2 through 6 over time. ALL, acute lymphoblastic leukemia; mo, month; SCID, severe combined immunodeficiency syndrome; yo, year old. Treatment of hMPV Nine patients were treated for their hMPV infections (median age 9 years; range, 5 months–19 years). These patients included 5 HSCT recipients, 2 patients with hematologic malignancy, and 2 patients with solid tumors. Indications for HSCT included aplastic anemia (n = 2, 22%), SCID (n = 2, 22%), and Ewing sarcoma (n = 1, 11%). Eight (89%) of these treated patients had fever, 6 (75%) had cough, and 7 (78%) had abnormal chest imaging. Five (56%) had a copathogen identified. Five (56%) were hospitalized in the ICU, and 5 (56%) received supplemental oxygen. Five (56%) patients received both ribavirin and IVIG, 2 (22%) received ribavirin alone, and 2 (22%) received IVIG alone.

tients had fever, 6 (75%) had cough, and 7 (78%) had abnormal chest imaging. Five (56%) had a copathogen identified. Five (56%) were hospitalized in the ICU, and 5 (56%) received supplemental oxygen. Five (56%) patients received both ribavirin and IVIG, 2 (22%) received ribavirin alone, and 2 (22%) received IVIG alone. Of the 5 HSCT recipients, 3 (60%) had hMPV detected prior to transplant (median time prior to transplant: 43 days; range, 93–21 days), and 1 detected 11 days after transplant. Treatment with ribavirin and IVIG was initiated a median of 5 days after hMPV diagnosis (range, 2–46 days), and several patients received multiple courses of ribavirin and IVIG. Treatment with ribavirin alone (n = 2) was initiated 2 and 4 days after diagnosis, respectively. Of the 7 patients who received ribavirin, 6 (86%) received inhaled ribavirin at a dose of 2 g 3 times daily for a 5-day course, and 1 (14%) received an 11-day course of intravenous ribavirin.

nly 1 severe case, with no related mortality [19]. Prospective studies of hMPV in adults with hematologic malignancies demonstrated that 9% of respiratory tract infections were attributed to hMPV, and that the majority of these were in HSCT recipients [13]. Among HSCT recipients, hMPV was associated with 19% mortality. Our study is one of the first to describe the presentation and management of symptomatic hMPV infection in immunocompromised children. The presentation of hMPV in pediatric solid organ transplant recipients was shown to be similar to patients with other immunocompromising conditions, with fever and cough as the most common symptoms. We also show that the majority of immunocompromised patients diagnosed with hMPV are subsequently admitted to the hospital for further evaluation, but that only 2% of those initially presenting with URTI symptoms go on to develop LRTI. However, there were no clear differences in the presentation of patients with URTI or LRTI in terms of age, symptoms (fever or cough), laboratory abnormalities, or underlying condition that would clearly identify risk factors for LRTI disease and guide decision-making. Interestingly, 2 of the 3 patient deaths were in patients with hMPV pneumonia diagnosed prior to HSCT. Both of these patients, however, had underlying diseases (aplastic anemia and SCID), which made it extremely unlikely that they would be able to clear their viral infection without reconstitution of their immune system through HSCT. These patients both underwent HSCT, and subsequently died of hMPV pneumonia despite use of ribavirin and IVIG and supportive care including mechanical ventilation and ICU stay. These 2 patients continued to have detectable virus within days of death.

infection without reconstitution of their immune system through HSCT. These patients both underwent HSCT, and subsequently died of hMPV pneumonia despite use of ribavirin and IVIG and supportive care including mechanical ventilation and ICU stay. These 2 patients continued to have detectable virus within days of death. At our institution as well as other transplant centers, all pediatric HSCT recipients are screened by respiratory viral PCR prior to transplant. Pretransplant screening for RSV and delay of transplant in adults is effective in reducing rates of pneumonia [20]. Our policy in pediatric patients is to delay transplant if patients have evidence of infection with RSV, influenza, adenovirus, or hMPV, even if patients are asymptomatic. Transplantation is not generally delayed for asymptomatic shedding of rhinovirus, bocavirus, or coronavirus. This study further enforces the risks associated with transplantation during documented hMPV viral shedding or disease. Because infection may result in such serious disease, potential modalities to treat or at least stabilize patients while white blood cells and/or immune function can return are considered and utilized in these patients. Treatment with IVIG and ribavirin is currently being utilized at our institution for severely immunocompromised individuals [21, 22]. Among the patients who received treatment, we demonstrated a 22% mortality rate as compared to a 2% mortality rate in untreated individuals. This, however, is almost certainly a reflection of clinical decision-making to initiate treatment in individuals with more severe disease.

immunocompromised individuals [21, 22]. Among the patients who received treatment, we demonstrated a 22% mortality rate as compared to a 2% mortality rate in untreated individuals. This, however, is almost certainly a reflection of clinical decision-making to initiate treatment in individuals with more severe disease. In this retrospective study, we identified hMPV-infected immunocompromised children through screening of laboratory results from our virology laboratory. We did not screen asymptomatic children and may not have captured complete data on children who were evaluated solely as outpatients at other private clinics or hospitalized in other institutions. As the regional pediatric referral center, most immunocompromised patients are seen and followed at our institution but because of our large geographic referral area, it is possible that some children were seen elsewhere. Therefore, our study could be biased towards detection of more severe hMPV disease. Also, it is possible that a subset of earlier hMPV-positive cases were missed through use of DFA as compared to PCR, a more sensitive detection technique [12]. Furthermore, we were unable to capture all data at the time of clinical presentation, or to obtain full information regarding the clinical decision-making for the administration of ribavirin and/or IVIG. Therefore, the comparison of outcomes in patients who did and did not receive treatment is problematic, and the potential benefits of therapy are not readily evaluable. Unlike a previous hMPV study conducted in adult HSCT recipient patients in Italy [23], we did not assess hMPV shedding in asymptomatic patients and unfortunately cannot comment on rates of symptomatic versus asymptomatic shedding of hMPV in our patients. Finally, we were limited in our ability to classify disease severity based on hypoxemia, as some patients did not have oxygen saturations charted. We suspect that a higher proportion of our patients would be classified as having severe disease using criteria such as that used by Papenburg et al [5]. Although only 8 (15%) of our patients were identified as having acquired their hMPV infection nosocomially, the vast majority of the patients were seen frequently on an outpatient basis in clinic for blood draws, interventions, and chemotherapy, making it difficult to differentiate true community-acquired disease from that acquired in the hospital setting.

were identified as having acquired their hMPV infection nosocomially, the vast majority of the patients were seen frequently on an outpatient basis in clinic for blood draws, interventions, and chemotherapy, making it difficult to differentiate true community-acquired disease from that acquired in the hospital setting. A prospective randomized study of ribavirin and IVIG administration, or of newer therapeutic agents under development for treatment of paramyxovirus infections [24, 25], would be helpful given the high morbidity associated with hMPV in the immunocompromised population. In particular, a study of the use of novel agents, including fusion inhibitors [26], could be studied in this population given their potential to serve as postexposure prophylaxis in high-risk individuals. The documentation of severe hMPV disease in immunocompromised children supports previous data documenting severe and fatal hMPV disease reported in adult HSCT recipients [9, 13] and highlights the need for treatment options in this population. Acknowledgments Financial support. This work was supported by the National Institutes of Health (1K23AI103105 to H.Y.C.). Potential conflicts of interest. J.A.E. has received research support from Roche, Gilead, GlaxoSmithKline, and Chimerix and served as a consultant to GlaxoSmithKline. All other authors declare no conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

Potential conflicts of interest. J.A.E. has received research support from Roche, Gilead, GlaxoSmithKline, and Chimerix and served as a consultant to GlaxoSmithKline. All other authors declare no conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Author contributions. C.R. and J.A.E. jointly conceived the study. E.F., H.Y.C., and C.R. performed the chart review. J.K. provided the virologic data. H.Y.C. performed the data analysis and wrote the manuscript. All authors discussed the results and implications and commented on the manuscript at all stages. Prior presentation of results. These results have been presented in part at the International Respiratory Syncytial Virus Symposium in Santa Fe, New Mexico in 2012.

To the editor—Borse et al. [1] describe an economic analysis of respiratory syncytial virus (RSV) prophylaxis for infants in Alaska's Yukon-Kuskokwim Delta. The authors should be congratulated for evaluating this important question with a robust analysis. As these infants are almost entirely insured by Medicaid, we wish to clarify the net Medicaid cost of palivizumab. Based on the 2010 average wholesale price (AWP), Alaska Medicaid's published reimbursement rate, and the minimum Medicaid rebate, the authors estimated a cost of $1055 per 50 mg [1]. However, using Alaska Medicaid's reported pre-rebate expenditures for palivizumab and incorporating all components of the Medicaid rebate yields a more accurate estimate, which is substantially lower at $588 per 50 mg [2, 3]. AWP is determined by drug reference companies and does not reflect a price at which manufacturers sell products to wholesalers. The US Office of the Inspector General has described AWP-based reimbursement as “fundamentally flawed” and has recommended payment based on a single national pricing benchmark based on average drug acquisition costs [4]. Given this, a better source is wholesale acquisition cost (WAC), or the list price for a drug sold by a manufacturer to wholesalers. The 2010 WAC for 50 mg of palivizumab was $1074. For Alaska Medicaid specifically, the reported pre-rebate palivizumab expenditure for 2010–2011 was $1082 per 50 mg [2].

sition costs [4]. Given this, a better source is wholesale acquisition cost (WAC), or the list price for a drug sold by a manufacturer to wholesalers. The 2010 WAC for 50 mg of palivizumab was $1074. For Alaska Medicaid specifically, the reported pre-rebate palivizumab expenditure for 2010–2011 was $1082 per 50 mg [2]. Precise estimates of Medicaid rebates for drugs are difficult to calculate because rebate payments are not publicly available. However, they can be estimated based on the two components of the Medicaid rebate [5]. The authors note that the first component is 17.1% of average manufacturer price. However, there is a second consumer price index (CPI)–based component, which effectively precludes drug cost increases in excess of changes in CPI [5]. Based on data from 10 large states, these two rebate components were estimated to result in a 41% reduction in the 2010 palivizumab cost for Medicaid [6]. Other drugs have rebates of similar magnitude; Medicaid rebates recouped 45% of expenditures on 100 brand-name drugs in 2009 [4]. For Alaska Medicaid specifically, the estimated net 2010–2011 cost of palivizumab after rebates is $588 per 50 mg [2, 3]. This cost is considerably lower than the Borse et al. estimate and approaches one of the modeled cost-neutral thresholds of $486 [1].

ecouped 45% of expenditures on 100 brand-name drugs in 2009 [4]. For Alaska Medicaid specifically, the estimated net 2010–2011 cost of palivizumab after rebates is $588 per 50 mg [2, 3]. This cost is considerably lower than the Borse et al. estimate and approaches one of the modeled cost-neutral thresholds of $486 [1]. Lastly, the authors incorrectly state that palivizumab AWP increased by 184% from 2001–2010 (20% annually), referencing Red Book data [7, 8]. Red Book [7, 8] reports a palivizumab AWP increase of 78% from 2001 to 2010, a 7% annual increase. After Medicaid rebates, the net US cost increase was approximately 60% from 2001 to 2010, or 5.5% annually. This increase is lower than the 83% inflation rate for US inpatient hospital services from 2001 to 2010 [9]. Similarly, the cost of an RSV hospitalization among US infants increased by 89% from 2000 to 2009 [10]. In closing, we congratulate the authors on a robust analysis and hope that this additional information regarding the net Medicaid costs of palivizumab will help inform policy decisions and future research regarding RSV prophylaxis in the United States. Acknowledgments Financial support. This letter was supported by MedImmune. Potential conflicts of interest. C.A. is an employee of MedImmune, Gaithersburg, MD; K.M. is an employee of AstraZeneca, Gaithersburg, MD. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

BACKGROUND Human bocavirus (HBoV) is a parvovirus that was first discovered in 2005 [1]. To date, 4 different HBoV species, HBoV 1–4, have been reported and associated with different clinical manifestations [1–7]. The HBoV1 is primarily a respiratory virus; the other species [2–4] are more commonly related to gastrointestinal tract [7]. Acute otitis media (AOM) is one of the most common infections in children. Acute otitis media usually occurs concurrently with or just after viral upper respiratory tract infection (URI), and certain respiratory viruses, eg, respiratory syncytial virus (RSV), adenovirus, and rhinovirus have commonly been associated with AOM [8–10]. Beder et al. [11] detected HBoV in 6.3% of nasopharyngeal secretions (NPS) from children with AOM, and HBoV-DNA was also found in 2.7% of middle ear fluids (MEFs). In AOM with otorrhea, 4% of MEF samples were positive for HBoV [12]. Two studies with hospitalized patients reported that 33%–44% of children with respiratory infection and HBoV detection had AOM [4, 13]. In a recent study, HBoV1 infection, confirmed by serology, was associated with AOM in children [14].

fluids (MEFs). In AOM with otorrhea, 4% of MEF samples were positive for HBoV [12]. Two studies with hospitalized patients reported that 33%–44% of children with respiratory infection and HBoV detection had AOM [4, 13]. In a recent study, HBoV1 infection, confirmed by serology, was associated with AOM in children [14]. Human bocavirus-DNA has been detected in symptomatic and asymptomatic children [13, 15] and repeatedly from the same subjects [15]. The cause-and-effect relationship of HBoV detection in respiratory specimens and the presence of HBoV in children over time still need to be studied further. We investigated the presence of HBoV1-DNA in NPS from young children with URI to determine the role of HBoV1 in URI and AOM, and we compared the rate of AOM complicating HBoV1-URI to that of URI associated with other viruses.

ory specimens and the presence of HBoV in children over time still need to be studied further. We investigated the presence of HBoV1-DNA in NPS from young children with URI to determine the role of HBoV1 in URI and AOM, and we compared the rate of AOM complicating HBoV1-URI to that of URI associated with other viruses. STUDY DESIGN Study Design and Subjects Specimens tested for HBoV1 were archived specimens from a prospective, longitudinal study performed between January 2003 and March 2007 at the University of Texas Medical Branch (Galveston, TX) [10]. Constitutionally healthy children were enrolled at the ages of 6–35 months and followed for 1 year for occurrences of URI and AOM. The parents informed the study personnel when the child developed URI symptoms. Children were seen by a study physician and followed after URI onset for the occurrence of AOM. At each visit, otoscopic and physical examinations and tympanometry were performed. Acute otitis media was considered to have complicated URI if it occurred within 28 days of URI onset. Acute otitis media was defined as acute onset of symptoms, signs of tympanic membrane inflammation (erythema, opacification, or bugling), and the presence of fluid in the middle ear. The study was approved by the Institutional Review Board of University of Texas Medical Branch, and informed consent was obtained from the guardians of all study children.

acute onset of symptoms, signs of tympanic membrane inflammation (erythema, opacification, or bugling), and the presence of fluid in the middle ear. The study was approved by the Institutional Review Board of University of Texas Medical Branch, and informed consent was obtained from the guardians of all study children. Virologic Studies Respiratory specimens for viral studies were collected at the initial URI visit and when AOM was diagnosed. Nasal swabs were collected for viral culture and NPS for other viral studies. The NPS sample was collected by vacuum suction catheter with mucus trap, which were rinsed with 1 mL phosphate-buffered saline. The total secretion volume was recorded to provide the dilution factor of the original sample.

AOM was diagnosed. Nasal swabs were collected for viral culture and NPS for other viral studies. The NPS sample was collected by vacuum suction catheter with mucus trap, which were rinsed with 1 mL phosphate-buffered saline. The total secretion volume was recorded to provide the dilution factor of the original sample. Respiratory specimens were collected during 864 URI episodes in the original study [10]. The NPS samples collected during RSV season were also analyzed for RSV antigen detection by enzyme immunoassay (EIA). Culture and RSV-EIA-negative samples were tested by real-time polymerase chain reaction (PCR) for adenovirus, enterovirus, rhinovirus, and coronavirus (OC43, 229E, and NL63) and by microarray PCR for RSV A and B, parainfluenza viruses 1–3, and influenza viruses A and B, performed at the Medical College of Wisconsin (Milwaukee, WI). The HBoV1 and human metapneumovirus (hMPV) were not targeted in the assay performed and reported in the original study. Specific to this report, 707 frozen archived NPS specimens were available for testing by quantitative PCR (qPCR) for hMPV, HBoV, and RSV; these represent 81% of the URI samples collected for viral studies. All of the results from previous and new virological analysis of these 707 samples were included in this study.

y. Specific to this report, 707 frozen archived NPS specimens were available for testing by quantitative PCR (qPCR) for hMPV, HBoV, and RSV; these represent 81% of the URI samples collected for viral studies. All of the results from previous and new virological analysis of these 707 samples were included in this study. Nucleic acid extraction and qPCR were performed in a “clean room” facility within the Galveston National Laboratory (University of Texas Medical Branch) using MagMAX Total Nucleic Acid isolation kits (Ambion/Applied Biosystems, Austin, TX) and a Biosprint 96 (QIAGEN, Valencia, CA). A customized script directed the extraction protocol to optimally recover RNA and DNA from each sample. After extraction, the elution volume (200 µL) was diluted 1:1 with nuclease-free 0.1 mM EDTA (Ambion/Applied Biosystems, Austin, TX) and distributed to daughter plates for subsequent analyses.

(QIAGEN, Valencia, CA). A customized script directed the extraction protocol to optimally recover RNA and DNA from each sample. After extraction, the elution volume (200 µL) was diluted 1:1 with nuclease-free 0.1 mM EDTA (Ambion/Applied Biosystems, Austin, TX) and distributed to daughter plates for subsequent analyses. DNA from each sample was evaluated using a duplex qPCR assay with primers that amplified targets within the HBoV1 NS-1 region [16] and human glyceraldehyde 3-phosphate dehydrogenase (hGAPDH) [17]. Glyceraldehyde 3-phosphate dehydrogenase was used as an indicator of sample integrity and extracted nucleic acid quality. TaqMan probes were used to track the specific amplification in the duplex. Quantitative PCR was completed in a C1000 thermocycler equipped with a CFX reaction module (Bio-Rad) using the following parameters: Cycle 1: 95°C (3 minutes); Cycle 2, Step 1: 95°C (15 seconds), Step 2: 60°C (45 seconds), repeated 50 times. Fluorescent signal data were collected at the end of each annealing and extension step. Viral genomic titers were extrapolated from standard curves of plasmids harbouring the PCR targets generated in parallel for each run. A parallel qPCR reaction for GAPDH [17] was completed on every clinical sample to evaluate RNA and cDNA quality. Detection of hGAPDH at less than 500 copies/reaction from the NPS sample suggested inadequacy of the specimen and led to the exclusion of the sample from further analysis. Viral load was calculated as HBoV1-DNA copies/mL of the original specimen based on the original dilution of the specimens and subsequent assay dilutions. Due to unknown dilution of 58 NPS samples, viral load analysis was available in NPSs from 649 URI episodes.

led to the exclusion of the sample from further analysis. Viral load was calculated as HBoV1-DNA copies/mL of the original specimen based on the original dilution of the specimens and subsequent assay dilutions. Due to unknown dilution of 58 NPS samples, viral load analysis was available in NPSs from 649 URI episodes. In addition to HBoV1 qPCR, duplex hMPV-RSV-qPCR was also performed; hMPV data have been reported previously [18]. Virology results reported here combined data from qPCR and data from the original study [10]. For RSV, previous PCR data were performed only in culture-negative and EIA-negative samples by microarray, which is less sensitive than real-time PCR [19]. As a result, new detection of RSV was made by qPCR in 71 URI episodes, including 38 episodes with RSV as the single virus. Statistics Comparisons were made using Fisher's exact test for 2 × 2 tables and t tests. For viral load, log transformations were applied to make standard deviations comparable. To compare number of URI episodes, Poisson regression with a categorical predictor was used and assessed for goodness-of-fit. All calculations were carried out in R, a software environment for statistical computing and graphics (http://cran.r-project.org).

formations were applied to make standard deviations comparable. To compare number of URI episodes, Poisson regression with a categorical predictor was used and assessed for goodness-of-fit. All calculations were carried out in R, a software environment for statistical computing and graphics (http://cran.r-project.org). RESULTS The HBoV1 testing was performed in 707 NPS samples collected during URI from 201 children. Demographic characteristics and risk factor data are presented in Table 1, which also compares data from children who had 1 or more URI episode with HBoV1 detection with those without. Children with HBoV1 infection had significantly more frequent URI episodes, compared with children without HBoV1 infection; they also have more URI episodes associated with multiple viruses. Of 94 children with HBoV1 infection, HBoV1 was detected more than once in 42 (45%) children during the year. In 23 children, HBoV1 was detected twice: in 10 children, 3 times; in 5 children, 4 times; in 2 children, 5 times; in 1 child, 6 times; and 1 child had 8 HBoV-positive URI episodes during the study period. The pattern of multiple HBoV1 detections varied in the 42 children: multiple detection in consecutive samples, which suggests persistence (n = 23); 2 or more HBoV1 detections with at least 1 negative detection in between, which suggests either reinfection or periodic shedding (n = 19). The interval between HBoV1-positive URI episodes ranged from 8 to 301 days (with or without HBoV1-negative episodes in between). Table 1. Demographic and Individual Characteristics of 201 Study Children

tions with at least 1 negative detection in between, which suggests either reinfection or periodic shedding (n = 19). The interval between HBoV1-positive URI episodes ranged from 8 to 301 days (with or without HBoV1-negative episodes in between). Table 1. Demographic and Individual Characteristics of 201 Study Children Children With HBoV1 Infectiona (patients, n = 94) % Children Negative for HBoV1a (patients, n = 107) % Female 45 48 54 51 Median age at enrollment (mo) 12 (range, 6–34) 12 (range, 6–35) Number of URI episodes/child year (median)b 4.8 2.4 Number of URI episodes with ≥2 viruses/child year (median)b 2.1 0.4 URI episodes with ≥2 viruses/all URI episodesb 197 of 448 44 42 of 259 16 Race Asian 3 3 3 3 Black 28 30 29 27 Biracial 8 9 11 10 White 55 59 64 60 Ethnicity: Hispanic/Latino 37 39 52 49 Childcare arrangement Home 60 64 72 67 Home day care 7 7 9 8 Day care center 26 28 26 24 Breast feedingc 50 53 55 51 Cigarette smoke exposure 34 36 29 27 History of prior otitis media episodesb 65 69 48 45 Abbreviations: HBoV1, human bocavirus 1; URI, upper respiratory tract infection. aOne or more HBoV1-positive result(s) during 1-year study period. bSignificant difference between the groups, Fisher's exact test for 2 × 2 tables, P < .001. cAny breast feeding irrespective of the duration.

Children With HBoV1 Infectiona (patients, n = 94) % Children Negative for HBoV1a (patients, n = 107) % Female 45 48 54 51 Median age at enrollment (mo) 12 (range, 6–34) 12 (range, 6–35) Number of URI episodes/child year (median)b 4.8 2.4 Number of URI episodes with ≥2 viruses/child year (median)b 2.1 0.4 URI episodes with ≥2 viruses/all URI episodesb 197 of 448 44 42 of 259 16 Race Asian 3 3 3 3 Black 28 30 29 27 Biracial 8 9 11 10 White 55 59 64 60 Ethnicity: Hispanic/Latino 37 39 52 49 Childcare arrangement Home 60 64 72 67 Home day care 7 7 9 8 Day care center 26 28 26 24 Breast feedingc 50 53 55 51 Cigarette smoke exposure 34 36 29 27 History of prior otitis media episodesb 65 69 48 45 Abbreviations: HBoV1, human bocavirus 1; URI, upper respiratory tract infection. aOne or more HBoV1-positive result(s) during 1-year study period. bSignificant difference between the groups, Fisher's exact test for 2 × 2 tables, P < .001. cAny breast feeding irrespective of the duration. Respiratory viruses were detected in 542 (77%) NPS samples; of these, 303 (43%) contained a single virus. HBoV1 was detected in 172 (24%) URI episodes (Table 2). In 44 URI episodes (6% of all episodes), HBoV1 was the only respiratory virus detected; in 128, another virus was also detected, most often adenovirus, enterovirus, and rhinovirus (46, 40, and 36 episodes, respectively). The HBoV1 was detected year-round, with 63% between October and March. There was no significant difference in signs and symptoms at the time of sick visit between the children with HBoV1 as a single virus and the children without HBoV1 (data not shown). Table 2. Respiratory Viruses Detected During 707 Upper Respiratory Tract Infection Episodes

ar-round, with 63% between October and March. There was no significant difference in signs and symptoms at the time of sick visit between the children with HBoV1 as a single virus and the children without HBoV1 (data not shown). Table 2. Respiratory Viruses Detected During 707 Upper Respiratory Tract Infection Episodes Virus URI Episodes, n (%a) Episodes With Single Virus, n Percentage of AOM Related to Single Virus HBoV1 172 (24) 44 52% Adenovirus 164 (23) 50 48% Rhinovirus 145 (21) 59 25% RSV 105 (15) 41 44% Enterovirus 103 (15) 31 32% Coronavirus 56 (8) 11 45% hMPV 48 (7) 25 24% Parainfluenzavirus 43 (6) 26 27% Influenzavirus 31 (4) 16 31% Percentage of AOM Overall Single virus 303 (43) 37% Combined viruses 239 (34) 41% Virus-negative 165 (23) 33% Total 707b 37% Abbreviations: AOM, acute otitis media; HBoV, human bocavirus; hMPV, human metapneumovirus; RSV, respiratory syncytial virus; URI, upper respiratory tract infection. a Percentage of total number of URI episodes. bTotal number of viruses = 867.

Virus URI Episodes, n (%a) Episodes With Single Virus, n Percentage of AOM Related to Single Virus HBoV1 172 (24) 44 52% Adenovirus 164 (23) 50 48% Rhinovirus 145 (21) 59 25% RSV 105 (15) 41 44% Enterovirus 103 (15) 31 32% Coronavirus 56 (8) 11 45% hMPV 48 (7) 25 24% Parainfluenzavirus 43 (6) 26 27% Influenzavirus 31 (4) 16 31% Percentage of AOM Overall Single virus 303 (43) 37% Combined viruses 239 (34) 41% Virus-negative 165 (23) 33% Total 707b 37% Abbreviations: AOM, acute otitis media; HBoV, human bocavirus; hMPV, human metapneumovirus; RSV, respiratory syncytial virus; URI, upper respiratory tract infection. a Percentage of total number of URI episodes. bTotal number of viruses = 867. Overall, 37% of all URI episodes, 39% of virus-positive URI episodes (single or multiple viruses), and 45% of HBoV1-positive episodes (single or multiple viruses) were complicated by AOM. Of URI associated with the presence of a single virus, the rate of AOM complicating URI was 52% in HBoV1-positive episodes. The rates of AOM complicating URI for other viruses are presented in Table 2. Although presence of HBoV1 alone in the child with acute onset of URI, without other viruses detected, suggests the association between HBoV1 and acute URI symptoms, persistence of HBoV1 from previous episodes could not be ruled out. We used available longitudinal data to determine whether these children had shed HBoV1 previously. In 44 URI episodes (35 children), 15 episodes were associated with previous shedding of HBoV1 10–89 days previously. Of the remaining 29 episodes, there was no HBoV1 detected in the previous URI episode in 15; previous data were not available in 14 episodes (first URI episode in the study). The rate of AOM after URI was 52% (15 of 29) in cases without documented previous HBoV1 detection and 40% (6 of 13) in cases in which available data excluded HBoV1 persistence.

here was no HBoV1 detected in the previous URI episode in 15; previous data were not available in 14 episodes (first URI episode in the study). The rate of AOM after URI was 52% (15 of 29) in cases without documented previous HBoV1 detection and 40% (6 of 13) in cases in which available data excluded HBoV1 persistence. Viral load determination was available in samples collected during 157 of 172 (91%) episodes that were positive for HBoV1. The median HBoV1 viral load of all positive samples was 2.7 × 106 copies/mL of the original volume; the mean viral load was 2.7 × 1010 copies/mL (range, 3.2 × 103 to 1.7 × 1012 copies/mL). The HBoV1 viral load did not correlate with any URI sign or symptom or development of AOM (data not shown). DISCUSSION In this longitudinal study to determine URI and AOM occurrences in young children, we frequently detected HBoV1 alone or in combination with other viruses during URI. The unique feature of our study is the comparison of the rate of AOM complicating URI associated with single respiratory virus. We found that a high proportion of HBoV1-associated URI episodes were complicated by AOM. Our findings suggest that HBoV1 may play an important role in AOM pathogenesis, similar to other viruses such as RSV, adenoviruses, and coronaviruses [10, 20].

rate of AOM complicating URI associated with single respiratory virus. We found that a high proportion of HBoV1-associated URI episodes were complicated by AOM. Our findings suggest that HBoV1 may play an important role in AOM pathogenesis, similar to other viruses such as RSV, adenoviruses, and coronaviruses [10, 20]. The HBoV1 is now recognized as a respiratory virus [5]; it has been detected in the MEF of children with AOM [12] and active HBoV1 infection documented by serology significantly associate with AOM development [14]. However, it is known that HBoV-DNA can be detected in asymptomatic children [15] due to prolonged presence of the viral nucleic acids. Therefore, detection of HBoV1-DNA alone in the NPS might not be sufficient to document active HBoV1 infection. Unfortunately, we were unable to confirm active HBoV1 infection in our study by serology. Due to the longitudinal nature of the study and nonsevere nature of viral URI and AOM, we were not able to include blood drawing for acute and convalescent serology in our design. Nevertheless, indirect evidence exists to support the role of active HBoV1 infection in our cases with URI associated with HBoV1 alone. First, we used extensive viral diagnostic methods to detect a variety of common respiratory viruses during symptomatic URI, although we did not detect less common, newly described viruses such as polyomaviruses and parechoviruses. Negative findings for other viruses but positive finding for HBoV1 only suggested the association between HBoV1 (a recognized respiratory virus) and acute symptoms. Second, viral load in the cases of HBoV1 alone was relatively high (mean viral load, 1.8 × 1010 copies/mL of the original sample; range, 3.2 × 103 to 5.5 × 1011). Lastly, in our recently published study using NPS samples from the same cohort [21], concentrations of lactate dehydrogenase, a marker for cellular injury during inflammation, are associated with the rate of AOM complication after URI and with the presence of adenovirus, rhinovirus, and HBoV1. These findings together support the association between the presence of HBoV1 and its clinical significance during URI episodes for which no other virus was detected.

llular injury during inflammation, are associated with the rate of AOM complication after URI and with the presence of adenovirus, rhinovirus, and HBoV1. These findings together support the association between the presence of HBoV1 and its clinical significance during URI episodes for which no other virus was detected. The incidence of HBoV1 detection in our patients with URI (24%) is among the highest published; overall, the incidence of HBoV has ranged from 5% to 33% in children with respiratory infection [2–4, 15, 22–23]. The higher incidence of HBoV detection in longitudinal studies, compared with that in cross-sectional studies, is likely from prolonged presence of the virus in the respiratory tract. The same reason may also explain high frequencies of concurrent detection of HBoV with other viruses, which has been reported to occur up to 72% [13, 15, 22, 24–25]. In our study, 45% of children with HBoV1 in the NPS had the virus detected more than once. Prolonged shedding of HBoV1 in nasal secretions has been described up to 11 weeks in children attending day care [15] and up to 3 months in otitis-prone children [26]. Extended viral shedding has also been observed with other DNA-viruses; in the same study population, we studied genetic sequences of adenoviruses and found that repeated presence could be from the same viral serotype and strain detected continuously or intermittently, or different serotype or strain detected sequentially [27]. Further studies of the viral genome in children with sequential HBoV1 are required, to differentiate between prolonged presence of the viral and reinfection with different HBoV isolates. These types of studies will help elucidate the natural history and foster the understanding of the pathogenicity of HBoV1 infection.

Further studies of the viral genome in children with sequential HBoV1 are required, to differentiate between prolonged presence of the viral and reinfection with different HBoV isolates. These types of studies will help elucidate the natural history and foster the understanding of the pathogenicity of HBoV1 infection. In conclusion, HBoV1 was a common virus detected alone or in combination with other respiratory viruses during URI. Among cases of URI associated with presence of single virus, HBoV1-URI was associated with a high rate of AOM complication. Acknowledgments We thank Krystal Revai, Janak Patel, Ron L. Veselenak, Sangeeta Nair, M. Lizette Rangel, Kyralessa B. Ramirez, Syed Ahmad, Michelle Tran, Liliana Najera, Rafael Serna, Carolina Pillion, and Ying Xiong for assistance with study subjects and specimen collection and processing. We also thank Jane Kuypers and Janet Englund (University of Washington, Seattle, WA) for providing HBoV-positive controls. Financial support. This work was supported by the National Institute of Deafness and Other Communication Disorders (grant number R01 DC005841) and by the National Center for Advancing Translational Sciences (grant number UL1 TR000071), National Institutes of Health. Potential conflict of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria are causative pathogens of diarrhea, hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS), and these bacteria are also the most common cause of acute renal failure in children [1–4]. Human STEC isolates are also designated enterohemorrhagic E coli, and serotype O157:H7 is one of the predominant serotypes responsible for outbreaks worldwide [5, 6]. Enterohemorrhagic E coli O157 is the main focus for diagnostics; however, non-O157 serotypes, such as O26, O103, O111, and O145, contribute significantly to cases of HC and HUS [5]. In 2011, a large outbreak of E coli O104:H4 in Germany led to HUS in more than 800 patients and 53 deaths [7, 8]. The Stxs are divided into 2 major types, Stx1 and Stx2, and Stx2 is responsible for the most severe symptoms [9–11]. In addition, several different subtypes of each Stx have been described [12, 13], and some Stx2 subtypes seem to be associated with more severe disease [14]. In addition, the presence of the gene for the adhesion factor intimin (eaeA) is linked to disease severity [9]. The issue of STEC carriage after infection is significant, and steps should be taken to limit the spread from person-to-person. However, only limited data regarding a median duration of carriage (17–18 days) [15] are available.

esence of the gene for the adhesion factor intimin (eaeA) is linked to disease severity [9]. The issue of STEC carriage after infection is significant, and steps should be taken to limit the spread from person-to-person. However, only limited data regarding a median duration of carriage (17–18 days) [15] are available. Rapid STEC detection is important in outbreak management and patient treatment, including prompt parenteral hydration, monitoring for development of severe disease, and avoidance of antibiotics and antidiarrheal agents, which can exacerbate disease [16]. The detection of STEC by culture is challenging, and traditional culture methods detect mainly O157:H7 [17, 18]. The Centers for Disease Control and Prevention (CDC) has issued recommendations to test simultaneously for O157 and non-O157 STEC in all stool specimens from patients with acute community-acquired diarrhea [19]. This diagnostic regimen was also recently recommended in a study by Lefterova et al [20]. Proper clinical diagnosis and management of non-O157 STEC infections also depend on improved physician awareness [20]. Currently, serotype-independent polymerase chain reaction (PCR) assays are used to detect stxs and are widely used for accurate and rapid diagnostics [20, 21]. When comparing the prevalence of STEC in the Nordic countries, Sweden shows the highest rates, with the highest number of outbreaks occurring in 2005 on the west coast of the country [22]. The higher prevalence may also depend on differences in diagnostic regimens and the number of specimens that were tested for STEC.

Rapid STEC detection is important in outbreak management and patient treatment, including prompt parenteral hydration, monitoring for development of severe disease, and avoidance of antibiotics and antidiarrheal agents, which can exacerbate disease [16]. The detection of STEC by culture is challenging, and traditional culture methods detect mainly O157:H7 [17, 18]. The Centers for Disease Control and Prevention (CDC) has issued recommendations to test simultaneously for O157 and non-O157 STEC in all stool specimens from patients with acute community-acquired diarrhea [19]. This diagnostic regimen was also recently recommended in a study by Lefterova et al [20]. Proper clinical diagnosis and management of non-O157 STEC infections also depend on improved physician awareness [20]. Currently, serotype-independent polymerase chain reaction (PCR) assays are used to detect stxs and are widely used for accurate and rapid diagnostics [20, 21]. When comparing the prevalence of STEC in the Nordic countries, Sweden shows the highest rates, with the highest number of outbreaks occurring in 2005 on the west coast of the country [22]. The higher prevalence may also depend on differences in diagnostic regimens and the number of specimens that were tested for STEC. In this study, we evaluated a 10-year STEC PCR screening regimen in children with diarrhea in a Swedish county. The most common serotypes were correlated with clinical symptoms, stx type, and presence of eaeA. Furthermore, our goal was to add insights regarding stx shedding to the limited data available.

When comparing the prevalence of STEC in the Nordic countries, Sweden shows the highest rates, with the highest number of outbreaks occurring in 2005 on the west coast of the country [22]. The higher prevalence may also depend on differences in diagnostic regimens and the number of specimens that were tested for STEC. In this study, we evaluated a 10-year STEC PCR screening regimen in children with diarrhea in a Swedish county. The most common serotypes were correlated with clinical symptoms, stx type, and presence of eaeA. Furthermore, our goal was to add insights regarding stx shedding to the limited data available. MATERIALS AND METHODS Patients Our study comprised all routine diarrheal stool culture specimens from patients younger than 10 years of age (n = 10 342) from 1 May 2003 through April 2013 in the County of Jönköping, Sweden. All stool specimens were collected using swabs (Copan Diagnostics Inc., Brescia, Italy). Patients were divided in 1 group in which analyses of STEC were requested by the clinician (n = 2366) and 1 screening group (n = 7976). In addition, contact tracing around index cases was performed (n = 202). The STEC-positive patients were sampled weekly until they were negative, and the duration of stx shedding was defined as the time from the first positive sample to the first negative sample. Clinical data were collected from all patients (n = 191) who tested positive for STEC by PCR through a questionnaire and by reviewing medical records. Results from clinical chemistry analysis on peripheral blood done at the routine chemistry laboratory at Ryhov County Hospital were available from 60 children, mainly from patients who required hospitalization. Criteria for HUS included 3 primary symptoms: hemolytic anemia with fragmentocytes, low platelet count, and acute renal failure with a creatinine outside the reference range of normal for age.

emistry laboratory at Ryhov County Hospital were available from 60 children, mainly from patients who required hospitalization. Criteria for HUS included 3 primary symptoms: hemolytic anemia with fragmentocytes, low platelet count, and acute renal failure with a creatinine outside the reference range of normal for age. Shiga Toxin-Producing Escherichia coli Detection and Typing The presence of stx in diarrheal stool specimens was determined by real-time PCR on suspensions of overnight cultures on blood agar plates [21]. In total, 157 of 191 PCR-positive specimens were sent to the Karolinska University Hospital for confirmatory testing, isolation of STEC, and serotyping according to methods described by Svenungsson et al [23]. Statistical Analyses Statistical analyses were done with Statistica version 12. Fisher's exact test and Pearsons χ2 test were used when comparing proportions. Mean values were compared by Mann-Whitney U test (nonnormal distributed) and Student t test as well as Bonferroni test (normal distributed). A P value < .05 was considered statistically significant. RESULTS Stx was found in specimens from 191 patients (104 boys, 87 girls), and, of these cases, 162 (85%) were index cases. Confirmatory testing at the Karolinska University Hospital, including both stx and eaeA analysis, was performed on 157 of the total 191 specimens. In 153 (97%) of these 157 specimens, STEC was confirmed by stx detection and culture was successful in 88 (56%) cases. In 115 of 157 (73%) specimens, eaeA was detected.

ses. Confirmatory testing at the Karolinska University Hospital, including both stx and eaeA analysis, was performed on 157 of the total 191 specimens. In 153 (97%) of these 157 specimens, STEC was confirmed by stx detection and culture was successful in 88 (56%) cases. In 115 of 157 (73%) specimens, eaeA was detected. In total, 121 STEC cases were detected in the screening group, 41 in the requested group and 29 by contact tracing. The prevalence was 1.8% in the requested group and 1.5% in the screening group (P = .5), corresponding prevalence in the contact-tracing group was 14%. The numbers of children with STEC were 118 (62%), 40 (21%), and 33 (17%) in the age groups 0–3, 4–6, and 7–9 years, respectively. The annual incidence varied from 39 to 86 per 100 000 (Figure 1). Children below 10 years of age comprised 57.5% of the STEC cases in the County; however, when considering only STEC found in the requested group, children comprised approximately 20%. In comparison, culture for routine diagnostics revealed 200 cases of Campylobacter, 135 Salmonella, 76 Yersinia, and 18 Shigella, respectively. Figure 1. Annual incidence of Shiga toxin-producing Escherichia coli (STEC) in children below 10 years of age in the county of Jönköping, Sweden.

n comprised approximately 20%. In comparison, culture for routine diagnostics revealed 200 cases of Campylobacter, 135 Salmonella, 76 Yersinia, and 18 Shigella, respectively. Figure 1. Annual incidence of Shiga toxin-producing Escherichia coli (STEC) in children below 10 years of age in the county of Jönköping, Sweden. Clinical Characteristics and Laboratory Parameters Diarrhea was the most frequent symptom reported in 156 (82%) cases, and, of these, 29 (19%) had HC. Abdominal pain was the second most common symptom (34%), followed by vomiting (17%) and fever (17%). Seven children developed HUS (3.6%). Stx2 predominated in cases with HC (P < .0001) and HUS (P = .04). Hospitalization was necessary in 15 (7.8%) cases with a median length of stay of 15 days. In children with HC, elevated levels of leucocytes were detected (P < .0001). In HUS cases, elevated levels of leucocytes and creatinine were observed as well as low levels of thrombocytes (P < .0001) and erythrocyte volume fraction (P = .0001). Data Regarding stx Shedding Time, Serotypes, and Subtypes of stx Data on stx shedding time was available for 165 (86%) children, with a median duration of 20 days (1–256 days). For children with HC (n = 29), the median duration was 29 days (8–107 days); for children with uncomplicated diarrhea (n = 127), the median duration was 20 days (1–256 days) (P = .07) (Figure 2). The HUS cases had a median duration of 23 days (18–105 days). There was no difference in mean duration of stx shedding comparing stx types (P = .11) and presence of eaeA (P = .72).

9 days (8–107 days); for children with uncomplicated diarrhea (n = 127), the median duration was 20 days (1–256 days) (P = .07) (Figure 2). The HUS cases had a median duration of 23 days (18–105 days). There was no difference in mean duration of stx shedding comparing stx types (P = .11) and presence of eaeA (P = .72). In 88 cases, STEC isolation was successful, and the most common serotypes were O157 (n = 17), O26 (n = 14), O103 (n = 9), and O121 (n = 6). In addition, 19 other serotypes were found (Figure 3). In 3 of 7 HUS cases, isolation and serotyping was successful (two O121, one O157:H7), as well as in 15 of 29 HC cases (seven O157:H7, two O121, and two O103 and some singletons). Figure 2. Duration of stx shedding in children with no diarrhea (n = 35), diarrhea (n = 127), and hemorrhagic colitis (n = 29). STEC, Shiga toxin-producing Escherichia coli. Figure 3. Serotype distribution of isolated Shiga toxin-producing Escherichia coli (n = 88). The black bar illustrates non-O157 strains that we were not able to serotype further.

In 88 cases, STEC isolation was successful, and the most common serotypes were O157 (n = 17), O26 (n = 14), O103 (n = 9), and O121 (n = 6). In addition, 19 other serotypes were found (Figure 3). In 3 of 7 HUS cases, isolation and serotyping was successful (two O121, one O157:H7), as well as in 15 of 29 HC cases (seven O157:H7, two O121, and two O103 and some singletons). Figure 2. Duration of stx shedding in children with no diarrhea (n = 35), diarrhea (n = 127), and hemorrhagic colitis (n = 29). STEC, Shiga toxin-producing Escherichia coli. Figure 3. Serotype distribution of isolated Shiga toxin-producing Escherichia coli (n = 88). The black bar illustrates non-O157 strains that we were not able to serotype further. The distribution of stx types was 49%, 33%, and 18% for stx1, stx2, and stx1 + stx2, respectively. We found no differences regarding stx types, duration of stx shedding, and severity of symptoms between the screening group and the requested group. Stx1 was most frequent in serotypes O26 and O103, whereas stx2 was more frequent in O157 and O121. Serotype O157 had a higher probability of harboring eaeA (P = .004). Patients with stx2 were more often hospitalized (P ≤ .0001) and had HC (P ≤ .0001) or HUS (P = .04) compared with patients with stx1 or stx1 + stx2. eaeA showed no significant correlation with disease severity; however, HC cases showed a trend towards significance (P = .07). No significant difference in duration of stx in feces was seen between the stx types (P = .106–1.00), eaeA presence (P = .72), age groups (P = 1.00), or gender (P = .35). We found no difference between stx type and gender (P = .68) or between age groups (P = .25). In patients infected in Sweden, stx2 was more common than in patients infected abroad (P ≤ .0001).

x in feces was seen between the stx types (P = .106–1.00), eaeA presence (P = .72), age groups (P = 1.00), or gender (P = .35). We found no difference between stx type and gender (P = .68) or between age groups (P = .25). In patients infected in Sweden, stx2 was more common than in patients infected abroad (P ≤ .0001). Contact Tracing Contact tracing was performed in approximately 112 of 162 index cases, including 202 diarrheal stool culture specimens from children below 10 years of age. In this group, the STEC-positive rate was 14% (29 of 202), which was higher than in the other 2 groups (requested and screening) (each P < .00001). Source of transmission was determined at 5 occasions; in 4 cases, it was animal contact on a farm and in one case sausage. DISCUSSION In this study, we show comparable STEC prevalence and disease severity in children where the analysis was not requested and in those where STEC analysis was requested. Furthermore, we found that a high diversity of serotypes, including non-O157, caused severe disease. In addition, a great variation in the duration of stx shedding was shown, but no relation between shedding time and stx type or severity of symptoms was found.

t requested and in those where STEC analysis was requested. Furthermore, we found that a high diversity of serotypes, including non-O157, caused severe disease. In addition, a great variation in the duration of stx shedding was shown, but no relation between shedding time and stx type or severity of symptoms was found. The yearly incidence was relatively constant during the study except for 2005, which coincided with an outbreak on the west coast of Sweden [22]. The southern part of Sweden, including Jönköping, generally reports the highest annual STEC figures in the country. This may be explained by higher STEC-screening activities in these counties and more farms with a higher verotoxin-producing E coli frequency [24]. The Swedish annual STEC incidence is higher compared with other Nordic countries [25], which may be explained by differences in diagnostic regimens, screening activities, and differences in reporting of cases between countries. In Sweden, reporting of non-O157 serotypes was included in 2004, which also includes reporting based on stx detection only. One limitation of the present study is the fact that specimens were sent to another laboratory for STEC isolation, which may explain the low STEC isolation rates. However, the vast majority of stx-positive specimens were also detected by PCR at the Karolinska University Hospital, confirming our results. Because culture of non-O157 STEC is more challenging, we may have underestimated the importance of non-O157 STEC.

STEC isolation, which may explain the low STEC isolation rates. However, the vast majority of stx-positive specimens were also detected by PCR at the Karolinska University Hospital, confirming our results. Because culture of non-O157 STEC is more challenging, we may have underestimated the importance of non-O157 STEC. The majority of STEC cases (60%) were detected in the group of children between 0 and 4 years, which is in agreement with previous findings [26]. Children in this group often wear diapers and attend daycare, a combination that enhances the risk for transmission, as recently shown in an outbreak in Germany [27]. In accordance with previous findings, Stx2 was responsible for the most severe symptoms in our study as well [9-11]. In the present study, stx shedding was usually eliminated after less than 1 month; however, sometimes stx was excreted for several months (maximal duration 256 days). Proper follow-up of children is important to avoid further STEC spread and to prevent outbreaks. Furthermore, novel therapeutic strategies in the treatment for decolonization of long-term STEC carriers should be evaluated, and recent data indicate promising results for azithromycin [28].

hs (maximal duration 256 days). Proper follow-up of children is important to avoid further STEC spread and to prevent outbreaks. Furthermore, novel therapeutic strategies in the treatment for decolonization of long-term STEC carriers should be evaluated, and recent data indicate promising results for azithromycin [28]. In the present study, the majority of STEC cases were detected by screening of diarrhoeal stool culture specimens where analysis for stx was not requested. The frequent STEC detection in screening specimens underlines the lack of STEC awareness among physicians, who do not emphasize the need for STEC analysis. In recent studies, these same conclusions were reached by the CDC [19] and Lefterova et al [20]. Undetected cases of STEC infection may also be present in older individuals, and further studies are needed to determine its presence in diarrheal stool specimens of adults. Comparing culture data for routine diagnostics revealed that STEC was the second most common pathogen detected; however, because different methods are used for detection, this result should be interpreted carefully. Hence, molecular techniques can be used to detect diarrheal pathogens and affect the figures. Nevertheless, the high prevalence of STEC underlines its importance as a common diarrheal pathogen among children. The source of infection was only revealed in 5 cases, and the methods used presently in Sweden are cumbersome, focusing only on 5 serotypes in animals. Elimination of the source is crucial in outbreak situations, and better methods are needed to determine sources of infection.

In the present study, the majority of STEC cases were detected by screening of diarrhoeal stool culture specimens where analysis for stx was not requested. The frequent STEC detection in screening specimens underlines the lack of STEC awareness among physicians, who do not emphasize the need for STEC analysis. In recent studies, these same conclusions were reached by the CDC [19] and Lefterova et al [20]. Undetected cases of STEC infection may also be present in older individuals, and further studies are needed to determine its presence in diarrheal stool specimens of adults. Comparing culture data for routine diagnostics revealed that STEC was the second most common pathogen detected; however, because different methods are used for detection, this result should be interpreted carefully. Hence, molecular techniques can be used to detect diarrheal pathogens and affect the figures. Nevertheless, the high prevalence of STEC underlines its importance as a common diarrheal pathogen among children. The source of infection was only revealed in 5 cases, and the methods used presently in Sweden are cumbersome, focusing only on 5 serotypes in animals. Elimination of the source is crucial in outbreak situations, and better methods are needed to determine sources of infection. CONCLUSIONS In conclusion, most cases of STEC were found by PCR screening, with comparable prevalence and disease severity found in patients where STEC analysis was requested. Furthermore, a high diversity of serotypes (including non-O157) caused severe disease. Serotype-independent methods for STEC detection and improved physician awareness will more accurately detect the true number of infections and enhance patient safety. Prolonged shedding of STEC may be a risk factor for transmission, and therefore local guidelines for school-aged children should be reviewed to prevent further spread.

endent methods for STEC detection and improved physician awareness will more accurately detect the true number of infections and enhance patient safety. Prolonged shedding of STEC may be a risk factor for transmission, and therefore local guidelines for school-aged children should be reviewed to prevent further spread. Acknowledgments We thank the molecular diagnostic staff at the Department of Laboratory Services in County Council Jönköping, Sweden, for continuous support. Financial support. This work was supported by grants from Futurum, the Academy for Healthcare, County Council Jönköping, Sweden. Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest.

Human cytomegalovirus (HCMV) is a cause of serious disease in infancy particularly with immunosuppression from human immunodeficiency virus (HIV) [1]. Congenital HCMV, acquired in utero, is the main infectious cause of mental retardation and neurodevelopmental impairment in neonates [1]. Postnatal infection, mainly via breast milk, can cause severe morbidity in some preterm or low birthweight babies [2–10]. Human immunodeficiency virus and HCMV coinfected children have increased neurological and respiratory disease, acquired immune deficiency syndrome (AIDS) progression, and death [11–13]. In sub-Saharan Africa where HIV is endemic, maternal HCMV plasma deoxyribonucleic acid (DNA) was linked to increased mortality in both mother and child in Kenya [14]. Human cytomegalovirus secretion in milk is associated with infant HIV transmission in South Africa and Malawi [15, 16]. In maternally HIV-exposed Zambian children, who themselves remained HIV-negative, we showed that HCMV was associated with lower infant growth and psychomotor development [17]. In Zimbabwe, HCMV milk secretion was related to growth faltering in maternally HIV-exposed children [18]. West African children infected with HCMV already express the HCMV “aged” immune phenotype, present in older Europeans, which may alter immune responses to infections and vaccines [19, 20].

homotor development [17]. In Zimbabwe, HCMV milk secretion was related to growth faltering in maternally HIV-exposed children [18]. West African children infected with HCMV already express the HCMV “aged” immune phenotype, present in older Europeans, which may alter immune responses to infections and vaccines [19, 20]. Although maternal HIV exposure has been shown to increase HCMV congenital infection prevalence (from 1% to 4% [21–25]), the effects of maternal HIV on postnatal HCMV transmission via breastfeeding, the predominant route of infection, is unknown [26]. In women who are HIV-positive, a correlation has been made among milk HCMV loads, lower CD4 counts, reduced growth, and infant transmission [18, 27]. However, because comparisons have not been made to mothers who are HIV-negative, it is not known whether maternal HIV causes greater HCMV reactivation, load, or extended secretion in milk. In addition, the effects of breastfeeding duration, which varies greatly in women who are HIV-positive, are unknown. In Europe, breastfeeding of infants up to 3 months of age and raised HCMV viral load in breast milk increased risks of HCMV infection [3, 28]. In Africa, extended breastfeeding into the second year of life is common practice. In order to apply any intervention against HCMV, it is important to determine infection risk factors and their timing in HIV-positive versus HIV-negative women, especially in endemic regions of Africa where comorbidity is increased.

28]. In Africa, extended breastfeeding into the second year of life is common practice. In order to apply any intervention against HCMV, it is important to determine infection risk factors and their timing in HIV-positive versus HIV-negative women, especially in endemic regions of Africa where comorbidity is increased. In this study, we examined breast milk directly for HCMV DNA loads and genotypes. In addition, we examined the association of breastfeeding duration with HCMV infant infection risks in Zambia, where maternal HIV exposure is frequent. To our knowledge, there is no previous research comparing the effects of maternal HIV status on secretion and transmission of HCMV in breast milk. In this study, we have compared both HIV-positive and HIV-negative mothers in order to understand the effects of breastfeeding practice and HIV on secretion and transmission of HCMV. METHODS Ethical Approval This study was approved by the ethics committees of both the University of Zambia and the London School of Hygiene and Tropical Medicine, University of London.

In this study, we examined breast milk directly for HCMV DNA loads and genotypes. In addition, we examined the association of breastfeeding duration with HCMV infant infection risks in Zambia, where maternal HIV exposure is frequent. To our knowledge, there is no previous research comparing the effects of maternal HIV status on secretion and transmission of HCMV in breast milk. In this study, we have compared both HIV-positive and HIV-negative mothers in order to understand the effects of breastfeeding practice and HIV on secretion and transmission of HCMV. METHODS Ethical Approval This study was approved by the ethics committees of both the University of Zambia and the London School of Hygiene and Tropical Medicine, University of London. Study Population and Protocol Studies were conducted at Chilenje clinic in Lusaka, Zambia, in 2 cohorts: in the first cohort, studies examined extended HCMV secretion in breast milk; the second cohort focused on the physiological relevance in the longer term. Both cohorts were recruited from a similar region served by the clinic, many mothers participated in both cohorts, and there were similar antiretroviral therapy (ART) treatment protocols. At the time of the studies, local care standards included single-dose of nevirapine for HIV-positive mothers and their newborns. In the second, later cohort, ART was available to mothers with CD4 count <200 cells/μL, and towards the end of the study this was changed to <350 cells/μL, in accordance with revised National HIV treatment guidelines (Ministry of Health, Zambia). Only a few mothers in the study were on ART.

mothers and their newborns. In the second, later cohort, ART was available to mothers with CD4 count <200 cells/μL, and towards the end of the study this was changed to <350 cells/μL, in accordance with revised National HIV treatment guidelines (Ministry of Health, Zambia). Only a few mothers in the study were on ART. Breastfeeding Cohort The Breastfeeding and Postpartum Health study investigated postpartum health among 387 (198 HIV negative, 189 HIV positive) women, from 2001 to 2003 [29]. Breastfeeding was a recruitment criterion, and all women were breastfeeding exclusively or predominantly. Milk samples were collected on 11 scheduled visits during the first 16 postpartum weeks and stored at −80°C. Two hundred sixty-one milk samples (from 118 HIV-positive and 143 HIV-negative mothers), collected at postpartum week 16, were available for our study. For a subset of 40 women (20 HIV-negative and 20 HIV-positive), we also analyzed samples collected at 5 earlier time points: day 3, and weeks 2, 4, 9, and 12 postpartum. Maternal HIV serostatus was determined antenatally using a serial testing algorithm, per local care standards [28].

16, were available for our study. For a subset of 40 women (20 HIV-negative and 20 HIV-positive), we also analyzed samples collected at 5 earlier time points: day 3, and weeks 2, 4, 9, and 12 postpartum. Maternal HIV serostatus was determined antenatally using a serial testing algorithm, per local care standards [28]. Infant Cohort The Chilenje Infant Growth, Nutrition and Infection Study (CIGNIS) was a randomized double-blind controlled trial of micronutrient-fortified infant foods (ISRCTN37460449; www.controlled-trials.com/mrct) and was conducted from 2005 to 2009. Infants (n = 811) were enrolled at 6 months of age and observed for 12 months [30]. Socio-demographic information was obtained using a questionnaire at recruitment. At recruitment and monthly, mothers were asked whether they were still breastfeeding or when they stopped [31]. Infant venous blood was collected in plain vacutainers, serum was separated, and antibody was assayed for HIV and HCMV at study completion (18 months age). Human immunodeficiency virus serostatus was determined using the local Ministry of Health-approved serial testing algorithm as described previously [17]. The ETI-CYTOK-G PLUS ELISA Kit (DiaSorin) was used to test for HCMV immunoglobulin (Ig)G, with standard curves plotted using control sera provided and then used to interpolate each sample IgG titer; we considered HCMV IgG positive above a cutoff of 0.4 IU/mL.

roved serial testing algorithm as described previously [17]. The ETI-CYTOK-G PLUS ELISA Kit (DiaSorin) was used to test for HCMV immunoglobulin (Ig)G, with standard curves plotted using control sera provided and then used to interpolate each sample IgG titer; we considered HCMV IgG positive above a cutoff of 0.4 IU/mL. Deoxyribonucleic Acid Extraction, Qualitative and Quantitative Polymerase Chain Reaction Deoxyribonucleic acid was extracted from 200 µL homogenized milk using the QIAamp DNA Mini kit (QIAGEN) and eluted in 50 μL H2O. Human cytomegalovirus glycoprotein gB gene was used for qualitative polymerase chain reaction (PCR) screening and quantification, human GAPDH gene was used as internal control, and hypervariable marker HCMV glycoprotein gO (gO) was used for genotyping [17, 32, 33]. Human cytomegalovirus DNA copy numbers were computed by TaqMan real-time assay, performed in triplicate on the Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems Inc.) [32]. Standard curves were generated from 10-fold serial dilutions of plasmid-cloned amplification products (pGEM-T Easy Vector Systems, Promega), normalized with an internationally certified clinical reference standard (National Institute of Biological Standards and Control, UK Medicines and Healthcare products Regulatory Agency, Potters Bar, United Kingdom). Levels detected below the sensitivity of the virus standard were recorded at a value of half the limit of detection. Each quantitative PCR (qPCR) reaction had 10 μL KAPA PROBE FAST Universal qPCR Master Mix (Kapa Biosystems), 1 μL probe (5 mM), 1 μL each of both forward and reverse primer (10 mM), 0.4 μL ROX Low, 7 μL dH2O, and 5 μL template DNA. Cycling conditions were as follows: 95°C for 10 minutes, then 45 cycles of 95°C for 15 seconds, and 60°C for 1 minute. All PCR assays included both positive and negative controls (reagents and water-only).

be (5 mM), 1 μL each of both forward and reverse primer (10 mM), 0.4 μL ROX Low, 7 μL dH2O, and 5 μL template DNA. Cycling conditions were as follows: 95°C for 10 minutes, then 45 cycles of 95°C for 15 seconds, and 60°C for 1 minute. All PCR assays included both positive and negative controls (reagents and water-only). Genotyping Human cytomegalovirus gO (UL74 gene) genotyping was performed on a subset of 34 milk samples with sufficient remaining volume. Based on translated amino acid sequences, HCMV gO (UL74 gene) has 8 distinct genotypes: gO1a, gO1b, gO1c, gO2a, gO2b, gO3, gO4, and gO5 [32, 33]. We used PCR-based genotyping with the gOup/gOlw primers, which detected all 8 gO genotypes. Nucleotide sequences were determined using Sanger methods [32, 33] and compiled using ChromasPro software. Multiple alignments used CLUSTAL and phylogenetic analysis via maximum-likelihood methods in MEGA6 [34].

nd gO5 [32, 33]. We used PCR-based genotyping with the gOup/gOlw primers, which detected all 8 gO genotypes. Nucleotide sequences were determined using Sanger methods [32, 33] and compiled using ChromasPro software. Multiple alignments used CLUSTAL and phylogenetic analysis via maximum-likelihood methods in MEGA6 [34]. Statistical Analysis Programs Prism (version 6; GraphPad Software, Inc.) and SPSS (version 20.0; IBM Corp., Armonk, New York) were utilized to analyze milk HCMV DNA data by Student's t test and Mann–Whitney U test with 2-tailed P values and alpha = 0.05. Stata (version 11.1; StataCorp, College Station, Texas) was used in analyses of the infant cohort data, and odds ratios (ORs) and 95% confidence intervals (CIs) were obtained by logistic regression. We assessed associations of breastfeeding duration with HCMV antibody at 18 months, in a multivariable model. Analyses were adjusted for maternal education and socioeconomic status, as main effect modifiers as described [17], and stratified by maternal HIV status. To account for missing data, mainly from limited infant serum sample volumes, we also imputed missing HCMV results using multiple imputations with chained equations. The results imputed to account for missing data were similar to the main analyses (data not shown).

described [17], and stratified by maternal HIV status. To account for missing data, mainly from limited infant serum sample volumes, we also imputed missing HCMV results using multiple imputations with chained equations. The results imputed to account for missing data were similar to the main analyses (data not shown). RESULTS Increased Loads and Duration of Human Cytomegalovirus Milk Excretion in Human Immunodeficiency Virus-Positive Women Human cytomegalovirus DNA measurements were made in 405 breast milk samples from 261 mothers at week 16 postpartum and a subset of 40 mothers at multiple time points. In the longitudinal subset (Figure 1), all mothers screened were HCMV positive by qualitative assay from day 3, and the median HCMV DNA in day 3 milk (colostrum or transitional milk) was not significantly different in HIV-infected compared with HIV-uninfected mothers (3.9 and 4.1 log10 copies/mL, respectively; P = .90). Deoxyribonucleic acid lactia then increased in both groups, reaching peak levels at week 4. In the HIV-negative mothers, these loads declined gradually by week 16 to below day 3 levels, whereas in HIV-infected mothers, the DNA loads remained elevated. From week 4 to week 16, the median DNA loads had sustained increases in HIV-infected compared with the HIV-uninfected women (P < .001 by week 16), with over a 10-fold difference at peak levels recorded at week 4 (P = .026). Figure 1. Human cytomegalovirus (HCMV) shedding kinetics in milk of human immunodeficiency virus (HIV)-positive and HIV-negative women. Comparison of milk HCMV deoxyribonucleic acid (DNA) kinetics between HIV-positive (black lines) and negative women (gray lines) over the first 16 postpartum weeks (n = 40, 20 in each group). Human cytomegalovirus DNA load, log copies/mL milk, increased from comparable levels in the 2 groups from day 3 (week 1 [W1]) to peak levels by week 4 (W4) postpartum. Sensitivity cut-off is indicated by the lower dotted line. Milk DNA loads from HIV-positive women were raised compared with HIV-negative women from W4. Box plots show the median and interquartile range using a Mann–Whitney U test, **P = .026 and ***P < .001 indicate significant differences from W4 and week 16 (W16), respectively.

cut-off is indicated by the lower dotted line. Milk DNA loads from HIV-positive women were raised compared with HIV-negative women from W4. Box plots show the median and interquartile range using a Mann–Whitney U test, **P = .026 and ***P < .001 indicate significant differences from W4 and week 16 (W16), respectively. At week 16, HCMV was detected by the gB screening assay in 83.9% (99 of 118) of milk from HIV-infected women, versus 63.6% (91 of 143) in HIV-negative women (P < .001). Human cytomegalovirus DNA loads were quantified by real-time qPCR in 205 samples, all of which contained sufficient sample (with no demographic differences). Of these, there was a significantly higher proportion of HIV-infected women with DNA levels above the assay detection limit (88.0% [81 of 92] of HIV-infected versus 59.3% [67 of 113] among HIV-uninfected; P < .001). The mean DNA load was significantly higher in the HIV-infected group (7.9 × 104 copies/mL; 95% CI, 3.5 × 104 to 1.2 × 105) compared with the HIV-uninfected group (1.1 × 104 copies/mL; 95% CI, 0.6 × 104 to 1.5 ×104; P < .001) (Figure 2). Figure 2. Human cytomegalovirus (HCMV) deoxyribonucleic acid (DNA) loads in week 16 milk samples, stratified by human immunodeficiency virus (HIV) serostatus. Scatter plot showing HCMV DNA levels in all available milk samples, at week 16 (W16) (92 HIV-positive and 113 HIV-negative). Human immunodeficiency virus-positive women had a significantly higher mean milk HCMV DNA load, approaching 1 log higher, compared with their HIV-negative counterparts. Furthermore, a higher proportion of milk samples from the HIV-positive group remained with detectable HCMV at this late time point (88.0% [81 of 92] vs 59.3% [67 of 113]; P < .001), with correspondingly decreased proportions below the limit of detection, indicated by the dashed line, in the HIV-positive compared with HIV-negative group (12% [11 of 92] vs 40.7% [46 of 113]). Means were 7.9 × 104 and 1.1 × 104 copies/mL in HIV-positive and HIV-negative women, respectively (P < .001, 2 sample, 2 tailed Student's t test with 95% confidence interval indicated by error bars), with similar differences in the W16 values for the subset with the complete kinetics from Figure 1 as indicated by black circles, mean 8.2 × 104 and 0.5 × 104 copies/mL for HIV-positive compared with HIV-negative women.

P < .001, 2 sample, 2 tailed Student's t test with 95% confidence interval indicated by error bars), with similar differences in the W16 values for the subset with the complete kinetics from Figure 1 as indicated by black circles, mean 8.2 × 104 and 0.5 × 104 copies/mL for HIV-positive compared with HIV-negative women. Human Cytomegalovirus Glycoprotein O Genotypes in Breast Milk Human cytomegalovirus gO (UL74 gene) genotyping was performed in a subset of W4 (peak HCMV DNA loads) and W16 (latest time point) milk samples. This included all samples with sufficient DNA and 7 samples paired at both time points. On the basis of encoded amino acid sequences, all 8 HCMV gO distinct genotypes—gO1a, gO1b, gO1c, gO2a, gO2b, gO3, gO4, and gO5 [32, 33]—were detectable in the milk samples. These were predominantly genotypes 1a, 1b, and 5, as represented by reference strains AD169, TR, and Merlin, respectively (Figure 3); genotype prevalence differences in HIV-positive compared with HIV-negative women did not reach significance (Supplementary Table S1). There was no evidence for higher viral load with any one genotype, although all appeared raised in milk samples from HIV-positive women compared with HIV-negative women (Figure 4). Of the 7 paired samples, 3 had different genotypes detected at W4 and W16 (HIV-positive and HIV-negative). Figure 3. Phylogenetic analyses glycoprotein O (gO) genotypes. Representatives of genotype groups defined here were analyzed in comparison to reference strains, as described previously [32, 33]. Multiple alignments were performed using CLUSTAL in MEGA6 [34], followed by phylogenetic constructions inferred using the Maximum Likelihood method based on the JTT matrix-based model. The analysis involved 39 amino acid sequences and 154 positions in the final dataset. Reference strains for gO genotypes are indicated. Bootstrapping analyses indicate that major nodes are well supported.

wed by phylogenetic constructions inferred using the Maximum Likelihood method based on the JTT matrix-based model. The analysis involved 39 amino acid sequences and 154 positions in the final dataset. Reference strains for gO genotypes are indicated. Bootstrapping analyses indicate that major nodes are well supported. Figure 4. Genotype-independent increases in human cytomegalovirus (HCMV) load in milk from human immunodeficiency virus (HIV)-positive women. Viral loads per genotype were examined for HIV-positive and HIV-negative women. The main 3 glycoprotein O (gO) genotypes (gO1a, gO1b, and gO5) were plotted, and all the remaining genotypes were grouped together. All genotypes appear increased in HIV-positive women compared with HIV-negative women. Genotype gO1a (black circle), gO1b (gray circle), gO5 (white circle), other gO genotypes (crossed circle) are shown. Mean values are as follows: 2.6 × 105 and 5.9 × 104 for HIV-positive and HIV-negative women at both maximal and minimal secreted levels, at 4 and 16 weeks postpartum. Abbreviation: DNA, deoxyribonucleic acid.

ype gO1a (black circle), gO1b (gray circle), gO5 (white circle), other gO genotypes (crossed circle) are shown. Mean values are as follows: 2.6 × 105 and 5.9 × 104 for HIV-positive and HIV-negative women at both maximal and minimal secreted levels, at 4 and 16 weeks postpartum. Abbreviation: DNA, deoxyribonucleic acid. Prevalence of Infant Human Cytomegalovirus Antibody at 18 Months of Age and Breastfeeding Duration The effects of breastfeeding duration on infant HCMV infection were compared between HIV-positive and HIV-negative women in the CIGNIS infant cohort. Overall, 460 of 811 (57%) infant samples were available for HCMV antibody testing at 18 months age. Most infants were seropositive (384 of 460; 83%). As shown previously, we found no effect of micronutrient fortification (trial intervention) on HCMV antibody at 18 months, either overall or by maternal HIV status, but the prevalence of HCMV antibody significantly increased with decreasing maternal socioeconomic conditions or education and increased with longer breastfeeding duration, which were all measures adjusted in analyses of HCMV effects on growth [17]. Breastfeeding duration differed markedly between HIV-infected and HIV-uninfected women in this cohort, as shown previously [31]. Of the HCMV study subgroup analyzed here, only 3 HIV-negative women, compared with 29 HIV-positive women, never breastfed; and only an additional 6 HIV-negative women breastfed for less than 6 months. Therefore, to further analyze this result, we examined the effect of breastfeeding duration on HCMV antibody stratified by maternal HIV status. Among children of HIV-negative mothers, those who were still breastfeeding at 18 months had nearly 3 times the odds of HCMV antibody as those who had breastfed for <12 months (OR = 2.69; 95% CI, 0.84–8.59; P = .03) (Table 1). Even though it was a relatively small group who were uninfected by 18 months age, the children of HIV-positive mothers were now at significantly greater risk of early HCMV infection, as detected by antibody at 18 months, with prolonged breastfeeding (OR for breastfeeding >6 months compared with no breastfeeding =20.37; 95% CI, 3.71–111.70; P < .001) (Table 1). Table 1. Effects Maternal HIV and Breastfeeding Duration on HCMV Infection

s were now at significantly greater risk of early HCMV infection, as detected by antibody at 18 months, with prolonged breastfeeding (OR for breastfeeding >6 months compared with no breastfeeding =20.37; 95% CI, 3.71–111.70; P < .001) (Table 1). Table 1. Effects Maternal HIV and Breastfeeding Duration on HCMV Infection HCMV Infant Infection (Antibody) Months Breastfeeding Antibody N (%) Adjusted ORa (95% CI) P Value HIV-Negative Mothers <12b 25/32 (78.1%) 1 12–17 128/161 (79.5%) 0.94 (0.35–2.53) 18+ 110/119 (92.4%) 2.69 (0.84–8.59) .03 HIV-Positive Mothers Never 13/26 (50.0%) 1 <6 31/35 (88.6%) 6.83 (1.69–27.6) 6+ 42/44 (95.5%) 20.37 (3.71–111.7) <.001 Abbreviations: CI, confidence interval; HCMV, human cytomegalovirus; HIV, human immunodeficiency virus; OR, odds ratio. a Adjusted for socioeconomic status and maternal education. b Only 3 HIV-negative mothers never breastfed and only 6 for <6 months.

HCMV Infant Infection (Antibody) Months Breastfeeding Antibody N (%) Adjusted ORa (95% CI) P Value HIV-Negative Mothers <12b 25/32 (78.1%) 1 12–17 128/161 (79.5%) 0.94 (0.35–2.53) 18+ 110/119 (92.4%) 2.69 (0.84–8.59) .03 HIV-Positive Mothers Never 13/26 (50.0%) 1 <6 31/35 (88.6%) 6.83 (1.69–27.6) 6+ 42/44 (95.5%) 20.37 (3.71–111.7) <.001 Abbreviations: CI, confidence interval; HCMV, human cytomegalovirus; HIV, human immunodeficiency virus; OR, odds ratio. a Adjusted for socioeconomic status and maternal education. b Only 3 HIV-negative mothers never breastfed and only 6 for <6 months. DISCUSSION The study shows widespread HCMV infections in Zambian infants, from both HIV-positive and HIV-negative women. Human cytomegalovirus seropositivity was 83% by 18 months age, which is both higher and earlier than in many regions. Although similar to some other regions in Africa, this population has the added effect of endemic HIV. In Africa, the common practice of extended breastfeeding was identified as a risk for HCMV infection, which was increased for maternally HIV-exposed children. Furthermore, HIV-positive women had strikingly higher loads of HCMV secreted in their breast milk, with extended periods of raised levels, compared with HIV-negative women. There was some overlap so other factors may also influence secretion. This is comparable to studies from East Africa that reported high HCMV loads in HIV-infected women, although comparisons to HIV-negative women were not made [27]. Our studies have now compared both HIV-positive and HIV-negative mothers as well as considered breastfeeding duration in this sub-Saharan African setting. Similar to European and South East Asian surveys, our studies show peak HCMV DNA levels at 4 weeks postpartum but differences in shedding duration. More HIV-positive than HIV-negative women had detectable HCMV secretion in breast milk, with initial reactivated levels equal at day 3, then raised from 2 to 16 weeks postpartum. Recent studies show HCMV-susceptible CD14+ leukocytes increasing in breast milk at this time [35]. Thus, in milk secreted from HIV-positive women, there appears to be decreased immune regulation or increased susceptible cells, possibly from immune activation or inflammation. Studies in this cohort showed increased mastitis in HIV-positive women [29], and we have demonstrated increased secretion of HCMV. We further show that, in addition to increased risk of congenital infection from intrauterine HCMV infection [21-23], infants of HIV-infected women have increased odds of HCMV infant infection from breastfeeding. This can be a confounder for determining congenital infection, because diagnostic tests by saliva in those under 2 weeks of age may detect HCMV in saliva from breast milk.

l infection from intrauterine HCMV infection [21-23], infants of HIV-infected women have increased odds of HCMV infant infection from breastfeeding. This can be a confounder for determining congenital infection, because diagnostic tests by saliva in those under 2 weeks of age may detect HCMV in saliva from breast milk. Our studies of birth prevalence of HCMV in Zambia using newborn saliva show 1% (1 of 100) prevalence in normal labor ward (K.G.M. and U.A.G, unpublished), lower than studies in the neonatal intensive care unit where breast milk could also be a source of early infection [25].

l infection from intrauterine HCMV infection [21-23], infants of HIV-infected women have increased odds of HCMV infant infection from breastfeeding. This can be a confounder for determining congenital infection, because diagnostic tests by saliva in those under 2 weeks of age may detect HCMV in saliva from breast milk. Our studies of birth prevalence of HCMV in Zambia using newborn saliva show 1% (1 of 100) prevalence in normal labor ward (K.G.M. and U.A.G, unpublished), lower than studies in the neonatal intensive care unit where breast milk could also be a source of early infection [25]. Low socioeconomic status and level of education were associated with HCMV seroprevalence in analyses of risk factors, similar to those reported elsewhere [17, 36]. With adjustment for these risk factors, breastfeeding up to 18 months remained significantly associated with HCMV infection. Studies in other continents show that almost all HCMV-positive women excrete HCMV in breast milk from local tissue reactivation, which is distinct from detection in plasma. Even with breast milk secretion in HCMV-positive mothers, the transmission rate varies, and 58%–80% infants were found to be seropositive by 1 year of age [26]. In Europe and Asia, studies have shown breast milk HCMV secretion up to 2–3 months postpartum, with higher milk viral loads and prolonged secretion linked to infant transmission [3, 28, 37]. In Zambia, we showed sustained breast milk HCMV secretion for over 4 months. Furthermore, increased breastfeeding over 6 months among HIV-positive women, or over 18 months among HIV-negative women, increased risk of infant HCMV infection. This shows that HCMV is secreted (or possibly more transmissible in breast milk) for longer duration than that reported in Europe or Asia. In HIV-negative women, longer duration of lactation may increase HCMV local reactivation and secretion. In HIV-positive women, secreted levels may be further raised through both HIV and HCMV immune dysregulation and amplified with breastfeeding duration.

t milk) for longer duration than that reported in Europe or Asia. In HIV-negative women, longer duration of lactation may increase HCMV local reactivation and secretion. In HIV-positive women, secreted levels may be further raised through both HIV and HCMV immune dysregulation and amplified with breastfeeding duration. Increased transmission during breastfeeding also allows for reinfections with multiple strains and widens the total population exposure to HCMV infection. We previously showed complex mixtures of HCMV strains, a potential factor for severe HCMV disease (currently under assessment) and a marker for burden of infection in this population, from blood or lung samples of HIV-positive infants [32]. We used gO for genotyping, one of the most variable genes in HCMV. Our genotype analyses showed that all 8 gO genotypes were detected in breast milk, demonstrating that genotypes were not constrained in this tissue compartment. There was also evidence for mixed-infections indicative of reinfection, which was confirmed using gN genotyping (data not shown). In HIV-positive women, viral load seemed to be raised independent of the genotypes secreted. The main milk gO genotypes 1a, 1b, and 5 were similar to prevalences we previously described in blood and respiratory compartments of primarily HIV-infected children in the same region and in other tissues from different global sources; differences in minor genotypes did not reach significance, although gO3 was greater in other tissues (Supplementary Table 1 and Supplementary Figure S1) [32, 33]. The analyses were limited by the small proportion of samples available for genotype analyses. These genotype ratios require further study, because the gO trimeric or alternate pentameric complexes with gH/gL glycoproteins affect host transmission and candidate vaccines [38, 39].

1 and Supplementary Figure S1) [32, 33]. The analyses were limited by the small proportion of samples available for genotype analyses. These genotype ratios require further study, because the gO trimeric or alternate pentameric complexes with gH/gL glycoproteins affect host transmission and candidate vaccines [38, 39]. In the infant cohort, almost all HIV-negative women breastfed their infants, whereas a quarter of HIV-positive women never breastfed their infants, and overall HIV-positive women had significantly shorter breastfeeding durations than HIV-negative women (median 6 months vs 15 months, P < .01). Various reasons, including trying to limit infant HIV, were presented by the mothers [31]. This may have provided some protection from HCMV infection to children of HIV-positive mothers because those who never breastfed were only 50% HCMV positive, whereas breastfeeding at least to 6 months increased the prevalence of HCMV-positive children to 88.6% and over 6 months up to 95.5%. These differences in breastfeeding behavior between HIV-positive and HIV-negative women also meant that comparisons could only be made using different infant breastfeeding duration categories and are a limitation of the study. The 6-fold and 20-fold increase in odds of infection in adjusted analyses with breastfeeding to 6 months and over 6 months age, respectively, is in agreement with the viral loads measured in breast milk. Although limited from 2 different cohort studies, these were from the same residential area and study clinic. A study in Kenya showed that HCMV transmitters had a median of 5.4 compared with 4.5 logs copies/mL in milk for nontransmitters at 2 weeks postpartum [27]. Although it is difficult to apply exact thresholds, at similar times postpartum, in our Zambian cohort, HIV-positive compared with negative women had a median of 5.1 versus 4.0 logs copies/mL in breast milk. Furthermore, between day 3 and week 16, HIV-positive women had 23 measurements above 5.5 logs copies/mL compared with only 7 for HIV-negative women. This would indicate clinical relevance for the log differences in HCMV milk secretion of increased risk for transmission and at earlier times in the HIV-positive group.

k. Furthermore, between day 3 and week 16, HIV-positive women had 23 measurements above 5.5 logs copies/mL compared with only 7 for HIV-negative women. This would indicate clinical relevance for the log differences in HCMV milk secretion of increased risk for transmission and at earlier times in the HIV-positive group. In a separate analysis, we showed that HCMV adversely affected this infant cohort's growth and psychomotor development, particularly in maternally HIV-exposed children [17]. In Kenya, HCMV DNA in maternal plasma is a predictor of mortality in HIV-infected women and their infants [14], and lower CD4 levels were related to lower levels of milk HCMV required for transmission [27]. Although it was not measured, HCMV could be a factor in studies in Zambia where women with advanced HIV/AIDS who breastfed less than 4 months had improved infant survival [42]. In Zimbabwe, HIV-positive women with HCMV coinfection excreted both pathogens in breast milk, and HCMV infection correlated with higher levels of HIV ribonucleic acid [43]. Further analyses show HCMV milk secretion in HIV-positive mothers correlates with infant growth-faltering [18]. In United States, studies assessing earlier ART during pregnancy in HIV-positive women showed reductions in peri- or postnatal HCMV infection [44]. Although this was a non-breastfeeding population, the results support use of interventions to improve maternal health, such as earlier use of ART in HIV-positive women, which is now being applied in Zambia. However, recent studies in African breastfed populations showed that the use of antiretrovirals in HIV-positive mothers did not restrict HCMV transmission to their infants and therefore remained a risk for increasing numbers of HIV-exposed uninfected children [16, 18].

itive women, which is now being applied in Zambia. However, recent studies in African breastfed populations showed that the use of antiretrovirals in HIV-positive mothers did not restrict HCMV transmission to their infants and therefore remained a risk for increasing numbers of HIV-exposed uninfected children [16, 18]. There are several limitations to our studies. The HCMV serostatus of the mothers was not known, although breast milk DNA PCR showed that all mothers were HCMV positive. We did not have CD4 levels, and these can affect milk secretion of CMV, as shown in studies of only HIV-positive mothers in Kenya [27], and may have contributed to varying HCMV levels in the HIV-positive mothers. We did not include day care status as a possible source for infant transmission via saliva [40]; however, in Zambia, infant day care is minimal to nonexistent. Other HCMV secretory routes, particularly saliva and also urinary [41] from siblings, can affect transmission, and 50% of infants became HCMV seropositive from HIV-positive women who did not breastfeed. However, we could not compare this result to HIV-negative mothers because almost all of them breastfed. Furthermore, most of our cohort mothers had children under age 5 who could be secreting HCMV; therefore, in general, background exposure was equal. We did not screen for prenatal HCMV, but this result would be only approximately 1% based on previous studies on newborns. The strengths of this study are the size of the cohorts and the comparison between both HIV-negative and HIV-positive groups, which clearly show raised levels of HCMV milk secretion and duration, with increased risks for infant infection in HIV-positive compared with HIV-negative women.

on previous studies on newborns. The strengths of this study are the size of the cohorts and the comparison between both HIV-negative and HIV-positive groups, which clearly show raised levels of HCMV milk secretion and duration, with increased risks for infant infection in HIV-positive compared with HIV-negative women. The World Health Organization recommends 6 months exclusive breastfeeding for ideal infant nutrition and immune protection. In Zambia, Demographic Health Survey data show that 73% of infants are exclusively breastfed for 6 months, and, overall, 98% of children breastfed with a median duration of 20.1 months [45]. Possible interventions against HCMV need to retain breastfeeding benefits. Direct HCMV inactivation in milk using ultrashort heat treatment has been evaluated in Europe, particularly for at-risk, premature, and underweight infants, and was found to be effective while preserving the nutritional and immunological qualities of breast milk [46]. This intervention warrants assessment in the sub-Saharan Africa setting, where it could be useful for concurrent inactivation of HCMV and HIV in breast milk, for at risk groups. Administration of anti-HCMV drugs to mothers to lower milk HCMV load is another potential intervention, but this requires efficacy and safety trials; no anti-HCMV drug to date is licenced for use during lactation. Improved hygiene could lower complementary routes of transmission, including saliva or urine [47]. Furthermore, there are several promising vaccines currently in development [48, 49].

er potential intervention, but this requires efficacy and safety trials; no anti-HCMV drug to date is licenced for use during lactation. Improved hygiene could lower complementary routes of transmission, including saliva or urine [47]. Furthermore, there are several promising vaccines currently in development [48, 49]. CONCLUSIONS We conclude that longer breastfeeding duration over 6 to 18 months increases HCMV infant infection. We also showed that HIV-positive compared with HIV-negative women had both raised breast milk secretion and likelihood for infant infection. Interventions to reduce HCMV infection could be considered, particularly in countries with high HCMV and HIV prevalence, because breastfeeding remains critical for infant health. Supplementary Data Supplementary materials are available at the Journal of The Pediatric Infectious Diseases Society online (http://jpids.oxfordjournals.org). Acknowledgments We thank all of the mothers and children who had participated in these studies as well as the Lusaka District Health staff who gave their support. We are also grateful for the contributions of the Breastfeeding and Postpartum Health Study and the Chilenje Infant Growth, Nutrition and Infection Study team members, particularly Kathy Baisley and Andrea Rehman for statistical advice on breastfeeding duration effects and Molly Chisenga for milk sample collections.

t. We are also grateful for the contributions of the Breastfeeding and Postpartum Health Study and the Chilenje Infant Growth, Nutrition and Infection Study team members, particularly Kathy Baisley and Andrea Rehman for statistical advice on breastfeeding duration effects and Molly Chisenga for milk sample collections. Principal Investigator: Suzanne Filteau, London School of Hygiene and Tropical Medicine (LSHTM); Zambian Lead Investigator: Lackson Kasonka, University Teaching Hospital (UTH), Lusaka; Senior Investigators: Rosalind Gibson, University of Otago, New Zealand; Ursula A. Gompels, LSHTM; Shabbar Jaffar, LSHTM; Emmanuel Kafwembe, Tropical Diseases Research Centre, Ndola; Mwaka Monze, UTH; Moses Sinkala, Catholic Relief Services, Zambia; Andrew Tomkins, Institute of Child Health, University College, London; Rodah Zulu, National Institute of Science and Industrial Research, Zambia; Clinic Coordinator: Molly Chisenga; Clinical Officer: Joshua Siame; Data Manager: Hildah Banda Mabuda; Statisticians: Kathy Baisley, Helen Dale, Natasha Larke, Daniela Manno, Andrea Rehman; Research Fellows: Matthew Bates, Anne Mullen, Kunda Musonda, Marta Sanz-Ramos; Clinic Staff: Hellen Kangwa Bwalya, Margaret Chileshe, Priscilla Kangwa Kowa, Mabvuto Kumwenda, Munalula Likando, Sydney Mambwe, Mutinta Muzyamba, Anne Mwale, Lungowe Nyaywa; Laboratory Staff: Humphrey Bima, Julia Chibumbya, Laura Gosset, Louise Hackett, Abigail Jackson, Mirriam Kapambwe, Mazyanga Liewe, Sydney Mwanza, Ida Ndumba, Eric Njunju; Data Entry: Concillia Kabanga, Natalia Shampwaya; Drivers and Cleaners: John Chobo, Winford Kapumba, Charity Musonda, Philip Soko.

e, Lungowe Nyaywa; Laboratory Staff: Humphrey Bima, Julia Chibumbya, Laura Gosset, Louise Hackett, Abigail Jackson, Mirriam Kapambwe, Mazyanga Liewe, Sydney Mwanza, Ida Ndumba, Eric Njunju; Data Entry: Concillia Kabanga, Natalia Shampwaya; Drivers and Cleaners: John Chobo, Winford Kapumba, Charity Musonda, Philip Soko. Financial support. This work was funded by the Bill and Melinda Gates Foundation (Grant ID 37253) and the Commonwealth Scholarship Commission (reference number ZMCS-2012-643). Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest.

Influenza infection creates a high disease burden among children [1–4], with routine vaccination recommended in the United States [5] and elsewhere. Until recently, most programs used trivalent influenza vaccines (TIV) containing 2 influenza A strains (H1N1 and H3N2) and 1 B strain. Two antigenically distinct lineages of influenza B (Yamagata and Victoria) have cocirculated worldwide since 2000, and in the past decade, a substantial proportion of influenza B isolates from patients have been mismatched to the influenza B strain in the seasonal TIV. For example, during the 2012–2013 season, one third of influenza B viruses tested by the US Centers for Disease Control and Prevention were of the lineage absent from the seasonal vaccine [6]. During seasons where the predominant circulating influenza B virus is from the alternate lineage than the B strain included in the TIV, suboptimal vaccine protection can be expected [7–9]. A quadrivalent influenza vaccine (QIV) containing B strains derived from both lineages could offer broader protection by eliminating B lineage mismatch. This may be particularly important in children because, although vaccinated adults show moderate cross-reactive antibody responses against the alternative B lineage [10], children show poor cross-reactivity [11, 12]. Indeed, a meta-analysis of vaccine trials in young children found that efficacy was substantially reduced against influenza B strains of the alternative lineage to that contained in the vaccine [9].

e cross-reactive antibody responses against the alternative B lineage [10], children show poor cross-reactivity [11, 12]. Indeed, a meta-analysis of vaccine trials in young children found that efficacy was substantially reduced against influenza B strains of the alternative lineage to that contained in the vaccine [9]. Currently, the licensed dose in the United States for children between 6 and 35 months of age is 0.25 mL with 7.5 μg hemagglutinin (HA) of each influenza strain. This represents half of the 0.5 mL dose with 15 μg HA per strain that is licensed for older children and adults. The practice of administering the lower dose to young children began more than 30 years ago to reduce the fever and febrile convulsions associated with the whole virus vaccines available at the time [13]. However, studies have shown that children of this age mount a variable immune response to the 7.5 μg HA dose. Current split virus vaccines are better tolerated compared with whole virus vaccines and are associated with substantially lower rates of fever and febrile convulsions. Several studies have now evaluated these vaccines at the 15 μg HA dose in children 6–35 months of age. The higher dose elicits an increased immune response, particularly in children younger than 18 months, without increasing reactogenicity compared with the 7.5 μg HA dose [14–16].

ates of fever and febrile convulsions. Several studies have now evaluated these vaccines at the 15 μg HA dose in children 6–35 months of age. The higher dose elicits an increased immune response, particularly in children younger than 18 months, without increasing reactogenicity compared with the 7.5 μg HA dose [14–16]. The QIV manufactured by GSK Vaccines has been developed for use at the 15 μg HA dose regardless of age. It is licensed in Canada and Mexico for children from the age of 6 months. However, in the United States, it is only licensed from the age of 3 years. Herein, we describe a phase II study evaluating immunogenicity and safety of the QIV at a 15 μg HA dose in children between 6 and 35 months of age in the United States. Ideally, we would have compared the investigational QIV with a licensed QIV at the same 15 μg HA dose. Unfortunately, this was not possible. The TIV and QIV manufactured by Sanofi Pasteur are the only influenza vaccines licensed in the United States in this age group, both of which are used at a dose of 7.5 μg HA. Trial access to the QIV could not be guaranteed. Hence, the TIV at a 7.5 μg dose was the only available choice of comparator for the study.

ible. The TIV and QIV manufactured by Sanofi Pasteur are the only influenza vaccines licensed in the United States in this age group, both of which are used at a dose of 7.5 μg HA. Trial access to the QIV could not be guaranteed. Hence, the TIV at a 7.5 μg dose was the only available choice of comparator for the study. METHODS This study was a phase II randomized, controlled, observer-blind, multicenter trial comparing the immunogenicity and safety profiles of an inactivated QIV versus TIV in children 6–35 months of age in the United States (identifier NCT01974895). The primary objective was to evaluate immunogenicity of the QIV according to the US Center for Biologics Evaluation and Research (CBER) criteria for seroconversion. This criterion, rather than the seroprotection rate, was selected because it is more relevant for children who may be both unvaccinated and unexposed previously to either or both influenza A and B infection. The trial was sponsored by GlaxoSmithKline Biologicals SA, and it was approved by independent ethics committees and/or institutional review boards, conducted in accordance with the Declaration of Helsinki, the International Conference on Harmonisation Good Clinical Practice guidelines, and US regulatory requirements. Parents or legally acceptable representatives provided written informed consent prior to participation of their child.

r institutional review boards, conducted in accordance with the Declaration of Helsinki, the International Conference on Harmonisation Good Clinical Practice guidelines, and US regulatory requirements. Parents or legally acceptable representatives provided written informed consent prior to participation of their child. Participants, Vaccines, and Study Design Healthy children and those with chronic illness who were not acutely ill at the time of enrollment (determined by the investigator's clinical examination and assessment of the child's medical history) were enrolled from 12 centers across the United States during the fall and winter of 2013–2014. Children were excluded if they were febrile (temperature ≥38.0°C), acutely ill, immunocompromised, allergic to any vaccine component, had a history of Guillain-Barré syndrome within 6 weeks of prior influenza vaccination, had a known coagulation disorder, had received any influenza vaccine within the last 6 months, had received any immunoglobulin or blood product within the last 3 months, or had received an investigational product within the last 30 days. In accordance with the US Advisory Committee on Immunization Practices definition, children were considered vaccine-primed if they had received 2 or more doses of seasonal influenza vaccine since July 1, 2010; all others were considered vaccine-unprimed.

Participants, Vaccines, and Study Design Healthy children and those with chronic illness who were not acutely ill at the time of enrollment (determined by the investigator's clinical examination and assessment of the child's medical history) were enrolled from 12 centers across the United States during the fall and winter of 2013–2014. Children were excluded if they were febrile (temperature ≥38.0°C), acutely ill, immunocompromised, allergic to any vaccine component, had a history of Guillain-Barré syndrome within 6 weeks of prior influenza vaccination, had a known coagulation disorder, had received any influenza vaccine within the last 6 months, had received any immunoglobulin or blood product within the last 3 months, or had received an investigational product within the last 30 days. In accordance with the US Advisory Committee on Immunization Practices definition, children were considered vaccine-primed if they had received 2 or more doses of seasonal influenza vaccine since July 1, 2010; all others were considered vaccine-unprimed. The investigational QIV (GSK Vaccines, dba ID Biomedical Corporation, Sainte-Foy, Quebec, Canada) contained 15 μg HA of each of 4 strains (A/California/7/2009 [A/H1N1], A/Texas/50/2012 [A/H3N2], B/Brisbane/60/2008 [B/Victoria], and B/Massachusetts/2/2012 [B/Yamagata]), in a 0.5 mL dose. The HA content was measured by a validated Single Radial Immunodiffusion assay. The licensed comparator TIV (Sanofi Pasteur, Swiftwater, PA) contained 7.5 μg of each of the same A/H1N1, A/H3N2, and B Yamagata strains in a 0.25 mL dose. Vaccine-primed children received 1 vaccine dose on study day 0. Vaccine-unprimed children received a vaccine dose on days 0 and 28. Administration was via intramuscular injection in the deltoid muscle of the nondominant arm for children ≥12 months of age or the anterolateral region of the left thigh for children <12 months of age.

ed children received 1 vaccine dose on study day 0. Vaccine-unprimed children received a vaccine dose on days 0 and 28. Administration was via intramuscular injection in the deltoid muscle of the nondominant arm for children ≥12 months of age or the anterolateral region of the left thigh for children <12 months of age. Children were randomized 1:1 to receive either QIV or TIV according to an internet-based randomization system. A minimization algorithm was used to balance enrollment by group accounting for age (6–17 and 18–35 months), center, and priming status. The randomization system provided the unique treatment number to be used for each dose. GSK Vaccines performed the randomization. Study Endpoints and Procedures Each child had a blood sample taken at baseline and again 28 days after completion of the 1- or 2-dose vaccine series (depending on vaccine-priming status). Hemagglutination inhibition (HI) titers were determined from serum obtained pre- and postvaccination. Results were reported for each vaccine group as (1) geometric mean titers (GMTs), (2) seropositivity rates, (3) seroconversion rates (SCRs), (4) seroprotection rates (SPRs), and (5) mean geometric increases (MGIs). The limit of quantitation for the HI assay was 1:10; samples <1:10 were considered seronegative and were given an arbitrary value of 5 for the GMT calculation.

ometric mean titers (GMTs), (2) seropositivity rates, (3) seroconversion rates (SCRs), (4) seroprotection rates (SPRs), and (5) mean geometric increases (MGIs). The limit of quantitation for the HI assay was 1:10; samples <1:10 were considered seronegative and were given an arbitrary value of 5 for the GMT calculation. Seropositivity rate was defined as the percentage of participants with reciprocal HI titer ≥1:10. Seroconversion rate was defined as the percentage of participants with either (a) prevaccination reciprocal HI titer <1:10 and postvaccination reciprocal titer ≥1:40 or (b) prevaccination reciprocal titer ≥1:10 and at least a 4-fold increase in postvaccination reciprocal titer. Seroprotection rate was defined as the percentage of participants who attained reciprocal HI titers of ≥1:40. Mean geometric increase was defined as the geometric mean of the within-subject ratios of the postvaccination/prevaccination reciprocal HI titer. Solicited injection site and general adverse events (AEs) were recorded in diary cards on the day of vaccination and for 6 days afterwards. Spontaneously reported symptoms were recorded until 28 days after vaccination. Serious AEs (SAEs), potential immune-mediated disorders (pIMDs), and medically attended AEs (MAEs) were recorded until the final telephone contact on day 180. Fever was defined as temperature ≥38.0°C by any route (axillary, rectal, oral, or tympanic).

sly reported symptoms were recorded until 28 days after vaccination. Serious AEs (SAEs), potential immune-mediated disorders (pIMDs), and medically attended AEs (MAEs) were recorded until the final telephone contact on day 180. Fever was defined as temperature ≥38.0°C by any route (axillary, rectal, oral, or tympanic). Study Objectives The primary objective was to evaluate whether the QIV elicited an immune response against each vaccine strain that met the CBER target for SCR 28 days after completion of the vaccine course, ie, lower limit (LL) of the 95% confidence interval (CI) ≥40%. Secondary objectives included: (1) demonstrating superior immunogenicity of QIV versus TIV against the B/Victoria strain 28 days after completion of the vaccine series (criteria: LL of the 95% CI for GMT ratio [QIV/TIV] >1.5 and for SCR difference [QIV minus TIV] >10%); (2) describing GMT, SPR, SCR, and MGI; (3) describing safety and reactogenicity; and (4) evaluating the relative risk of fever (≥38.0°C) in the QIV group versus the TIV group over the 4-day postvaccination period. The tertiary objective was to describe the GMT ratio (TIV/QIV) and SCR difference (TIV minus QIV) for the H1N1, H3N2, and B/Yamagata strains.

GI; (3) describing safety and reactogenicity; and (4) evaluating the relative risk of fever (≥38.0°C) in the QIV group versus the TIV group over the 4-day postvaccination period. The tertiary objective was to describe the GMT ratio (TIV/QIV) and SCR difference (TIV minus QIV) for the H1N1, H3N2, and B/Yamagata strains. Statistics The primary immunogenicity analysis was based on the per-protocol cohort, which included children who met all eligibility criteria, complied with the protocol and vaccine schedule, and had data available for antibodies against at least 1 vaccine strain postvaccination. Immunogenicity analyses excluded participants with missing or nonevaluable measurements. The safety analysis was based on the intent-to-treat cohort.

ho met all eligibility criteria, complied with the protocol and vaccine schedule, and had data available for antibodies against at least 1 vaccine strain postvaccination. Immunogenicity analyses excluded participants with missing or nonevaluable measurements. The safety analysis was based on the intent-to-treat cohort. A point estimate and its 2-sided exact 95% CI were calculated using Proc StatXact for GMT, SCR, SPR, and MGI by vaccine group for all children. In addition, subgroup analyses were conducted to explore potential differences between 2 age strata (6–17 and 18–35 months). The 95% CIs for the mean of log-transformed antibody titers were first obtained assuming that log-transformed values were normally distributed with unknown variance. The 95% CIs for GMT were then computed by exponential-transformation of the 95% CI for the mean of log-transformed titer. The GMT ratio (QIV/TIV) was computed using an analysis of covariance model on the log-transformed titers, including the vaccine group as a fixed effect and prevaccination HI titers, age, and vaccine-priming status as covariates. The asymptotic standardized 95% CI for the difference in SCR between QIV and TIV was computed using Proc StatXact [17].

computed using an analysis of covariance model on the log-transformed titers, including the vaccine group as a fixed effect and prevaccination HI titers, age, and vaccine-priming status as covariates. The asymptotic standardized 95% CI for the difference in SCR between QIV and TIV was computed using Proc StatXact [17]. Study power was calculated using PASS 2005. Assuming 50 evaluable children in the QIV group, the overall power of the study was 99.34% to meet CBER criteria for SCR simultaneously for all 4 strains (primary objective). Assuming 50 evaluable children in each vaccine group, the power to demonstrate superiority of the QIV over the TIV was 99.17% in terms of GMT ratio and >99.99% in terms of SCR difference (a secondary objective). No power calculation was performed for other secondary and tertiary objectives. However, the study planned to enroll 250 participants per group to assess the SCR difference for the common strains between the 2 groups with reasonably adequate power and to increase the probability of detecting fever, a potential consequence of the increased antigen content in the QIV.

ondary and tertiary objectives. However, the study planned to enroll 250 participants per group to assess the SCR difference for the common strains between the 2 groups with reasonably adequate power and to increase the probability of detecting fever, a potential consequence of the increased antigen content in the QIV. RESULTS The first participant was enrolled in October 2013 and the last study contact was in July 2014. The initial enrollment target was 500 children, but 1 major planned center was unable to participate; thus, 316 children were enrolled. Although the planned enrollment of 500 children was not attained, the 316 children enrolled assured adequate power for the analysis of the primary and secondary confirmatory objectives. A total of 314 and 280 children were included in the intent-to-treat cohort (Total Vaccinated Cohort) and per-protocol cohort for immunogenicity, respectively (Figure 1). Demographics were similar between groups (Table 1). In the QIV group, 64 (40.5%) children received 1 vaccine dose, whereas 94 (59.5%) received 2 doses. Corresponding values in the TIV group were 64 (41.0%) and 92 (59.0%). Table 1. Participant Demographics (Intent-to-Treat Cohort)

mmunogenicity, respectively (Figure 1). Demographics were similar between groups (Table 1). In the QIV group, 64 (40.5%) children received 1 vaccine dose, whereas 94 (59.5%) received 2 doses. Corresponding values in the TIV group were 64 (41.0%) and 92 (59.0%). Table 1. Participant Demographics (Intent-to-Treat Cohort) Characteristic QIV N = 158 TIV N = 156 Total N = 314 Mean age (SD), months 19.6 (8.8) 19.8 (8.9) 19.7 (8.9) Female gender, n (%) 74 (46.8) 82 (52.6) 156 (49.7) Geographic ancestry, n (%) White (Caucasian or European heritage) 86 (54.4) 88 (56.4) 174 (55.4) African/African American 56 (35.4) 56 (35.9) 112 (35.7) American Indian or Alaskan Native 1 (0.6) 0 (0) 1 (0.3) Asian 1 (0.6) 1 (0.6) 2 (0.6) White (Arabic or North African) 1 (0.6) 0 (0) 1 (0.3) Native Hawaiian or other Pacific Islander 0 (0) 0 (0) 0 (0) Other 13 (8.2) 11 (7.1) 24 (7.6) Abbreviations: QIV, quadrivalent influenza vaccine; SD, standard deviation; TIV, trivalent influenza vaccine. Figure 1. Participant disposition. QIV, quadrivalent influenza vaccine; TIV, trivalent influenza vaccine.

T initiation and 1 with 2 tuberculosis episodes after ART initiation). Eight were bacteriologically confirmed and 69 were clinically diagnosed. There were 56 pulmonary, 12 lymphadenitis, 2 meningitis, 2 abdominal, 1 pulmonary plus meningitis, 1 parotid and 1 cutaneous tuberculosis, and 2 unspecified tuberculosis sites. During tuberculosis treatment, 54 received an efavirenz-based ART regimen, 8 received a nevirapine-based regimen, 7 received a protease inhibitor-based regimen, and 4 received a zidovudine plus didanosine regimen; 3 discontinued ART before tuberculosis treatment; and 1 died before starting tuberculosis treatment. Based on the data available, which were mostly collected before consensus definitions of immune reconstitution inflammatory syndrome (IRIS) were proposed [16], the physician in charge of the retrospective review suspected that 8 children (3 with a prevalent tuberculosis and 5 with an incident tuberculosis) developed an IRIS between 10 days and 4 months after ART initiation. However, the information available was not sufficient to definitely classify these cases.

Characteristic QIV N = 158 TIV N = 156 Total N = 314 Mean age (SD), months 19.6 (8.8) 19.8 (8.9) 19.7 (8.9) Female gender, n (%) 74 (46.8) 82 (52.6) 156 (49.7) Geographic ancestry, n (%) White (Caucasian or European heritage) 86 (54.4) 88 (56.4) 174 (55.4) African/African American 56 (35.4) 56 (35.9) 112 (35.7) American Indian or Alaskan Native 1 (0.6) 0 (0) 1 (0.3) Asian 1 (0.6) 1 (0.6) 2 (0.6) White (Arabic or North African) 1 (0.6) 0 (0) 1 (0.3) Native Hawaiian or other Pacific Islander 0 (0) 0 (0) 0 (0) Other 13 (8.2) 11 (7.1) 24 (7.6) Abbreviations: QIV, quadrivalent influenza vaccine; SD, standard deviation; TIV, trivalent influenza vaccine. Figure 1. Participant disposition. QIV, quadrivalent influenza vaccine; TIV, trivalent influenza vaccine. Immunogenicity Seroconversion rates at 28 days after vaccination with the QIV were 80.4% for A/H1N1, 72.0% for A/H3N2, 86.0% for B/Yamagata, and 66.4% for B/Victoria (Table 2). The LL of the 95% CI for SCR was >40% for each vaccine strain in the overall population and in both age groups (Table 2), demonstrating that QIV met CBER criterion for seroconversion. In addition, QIV immunogenicity was superior to TIV for the B/Victoria strain overall and in both age strata: the LL of the 95% CI was >1.5 for adjusted GMT ratio and >10% for the difference in SCR (Figure 2A and B). Table 2. SCR, SPR, and MGI Against Each Vaccine Strain Overall and According to Age Stratum at 28 Days After Completion of Vaccination Series (Per-Protocol Cohort for Immunogenicity)

verall and in both age strata: the LL of the 95% CI was >1.5 for adjusted GMT ratio and >10% for the difference in SCR (Figure 2A and B). Table 2. SCR, SPR, and MGI Against Each Vaccine Strain Overall and According to Age Stratum at 28 Days After Completion of Vaccination Series (Per-Protocol Cohort for Immunogenicity) Variable All Ages 6–17 Months 18–35 Months QIV N = 143 TIV N = 137 QIV N = 53 TIV N = 53 QIV N = 90 TIV N = 84 SCR, % (95% CI) A/H1N1 80.4 (73.0–86.6) 71.5 (63.2–78.9) 64.2 (49.8–76.9) 66.0 (51.7–78.5) 90.0 (81.9–95.3) 75.0 (64.4–83.8) A/H3N2 72.0 (63.9–79.2) 68.6 (60.1–76.3) 56.6 (42.3–70.2) 67.9 (53.7–80.1) 81.1 (71.5–88.6) 69.0 (58.0–78.7) B/Yamagata 86.0 (79.2–91.2) 83.9 (76.7–89.7) 71.7 (57.7–83.2) 73.6 (59.7–84.7) 94.4 (87.5–98.2) 90.5 (82.1–95.8) B/Victoria 66.4 (58.1–74.1) 12.4 (7.4–19.1) 60.4 (46.0–73.5) 1.9 (0.0–10.1) 70.0 (59.4–79.2) 19.0 (11.3–29.1) Post-vaccination SPR, % (95% CI) A/H1N1 87.4 (80.8–92.4) 81.0 (73.4–87.2) 67.9 (53.7–80.1) 69.8 (55.7–81.7) 98.9 (94.0–100) 88.1 (79.2–94.1) A/H3N2 82.5 (75.3–88.4) 80.3 (72.6–86.6) 60.4 (46.0–73.5) 69.8 (55.7–81.7) 95.6 (89.0–98.8) 86.9 (77.8–93.3) B/Yamagata 94.4 (89.3–97.6) 90.5 (84.3–94.9) 84.9 (72.4–93.3) 79.2 (65.9–89.2) 100 (96.0–100) 97.6 (91.7–99.7) B/Victoria 70.6 (62.4–77.9) 19.7 (13.4–27.4) 60.4 (46.0–73.5) 3.8 (0.5–13.0) 76.7 (66.6–84.9) 29.8 (20.3–40.7) MGI (95% CI) A/H1N1 13.7 (11.1–17.0) 9.1 (7.3–11.3) 11.2 (7.3–17.1) 8.3 (5.7–12.2) 15.5 (12.4–19.5) 9.6 (7.4–12.6) A/H3N2 9.1 (7.7–10.8) 7.5 (6.4–8.9) 7.3 (5.4–9.8) 8.6 (6.5–11.3) 10.4 (8.5–12.7) 6.9 (5.6–8.6) B/Yamagata 14.6 (11.7–18.2) 11.4 (9.1–14.2) 9.8 (6.2–15.5) 8.4 (5.5–12.9) 18.4 (14.9–22.9) 13.7 (10.8–17.5) B/Victoria 8.9 (7.3–10.9) 1.9 (1.7–2.2) 8.2 (5.8–11.6) 1.4 (1.2–1.6) 9.4 (7.4–12.0) 2.4 (2.0–2.8) Abbreviations: CI, confidence interval; MGI, mean geometric increase; QIV, quadrivalent influenza vaccine; SCR, seroconversion rate; SPR, seroprotection rate; TIV, trivalent influenza vaccine.

(14.9–22.9) 13.7 (10.8–17.5) B/Victoria 8.9 (7.3–10.9) 1.9 (1.7–2.2) 8.2 (5.8–11.6) 1.4 (1.2–1.6) 9.4 (7.4–12.0) 2.4 (2.0–2.8) Abbreviations: CI, confidence interval; MGI, mean geometric increase; QIV, quadrivalent influenza vaccine; SCR, seroconversion rate; SPR, seroprotection rate; TIV, trivalent influenza vaccine. Figure 2. Adjusted geometric mean titer (GMT) ratio and difference in seroconversion rate (SCR) for quadrivalent influenza vaccine (QIV) versus trivalent influenza vaccine (TIV) against B/Victoria overall and according to age stratum at 28 days after completion of vaccination series (per-protocol cohort for immunogenicity). (A) GMT ratio (QIV/TIV); (B) difference in SCR (QIV minus TIV). CI, confidence interval; LL, lower limit.

t influenza vaccine (QIV) versus trivalent influenza vaccine (TIV) against B/Victoria overall and according to age stratum at 28 days after completion of vaccination series (per-protocol cohort for immunogenicity). (A) GMT ratio (QIV/TIV); (B) difference in SCR (QIV minus TIV). CI, confidence interval; LL, lower limit. Quadrivalent influenza vaccine and TIV had similar immunogenicity expressed as postvaccination GMT against the A/H1N1, A/H3N2, and B/Yamagata strains (Figure 3A). Quadrivalent influenza vaccine, but not TIV, was immunogenic against the B/Victoria strain. Geometric mean titers were numerically higher in children 18–35 months of age compared with children 6–17 months of age, but with no notable treatment group differences within each age stratum for the 3 common strains (Figure 3B and C). Robust immunogenicity against all 4 strains was shown for QIV in terms of SPR and MGI, and TIV was immunogenic against the A/H1N1, A/H3N2, and B/Yamagata strains (Table 2). In a planned exploratory analysis, QIV and TIV were similarly immunogenic with regard to GMT ratio and SCR difference for the 3 common strains (Figure 4A and B). Figure 3. Geometric mean titer (GMT) overall and according to age stratum prevaccination and 28 days after completion of vaccination series (per-protocol cohort for immunogenicity). (A) All ages; (B) 6–17 months; (C) 18–35 months. CI, confidence interval; Pre, pre-vaccination; Post, 28 days following final vaccination; QIV, quadrivalent influenza vaccine; TIV, trivalent influenza vaccine.

um prevaccination and 28 days after completion of vaccination series (per-protocol cohort for immunogenicity). (A) All ages; (B) 6–17 months; (C) 18–35 months. CI, confidence interval; Pre, pre-vaccination; Post, 28 days following final vaccination; QIV, quadrivalent influenza vaccine; TIV, trivalent influenza vaccine. Figure 4. Adjusted geometric mean titer (GMT) ratio and difference in seroconversion rate (SCR) for quadrivalent influenza vaccine (QIV) versus trivalent influenza vaccine (TIV) against the common vaccine strains (A/H1N1, A/H3N2, and B/Yamagata) at 28 days after completion of vaccination series (per-protocol cohort for immunogenicity). (A) Adjusted GMT ratio (TIV/QIV); (B) SCR difference (TIV minus QIV). Safety and Reactogenicity Pain at the injection site was the most common AE, occurring in approximately one third of both QIV and TIV recipients (Table 3). Irritability or fussiness was the most common solicited general symptom, occurring in 50.3% and 45.3% of children receiving QIV and TIV, respectively. Ten children in each group experienced fever ≥38.0°C during the 7-day postvaccination period (Table 3), of whom 7 and 8 children experienced fever within 4 days postvaccination in the QIV and TIV groups, respectively. There was no difference between groups in relative risk of fever ≥38.0°C (0.86 [95% CI, 0.33–2.23]) over 4 days postvaccination. Table 3. Safety Outcomes Reported Throughout the Study (Intent-to-Treat Cohort)

whom 7 and 8 children experienced fever within 4 days postvaccination in the QIV and TIV groups, respectively. There was no difference between groups in relative risk of fever ≥38.0°C (0.86 [95% CI, 0.33–2.23]) over 4 days postvaccination. Table 3. Safety Outcomes Reported Throughout the Study (Intent-to-Treat Cohort) Variable Number (%) Children Reporting Outcome QIV TIV Soliciteda injection site symptoms during 7-day postvaccination period (N = 151 QIV, N = 148 TIV)b Pain 48 (31.8) 48 (32.4) Redness 2 (1.3) 0 (0) Swelling 0 (0) 1 (0.7) Solicited general symptoms during 7-day postvaccination period (N = 151 QIV, N = 148 TIV)b Drowsiness 60 (39.7) 56 (37.8) Fever (≥38.0°C) 10 (6.6) 10 (6.8) Irritability/fussiness 76 (50.3) 67 (45.3) Loss of appetite 49 (32.5) 46 (31.1) Unsolicited (spontaneously reported) symptoms during 28-day postvaccination period (N = 158 QIV, N = 156 TIV) All 77 (48.7) 75 (48.1) Related to vaccine 11 (7.0) 7 (4.5) Serious adverse event during entire study period (N = 158 QIV, N = 156 TIV) All 5 (3.2) 4 (2.6) Medically attended eventc during entire study period (N = 158 QIV, N = 156 TIV) All 77 (48.7) 89 (57.1) Abbreviations: AEs, adverse events; QIV, quadrivalent influenza vaccine; TIV, trivalent influenza vaccine. aAll solicited injection site symptoms were considered related to vaccination. bOnly subjects who have documented safety data were included in the calculation of solicited AEs. cHospitalization, emergency room visit, medical practitioner visit.

Variable Number (%) Children Reporting Outcome QIV TIV Soliciteda injection site symptoms during 7-day postvaccination period (N = 151 QIV, N = 148 TIV)b Pain 48 (31.8) 48 (32.4) Redness 2 (1.3) 0 (0) Swelling 0 (0) 1 (0.7) Solicited general symptoms during 7-day postvaccination period (N = 151 QIV, N = 148 TIV)b Drowsiness 60 (39.7) 56 (37.8) Fever (≥38.0°C) 10 (6.6) 10 (6.8) Irritability/fussiness 76 (50.3) 67 (45.3) Loss of appetite 49 (32.5) 46 (31.1) Unsolicited (spontaneously reported) symptoms during 28-day postvaccination period (N = 158 QIV, N = 156 TIV) All 77 (48.7) 75 (48.1) Related to vaccine 11 (7.0) 7 (4.5) Serious adverse event during entire study period (N = 158 QIV, N = 156 TIV) All 5 (3.2) 4 (2.6) Medically attended eventc during entire study period (N = 158 QIV, N = 156 TIV) All 77 (48.7) 89 (57.1) Abbreviations: AEs, adverse events; QIV, quadrivalent influenza vaccine; TIV, trivalent influenza vaccine. aAll solicited injection site symptoms were considered related to vaccination. bOnly subjects who have documented safety data were included in the calculation of solicited AEs. cHospitalization, emergency room visit, medical practitioner visit. Spontaneously reported AEs considered related to vaccination occurred in 7.0% and 4.5% of children in the QIV and TIV group, respectively (Table 3). Two grade 3 AEs (defined as severe enough to prevent everyday activity) considered possibly related to vaccination occurred in the QIV group (diarrhea and upper respiratory tract infection) and 1 in the TIV group (nasopharyngitis). Medically attended AEs occurred in 48.7% of children in the QIV group and 57.1% in the TIV group (Table 3).

fined as severe enough to prevent everyday activity) considered possibly related to vaccination occurred in the QIV group (diarrhea and upper respiratory tract infection) and 1 in the TIV group (nasopharyngitis). Medically attended AEs occurred in 48.7% of children in the QIV group and 57.1% in the TIV group (Table 3). Five children in the QIV group experienced an SAE: unspecified viral infection (1), respiratory syncytial virus infection (2), dehydration (1), and sleep apnea syndrome (1). Four children in the TIV group experienced SAEs: respiratory syncytial virus bronchiolitis (1), convulsion (1), failure to thrive (1), and neck abscess (1). None of the SAEs reported from either group was considered related to vaccination. No children experienced a pIMD, and there were no deaths during the study.

(1). Four children in the TIV group experienced SAEs: respiratory syncytial virus bronchiolitis (1), convulsion (1), failure to thrive (1), and neck abscess (1). None of the SAEs reported from either group was considered related to vaccination. No children experienced a pIMD, and there were no deaths during the study. DISCUSSION The 2013–2014 influenza season was the first time the World Health Organization selected a second influenza B virus for inclusion in QIV formulations, reflecting their recognition of the potential benefit of QIV for reducing the risk of influenza B disease [18, 19]. Although QIV eliminates the risk of reduced vaccine effectiveness due to influenza B virus lineage mismatch for all ages, children lacking prior exposure to both influenza B lineages may especially benefit from QIV. The QIV manufactured by GSK Vaccines and studied in the present trial is licensed for children from 6 months of age in Mexico and Canada but only from 3 years of age in the United States. To license GSK's QIV in younger children in the United States, a study demonstrating immunological noninferiority and an acceptable safety profile versus a licensed product is required. After the positive results of this phase II trial, a phase III trial (identifier NCT02242643) comparing GSK's QIV at a dose of 15 μg HA with the licensed QIV manufactured by Sanofi Pasteur is now underway at more than 60 sites in the United States and Mexico in children 6–35 months of age.

licensed product is required. After the positive results of this phase II trial, a phase III trial (identifier NCT02242643) comparing GSK's QIV at a dose of 15 μg HA with the licensed QIV manufactured by Sanofi Pasteur is now underway at more than 60 sites in the United States and Mexico in children 6–35 months of age. The present study showed that the QIV is immunogenic in children 6–35 months of age in stable health, with SCRs of 80.4%, 72.0%, 86.0%, and 66.4% against the A/H1N1, A/H3N2, B/Yamagata and B/Victoria strains, respectively. Because the LL of the 95% CI was ≥40% for each strain, these results met CBER's SCR licensure criterion. In addition, the QIV had superior immunogenicity against the B/Victoria strain compared with TIV. Immunogenicity against the 3 strains common to both vaccines was similar, indicating that addition of the second B strain does not affect immunogenicity of the other influenza strains. A study of Sanofi Pasteur's QIV in young children also supported that the addition of a second B strain has no negative impact on the immune responses to other strains, albeit at a dose of 7.5 μg HA [20]. There were no treatment group differences within either age strata. This is an important finding because in a previous study of an investigational inactivated TIV given at doses of either 7.5 μg or 15 μg HA, the investigational TIV elicited a lower immune response in children <18 months of age than Sanofi Pasteur's TIV [14], the same comparator as used in our study. In contrast, in our study, the immune response against the common vaccine strains was similar with the QIV and TIV in children 6–17 months of age.

or 15 μg HA, the investigational TIV elicited a lower immune response in children <18 months of age than Sanofi Pasteur's TIV [14], the same comparator as used in our study. In contrast, in our study, the immune response against the common vaccine strains was similar with the QIV and TIV in children 6–17 months of age. Immunogenicity and safety of inactivated QIVs have been evaluated in adults [21–24] and children [20, 25–27]. In all studies, the QIVs produced superior immune responses to the B lineage not contained in the control TIV and a comparable response to the common vaccine strains. One open-label [25] and 1 TIV-controlled study [27] evaluated GSK's QIV in children 6–35 months of age during the 2010–2011 and 2012–2013 influenza seasons. The results of those studies, taken together with the results described here, show that the QIV produces comparable immunogenicity across seasons. The GMT ratio and SCR difference versus TIV for the B/Victoria strain were of similar magnitude in the present study to those in the above-mentioned TIV-controlled study of GSK's QIV (GMT ratio 6.3 and SCR difference 64.2%) [27] and a study of Sanofi Pasteur's QIV (GMT ratio 4.4 and SCR difference 51.8%) [20].

seasons. The GMT ratio and SCR difference versus TIV for the B/Victoria strain were of similar magnitude in the present study to those in the above-mentioned TIV-controlled study of GSK's QIV (GMT ratio 6.3 and SCR difference 64.2%) [27] and a study of Sanofi Pasteur's QIV (GMT ratio 4.4 and SCR difference 51.8%) [20]. The QIV was given at the full adult dose of 15 μg HA per influenza strain in the present study, rather than the lower 7.5 μg HA dose traditionally recommended for infants in the United States. Historically, the lower dose was recommended because of the increase in fever and febrile convulsions observed in young children given a full dose of the whole virus vaccines available at the time [13]. However, studies of the TIV have shown variable immune responses in young children to the 7.5 μg HA dose, particularly to the vaccine B strain [11, 28, 29]. In contrast, studies using the full 15 μg HA dose as a split virion vaccine in young children have shown a consistently robust immune response with no increase in reactogenicity or fever [14–16].

shown variable immune responses in young children to the 7.5 μg HA dose, particularly to the vaccine B strain [11, 28, 29]. In contrast, studies using the full 15 μg HA dose as a split virion vaccine in young children have shown a consistently robust immune response with no increase in reactogenicity or fever [14–16]. Importantly, our study showed that the QIV and the TIV have similar reactogenicity and AE profiles, with no apparent adverse effect on tolerability of the higher antigen content in the QIV (60 μg HA for 4 strains compared with 22.5 μg for 3 strains in the TIV). Some previous studies have suggested that pain at the injection site may be modestly increased with QIV compared with TIV or hepatitis A vaccine [25, 30], whereas others report similar levels of pain [21, 23, 26, 27]. The incidence of fever was similar with both vaccines, consistent with previous published studies [25, 30].

previous studies have suggested that pain at the injection site may be modestly increased with QIV compared with TIV or hepatitis A vaccine [25, 30], whereas others report similar levels of pain [21, 23, 26, 27]. The incidence of fever was similar with both vaccines, consistent with previous published studies [25, 30]. Quadrivalent influenza vaccines in young children are expected to be particularly valuable during seasons in which both B lineages are cocirculating or there is an unexpected shift from one lineage to another. Influenza B is reported to cause a disproportionate number of influenza-related deaths in children [31]. Furthermore, it is well recognized, particularly in children, that vaccine efficacy and immunogenicity against influenza B is reduced if the B strain in the TIV is of a different lineage to the circulating B strain [7–9, 11, 12]. Mismatching of the vaccine and circulating B strain has occurred frequently. In the United States, the B strain in the seasonal TIV was mismatched to the circulating strain in 6 of the last 14 seasons [32]. Use of QIV in place of TIV is predicted to reduce the number of influenza cases, influenza-related hospitalizations, and influenza-related deaths in the overall population [33–35].

equently. In the United States, the B strain in the seasonal TIV was mismatched to the circulating strain in 6 of the last 14 seasons [32]. Use of QIV in place of TIV is predicted to reduce the number of influenza cases, influenza-related hospitalizations, and influenza-related deaths in the overall population [33–35]. CONCLUSIONS In conclusion, the investigational QIV was immunogenic with an acceptable safety profile in children 6–35 months of age. Compared with the licensed TIV, QIV had superior immunogenicity against the B/Victoria strain and comparable immunogenicity against the 3 strains common to both vaccines. The next phase of the QIV's development in children 6–35 months of age is a phase III trial in countries where it is not yet licensed. In countries where it is already licensed for this age group, a switch from TIV to QIV would provide broader protection in this vulnerable group.

the 3 strains common to both vaccines. The next phase of the QIV's development in children 6–35 months of age is a phase III trial in countries where it is not yet licensed. In countries where it is already licensed for this age group, a switch from TIV to QIV would provide broader protection in this vulnerable group. Acknowledgments We are indebted to the participating study volunteers and their parents, clinicians, nurses, and laboratory technicians at the study sites, as well as to the sponsor's project staff for their support and contributions throughout the study. In particular, we thank W. Seger, A. Moskow, S. Moskow, R. Hines, and E. Zissman (investigators); and W. Jiang (Central Safety Contact), C. Probst (Regulatory Affairs representative), B. Corsaro (Project Manager), E. Praet (Study Delivery Lead), C. Stalens (Study Manager), S. Ravault (Clinical Regulatory Affairs), and B. Pereira (Scientific Writer) from GSK Vaccines. Finally, we thank M. L. Greenacre (An Sgriobhadair, UK, on behalf of GSK Vaccines) for providing medical writing services and B. Dumont (Business & Decision Life Sciences, on behalf of GSK Vaccines) for editorial assistance and manuscript coordination.

ory Affairs), and B. Pereira (Scientific Writer) from GSK Vaccines. Finally, we thank M. L. Greenacre (An Sgriobhadair, UK, on behalf of GSK Vaccines) for providing medical writing services and B. Dumont (Business & Decision Life Sciences, on behalf of GSK Vaccines) for editorial assistance and manuscript coordination. Author contributions. V. K. J. acquired the funding and participated in the choice of centers and recruitment of the investigators. All authors participated in the conception, design, and planning of the study. J. B. D. and V. K. J. provided subjects or study materials. L. W., V. C., J. B. D., and P. L. participated to the acquisition or the assembling of the data. L. W., V. C., P. L., and V. K. J. performed or supervised the analysis, and L. W., V. C., B. L. I., and V. K. J. performed the quality check. All authors contributed to the interpretation of results. V. C. and P. L. provided statistical expertise. L. W., P. L., B. L. I., and V. K. J. supervised of the study group. All authors read and approved the final manuscript. Financial support. This work was supported by GlaxoSmithKline Biologicals SA. GlaxoSmithKline Biologicals SA paid for all costs associated with the development of this manuscript.

Author contributions. V. K. J. acquired the funding and participated in the choice of centers and recruitment of the investigators. All authors participated in the conception, design, and planning of the study. J. B. D. and V. K. J. provided subjects or study materials. L. W., V. C., J. B. D., and P. L. participated to the acquisition or the assembling of the data. L. W., V. C., P. L., and V. K. J. performed or supervised the analysis, and L. W., V. C., B. L. I., and V. K. J. performed the quality check. All authors contributed to the interpretation of results. V. C. and P. L. provided statistical expertise. L. W., P. L., B. L. I., and V. K. J. supervised of the study group. All authors read and approved the final manuscript. Financial support. This work was supported by GlaxoSmithKline Biologicals SA. GlaxoSmithKline Biologicals SA paid for all costs associated with the development of this manuscript. Potential conflicts of interest. L. W., V. C., P. L., B. L. I., and V. K. J. are employees of the GSK group of companies. L. W., P. L., B. L. I., and V. K. J. report ownership of stock options and/or restricted shares. J. B. D. reports payments from the GSK group of companies for the conduct of the study; and grants from Pfizer and from the GSK group of companies, outside the submitted work. J. B. D. also provides consulting in the area of immunizations for Sanofi Pasteur, Novartis, Merck, the GSK group of companies, and Medimmune; he has also served on advisory boards for the GSK group of companies and Medimmune.

tudy; and grants from Pfizer and from the GSK group of companies, outside the submitted work. J. B. D. also provides consulting in the area of immunizations for Sanofi Pasteur, Novartis, Merck, the GSK group of companies, and Medimmune; he has also served on advisory boards for the GSK group of companies and Medimmune. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

In 2014, 9.6 million people developed active tuberculosis worldwide, including an estimated 1.2 million living with human immunodeficiency virus (HIV) [1]. Tuberculosis remains the primary cause of death among HIV-infected people (390 000 deaths) [1, 2]. Thailand is one of the 22 high tuberculosis burden countries listed by the World Health Organization (WHO), with an incidence of 171 per 100 000 in the general population in 2014, whereas the incidence reported in the HIV-infected population is more than 20 times higher [1, 3]. Approximately half of the 12 000 people who died of tuberculosis in Thailand in 2014 were infected with HIV [1]. In Thailand, data on tuberculosis incidence and associated mortality are limited, particularly in children living with HIV [4–6], and the long-term risk of tuberculosis in children on antiretroviral therapy (ART) is unknown. Within a large multicenter cohort of HIV-infected children on ART in Thailand, we estimated tuberculosis incidence, the impact of different risk factors on incident tuberculosis, and the contribution of tuberculosis on the risk of death. METHODS Study Population This analysis included all HIV-infected children less than 15 years of age who initiated first-line ART between January 1, 1999 and December 31, 2012 and were followed in the Program for HIV Prevention and Treatment (PHPT) cohort (ClinicalTrials.gov: NCT00433030) in a network of public hospitals throughout the country, as previously described [7].

V-infected children less than 15 years of age who initiated first-line ART between January 1, 1999 and December 31, 2012 and were followed in the Program for HIV Prevention and Treatment (PHPT) cohort (ClinicalTrials.gov: NCT00433030) in a network of public hospitals throughout the country, as previously described [7]. Baseline Data and Follow-Up Children were followed-up at 2 weeks, 1 month, and 3 months after initiating ART and every 3 months thereafter for physical examination, complete blood count (hemoglobin, hematocrit, white blood count, red blood count, platelets, lymphocytes, neutrophils), clinical chemistries (plasma glucose, alanine transaminase, creatinine), drug refills, and adherence counseling. CD4 percentage and HIV ribonucleic acid (RNA) viral load were measured 3 and 6 months after ART initiation and every 6 months thereafter. The cohort study protocol included screening for tuberculosis before initiating ART and during follow-up in case of presumptive symptoms, based on interview, medical history, clinical examination, chest x-ray, tuberculin skin test, sputum acid-fast bacillus smear and/or gastric aspirate if indicated. Children who never came back for follow-up during the study period were considered lost to follow-up. In the event of a participant’s death, the immediate cause and underlying conditions were reported by the attending physicians. All death reports were reviewed, and causes of death were classified by 2 independent physicians based on the International Classification of Diseases [8].

d were considered lost to follow-up. In the event of a participant’s death, the immediate cause and underlying conditions were reported by the attending physicians. All death reports were reviewed, and causes of death were classified by 2 independent physicians based on the International Classification of Diseases [8]. Tuberculosis Case Definition A physician (C. D.) reviewed all available records of cases with suspected tuberculosis infection and classified each tuberculosis case as clinically diagnosed with or without bacteriologic confirmation, following the WHO reporting guidelines [9]. Children were classified in 4 groups according to their tuberculosis infection status: “prevalent tuberculosis” for children with ongoing tuberculosis treatment at the time of ART initiation or starting within the following 30 days; “incident tuberculosis” for children with a first or recurrent tuberculosis diagnosis at least 30 days after ART initiation; “history of treated tuberculosis” for children who had recovered from tuberculosis before ART initiation; and “no tuberculosis” for children who were not diagnosed with tuberculosis before ART initiation or during follow-up. The choice of the cutoff (30 days) to define “prevalent” and “incident” tuberculosis was driven by the literature [10, 11], although it is likely that not all tuberculosis infections already established at time of ART initiation were diagnosed within 30 days.

with tuberculosis before ART initiation or during follow-up. The choice of the cutoff (30 days) to define “prevalent” and “incident” tuberculosis was driven by the literature [10, 11], although it is likely that not all tuberculosis infections already established at time of ART initiation were diagnosed within 30 days. Statistical Analysis The follow-up period was from the date of ART initiation to the date of death, date of last clinic visit, or December 31, 2013, whichever occurred first. Children with a history of treated tuberculosis at time of ART initiation were included in the incidence and risk factor analyses, but those with prevalent tuberculosis were excluded. The incidence of tuberculosis infections diagnosed during follow-up was calculated using person-time incidence rates. Overall and stratified incidence rates were defined as the number of incident tuberculosis cases divided by the number of person-years of follow-up (PYFU). The 95% confidence intervals (CIs) of the incidence rates were calculated based on the Poisson distribution. Risk factors for incident tuberculosis were identified using univariable and multivariable Fine and Gray’s competing risks proportional subhazards models [12], with death from other causes treated as a competing event. The association between diagnosis of incident tuberculosis and mortality was evaluated using univariable and multivariable Cox proportional hazards regression models, tuberculosis diagnosis being dichotomized and treated as a time-dependent variable (never or ever been diagnosed with tuberculosis).

s a competing event. The association between diagnosis of incident tuberculosis and mortality was evaluated using univariable and multivariable Cox proportional hazards regression models, tuberculosis diagnosis being dichotomized and treated as a time-dependent variable (never or ever been diagnosed with tuberculosis). Potential risk factors for incident tuberculosis and mortality assessed at ART initiation were as follows: sex, age, body mass index (BMI)-for-age [13, 14], calendar year of enrollment, and age-adjusted HIV RNA viral load, CD4 percentage, hemoglobin, hematocrit, neutrophils, and lymphocytes. Missing values at ART initiation were imputed using multiple imputation by chained equations with predictive mean matching (10 imputations) [15]. Except for BMI-for-age and calendar year of enrollment, all continuous variables were first categorized into quartiles rounded to the nearest clinically meaningful values and then put into smaller groups determined by the magnitude of the model coefficients in the univariable analyses. Multivariable models included all variables with P < .25 in the univariable analyses, and a forward selection procedure was used to identify risk factors independently associated with incident tuberculosis and mortality. The proportional hazards assumption of the models was tested using an interaction term between independent variables and time. Data were censored at the date of last clinic visit or December 31, 2013, whichever occurred first.

d to identify risk factors independently associated with incident tuberculosis and mortality. The proportional hazards assumption of the models was tested using an interaction term between independent variables and time. Data were censored at the date of last clinic visit or December 31, 2013, whichever occurred first. All reported P values are 2-sided, and P < .05 was considered statistically significant. Missing data imputation and statistical analyses were performed using Stata 13.0 (StataCorp, College Station, TX). Ethical Considerations The PHPT Cohort protocol and subsequent amendments were approved by the Ethics Committees of the Thai Ministry of Public Health, the Faculty of Associated Medical Sciences at Chiang Mai University, and local hospital ethics committees. RESULTS Study Population Characteristics A total of 670 children were eligible for inclusion (Figure 1), including 367 (55%) females. At ART initiation, median age was 6.4 years (interquartile range [IQR], 2.0 to 9.6), BMI-for-age z-score was −0.8 (IQR, −1.9 to 0.0), HIV RNA viral load was 5.1 log10 copies/mL (IQR, 4.6 to 5.6), and CD4 was 9% (IQR, 3 to 17) (Table 1). Fifty-five children (8%) received isoniazid preventive therapy at some point during the study period. The median duration of follow-up from ART initiation was 7.7 years (IQR, 3.6 to 9.8). Of the 670 children, 14 had a history of treated tuberculosis. At the end of the study, 377 (56%) children were no longer on follow-up: 161 voluntarily withdrew from the study, 160 were lost to follow-up, and 56 died.

he study period. The median duration of follow-up from ART initiation was 7.7 years (IQR, 3.6 to 9.8). Of the 670 children, 14 had a history of treated tuberculosis. At the end of the study, 377 (56%) children were no longer on follow-up: 161 voluntarily withdrew from the study, 160 were lost to follow-up, and 56 died. Figure 1. Study population flowchart. ART, antiretroviral therapy; HIV, human immunodeficiency virus; PHPT, Program for HIV Prevention and Treatment. Table 1. Study Population Characteristics at ART Initiation

he study period. The median duration of follow-up from ART initiation was 7.7 years (IQR, 3.6 to 9.8). Of the 670 children, 14 had a history of treated tuberculosis. At the end of the study, 377 (56%) children were no longer on follow-up: 161 voluntarily withdrew from the study, 160 were lost to follow-up, and 56 died. Figure 1. Study population flowchart. ART, antiretroviral therapy; HIV, human immunodeficiency virus; PHPT, Program for HIV Prevention and Treatment. Table 1. Study Population Characteristics at ART Initiation Characteristics at ART Initiation Prevalent Tuberculosis (n = 47a) n (%) Incident Tuberculosis (n = 30a) n (%) No Tuberculosis (n = 593a) n (%) Overall (n = 670a) n (%) Female sex 26 (55) 23 (77) 318 (54) 367 (55) Age <2 years 6 (13) 7 (23) 153 (26) 166 (25) 2 to 6 years 10 (21) 4 (13) 138 (23) 152 (23) 6 to 10 years 19 (40) 8 (27) 180 (30) 207 (31) ≥10 years 12 (26) 11 (37) 122 (21) 145 (22) BMI-for-age (n = 43) (n = 27) (n = 527) (n = 597) z-score < −2 16 (37) 9 (33) 131 (25) 156 (26) z-score between −2 and +1 24 (56) 17 (63) 347 (66) 388 (65) z-score ≥ +1 3 (7) 1 (4) 49 (9) 53 (9) Calendar Year of Enrollment 1999 to 2002 5 (11) 4 (13) 111 (19) 120 (18) 2003 to 2005 33 (70) 22 (73) 331 (56) 386 (58) 2006 to 2012 9 (19) 4 (13) 151 (25) 164 (24) HIV RNA viral load (n = 37) (n = 23) (n = 460) (n = 520) <4.5 log10 copies/mL 2 (5) 4 (17) 91 (20) 97 (19) 4.5 to 5.0 log10 copies/mL 7 (19) 4 (17) 88 (19) 99 (19) 5.0 to 5.5 log10 copies/mL 13 (35) 5 (22) 131 (28) 149 (29) ≥5.5 log10 copies/mL 15 (41) 10 (43) 150 (33) 175 (34) CD4 (n = 45) (n = 29) (n = 562) (n = 636) <5% 19 (42) 18 (62) 192 (34) 229 (36) 5% to 10% 9 (20) 2 (7) 100 (18) 111 (17) 10% to 15% 12 (27) 3 (10) 95 (17) 110 (17) ≥15% 5 (11) 6 (21) 175 (31) 186 (29) Hemoglobin (n = 43) (n = 28) (n = 529) (n = 600) <9 g/dL 7 (16) 7 (25) 83 (16) 97 (16) 9 to 10 g/dL 8 (19) 8 (29) 102 (19) 118 (20) 10 to 11 g/dL 9 (21) 7 (25) 135 (26) 151 (25) ≥11 g/dL 19 (44) 6 (21) 209 (40) 234 (39) Hematocrit (n = 44) (n = 28) (n = 539) (n = 611) <30% 9 (20) 12 (43) 146 (27) 167 (27) 30% to 32% 7 (16) 6 (21) 84 (16) 97 (16) 32% to 34% 10 (23) 4 (14) 110 (20) 124 (20) ≥34% 18 (41) 6 (21) 199 (37) 223 (36) Neutrophils (n = 43) (n = 28) (n = 509) (n = 580) <3000 cells/mm3 28 (65) 7 (25) 222 (44) 257 (44) 3000 to 4000 cells/mm3 3 (7) 8 (29) 117 (23) 128 (22) 4000 to 5000 cells/mm3 4 (9) 3 (11) 59 (12) 66 (11) ≥5000 cells/mm3 8 (19) 10 (36) 111 (22) 129 (22) Lymphocytes (n = 43) (n = 28) (n = 509) (n = 580) <2000 cells/mm3 22 (51) 18 (64) 191 (38) 231 (40) 2000 to 3000 cells/mm3 7 (16) 3 (11) 115 (23) 125 (22) 3000 to 4000 cells/mm3 7 (16) 4 (14) 56 (11) 67 (12) ≥4000 cells/mm3 7 (16) 3 (11) 147 (29) 157 (27) Abbreviations: ART, anti

ls/mm3 8 (19) 10 (36) 111 (22) 129 (22) Lymphocytes (n = 43) (n = 28) (n = 509) (n = 580) <2000 cells/mm3 22 (51) 18 (64) 191 (38) 231 (40) 2000 to 3000 cells/mm3 7 (16) 3 (11) 115 (23) 125 (22) 3000 to 4000 cells/mm3 7 (16) 4 (14) 56 (11) 67 (12) ≥4000 cells/mm3 7 (16) 3 (11) 147 (29) 157 (27) Abbreviations: ART, anti retroviral therapy; BMI, body mass index; HIV, human immunodeficiency virus; RNA, ribonucleic acid. aUnless otherwise specified. Description of Tuberculosis Cases Of the 670 children, 77 (11%) experienced tuberculosis. Of these 77 cases, 47 were prevalent (none of them experienced recurrent tuberculosis after ART initiation) and 30 incident (including 2 with a history of treated tuberculosis before ART initiation and 1 with 2 tuberculosis episodes after ART initiation). Eight were bacteriologically confirmed and 69 were clinically diagnosed. There were 56 pulmonary, 12 lymphadenitis, 2 meningitis, 2 abdominal, 1 pulmonary plus meningitis, 1 parotid and 1 cutaneous tuberculosis, and 2 unspecified tuberculosis sites.

roposed [16], the physician in charge of the retrospective review suspected that 8 children (3 with a prevalent tuberculosis and 5 with an incident tuberculosis) developed an IRIS between 10 days and 4 months after ART initiation. However, the information available was not sufficient to definitely classify these cases. Tuberculosis Incidence After exclusion of the 47 prevalent tuberculosis cases, 623 children contributed to 4068 PYFU. There were 30 incident tuberculosis cases, leading to an overall crude tuberculosis incidence rate of 7 per 1000 PYFU (95% CI, 5–11) (when including the 47 prevalent tuberculosis cases in the calculation, the incidence was similar: 7 per 1 000 PYFU [95% CI, 5–10]). The incidence rate was 4 per 1000 PYFU in males (95% CI, 2–8) and 10 per 1000 PYFU in females (95% CI, 7–16). Median duration of follow-up between ART initiation and tuberculosis diagnosis was 15 months (IQR, 3–65). Incidence rates sharply decreased with the duration of follow-up, from 60 per 1000 PYFU (95% CI, 31–115) from 1 to 3 months after ART initiation to 5 per 1000 PYFU (95% CI, 2–10) after 5 years (Figure 2). Figure 2. Incidence rates of tuberculosis after antiretroviral therapy (ART) initiation, stratified by follow-up duration. Circles: estimated incidence rates per stratum of follow-up duration since ART initiation. Segments: 95% confidence intervals of the incidence rates per stratum, calculated based on the Poisson distribution. Dotted line: overall incidence rate.