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Introduction Schistosomiasis mansoni was first described in Uganda in Arua district, in West Nile, the area of the present study in 1902. This was the first time ever S.mansoni was observed (Castellani, 1903; Castellani and Low 1904). Since then the Districts of West Nile, the areas for this study drew a lot of attention in the study of clinical, epidemiology and pathology of S.mansoni (Nelson 1958; Ongom and Bradley. 1972., Ongom et al., 1997). Nelson was the first to make a complete assessment of the incidence, distribution and importance of schistosomiasis mansoni as a health problem in Uganda especially in West Nile (Nelson, 1958a). He also found that the prevalence and intensity of infection was highest immediately along the bank of the River Nile and decrease with altitude and distance from the bank of the Rive Nile. Nelson further observed that enlarged spleens and anaemia was common clinical feature among young children of ten years old with intense infections with Bilharzia (Nelson, 1958b). In resent studies in Rhino Camp, Adama showed deterioration in the morbidity due to S.mansoni infection (dissertation for Health Visitor Collage Kampala-Uganda, 1986). In the same year, Bukenya and Adama reported a prevalence of 37% in Rhino Camp (Bukenya and Adama, 1986).

nse infections with Bilharzia (Nelson, 1958b). In resent studies in Rhino Camp, Adama showed deterioration in the morbidity due to S.mansoni infection (dissertation for Health Visitor Collage Kampala-Uganda, 1986). In the same year, Bukenya and Adama reported a prevalence of 37% in Rhino Camp (Bukenya and Adama, 1986). Previously in Uganda and elsewhere in the tropical countries where S.mansoni is endemic, the study on the morbidity of S. mansoni infections was based on the intensity of the egg excretion, and clinical methods to describe the pathological sequelae induced by the parasites. These methods included the quantitative estimation of S.mansoni eggs in the stool specimens, palpation of liver and spleen, biopsy of liver and kidney, X-ray and autopsy of the liver, kidneys spleen and other internal organs. Any of these methods or in combination as applicable were used to detect the morbidity and pathology due to S. mansoni. By these methods it was difficult to accurately assess the degree of pathology in these organs (Nelson 1958a or 1958b//; Ongom et al. 1972, 1997). Beside, the invasive needle biopsy of these organs in inexperience hands was risky and sometimes fatal. This made monitoring of reversibility of pathology in these organs after schistosomal chemotherapy difficult (Ongom et al., 1997). Abdel Wahab introduced a non-invasive ultrasound as a tool to detect pathology due to S. mansoni. These pathological lesions are common in most internal organs. The sexually mature female and male worms pair in the liver and move out into the mesenteric veins in the small intestine where they dwell and start to lay eggs. This is true in cases of S. mansoni, S. japonicum, S. intercalatum, and S. mekongi. Nevertheless, S. haematobium move to the wall of the bladder where they reside. One to two months after initial infection, clinical signs and symptoms develop. These symptoms include clinical toxaemic fever, Katayama fever common in schistosomiasis mansoni and schistosomiasis japonicum (Ana et al,. 1995), weakness, weight loss, diarrhoea, abdominal pain, urticaria and marked eosinophilia are clinical symptoms commonly seen in the acute phase of infection that progresses to the chronic phase. After three to five months, the chronic intestinal phase (IN) sets in. During IN stage schistosome eggs are present in stool. This stage is followed by hepatointestinal (HI) stage. In this stage S. mansoni eggs increase in stool and the liver becomes palpable (Chen and Mott, 1988).

that progresses to the chronic phase. After three to five months, the chronic intestinal phase (IN) sets in. During IN stage schistosome eggs are present in stool. This stage is followed by hepatointestinal (HI) stage. In this stage S. mansoni eggs increase in stool and the liver becomes palpable (Chen and Mott, 1988). Hepatosplenic (HS) schistosomiasis is the established chronic phase of schistosomiasis usually resulting from heavy S. mansoni infection. In the HS phase, the clinical symptoms observed in acute phase are apparent and both the liver and spleen are palpable. This stage is closely associated with periportal fibrosis, portal hypertension, collateral circulation, oesophageal varices, ascites and heamatemesis (Gazzinelli et al,. 1985, 1992; Gerspacher-Lara et al,. 1998). In human schistosomiasis, the eggs laid by female schistosome cause morbidity, which is the severity of the disease (Gryseels and Poldernan, 1991). The circulating eggs can reach nearly all organs like the spleen, kidney, heart and occasionally the brain. These eggs are immunogenic. They elicit immune responses in the host (Amelia et al., 2000) with granuloma formations around the eggs which later become fibrotic and calcified in the venous wall. Portal veins and the surrounding periportal veins are commonly affected.

he spleen, kidney, heart and occasionally the brain. These eggs are immunogenic. They elicit immune responses in the host (Amelia et al., 2000) with granuloma formations around the eggs which later become fibrotic and calcified in the venous wall. Portal veins and the surrounding periportal veins are commonly affected. This non-invasive repeatable method of Abdel Wahab for detecting periportal fibrosis due to S. mansoni led to more detailed revelation of pathological feature in internal organs of patients infected with S. mansoni and allows the monitoring of the reversibility of the liver periportal fibrosis after schistosomal therapy (Houston et al., 1993). In most S. mansoni endemic areas longitudinal observations of reversibility of the periportal fibrosis after treatment with praziquental 40mg/kg body weight are still scarce (Doehring -Schwedtfeger et al., 1992). Study of this kind would help to determine the epidemiology and praziquantel chemotherapy regimen required for control of schitosomiasis morbidity in hyper endemic areas (Homeida et al, .1988; Doerhing Schwerdtfeger et al,. 1990; Abdel- Wahab et al,. 1990).

weight are still scarce (Doehring -Schwedtfeger et al., 1992). Study of this kind would help to determine the epidemiology and praziquantel chemotherapy regimen required for control of schitosomiasis morbidity in hyper endemic areas (Homeida et al, .1988; Doerhing Schwerdtfeger et al,. 1990; Abdel- Wahab et al,. 1990). The aim of this study was to evaluate the effect of treatment with praziquantel, 40mg/kg body weight one year later and the reduction of the eggs excretions, on the reversibility of S. mansoni induced liver periportal fibrosis and to observe specific dynamic developments of S mansoni induced liver parenchyma alterations among the 1273 patients previously studied in 2005 (Houston et al., 1993; Doehring -Schwedtfeger et al., 1992, Homeida et al., 1988; Doerhing Schwerdtfeger et al., 1990; Abdel- Wahab, et al., 1990).

liver periportal fibrosis and to observe specific dynamic developments of S mansoni induced liver parenchyma alterations among the 1273 patients previously studied in 2005 (Houston et al., 1993; Doehring -Schwedtfeger et al., 1992, Homeida et al., 1988; Doerhing Schwerdtfeger et al., 1990; Abdel- Wahab, et al., 1990). Materials and Methods In 2005, 1562 people including fishermen and women, school pupils, teachers, and civil servants were enrolled and studied in Rhino Camp and Obongi fishing villages in Northern Uganda for S. mansoni using Kato/Katz stool smear method. Each village had a population between 4,000 and 6,000 people. Rhino Camp is about 60 Kilometres East of Arua town, and it is located 30 North and 310 East. Here (n=733) 46.9% people were screened microscopically for S. mansoni infection. Obongi is located about 3.50 North and 31.50 East. Likewise in Obongi (n=829) 53.1% people were examined microscopically for S. mansoni infections. Obongi is about 100 kilometres north of Arua town (Odongo-Aginya et al,. 2002). The main activity in Rhino Camp and Obongi villages is fishing, although, there is low scale cultivation along the Nile where the inhabitants plant food crops like vegetables, cassava and potatoes. There is a poor earth surface road system connecting the two villages used by few lorries which come to these villages on market days. Therefore, the inhabitants of the two villages mainly depend on canoes as their means of transportation.

le where the inhabitants plant food crops like vegetables, cassava and potatoes. There is a poor earth surface road system connecting the two villages used by few lorries which come to these villages on market days. Therefore, the inhabitants of the two villages mainly depend on canoes as their means of transportation. The stratification of the study groups in the two fishing villages was similar. Firstly, the registered and licensed fishermen and women operating fishing canoes at the landing sites including their families were all included in the study. Secondly, the pupils in primary schools situated about 100 meters to the river were all studied. Thirdly, a small number of civil servants including teachers, police, local administrators, and priests, people with low water contact were also registered in the study. The fourth group included volunteers from the community who came and enrolled in the study every morning. Stool examination for parasites Each patient participating in the study was given a stool container and was asked to return it with about 10 gram of his or her stool the next day. Three slides containing 41.7mg of sieved stool were processed using Kato/Katz method (Katz, et al., 1994). The arithmetic mean egg count of the three slides examined was multiplied by a factor of 24 to obtain the egg count per gram faeces.

and was asked to return it with about 10 gram of his or her stool the next day. Three slides containing 41.7mg of sieved stool were processed using Kato/Katz method (Katz, et al., 1994). The arithmetic mean egg count of the three slides examined was multiplied by a factor of 24 to obtain the egg count per gram faeces. Pathological study using portable ultrasound machine Ultrasound observers were blinded of the result of all the tests, especially, egg count in both studies in 2005 and 2006, and Pf stages results from the previous year 2005. Ultrasound was performed using the same high quality Aloka portable ultrasound machine (SSD-500, Hellige-Aloka, Freiburg, Germany) in both studies in 2005 and 2006. It was equipped with 3.5 MHZ convex transducer. Photo documentation was done using a Mitsubishi p 66 E-Thermal printers. Abdominal ultrasound was done with the patient lying in dorsal position according to the most recently proposed technique (Jenkins et al., (1992). The sonomorphological abnormalities of periportal fibrosis were categorised according to the Managil score (Cairo working group, 1992). Organomorphometry of liver and spleen was done according to Dittrich et al. (1983) that is, liver length in three different sections, external diameter of extrahepatic portal veins, and three PF branches were measured in millimetres. The grading of the liver fibrosis was done according to Doerhing Schwerdtfeger et al. (1990) as follows: -

liver and spleen was done according to Dittrich et al. (1983) that is, liver length in three different sections, external diameter of extrahepatic portal veins, and three PF branches were measured in millimetres. The grading of the liver fibrosis was done according to Doerhing Schwerdtfeger et al. (1990) as follows: - 0 No echogenicity with smooth texture around the portal veins. This represents grade (0). 1 Echogenicity with smooth irregular texture around periportal veins and gall bladder neck. This represents grade (I) PF. 2 Broad echogenicity >10mm in the central and around periportal veins indicate grade (II) PF. 3 Involvement of complete liver characterized by echogenicities streaks not confined to periportal veins only.

0 No echogenicity with smooth texture around the portal veins. This represents grade (0). 1 Echogenicity with smooth irregular texture around periportal veins and gall bladder neck. This represents grade (I) PF. 2 Broad echogenicity >10mm in the central and around periportal veins indicate grade (II) PF. 3 Involvement of complete liver characterized by echogenicities streaks not confined to periportal veins only. This indicates grade (III) PF. The same people studied in 2005 who had S. mansoni eggs in their stool came back for review in 2006 one year later. Nine hundred and thirty nine n=939 (73.8%) out of n=1273 people were not excreting S. mansoni eggs in their stool detected using Kato/Katz method Katz et al. (1994) while 334 (26.2) were still excreting S.mansoni in their stool. The intensity of the eggs excreted in 2006 was lower than the previous year. Nevertheless the abdominal ultrasound scan was done on all 1273 again in 2006. The recovery of all the patients studied in previous year has been possible because of the short period, the good mobilisation of the community by the local authorities and the willingness of the patients to participate in the study because of the free treatments and inducement offered to them during the study. Because of this, some of the participants walked long distances to come to be examined and treated. The same procedures used in the previous year were repeated during the re-evaluation study. Detailed clinical investigation was done with the help of the health workers .The clinical questions asked were, information on diarrhoea, blood in stool, haematemesis, and abdominal pains.

es to come to be examined and treated. The same procedures used in the previous year were repeated during the re-evaluation study. Detailed clinical investigation was done with the help of the health workers .The clinical questions asked were, information on diarrhoea, blood in stool, haematemesis, and abdominal pains. Treatment Stool for microscopic examinations were all done in the morning hr when the patients had just brought in their stool specimens. Those who were found excreting eggs of S. mansoni in their stool were treated with a single oral dose of Praziquantel in the field clinic the next day in the morning by nurses who were trained in the procedures for the treatment. The patients were advised to eat before coming for treatment. Their weights were taken and interpolated against pre-calculated dosages chart containing the number of the 600 mg tablets of Praziquantel (FROM MEDOCHEMIE LTD.LIMASSOL-CYPRUS EUROPE) required to achieve 40 mg/kg body weight. Pregnant mothers were not treated with praziquantel until after birth. Other intestinal Helminths and protozoa were treated accordingly. In addition, ailments which could not be treated in the study clinic were referred to other Health facilities and Arua Hospital. Inclusion criteria Permanent residents of Rhino Camp and Obongi fishing villages. Residents who have fully consented to participate in the study. Children between 5 and 18 years old who were granted full permission to participate in the study by their parents/ guardians and who signed the ethical form on behalf of their children.

Inclusion criteria Permanent residents of Rhino Camp and Obongi fishing villages. Residents who have fully consented to participate in the study. Children between 5 and 18 years old who were granted full permission to participate in the study by their parents/ guardians and who signed the ethical form on behalf of their children. Individuals who had not had antischistosomal treatment six months prior the study. Exclusion criteria Non-residents of Rhino Camp and Obongi fishing villages. Residents who declined to participate in the study. Individuals who had had treatments with antischistosomal drugs six months before the study. Those individual who were very ill with chronic ailments. Ethical consideration This study received clearance from the Uganda Government Ministry of Health ethical committee and approval of Uganda National Council of Science and Technology all based in Kampala Uganda. Informed consent was obtained from the adults but in cases of children under eighteen years of age, their parents' consent was requested. The community was educated on the scope of the study in the local languages and they were asked to decide on the participation of their children.

hnology all based in Kampala Uganda. Informed consent was obtained from the adults but in cases of children under eighteen years of age, their parents' consent was requested. The community was educated on the scope of the study in the local languages and they were asked to decide on the participation of their children. Results A total of 1,562 people from Rhino Camp and Obongi fishing villages along the Albert Nile in Northern Uganda were examined parasitologically for S.mansoni infection. These were from Rhino Camp, seven hundred and thirty three (n=733) 46.9% and Obongi (n=829) 53.1%. A total of (n=1273) 81.5% had S. mansoni eggs in their stools and (n=289) 18.5% were negative for S.mansoni infection in the field. The 289 negative specimens were further processed in the laboratory in Borne Germany using formal either concentration technique. Seven of them were positive with low egg count of an average of two eggs per low power microscopic field. Therefore, the true prevalence of S.mansoni infection is higher than 81.5% recorded in the field. The intensity of infection was classified as follows: Low 1–99epg, medium 100–499epg and high ≥500 epg (Sleigh et al. 1958). The distribution of different PFs in the different egg intensity in the Rhino Camp and Obongi fishing villages is shown in Figure 1. Age related intensity and the distribution in different grades of PF is shown in Figure 2. The distribution of PFs according to sex in the study patients was similar but their occupational activities were shown to influence the prevalence and the intensities of infections and consequentially the pathologies of the disease (Figure 3). The patterns of percentage of people with PFs in Rhino Camp and Obongi were similar in both 2005 and 2006 (Figure 4).

n the study patients was similar but their occupational activities were shown to influence the prevalence and the intensities of infections and consequentially the pathologies of the disease (Figure 3). The patterns of percentage of people with PFs in Rhino Camp and Obongi were similar in both 2005 and 2006 (Figure 4). Figure 1 The distribution of periportal fibrosis according to the intensity of infection in 2005 Relationship between different levels of intensity of infections and grades of periportal fibrosis among patients studied in Rhino Camp and Obongi in Northern Uganda, 1–99epg ▪; 100– 499epg □; ≥ 500epg Figure 2 Periportal fibrosis according to age group in Rhino Camp and Obongi in 2005 Periportal fibrosis in different age groups in years in Rhino Camp and Obongi in Northern Uganda 1–10 ▪; 11–20 □; ≥30 Figure 3 showing relative influence of occupational activities of Periportal Fibrosis in Rhino Camp and Obongi 2005. Periportal fibrosis in different categories of patients studied at Rhino Camp and Obongi in Northern Uganda according to their occupations; Pfs I ; Pfs II □; Pfs III ▪ Figure 4 Comparing different grades of periportal fibrosis in Rhino Camp and Obongi fishing villages in 2005. Percentage comparison of periportal fibrosis at Rhino Camp and Obongi in northern Ugandan. Periportal fibrosis in Rhino Camp ○ Periportal fibrosis in Obongi ▪

Periportal fibrosis in different categories of patients studied at Rhino Camp and Obongi in Northern Uganda according to their occupations; Pfs I ; Pfs II □; Pfs III ▪ Figure 4 Comparing different grades of periportal fibrosis in Rhino Camp and Obongi fishing villages in 2005. Percentage comparison of periportal fibrosis at Rhino Camp and Obongi in northern Ugandan. Periportal fibrosis in Rhino Camp ○ Periportal fibrosis in Obongi ▪ Percentage of reversibility of Pfs of people who had Abdominal ultrasound done on them in both years indicated that out of 506 patients found with no pathology in their livers in 2005 (Pf= 0); 395 (78%) remained at the same level. However, 98 (19%) of them developed light liver parenchyma changes, Pf grade (I). A small number of the patients, 13 (3%) presented moderate Pf grade (II). Two hundred fifty nine (259) patients showed Pf grade (I) in 2005 out of whom 169 (65%) became Pf (0) in 2006. Seventy four (23.6%) remained in Pf grade (I) and 16 (6.2%) digressed to Pf grade (II). Out of the 147 (86%) patients with Pf grade (II) in 2005, 90 (61%) ameliorated their liver parenchyma damage to Pf grade (0) and 45 (30.6%) had light changes of their liver parenchyma to Pf grade (I), 6 (4.1%) remained in Pf grade (II) and another 6 (4.1%) of them digress to Pf grade (III). Among the 27 severe Pf grade (III) detected in the previous year, 8 (29.6%) reversed to Pf grade (0), another 8 (29.6%) improved to Pf grade (II) and 7 (25.9%) reverted to Pf grade (I). But 4 (14.8%) remained in Pf grade (III)

), 6 (4.1%) remained in Pf grade (II) and another 6 (4.1%) of them digress to Pf grade (III). Among the 27 severe Pf grade (III) detected in the previous year, 8 (29.6%) reversed to Pf grade (0), another 8 (29.6%) improved to Pf grade (II) and 7 (25.9%) reverted to Pf grade (I). But 4 (14.8%) remained in Pf grade (III) In general, in 2006, after a single dose treatment with Praziquantel 40mg/kg body weight, there were more people who were negative for S.mansoni infections (n=939) 73.8% unlike in the previous year where only (n=128) 18.5% had no S. manson in their stool. There were also improvement in the sonomorphological abnormalities of periportal fibrosis and organomorphometry of livers and spleens. In 2005, there were 433 patients with various grades of Pfs (I to III) versus 277 patients in 2006, a cure proportion of 156 (64%). Even of more significant one in 2005 grade (II) and grade (III) were observed in 174 patients but only 53 one year later, cure proportion of 121 (70%). Those 53 patients with grade (II) and grade (III) of liver involvement showed deterioration in PFs after treatment with praziquantel 40mg/kg body weight while living in the same environment.

nt one in 2005 grade (II) and grade (III) were observed in 174 patients but only 53 one year later, cure proportion of 121 (70%). Those 53 patients with grade (II) and grade (III) of liver involvement showed deterioration in PFs after treatment with praziquantel 40mg/kg body weight while living in the same environment. Discussion According to the data presented, a positive influence of treatment with praziquantel 40mg/kg body weight on reversibility of liver PF changes due to S.mansoni infections in West Nile Uganda was observed. These observations agree with earlier works (Houston et al., 1993; Doehring-Schwedtfeger et al., 1992; Homeida et al., 1988). The positive trend was found in both young and old patients, with fibrosis of the liver. A similar finding was made by Homeida et al. (1988). These were more pronounced in patients with light liver PFs grade (I) and grade (II) as was earlier noted by Katz, et al. (1994). But patients with severe liver PFs grade (III) showed low reversibility of PFs. Since West Nile in Uganda is a hyper-endemic area for S. mansoni, this finding indicate that to sustain the reversibility of PFs, half yearly therapy with praziquaantel 40mg/kg body weight to the infected patients would prevent the development of severe PFs of the liver and encouraged reversibility of the lower PF grades. This phenomenon of sustainability of reversibility using repeated treatments has been demonstrated in previous studies (Homeida et al., 1988; Doerhing Schwerdtfeger et al., 1990; Abdel- Wahab et al., 1990).

nts would prevent the development of severe PFs of the liver and encouraged reversibility of the lower PF grades. This phenomenon of sustainability of reversibility using repeated treatments has been demonstrated in previous studies (Homeida et al., 1988; Doerhing Schwerdtfeger et al., 1990; Abdel- Wahab et al., 1990). Lakwo et al. (1994) in the same area of study showed that the water contact activities varied among the school children, fishermen and women but the overall frequencies of getting in contact with the water body at the Nile were similar. These different water contact activities included, playing and swimming in the water, washing utensils and clothing, collecting papyrus rids for making mats and fishing. These frequent water contact activities allow reinfection to take place. This could explain the irreversibility and deterioration of some of the PFs observed in this study. Because of ethical reason, there was no untreated group of patients set parallel to the study population to demonstrate natural PFs reversibility phenomenon. Jenkins et al., editor (1992) showed that beside careful evaluation of confounding factors such as improvement on the standard of living, changes of habits like water contamination with human excreta which encourage transmission of S.mansoni due to institutionalisation of health education, that is provision safe water and sanitation, also needed to be followed to ascertain their involvement in the reversibility of the liver damage in various grades of PFs. Mass chemotherapy treatment with antischistosomal drug is not considered as the only solution for those living in hyper-endemic areas for schistosome, but these data suggest that the life threatening complication of S. mansoni, liver fibrosis and consecutive portal hypertension easily observed with portable ultrasound machine, have been positively reduced one year after treatment with praziquantel as was also demonstrated earlier elsewhere (Homeida et al., 1988; Doerhing Schwerdtfeger et al., 1990; Abdel- Wahab et al., 1990).

n of S. mansoni, liver fibrosis and consecutive portal hypertension easily observed with portable ultrasound machine, have been positively reduced one year after treatment with praziquantel as was also demonstrated earlier elsewhere (Homeida et al., 1988; Doerhing Schwerdtfeger et al., 1990; Abdel- Wahab et al., 1990). Acknowledgement We gratefully acknowledged the assistance rendered to us by the Ministry of Health staff of Rhino Camp and Obongi Health centres. Patrick Okello, Statistician, Uganda Bureau of Statistics assisted us with data analysis. Last but not least, without the grants (grant nr ID 05090 and grant nr .ID 900286) from Edna McConnell Clark Foundation, New York and UNDP/World Bank/WHO Special Programme for Research and training in Tropical Diseases respectively, this study would not have been accomplished.

Introduction Internationally, deafness and hearing impairment as a consequence of bacterial meningitis has been reported to be the most common serious complication of bacterial meningitis in the paediatric population; occurring in 6 to 31% of cases depending on the type of meningitis investigated and the type and severity of hearing impairment included in the sample (Brookhouser et al., 1988; Fortnum and Hull, 1992; Dodd et al., 1997; Woolley et al., 1999; Asadi-Pooya et al., 2008). Meningitis is also the most common identifiable cause of acquired profound hearing loss in children and adults (Dodds et al., 1997). Asadi-Pooya, Asadi-Pooya and Rosin (2008) assert that deafness or some degree of hearing impairment occurs in 3.5% to 37% of survivors of meningitis. The incidence may be higher in developing countries than in first world countries since access to resources such as adequate health care is vastly different to that in the developed countries; and also because of the high incidence of HIV/AIDS in these third world countries. Cultural and linguistic diversity influences on health service provision may also be a contributing factor to the incidence and management of meningitis in developing countries, such as South Africa.

t to that in the developed countries; and also because of the high incidence of HIV/AIDS in these third world countries. Cultural and linguistic diversity influences on health service provision may also be a contributing factor to the incidence and management of meningitis in developing countries, such as South Africa. It is reported that in developed countries approximately 10% of survivors of bacterial meningitis experience permanent hearing loss while others experience a transient loss of hearing (Richardson et al., 1997). Both permanent and transient losses of hearing are reported to develop in the first few days of illness (Richardson et al., 1997). The onset of the hearing loss associated with bacterial meningitis most likely occurs early in the illness perhaps before other symptoms and signs are evident (Woolley et al., 1999). Furthermore, the phenomenon of fleeting hearing loss may occur in a critical period around the second day of illness during which hearing loss may be reversed (Richardson et al., 1997). This highlights the importance of early referrals and prompt diagnosis which may facilitate performance of appropriate hearing assessment so that hearing loss may be prevented, reversed or further damage is retarded (Woolley et al., 1999).

of illness during which hearing loss may be reversed (Richardson et al., 1997). This highlights the importance of early referrals and prompt diagnosis which may facilitate performance of appropriate hearing assessment so that hearing loss may be prevented, reversed or further damage is retarded (Woolley et al., 1999). Research by Woolley et al. (1999) indicates that, in majority of cases, if hearing loss is present at discharge from hospital, recovery of hearing does not occur. Therefore, referral of patients for audiological services is essential in order for effective early intervention services to be provided. “All children recovering from bacterial meningitis should be referred for audiologic assessment before discharge from the hospital” [4 p. 513]. This referral is recommended by some authors to be appropriate only 2 to 4 weeks after discharge to allow for any middle ear involvement to resolve (Dodds et al., 1997), and to ensure that profound hearing impairments are detected early enough to enable a cochlear implant to be a viable option (Fortnum and Hull, 1992). However, hearing loss post meningitis has been found to fluctuate or deteriorate over time and therefore it may be insufficient to assess hearing only once after discharge (Koomen et al., 2003). Nevertheless, the importance of early identification and monitoring of this hearing impairment is highlighted.

d Hull, 1992). However, hearing loss post meningitis has been found to fluctuate or deteriorate over time and therefore it may be insufficient to assess hearing only once after discharge (Koomen et al., 2003). Nevertheless, the importance of early identification and monitoring of this hearing impairment is highlighted. In a retrospective audit performed by Riordan et al. (1993), the major reason for hearing not being assessed in this population was lack of referral. This is different to findings by Fortnum and Hull (1992) which indicated that, in their study, reasons for non-referral included factors such as the belief held by some paediatricians that it is generally unnecessary to do formal hearing tests following bacterial meningitis; the assertion that only in children where there was already some concern over their hearing should be referred; and the belief that only children who met some other criterion should have their hearing tested. Riordan et al. (1993) suggested that routine referral for audiological assessment at discharge in conjunction with re-referral at out-patient attendance may assist in increasing the number of children assessed after bacterial meningitis, while Fortnum and Hull (1992) asserted that to refer postmeningitic children for hearing testing should not be in question, but that referral should be made while the child is still in hospital and that the importance of the assessment be emphasized to the parents. In a qualitative analysis of parents' experiences with early detection of hearing loss, research found that powerful emotions experienced by the parents at diagnosis included denial and shock as well as frustration arising from the delays in diagnosis (Russ et al., 2004), hence early referrals are of paramount importance to the emotional adjustment as well as compliance to treatment strategies of the parents. More recently, Koomen et al. (2003) advocate that hearing loss following meningitis can be predicted satisfactorily, hence when the hearing of children who are predicted to be at risk is tested as part of their routine follow-up, no children with hearing loss need be missed.

ance to treatment strategies of the parents. More recently, Koomen et al. (2003) advocate that hearing loss following meningitis can be predicted satisfactorily, hence when the hearing of children who are predicted to be at risk is tested as part of their routine follow-up, no children with hearing loss need be missed. The nature of the hearing loss in postmeningitic children is most commonly sensorineural (Richardson et al., 1997), with the exact mechanism of the hearing loss still not very well understood. However, the mechanism is thought to be likely due to multiple factors which include direct labyrinth involvement, cochlear neuroepithelial damage, and vascular insult (Kutz et al., 2006). There is some evidence indicating possible lesions to the brainstem or higher cortical areas, but the site of auditory lesion as a result of damage following meningitis is almost always to the cochlear and the organ of corti, thus providing exclusion of damage to the cochlear nerve (Richardson et al., 1997). This survival of the cochlear nerve makes these children good candidates for cochlear implantation (Richardson et al., 1997). However, Wilson et al., 2003) describe how the cochlear duct may be obliterated by osteoneogenesis within a few months of meningitis which may make cochlear implantation ineffective or impossible. Therefore, early identification is vital (Dodds et al., 1997).

didates for cochlear implantation (Richardson et al., 1997). However, Wilson et al., 2003) describe how the cochlear duct may be obliterated by osteoneogenesis within a few months of meningitis which may make cochlear implantation ineffective or impossible. Therefore, early identification is vital (Dodds et al., 1997). Failure to detect hearing loss may result in significant consequences for the child's speech and language acquisition, academic performance as well as social and emotional wellbeing. Therefore the identification of hearing loss in these children is crucial in order to ensure early habilitation and minimising of the long term educational, social and emotional difficulties that these children may experience (Bush, 2003; Kutz et al., 2006). Research has shown that children who are deprived of sufficient amounts and/or quality of language input in their earliest years are at risk for poor outcomes in both language and academic endeavours later in childhood (Nicholas and Geers, 2006). In addition, children detected late may never catch up with their normal hearing peers in academic, social and emotional domains of development (Olusanya, 2008). On the other hand, evidence in developed countries indicate that early hearing detection and intervention programs leads to linguistic, speech and cognitive development that is comparable to normal hearing peers (Hatzopoulos et al., 2007). Thus, effectively addressing the developmental constraints commonly associated with childhood hearing loss becomes crucial.

untries indicate that early hearing detection and intervention programs leads to linguistic, speech and cognitive development that is comparable to normal hearing peers (Hatzopoulos et al., 2007). Thus, effectively addressing the developmental constraints commonly associated with childhood hearing loss becomes crucial. When investigating the impact of hearing loss following meningitis, deficits in the domain of speech and language development become clearly evident. Research by Nicholas and Geer (2006) on the effects of early auditory experience on the spoken language of deaf children illustrated that profoundly deaf children demonstrated a much lower level of lexical and syntactic skills. This low level of lexical and grammatical proficiency places profoundly deaf children at a disadvantage when compared to their chronologic age-mates. This results in the inability of these children to fully participate in, and benefit from typical preschool activities without a high degree of communicative support (Nicholas and Geers, 2006). These speech-language difficulties may be further exacerbated beyond childhood, as these individuals may not be accepted, with a lack of effort being made to communicate with them. They may also demonstrate poor involvement in social activities due to a fear of stigmatisation or rejection by community (Hatzopoulos et al., 2007; Moeller, 2000; Olusanya, 2008; Yoshinaga-Itano, 2004).

yond childhood, as these individuals may not be accepted, with a lack of effort being made to communicate with them. They may also demonstrate poor involvement in social activities due to a fear of stigmatisation or rejection by community (Hatzopoulos et al., 2007; Moeller, 2000; Olusanya, 2008; Yoshinaga-Itano, 2004). A follow up study performed by Bedford et al. (2001) at five years post meningitis indicated severe communication problems or absence of speech in 55 of 1584 subjects, with a further 9.4 % of the children having speech or language delay. Research by Woolley et al. (1999) indicated that 21% of school age survivors of meningitis had functionally important disabilities including central auditory perceptual dysfunction that adversely affects learning ability. Decreased academic performance and behavioural disabilities were also indicated by these authors. Had intervention been implemented early following the diagnosis of meningitis, these long-term sequelae could have been prevented or minimised.

ntral auditory perceptual dysfunction that adversely affects learning ability. Decreased academic performance and behavioural disabilities were also indicated by these authors. Had intervention been implemented early following the diagnosis of meningitis, these long-term sequelae could have been prevented or minimised. In an Australian study tracking 12 year outcomes following bacterial meningitis, results indicated that these children remain at significant risk for neurological abnormalities (Grimwood et al., 2000). Central auditory function was found to have improved with time, however, difficulty with language based tasks remained. In the same study, findings regarding audiological outcomes indicated that while most sensorineural hearing loss following meningitis remains stable, spontaneous fluctuations or even progression may occur more than twelve years after recovery (Grimwood et al., 2000). These findings raise the need for audiologists to be involved in the initial diagnosis and management, as well as long term monitoring of post meningitis paediatric populations. By ensuring that early diagnosis and early intervention occurs these difficulties may be averted or at least appropriate intervention may be provided thus decreasing the negative impact of disability in this population group. This early diagnosis can only be achieved if sensitive and comprehensive audiological tests form part of the assessment and management protocol of these patients.

difficulties may be averted or at least appropriate intervention may be provided thus decreasing the negative impact of disability in this population group. This early diagnosis can only be achieved if sensitive and comprehensive audiological tests form part of the assessment and management protocol of these patients. Research indicates that otoacoustic emissions (OAEs) combined with tympanometry followed by an auditory brainstem response (ABR) if indicated is the best test battery that should form part of hearing evaluation guidelines and protocols for this population (Woolley et al., 1999). Behavioural audiometry is also recommended as soon as possible in order to establish the degree and configuration of the hearing loss (Woolley et al., 1999). In a study by Faraji et al. (2004) ABR, OAE and Behavioural Audiometry performed in children with meningitis during the critical period and recovery period was found to diagnose any degree of hearing loss with a high level of accuracy. The efficacy of OAEs as a screening tool for children recovering from acute bacterial meningitis was researched by Richardson, Williamson, Reid, Tarlow and Rudd (1998), and findings indicated that OAE screening was both feasible and effective. OAEs were found to be highly sensitive and relatively specific. Furthermore the technique was found to be well tolerated by patients. Therefore, in-patient OAE screening has been identified as a method of early diagnosis of hearing loss and prompt auditory rehabilitation (Richardson et al., 1998). However, it must be considered that retro-cochlear hearing loss may be missed by an OAE screening as the pathologic lesion is in the auditory nerve or higher centre (Richardson et al., 1998). This highlights the importance of cross check principles and holistic test batteries used in audiology diagnosis.

et al., 1998). However, it must be considered that retro-cochlear hearing loss may be missed by an OAE screening as the pathologic lesion is in the auditory nerve or higher centre (Richardson et al., 1998). This highlights the importance of cross check principles and holistic test batteries used in audiology diagnosis. Audiologists should also be aware of the symptomatology that occurs during meningitis as these may be useful to take into consideration when examining risk factors for paediatric populations who may form part of their case load. Information regarding symptomatology experienced during meningitis is of great importance as Kutz et al (2006) identified factors such as the length of hospitalization, development of seizures, fever, elevated cerebrospinal fluid protein and decreased cerebrospinal fluid glucose as significant predictors for hearing loss in children with bacterial meningitis. Grimwood et al. (1996) identified that age (children under 12 months), seizures developing or persisting after 72 hrs of appropriate treatment in hospital, and the presence of focal neurological signs are each important risk factors for adverse outcomes of bacterial meningitis.

ss in children with bacterial meningitis. Grimwood et al. (1996) identified that age (children under 12 months), seizures developing or persisting after 72 hrs of appropriate treatment in hospital, and the presence of focal neurological signs are each important risk factors for adverse outcomes of bacterial meningitis. By identifying and acknowledging symptomatology during illness professionals may identify factors associated with long term sequelae. In doing so the professionals may ensure appropriate additional follow up treatment as well as parental counselling Grimwood et al., 1996). Therefore, having knowledge of these prediction factors may be useful when establishing referral protocols for audiology assessments. In order to identify children with meningitis who have acquired a hearing loss it is recommended that all children at risk have a reliable evaluation of their hearing. Referral for a hearing assessment should be an essential part of the treatment protocol for all such children. As postulated by Wilson, Roberts and Stephens (2003), a well defined referral pathway can improve assessment and referral rates post meningitis.

ll children at risk have a reliable evaluation of their hearing. Referral for a hearing assessment should be an essential part of the treatment protocol for all such children. As postulated by Wilson, Roberts and Stephens (2003), a well defined referral pathway can improve assessment and referral rates post meningitis. Despite South Africa having a relatively well-developed infrastructure compared to other regions in Sub-Saharan Africa, wide scale access to health care (including audiological services) for the majority of its citizens is far from common practice. Universal newborn hearing screening and routine hearing testing of children who present with risk factors for hearing loss is not performed as part of routine paediatric management. This may be due to the limited state resources, but may also be due to the lack of contextual research on paediatric hearing impairments. The high incidence of HIV/AIDS, which presents as an overwhelming burden to the health resources of the country, may also be a factor in that available resources may be directed toward management of HIV/AIDS at the possible expense of other conditions. The fact that meningitis is one of the main opportunistic infections seen with HIV/AIDS, its general management requires investigating; and that includes management of the known consequences of meningitis, including hearing impairment. Therefore research in a South African context is vital for the collation and development of appropriate and efficient hearing evaluation guidelines and protocols for the post meningitis population, which highlights once more the importance of this study.

e known consequences of meningitis, including hearing impairment. Therefore research in a South African context is vital for the collation and development of appropriate and efficient hearing evaluation guidelines and protocols for the post meningitis population, which highlights once more the importance of this study. Method Aim of the study The primary aim of this study was to establish what the audiological referral protocols are for post meningitis paediatric populations in Johannesburg, Gauteng. There were four specific sub-aims to the study: To determine if audiological assessment referrals were made following infection. To determine the time of referral post meningitis diagnosis. To determine what audiological assessments were conducted on this population. To determine if a correlation exists between the presenting signs and symptoms and the audiology referrals

Method Aim of the study The primary aim of this study was to establish what the audiological referral protocols are for post meningitis paediatric populations in Johannesburg, Gauteng. There were four specific sub-aims to the study: To determine if audiological assessment referrals were made following infection. To determine the time of referral post meningitis diagnosis. To determine what audiological assessments were conducted on this population. To determine if a correlation exists between the presenting signs and symptoms and the audiology referrals Design of the Study This study was a record review and was therefore retrospective in nature. The adopted design allowed for examination of data already on file, which meant that the researchers could make observations and provide descriptive statistics from this data (Schiavetti and Metz, 2006). Record reviews are useful in instances where it is logistically infeasible to conduct an experiment relating the variables of interest (McBurney & White, 2007), as in the current study. However, an acknowledged limitation of this design is that the researcher is limited to the information exclusively provided by the agency that collected the data and by any biases or inaccuracies present in the collection procedure (McBurney & White, 2007). Participants Participant selection criteria - Inclusion Criteria The participant inclusion criteria included the following: All files reviewed had to be of children between the ages of birth to 6 years as the focus was on early intervention.

Design of the Study This study was a record review and was therefore retrospective in nature. The adopted design allowed for examination of data already on file, which meant that the researchers could make observations and provide descriptive statistics from this data (Schiavetti and Metz, 2006). Record reviews are useful in instances where it is logistically infeasible to conduct an experiment relating the variables of interest (McBurney & White, 2007), as in the current study. However, an acknowledged limitation of this design is that the researcher is limited to the information exclusively provided by the agency that collected the data and by any biases or inaccuracies present in the collection procedure (McBurney & White, 2007). Participants Participant selection criteria - Inclusion Criteria The participant inclusion criteria included the following: All files reviewed had to be of children between the ages of birth to 6 years as the focus was on early intervention. All files reviewed had to be of children who were admitted to the hospital and treated at the hospital for meningitis. Files included records which dated back to 3 years to ensure that the findings would not be influenced by short term factors such as staffing shortages, strikes, and so on. All files reviewed had to be of children who had normal hearing function pre-morbidly, based on the case history data in the files. Participant selection criteria - Exclusion Criteria The participant exclusion criteria included the following: Admission for conditions other than meningitis. Pre-morbid hearing impairment. Age older than 6 years.

All files reviewed had to be of children who had normal hearing function pre-morbidly, based on the case history data in the files. Participant selection criteria - Exclusion Criteria The participant exclusion criteria included the following: Admission for conditions other than meningitis. Pre-morbid hearing impairment. Age older than 6 years. Sampling Procedure A non probability convenience sampling technique was adopted in the current study since the sample was restricted to a part of the population that was readily available and random sampling would have been difficult to achieve (Schiavetti and Metz, 2006). Participants were recruited by consulting the paediatric medical ward discharge summaries. These discharge summaries provided hospital codes which allowed for full medical records including bed letters to be obtained. Consequently, clinical information for review was obtained both from discharge summaries as well as from full medical records. Participants with the diagnosis of meningitis were recruited to participate in the study. Participant Description A total of 100 participants with a positive history of meningitis including both males and females comprised the research sample at the initial stages of this study. Of these 100 participants only 47 passed the inclusion criteria stipulated above and therefore comprised the final sample size. These participants were from two tertiary academic state hospitals that had both paediatric medical wards as well as Audiology departments.

research sample at the initial stages of this study. Of these 100 participants only 47 passed the inclusion criteria stipulated above and therefore comprised the final sample size. These participants were from two tertiary academic state hospitals that had both paediatric medical wards as well as Audiology departments. Research procedures and materials All files that met the inclusion criteria were reviewed and data was captured on a spreadsheet. The spreadsheet was designed by the researchers with the aim of capturing the following data: Hospital Code (A or B) Patient code number Age: The age of the child at the time of infection is of vital importance as evidence suggests that there is a critical, or at least a sensitive, period for language acquisition (Hurford, 1991). Gender Type of meningitis: In a study by Kutz et al. (2006) on the clinical predictors for hearing loss in children with bacterial meningitis, the incidence of hearing loss was greater in patients with Streptococcus Pneumoniae meningitis than in patients with Neisseria Meningitidis thus illustrating the importance of establishing the type of meningitis. Symptomatology: By identifying and characterising symptomatology during illness, professionals may identify factors associated with long term sequelae following meningitis. In doing so the professionals may ensure appropriate additional follow up treatment as well as parental counselling (Grimwood et al., 1996). Therefore, symptomatology was included in the data spreadsheet. Treatment

Symptomatology: By identifying and characterising symptomatology during illness, professionals may identify factors associated with long term sequelae following meningitis. In doing so the professionals may ensure appropriate additional follow up treatment as well as parental counselling (Grimwood et al., 1996). Therefore, symptomatology was included in the data spreadsheet. Treatment Duration of hospital stay: Research by Kutz et al. (2006) identified the length of hospitalization as a significant predictor for hearing loss in children with bacterial meningitis. Was audiological assessment recommended? Time of referral Type of audiological tests conducted Other referrals made: The collaboration among professionals and families in the effective delivery of service to infants and children with hearing loss is imperative in ensuring appropriate services are received. Therefore, other referrals were included in the data spreadsheet in order to ensure this information was captured.

eferrals made: The collaboration among professionals and families in the effective delivery of service to infants and children with hearing loss is imperative in ensuring appropriate services are received. Therefore, other referrals were included in the data spreadsheet in order to ensure this information was captured. Validity and reliability The information collated for the current study was thought to be fairly reliable since this information was obtained from medical records as opposed to patient self reports. Turkkan (1999) asserts that even straightforward questioning on self reports is fraught with potential for misunderstandings thus illustrating a limitation of self reports. Furthermore, special ethical considerations need to be taken into account during self report data collection as individuals may not be fully capable of understanding the research protocols (Turkkan, 1999). The researcher reviewed the files personally to ensure that errors in capturing were eliminated, and half of the files were re-reviewed by the co-researcher to ascertain reliability of the data captured. External validity of the current study was however compromised by the fact that systematic randomised sampling was not the procedure used, and the data was only pooled from two hospitals in Gauteng.

re eliminated, and half of the files were re-reviewed by the co-researcher to ascertain reliability of the data captured. External validity of the current study was however compromised by the fact that systematic randomised sampling was not the procedure used, and the data was only pooled from two hospitals in Gauteng. Data analysis and statistical procedures This study made use of descriptive and inferential statistics. Descriptive statistics was used as it allows for group differences to be observed; and developmental trends or relationships among variables to be measured by the researcher (Schiavetti and Metz, 2006). Inferential statistics was also adopted as it makes use of mathematical methods (such as hypothesis development) that employ probability theory for deducing/ inferring the properties of a population from the analysis of the properties of a set of data drawn from it (Fife-Shaw, 2002). Inferential statistics, in the form of regression analysis, was conducted (McBurney & White, 2007). Logistic regression was chosen as audiological assessment in the current study is a binary response variable (i.e. the child was either referred or not) and the predictor variables were a mix of discrete and continuous variables (Fife-Shaw, 2002). Age, gender, fever, headache, stiff neck, vomiting, stomach ache, dizziness, cough, seizure, duration of hospital stay, and type of meningitis were the variables assessed. The null hypothesis for the current study was that there is no relationship between these variables and referral for audiology services, while the alternative hypothesis was that at least one of these factors is related to whether or not an audiology referral is made. To statistically test the hypothesis a significance level (alpha) of 0.05 was selected which meant that there would be a 95% confidence level that results were not due to chance (Howell, 2007).

the alternative hypothesis was that at least one of these factors is related to whether or not an audiology referral is made. To statistically test the hypothesis a significance level (alpha) of 0.05 was selected which meant that there would be a 95% confidence level that results were not due to chance (Howell, 2007). Ethical consideration Ethical clearance was obtained from the University of the Witwatersrand, Human Research Ethics Committee (medical) before the study was conducted (protocol number: M080345). The researchers ensured that permission was obtained from the relevant authorities (hospital superintendents and heads of the wards) at the research sites. Written informed consent to participate in the study was obtained before the record reviews were conducted with an assurance that confidentiality of all records would be maintained. Furthermore, to ensure anonymity, researchers ensured that no personal or identifying information was included in the research report and research coding numbers instead of identifying information were used. The current study also reduced risks to the participants to a minimum since it was a record review. Lastly, the hospitals involved were given the opportunity to request to see the research results if they were interested (South African Medical Research Council, 2008).

mbers instead of identifying information were used. The current study also reduced risks to the participants to a minimum since it was a record review. Lastly, the hospitals involved were given the opportunity to request to see the research results if they were interested (South African Medical Research Council, 2008). Results The results obtained from this study are presented in accordance with the specific aims of the study. It should be noted that the results are not discussed separately for the two participating hospitals because the researchers believed that no additional information would be gained from that process, particularly because of the final sample size. A demographic profile of the participants is displayed in Table 1. Table 1 Demographic profile of all participants in the study (N=47) Factor Sub-Category Number Percentage Age Range 4days to 6 years 47 (mean age: 1.9years) Gender Male Female 25 22 53% 47% There were 47 participants in the study (as seen in Table 1) ranging in age from 4 days to 6 years with a mean age of 1.9 years. Participants included both males and females. As illustrated in Table 2, various forms of meningitis were identified in the current study with meningococcal meningitis being the most prevalent with 45% of the sample presenting with this form, while Haemophilus. H influenzae was the least presenting type of meningitis (4%). Table 2 Medical profile of all participants in the study (N=47) Factor Sub-Category Number Percentage Type of meningitis Meningococcal Strep. Pneumonia Group B Strep Tuberculosis Haemophilus H. Influenza Bacterial Unspecified Pathogen 21 5 5 5 2

As illustrated in Table 2, various forms of meningitis were identified in the current study with meningococcal meningitis being the most prevalent with 45% of the sample presenting with this form, while Haemophilus. H influenzae was the least presenting type of meningitis (4%). Table 2 Medical profile of all participants in the study (N=47) Factor Sub-Category Number Percentage Type of meningitis Meningococcal Strep. Pneumonia Group B Strep Tuberculosis Haemophilus H. Influenza Bacterial Unspecified Pathogen 21 5 5 5 2 2 7 45% 11% 11% 11% 4% 4% 15% Presenting symptomatology Fever Vomiting Seizures Stiff neck Diarrhoea Stomach cramps Bulging fontanel Cough Irritability Headache Increased tone Inability to sit/walk Oral candida Oral ulcers Photophobia 28 19 18 17 11 10 9 7 6 5 4 2 1 1 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Duration of Hospital stay Range Average Standard deviation 5–21 days 10.3 days 4.4 days N/A Key: N/A=not applicable The most commonly presenting symptomatology in the current sample (Table 2) included fever, vomiting, seizures and neck stiffness, with the duration of hospital stay ranging from 5 days to 21 days (with an average of 10.3 days). Audiology referrals made The first objective of this study was to determine if audiological assessment referrals were made following meningitis. Findings from the current sample indicated that referrals were made in 28 cases indicating a referral rate of 60% as depicted in Table 3. Almost half (40%) of the cases reviewed were not referred. Table 3 Audiological referral profile of all participants in the study (N=47)

Audiology referrals made The first objective of this study was to determine if audiological assessment referrals were made following meningitis. Findings from the current sample indicated that referrals were made in 28 cases indicating a referral rate of 60% as depicted in Table 3. Almost half (40%) of the cases reviewed were not referred. Table 3 Audiological referral profile of all participants in the study (N=47) Factor Sub-Category Number Percentage Was the referral made? Yes No 28 19 60% 40% Time of referral (n=28) As in-patients As out-patients soon after discharge Later than 3 months 25 3 0 89% 11% 0% What audiological services were conducted? (n=28) Otoscopy Tympanometry Basic Pure tone audiometry Speech Audiometry OAEs ABR ASSR 28 28 0 0 9 0 0 N/A N/A N/A N/A N/A N/A N/A Other referrals Physiotherapy Occupational therapy Speech-Language Therapy Dietician Follow-up clinic 8 4 4 3 4 N/A N/A N/A N/A N/A Key: N/A=not applicable Time of referral for audiology testing The second objective of this study was to determine the time of audiological referral in relation to the onset of infection. Of the 28 cases that were referred, 25 (89%) were referred as in-patients before discharge from the hospital, with the other 3 cases being referred after discharge (but within three months). It must be noted that in the sample reviewed, no cases were referred three months post discharge and onwards (Table 3).

nfection. Of the 28 cases that were referred, 25 (89%) were referred as in-patients before discharge from the hospital, with the other 3 cases being referred after discharge (but within three months). It must be noted that in the sample reviewed, no cases were referred three months post discharge and onwards (Table 3). Audiological assessments conducted The third aim of this study was to establish which audiological tests were conducted on this population. Of the 28 cases referred for audiological assessment (Table 3), all cases had otoscopic examinations and tympanometry conducted, and only 9 cases had OAEs performed. No participants had basic pure tone audiometry, speech audiometry, auditory brainstem response, and auditory steady state response.

ed on this population. Of the 28 cases referred for audiological assessment (Table 3), all cases had otoscopic examinations and tympanometry conducted, and only 9 cases had OAEs performed. No participants had basic pure tone audiometry, speech audiometry, auditory brainstem response, and auditory steady state response. Correlation between signs and symptoms and audiology referrals The final aim of the study was to determine if there was any correlation between signs and symptoms and audiology referrals. Logistic regression was utilized to determine what predictive factors can be used to model whether or not a child will be sent for an audiological assessment based on symptomatology. Of all the variables analysed, the only significant predictor variable where p< 0.01 was fever (whether the child presented with it or not). This infers that if one were to model predictors that could determine whether a child will be referred for an audiological assessment, fever was the only variable indicator. This finding can be supported within the current study, particularly since it is consistent with previous findings on fever in meningitis being a predictive factor for hearing loss. However, because of the small sample size in the current study, this finding cannot be put forward as the only recommendation to be followed in post meningitis hearing referral. Although fever can be considered as a variable indicator, other indicators should still be used to ensure that no cases of post meningitic hearing loss are missed. Nevertheless, current findings indicate that fever must be acknowledged as a crucial factor to be included during the development of audiology referral protocols post meningitis.

considered as a variable indicator, other indicators should still be used to ensure that no cases of post meningitic hearing loss are missed. Nevertheless, current findings indicate that fever must be acknowledged as a crucial factor to be included during the development of audiology referral protocols post meningitis. Additional Analysis Length of Hospital Stay The average duration of hospital stay in the current study was 10.3 days ±4.4 days. The shortest duration of stay was 5 days and the longest duration of stay was 21 days. Other Referrals Although the audiologist is typically the first professional to identify hearing loss in infants and children, other professionals may also identify risk factors for hearing loss. Therefore, information regarding referrals to other professionals was included in this study. Eight children were referred for physical therapy, four children were referred for occupational therapy, four children were referred for speech-language therapy, four children were referred to a follow up clinic and three children were referred to a dietician as depicted in Table 3.

ssionals was included in this study. Eight children were referred for physical therapy, four children were referred for occupational therapy, four children were referred for speech-language therapy, four children were referred to a follow up clinic and three children were referred to a dietician as depicted in Table 3. Discussion As can be seen in Table 1, the sample for the current study seemed to be fairly representative of the South African paediatric population infected with meningitis as there were slightly more males than females in the sample; as seen in the reported distribution of meningococcal meningitis by gender in South Africa between the year 2001 and 2005 (Department of Health: Republic of South Africa, 2006). Furthermore, the fact that this sample consisted of 34 cases (72%) under the age of three years is consistent with data released by the South African Department of Health (Department of Health: Republic of South Africa, 2006) which indicate that in South Africa between 2001 and 2004, 46% of notified cases of meningitis with known age were children under the age of five years. This age of presentation highlights the need for more vigilant audiologic management of these cases as this is the critical period for speech and language development. Furthermore, this is the stage when cognitive skills are still immature, and hence undetected deficits post meningitis may remain until the child has started school; which is not of benefit to that child (Grimwood et al., 1996).

nagement of these cases as this is the critical period for speech and language development. Furthermore, this is the stage when cognitive skills are still immature, and hence undetected deficits post meningitis may remain until the child has started school; which is not of benefit to that child (Grimwood et al., 1996). Meningococcal meningitis was the most commonly presenting meningitis with a positive diagnosis in 21 children (45%), with Haemophilus. H influenzae being the least presenting type of meningitis (4%) (Refer to Table 1). These findings are consistent with those found in Athens, Greece; where meningococcal meningitis was also the leading type of meningitis with 66% of the sample presenting with it (Theodoridou et al., 2007). Findings from the current study seem to imply that pathogens causing meningitis in South Africa may be similar to pathogens causing meningitis in other countries; possibly indicating the probability that the burden of disease might be experienced in a similar fashion; although debatably co-existence of other infectious diseases may be much higher in this context. This has great implications for the audiological management of this population as this indicates that adoption of management protocols established in developed countries may be appropriate for the management of the South African paediatric meningitis population.

r infectious diseases may be much higher in this context. This has great implications for the audiological management of this population as this indicates that adoption of management protocols established in developed countries may be appropriate for the management of the South African paediatric meningitis population. The most commonly occurring presenting symptoms in the current study were fever, vomiting, seizures and stiff neck. These are the classical features which are often documented in meningitis (Van de Beek et al., 2004). Again findings of the current study are consistent with the findings of a study by Valmari et al. (1987) in Finland and confirm that the sample for the current study seems to be representative of the general meningitis population, although Wubbel and McCracken (1998) suggest that presenting symptomatology of meningitis may differ according to the age of the individual as well as the type of meningitis. Information regarding symptomatology experienced during meningitis is of great importance as it was identified that age (that is children under 12 months), seizures developing or persisting after 72 hrs of appropriate treatment in hospital and the presence of focal neurological signs are each important risk factors for adverse outcomes of bacterial meningitis (Grimwood et al., 1996). Findings from the current study on symptomatology are particularly important since these symptoms have been identified as significant predictors for hearing loss in children with bacterial meningitis (Kutz et al., 2006). Knowledge of these predictive factors by the health care team involved in management of paediatric meningitis will not only facilitate the establishment of audiological referral protocols for this population; but may also aid professionals in identifying factors associated with long term sequelae. In doing so, these professionals may ensure that appropriate and comprehensive follow up treatment as well as parental counselling is in place for families that need it (Grimwood et al., 1996).

erral protocols for this population; but may also aid professionals in identifying factors associated with long term sequelae. In doing so, these professionals may ensure that appropriate and comprehensive follow up treatment as well as parental counselling is in place for families that need it (Grimwood et al., 1996). As far as referral for audiology services is concerned, the current study revealed that almost half (40%) of the cases were not referred for audiological assessment following meningitis. This is significantly higher than reports from developed countries such as England; where although some cases are not referred, only less than a quarter of these cases were not referred. Wilson, Roberts and Stephens (2003) indicated that between 7.7% and 27% of children in Wales were not referred for hearing testing post meningitis. In a study by Riordan et al. (1993) in England 75% of survivors of bacterial meningitis received audiological assessment between 1998 and 1999, a number that is significantly higher than that reflected in the current study. Riordan et al. (1993) identified that in their context, the major reason for children not being assessed was non-referral. Although reasons for children not being assessed were not explored in the current study, it is possible that non-referral also played a significant role. Over and above non-referral, other reasons specific to developing country contexts include insufficient resources such as lack of diagnostic equipment in most state hospitals, as well as insufficient numbers of audiologists in the country. Swanepoel (2006) described the major challenges to the provision of adequate audiological services in South Africa as being attributed to an insufficient number of audiologists which is unequally distributed between the private and public sector. Lack of referral may also indicate the differential priorities placed on presenting symptoms in any admitted child; that is, hearing loss may not be viewed as a priority during the early medical management. Non-referral of paediatric cases following meningitis has serious consequences for early detection of hearing loss and subsequent rehabilitation which may minimize the almost inevitable speech and language difficulties, academic difficulties, as well as social and emotional maladjustment that come with a hearing impairment.

eferral of paediatric cases following meningitis has serious consequences for early detection of hearing loss and subsequent rehabilitation which may minimize the almost inevitable speech and language difficulties, academic difficulties, as well as social and emotional maladjustment that come with a hearing impairment. When it came to the timing of referrals for audiological services, a second aim of the current study, findings indicate that almost all the cases that were referred (80%) were done so as in-patients before discharge from the hospital; with the remaining cases being referred within three months after discharge. These findings are consistent with previous reports which indicate that a large number of cases are referred before discharge. For example, in a retrospective audit of case notes of children notified to the Public Health Department of Wales, 50% of children were assessed before discharge with 30 % being assessed within the first 8 weeks following infection, while the remaining 19% had no record of hearing assessment (Wilson et al., 2003). Because in the current study no cases were referred after three months, it may be inferred that if patients are not referred as in-patients or within three months following discharge from hospital, they may never be referred for audiological assessment. This has implications for paediatricians managing these cases in that in-patient referral for hearing tests may need to be prioritized with a follow up appointment arranged as part of the medical follow up clinic. The in-patient referral is also important because researchers such as Woolley et al. (1999) assert that in a majority of cases, if hearing loss is present at discharge from hospital, recovery of hearing does not occur, hence early identification and management of this hearing loss becomes crucial. The follow up audiological assessment would then ensure that middle ear deafness that Dodds, Tyszkiewicz and Ramsden (1997) report only resolves 2–4 weeks after discharge, can also be taken into account and hearing function monitored. This referral pathway, if well-defined, can improve assessment/referral rates post meningitis as advocated by Wilson, Roberts and Stephens (2003).

middle ear deafness that Dodds, Tyszkiewicz and Ramsden (1997) report only resolves 2–4 weeks after discharge, can also be taken into account and hearing function monitored. This referral pathway, if well-defined, can improve assessment/referral rates post meningitis as advocated by Wilson, Roberts and Stephens (2003). Referrals for audiological services by paediatricians need to be met with appropriate and accurate audiology measures that allow for reliable and valid results to be obtained. Findings from the current study were disturbing in as far as audiological tests conducted were concerned. It is of great concern that of all the cases referred for hearing tests, no diagnostic testing (in the form of pure tone audiometry, speech audiometry, auditory brainstem response, and auditory steady state responses) was conducted. Only nine cases had OAEs performed, with the rest of the sample only having otoscopy and tympanometry. These findings could have been influenced by non-standardized record keeping protocols, an acknowledged limitation of the current study design. For example in some hospitals, audiology records are maintained separate from the hospital file, and going through these files as well could have yielded different results. This highlights the need for standardization of record keeping processes which may have significant medico-legal implications as well as implications for assurance of continuity of care (Hamilton et al., 2003). Woolley et al. (1999) advocate that OAEs combined with tympanometry followed by an ABR, if indicated, is the best test battery for the paediatric meningitis population. Furthermore, these authors emphasize the importance of conducting behavioural audiometry as soon as possible; in this way the degree and configuration of the hearing loss may be established (Woolley et al., 1999; Faraji et al., 2004). The fact that nine of the cases in the current study had OAEs performed is positive since studies on the efficacy of OAEs as a screening tool for children recovering from meningitis screening have found that OAEs are both feasible and effective (Faraji et al., 2004). These authors reported that the technique of OAEs was found to be well tolerated by patients and was considerably easier to perform than ABRs. Furthermore, OAEs were found to be highly sensitive and relatively specific and therefore in-patient OAE screening should assure early diagnosis of hearing loss and prompt auditory rehabilitation (Faraji et al., 2004).

of OAEs was found to be well tolerated by patients and was considerably easier to perform than ABRs. Furthermore, OAEs were found to be highly sensitive and relatively specific and therefore in-patient OAE screening should assure early diagnosis of hearing loss and prompt auditory rehabilitation (Faraji et al., 2004). Within a developing country with limited resources, it would seem that investing in OAE equipment for all paediatric wards would be a sound investment.

of OAEs was found to be well tolerated by patients and was considerably easier to perform than ABRs. Furthermore, OAEs were found to be highly sensitive and relatively specific and therefore in-patient OAE screening should assure early diagnosis of hearing loss and prompt auditory rehabilitation (Faraji et al., 2004). Within a developing country with limited resources, it would seem that investing in OAE equipment for all paediatric wards would be a sound investment. As far as determination of a correlation between signs and symptoms of meningitis and audiology referrals was concerned, the current study found fever to be the only significant predictor variable. This infers that if one were to model predictors that could determine whether a child will be referred for an audiological assessment in the current study, fever was the only variable indicator. Therefore, fever must be acknowledged as a crucial factor to be included during the development of audiology referral protocols post meningitis. This finding is particularly important where targeted hearing screening may be the only option available to audiologists due to limited resources. Fever has been previously reported as a predictor variable for hearing loss in children with bacterial meningitis (Kutz et al., 2006); hence it is possible that in the current study these were the patients that were referred because they presented with an obvious hearing impairment. Other predictor factors such as elevated cerebrospinal fluid (CSF) protein and decreased cerebrospinal fluid glucose (Kutz et al., 2006) were not explored in the current study and so it is not possible to say if CSF results would have been found to be a predictive factor since they did not form part of the logistic regression analysis, an acknowledged study design limitation.

id (CSF) protein and decreased cerebrospinal fluid glucose (Kutz et al., 2006) were not explored in the current study and so it is not possible to say if CSF results would have been found to be a predictive factor since they did not form part of the logistic regression analysis, an acknowledged study design limitation. It is important to note additional findings from the current study which reveal that significant referrals were made to the allied medical disciplines including physiotherapy, occupational therapy, speech-language therapy, as well as dieticians. These professionals can be targeted by audiologists as part of the referral pathway for audiological services for children post meningitis. Audiology referral rates may be increased by providing these professionals with information regarding risk factors for hearing loss, and presentation of hearing impairment. In this way, children who were not referred for audiology services as in-patients may be referred by allied medical disciplines on follow up visits. This, however, does not take the responsibility away from the doctors who should be the primary referring agents to the audiologists. The collaboration among professionals and families in the effective delivery of services to infants and children with hearing loss is imperative in ensuring that appropriate services are provided.

ver, does not take the responsibility away from the doctors who should be the primary referring agents to the audiologists. The collaboration among professionals and families in the effective delivery of services to infants and children with hearing loss is imperative in ensuring that appropriate services are provided. Additional findings also indicated that the average duration of hospital stay in the current study was 10.3 days ±4.4 days. The shortest duration of stay was 5 days and the longest 21 days. In a study on hospitalization during bacterial meningitis in Canada the overall median length of stay (LOS) was 11 to 12 days and remained stable over the study period from the year 1994 to 2001 (Canada Communicable Disease Report, 2005). It must be noted though that in this Canadian study, the duration of hospital stay varied by organism from 8 to 21 days. Although, the duration of hospital stay is not directly related to hearing loss or significantly associated with future hearing loss, as revealed in a study by Wolley et al. (1999); this information was thought to be of great relevance to the South African audiologist as the duration of hospital stay may present a challenge to the audiologist in terms of the development of audiology referral protocols under resource constraints. Audiologists need to be aware of the lack of consistency regarding the duration of hospital stay as this indicates the necessity for highly individualized assessment and treatment plans; that ensure that children are not discharged before a referral for audiology is made.

referral protocols under resource constraints. Audiologists need to be aware of the lack of consistency regarding the duration of hospital stay as this indicates the necessity for highly individualized assessment and treatment plans; that ensure that children are not discharged before a referral for audiology is made. Conclusions This study investigated the audiological referral protocols of populations post meningitis by means of a retrospective record review. The results of this study indicated that a significant number (40%) of cases were not referred for audiological assessment. The majority of the cases in the current study were referred as in-patients with very minimal referral occurring after discharge from the hospital. Those cases referred did not seem to have undergone comprehensive diagnostic audiological assessment with all cases only having otoscopy and tympanometry conducted, and only 9 having OAEs performed. The only variable that was a significant indicator for audiological referral in the current study was fever, although it is acknowledged that the list of variables included in the logistic regression analysis in the current study was not exhaustive.

otoscopy and tympanometry conducted, and only 9 having OAEs performed. The only variable that was a significant indicator for audiological referral in the current study was fever, although it is acknowledged that the list of variables included in the logistic regression analysis in the current study was not exhaustive. These findings highlight the need for establishment of referral protocols for this population with specific referral pathways drawn up, specific times of referrals, as well as specific audiological measures that need to be employed to ensure that the adverse consequences of hearing impairment following meningitis are minimized. These findings also indicate the need for the provision of training for allied medical disciplines as well as medical disciplines regarding risk factors for hearing loss which may improve referral rates for early detection and intervention of hearing impairment. The current study seems to indicate that improved hearing assessments of children following meningitis may be achieved if patients are referred while they are still in-patients with OAEs being an important tool in the battery of tests that need to be available in all paediatric wards.

ntervention of hearing impairment. The current study seems to indicate that improved hearing assessments of children following meningitis may be achieved if patients are referred while they are still in-patients with OAEs being an important tool in the battery of tests that need to be available in all paediatric wards. The current study did however have limitations including that of the fact that a small sample size from only two academic hospitals was recruited; hence generalization of the findings to other sites may be difficult. However, referrals may even be poorer in the primary and secondary level health care systems where resources are possibly more restricted. Furthermore, the possibility of findings being influenced by the methods of record keeping used at the two research sites should also be acknowledged. Lastly, because this study was based on record reviews, factors influencing referrals could not be explored; and these are important to document so that remediation strategies can be put in place; hence implications for future research where these actors can be taken into consideration on larger sample sizes.

Introduction Malaria, a tropical disease caused by protozoan parasites of the genus Plasmodium is one of the most important infectious diseases in the world (Breman, 2001). Malaria kills over a million people each year, with as many as 300–500 million people being infected, with extremely high fatality rates among young children below 5 years of age (WHO, 2005). Furthermore, anti-malarial drug resistance has become as one of the greatest challenges against malaria control, drug-resistance to Chloroquine and more recently quinine was responsible in the spread of malaria to new areas and occurrence of malaria in areas where the disease has been eradicated. Proper diagnosis of Plasmodium falciparum has played an important role in the occurrence, severity and management of malaria epidemics. Sensitive, accurate and specific methods of diagnosis are important in proper treatment.

malaria to new areas and occurrence of malaria in areas where the disease has been eradicated. Proper diagnosis of Plasmodium falciparum has played an important role in the occurrence, severity and management of malaria epidemics. Sensitive, accurate and specific methods of diagnosis are important in proper treatment. Light microscopy of thick and thin stained blood smears remains the gold standard method for diagnosing malaria (Moody and Chiodini, 2000). Thick smears are 20–40 times more sensitive than thin smears for screening of Plasmodium parasites, with a detection limit of 10–50 trophozoites/µl (Trampuz et al., 2003). Thin smears allow one to identify malaria species (including the diagnosis of mixed infections), quantify parasitemia, and assess for the presence of schizonts, gametocytes, and malarial pigment in neutrophils and monocytes (Trampuz et al., 2003). The diagnostic accuracy relies on the quality of the blood smear and experience of laboratory personnel. Although examination of the thick and thin blood smear is the ‘gold standard’ for diagnosing malaria, important advances have been made in diagnostic testing, including fluorescence microscopy of parasite nuclei stained with acridine orange, rapid dipstick immunoassay, and polymerase chain reaction (PCR) assays. Sensitivity and specificity of some of these methods exceed by far those of the thin and thick smear. Diagnosis based on polymerase chain reaction for species-specific Plasmodium genome are the most accurate, sensitive and specific compared to diagnostic methods, and are capable of detecting as few as 10 parasites/µl of blood (Hanschield, and Grobusch, 2002). WHO recommends that malaria be confirmed by parasite-based diagnosis before giving treatment (WHO, 2005). In developing world where malaria is highly prevalent, the resources to aid in proper diagnosis are lacking (Rafael et al., 2006). This lack of resources to aid proper and accurate diagnosis of P. falciparum has lead to improper administration of anti-malarials.

site-based diagnosis before giving treatment (WHO, 2005). In developing world where malaria is highly prevalent, the resources to aid in proper diagnosis are lacking (Rafael et al., 2006). This lack of resources to aid proper and accurate diagnosis of P. falciparum has lead to improper administration of anti-malarials. The present study compared the sensitivity, specificity and accuracy of microscopy with those of polymerase chain reaction (PCR) technique. This was prompted by a decline in the number of positive Plasmodial cases detected by microscopy, though clinical prescriptions for anti-malarial are on increase. This implies that treatment of malaria in epidemic areas should be carefully carried out following a proper diagnosis. Materials and Methods This present study was conducted from months of October to December, 2007 during in door residual spraying campaigns in the districts of Kisii, Narok and West Pokot in Western Province of Kenya. The malaria vectors in these areas are A. gambiae, A. funestus, and A.arabiensis (Castro et al., 2004). The malaria transmission in these districts is seasonal and epidemic. Generally, the rainfall pattern is bimodal, with a long rainy season between March and May and a short rain season between October and December.

. The malaria vectors in these areas are A. gambiae, A. funestus, and A.arabiensis (Castro et al., 2004). The malaria transmission in these districts is seasonal and epidemic. Generally, the rainfall pattern is bimodal, with a long rainy season between March and May and a short rain season between October and December. The study protocol was approved by the scientific steering committee and the ethical review committee of the Kenya Medical Research Institute (KEMRI), Kenya. Written and informed consent forms presented in native language and translated to the patients were obtained from adults and/ or parent or guardian of the children participating in the study. Blood samples were collected as dry blood spots on Whatman 3M filter paper from 129, 99 and 122 patients diagnosed as P. falciparum negative by microscopy from Kisii, Narok and West Pokot districts, respectively.

e patients were obtained from adults and/ or parent or guardian of the children participating in the study. Blood samples were collected as dry blood spots on Whatman 3M filter paper from 129, 99 and 122 patients diagnosed as P. falciparum negative by microscopy from Kisii, Narok and West Pokot districts, respectively. DNA Extraction DNA was extracted by Chelex method according to Snounou et al., 1993 with few modifications. Briefly, 4mm2 piece of filter paper with blood spot was cut with a sterile scalpel blade and incubated in 0.5% saponin in 1 × PBS overnight at 4° C. The brown solution was removed and replaced with 1 × PBS and then incubated for 20 mins. The solution was removed and 100µl of DNAse free water was added followed by 50µl of 20% Chelex. The tubes were placed into a heated block and voltexed every two mins. This was repeated up to 5 times. The solution was centrifuged and the supernatant carefully separated. The supernatant contains DNA and 30µl aliquots were taken into eppendorf tubes and stored at −20° C for PCR analysis.

lowed by 50µl of 20% Chelex. The tubes were placed into a heated block and voltexed every two mins. This was repeated up to 5 times. The solution was centrifuged and the supernatant carefully separated. The supernatant contains DNA and 30µl aliquots were taken into eppendorf tubes and stored at −20° C for PCR analysis. Screening P. falciparum microscopy negative samples by nested PCR Nested PCR was carried out targeting Pfmdr1 gene on MJ Thermocycler™. The following were the primers designed for outer PCR with primer 3.0 Software. Primers, MDR/A1 (TGT TGA AAG ATG GGT AAA GAG CAG AAA GAG) and MDR/A3 (TAC TTT CTT ATT ACA TAT GAC ACC ACA AAC) for forward and reverse amplification respectively were designed by primer 3.0 software. The reaction mixture comprised of 2µl of template DNA, 2.5 mM magnesium chloride, 100nM dNTP, 100nm of each primer and Taq polymerase at 1unit/reaction to a total volume of 30µl. The cycling conditions were set at 94°C for initial denaturation for 3 minutes followed by denaturation at 94°Cfor 1 min, annealing at 45°C for 3 seconds and extension at72°C for 1 minute. These conditions were repeated for 40 cycles.

100nm of each primer and Taq polymerase at 1unit/reaction to a total volume of 30µl. The cycling conditions were set at 94°C for initial denaturation for 3 minutes followed by denaturation at 94°Cfor 1 min, annealing at 45°C for 3 seconds and extension at72°C for 1 minute. These conditions were repeated for 40 cycles. The following primer pair was used in nested PCR was for forward and reverse amplifications respectively; MDR/A2 (GTC AAA CGT GCA TTT TTT ATT AAT GAC CAT TTA) and MDR/A4 (AAA GAT GGT AAC CTC AGT ATC AAA GAA GAG). Nest 1 PCR products were thawed on ice and 5µl from each tube was transferred into labeled sterilized PCR tube. The reaction mixture for nested PCR for Pfmdr1 comprised of 1X PCR Buffer, 2.5mM magnesium chloride, 100nM dNTPs, 100nm of each primer MDR/A2 and MDR/A4 and Taq polymerase at 1unit/reaction to a total volume of 30µl. The thermal cycling conditions were, denaturation at 94 ° C for 30 seconds followed by annealing at 45 ° C for 1 minute and extension at 72 ° C for 1minute. These steps were repeated for 40 cycles and then followed by a final extension at 72 ° C for 3 mins before halting the reaction at 4°C.

to a total volume of 30µl. The thermal cycling conditions were, denaturation at 94 ° C for 30 seconds followed by annealing at 45 ° C for 1 minute and extension at 72 ° C for 1minute. These steps were repeated for 40 cycles and then followed by a final extension at 72 ° C for 3 mins before halting the reaction at 4°C. PCR products analysis by gel electrophoresis The PCR products were confirmed and analysed on a1.5% agarose (Sigma) gel electrophoresis with 0.5µg/ml of ethidium bromide (Promega). The electrophoresis gel run for 30 minutes at 80 volts on a horizontal electrophoretic tank (Bio Rad) submerged with 1× TAE buffer. DNA amplification products were visualized under ultraviolet light against a 100 base pair marker (Roche) on a transilluminator (Vilber Lourmat) and the results documented using Polaroid® camera and Polaroid® instant films. Results and Discussions A total of 356 patients with ages between 4 to over 50 years participated in this study. There were 132 patients from Kisii, 99 from Narok and 125 from West Pokot district. These patients were examined for malaria by thick blood film by an experienced technician in field stations. It was found that 350 patients were P. falciparum negative and 6 positive by microscopy. For the P. falciparum negative, 129, 99 and 122 patients were from Kisii, Narok and west Pokot, respectively.

ot district. These patients were examined for malaria by thick blood film by an experienced technician in field stations. It was found that 350 patients were P. falciparum negative and 6 positive by microscopy. For the P. falciparum negative, 129, 99 and 122 patients were from Kisii, Narok and west Pokot, respectively. Seventy two (72) making up 20.57% of cases diagnosed as negative by microscopy were found to be positive for P. falciparum with nested-PCR. The percentages of P. falciparum positive patients were, 20.9% (n=27) in Kisii, 7.1% (n=7) in Narok and 31.1% (n=38) in west Pokot. Microscopy versus PCR The accuracy, sensitivity and specificity of PCR were evaluated against those of microscopy. These values were calculated using InStat® statistical software, taking microscopy as the gold standard while PCR as the test method (www.graphpad.com/instat3/instat.htm). The sensitivity, specificity and accuracy of PCR on all the samples collected were 100%, 79% and 39.9% respectively. Kisii samples had a sensitivity of 100%, specificity 79% and accuracy of 39.7%. Narok samples had a specificity 92.9% and accuracy level of 39.9%. West Pokot had sensitivity of 100%, specificity of 68.9 % and accuracy of 34%. These results are shown in Table 1 and Figure 1. Using Chi-square P< 0.0001 value was obtained at 95% confidence interval. Table 1 Shows the accuracy, sensitivity and specificity of PCR against microscopy for the samples collected from Kisii, Narok and west Pokot. These values were computed from www.graphpad.com/instat3/instat.htm taking microscopy as the gold standard STUDY

Microscopy versus PCR The accuracy, sensitivity and specificity of PCR were evaluated against those of microscopy. These values were calculated using InStat® statistical software, taking microscopy as the gold standard while PCR as the test method (www.graphpad.com/instat3/instat.htm). The sensitivity, specificity and accuracy of PCR on all the samples collected were 100%, 79% and 39.9% respectively. Kisii samples had a sensitivity of 100%, specificity 79% and accuracy of 39.7%. Narok samples had a specificity 92.9% and accuracy level of 39.9%. West Pokot had sensitivity of 100%, specificity of 68.9 % and accuracy of 34%. These results are shown in Table 1 and Figure 1. Using Chi-square P< 0.0001 value was obtained at 95% confidence interval. Table 1 Shows the accuracy, sensitivity and specificity of PCR against microscopy for the samples collected from Kisii, Narok and west Pokot. These values were computed from www.graphpad.com/instat3/instat.htm taking microscopy as the gold standard STUDY SITE SENSITIVITY % SPECIFICITY % ACCURACY OF THE TEST % KISII 100 79 39.7 NAROK *** 92.9 *** WEST POKOT 100 68.9 34 Legend: ***, no values for sensitivity and accuracy for the samples from Narok. Figure 1 Representative gel showing nested PCR products on 1.5% agarose gel electrophoresis of P. falciparum negative blood filters determined by microscopy. MM is the molecular weight marker, Lane 1, represents the negative control, Lane 2, the positive control and lanes 3–17 are the P. falciparum negative samples by microscopy from the epidemic areas.

nested PCR products on 1.5% agarose gel electrophoresis of P. falciparum negative blood filters determined by microscopy. MM is the molecular weight marker, Lane 1, represents the negative control, Lane 2, the positive control and lanes 3–17 are the P. falciparum negative samples by microscopy from the epidemic areas. For the sensitivity and accuracy of samples from Narok, there were no values because there were no positive samples by microscopy giving an infinite value. Diagnosis of malaria currently depends on the visualization of parasites by light microscopy of Giemsa-stained thick and thin blood smears. Although this method is cheap and simple it is labour intensive and requires a well trained personnel. Many studies have demonstrated that PCR method is more sensitive, accurate and specific compared to thick blood films diagnosis of P. falciparum. The detection of low P. vivax and P. falciparum parasitaemia by PCR, at levels undetectable by microscopy, has been reported (Snounou et al., 1993). Rapid dipstick immunoassays detect species-specific circulating parasite antigens targeting either the histidine-rich protein-2 of P. falciparum or a parasite-specific lactate dehydrogenase (Moody, 2002). Although the dipstick tests may enhance diagnostic speed, microscopic examination remains mandatory in patients with suspected malaria, because occasionally these dipstick tests are negative in patients with high parasitemia, and their sensitivity below 100 parasites/µl is low (Moody, 2002).

ehydrogenase (Moody, 2002). Although the dipstick tests may enhance diagnostic speed, microscopic examination remains mandatory in patients with suspected malaria, because occasionally these dipstick tests are negative in patients with high parasitemia, and their sensitivity below 100 parasites/µl is low (Moody, 2002). The present study compared microscopy with nested PCR method of detection. It was found that results obtained by PCR method were superior to those obtained by microscopy. This is because 72 (20.57%) out of 350 microscopy negative samples were found to be positive by PCR and all microscopy-positive samples were confirmed as positive by PCR. Hence PCR would be preferred to microscopy for the confirmation of clinical suspicion of malaria. However, PCR method is too cumbersome, expensive and not available in the local set ups where there are limited resources (Hanscheid and Grobusch, 2002). With the spread of P. falciparum resistant to anti-malarial drugs in various provinces and the increasing difficulty in controlling malaria, it is important to diagnose and treat malaria accurately. Microscopic observation of parasites stained with Giemsa in thick smears is an inexpensive and simple method that is still used.

e spread of P. falciparum resistant to anti-malarial drugs in various provinces and the increasing difficulty in controlling malaria, it is important to diagnose and treat malaria accurately. Microscopic observation of parasites stained with Giemsa in thick smears is an inexpensive and simple method that is still used. Several malaria infections from endemic countries are sub patent, with very low parasitemia, and our results also showed this has occurred in our study area (Hayder et al., 2005). Selection of drugs for treating malaria depends on species of Plasmodia detected in the blood of the patient. Delayed or missed diagnosis of falciparum malaria increases the risk of complicated or severe disease, which may be fatal, especially in non-immunes. Many isolates of P. falciparum are chloroquine resistant and thus would not be eradicated by the standard treatment for P. vivax. A missed diagnosis of P. vivax concurrent with P. falciparum is more problematic since these species could cause relapses, thereby compounding morbidity. Because of negative diagnosis by microscopy, untreated patients may be carriers of the Plasmodia parasites. These results suggest that, in malaria epidemic areas where there is transmission of P. falciparum, diagnosis of P. falciparum malaria by nested PCR is a very useful complement to microscopical examination. This is crucial in obtaining data on the incidences of each Plasmodium species and also for the follow-up of patients after specific treatment.

malaria epidemic areas where there is transmission of P. falciparum, diagnosis of P. falciparum malaria by nested PCR is a very useful complement to microscopical examination. This is crucial in obtaining data on the incidences of each Plasmodium species and also for the follow-up of patients after specific treatment. Microscopy has been found to have a great number of limitations. A superior and a more reliable diagnostic technique need to be put in place to enable proper treatment and control of malaria. The recent increase in population movement to and from areas of high malaria transmission intensity through domestic tourism, as well as migration due to conflicts and socioeconomic factors, has resulted in higher numbers of malaria cases, where a parallel increase of mortality, from 3.8 to 20%, has been mostly ascribed to late or incorrect diagnosis (Rubio et al., 1999). Despite treatment, between 1% and 4% of travelers who acquire P. falciparum malaria will die as a result of infection. This fatality rate increases to 20% or higher in patients who develop severe malaria, pregnant women or the elderly. Since 90% of people who contract malaria will not become ill until the level of parasitemia is really high (Kain et al., 1998). Sensitive routine laboratory techniques for rapid and accurate malaria are therefore desirable; first for diagnosis on admission, so as to initiate proper treatment and second during the follow up period in order to effectively manage and control malaria.

til the level of parasitemia is really high (Kain et al., 1998). Sensitive routine laboratory techniques for rapid and accurate malaria are therefore desirable; first for diagnosis on admission, so as to initiate proper treatment and second during the follow up period in order to effectively manage and control malaria. Although detection of Plasmodium parasite on Giemsa-stained blood smears by microscopy has been the reference standard for malaria diagnosis in laboratories for more than a century, it is an imperfect standard which depend mainly on the technical expertise and experience of the microscopist (Moody, 2002). The ability to maintain the required level of expertise in malaria diagnosis is a challenge particularly in periphery medical centers where the disease is not endemic (Drakeley et al., 2005). Microscopy is time consuming, can lead to misdiagnosis if the microscopist is inexperienced and /or when parasitemia is low (Hayder et al., 2005). Also, microscopy is not ideal for diagnosis of mixed infections.

hallenge particularly in periphery medical centers where the disease is not endemic (Drakeley et al., 2005). Microscopy is time consuming, can lead to misdiagnosis if the microscopist is inexperienced and /or when parasitemia is low (Hayder et al., 2005). Also, microscopy is not ideal for diagnosis of mixed infections. Alternative techniques for laboratory diagnosis of malaria have been developed for use in both endemic and epidemic areas (Hawkes, and Kain, 2007). Serological diagnostic methods and new rapid diagnostic test (RDT) for detection of antigen e.g. Parasight F (Becton Dickinson), ICT Malaria Pf/Pv (ICT Diagnostic) and OptiMAL (Flow Inc.) have been in use for detection of antigens. Though they offer an advantage in that results can be obtained within half an hour by non skilled technicians, they are tempered by three limitations (Moody, 2002). RDT methods do not offer improved sensitivity over microscopy; the sensitivity decreases as parasitemia fall below 100parasites/µl; false positives are observed particularly after treatment, as the parasites antigens detected can remain in the circulation following parasite clearance. Finally, current RDT are either specific to P. falciparum or they cannot distinguish between the parasite species present. Several PCR assays for malaria diagnosis have also been developed, based on species-specific sequences of the parasite's 18S subunit rRNA gene (Snounou et al., 1993). This make PCR assays ideal over microscopy.

ent RDT are either specific to P. falciparum or they cannot distinguish between the parasite species present. Several PCR assays for malaria diagnosis have also been developed, based on species-specific sequences of the parasite's 18S subunit rRNA gene (Snounou et al., 1993). This make PCR assays ideal over microscopy. The economic cost of misdiagnosed or wrongly treated malaria cannot be compared with the cost of introducing PCR diagnosis. Delays in recognition and appropriate treatment of malaria increase morbidity and mortality (Kain et al., 1998). Thick smears are 20–40 times more sensitive than thin smears for screening of Plasmodium parasites, with a detection limit of 10–50 trophozoites/µl. Thin smears allow one to identify malaria species (including the diagnosis of mixed infections), quantify parasitemia, and assess for the presence of schizonts, gametocytes and malarial pigment in neutrophils and monocytes. The diagnostic accuracy relies on the quality of the blood smear and experience of laboratory personnel. Tests based on PCR for species-specific Plasmodium genome are more sensitive and specific than are other tests, detecting as few as 10 parasites/µl of blood (Hanscheid, and Grobusch, 2002). Based on these diagnosis disparities and capabilities, it is clear that most malaria deaths are caused by misdiagnosis which leads to mistreatment.

R for species-specific Plasmodium genome are more sensitive and specific than are other tests, detecting as few as 10 parasites/µl of blood (Hanscheid, and Grobusch, 2002). Based on these diagnosis disparities and capabilities, it is clear that most malaria deaths are caused by misdiagnosis which leads to mistreatment. Conclusion In this study, an AmpliTaq-based PCR qualitative assay for the rapid detection and identification in clinical specimens of P. falciparum species of malaria parasite is described and evaluated using blood samples obtained from patients diagnosed by microscopy as negative. PCR-based methods have been demonstrated to be approximately 10-fold sensitive than microscopy. Putting into consideration the specificity and sensitivity of PCR-based assays, adoption of the technique as a routine diagnostic test- mainly aimed at patients presenting with mixed infection and febrile illness- will go a long way in eliminating cases of misdiagnosis and provide a window for early detection of malaria parasites which will enable the physician administer proper drugs and in good time. Again, the economic cost of misdiagnosis and mistreatment is over that of PCR diagnosis. This method should be adopted in rural settings to reduce mortality and morbidity caused by malaria due to poor diagnosis. Acknowledgement The authors wish to acknowledge the management of Kenya Medical Research Institute for allowing this work to be carried out in their laboratories.

Conclusion In this study, an AmpliTaq-based PCR qualitative assay for the rapid detection and identification in clinical specimens of P. falciparum species of malaria parasite is described and evaluated using blood samples obtained from patients diagnosed by microscopy as negative. PCR-based methods have been demonstrated to be approximately 10-fold sensitive than microscopy. Putting into consideration the specificity and sensitivity of PCR-based assays, adoption of the technique as a routine diagnostic test- mainly aimed at patients presenting with mixed infection and febrile illness- will go a long way in eliminating cases of misdiagnosis and provide a window for early detection of malaria parasites which will enable the physician administer proper drugs and in good time. Again, the economic cost of misdiagnosis and mistreatment is over that of PCR diagnosis. This method should be adopted in rural settings to reduce mortality and morbidity caused by malaria due to poor diagnosis. Acknowledgement The authors wish to acknowledge the management of Kenya Medical Research Institute for allowing this work to be carried out in their laboratories. Figure 2 Bar graph showing the number of positive by nested PCR from the samples diagnosed as negative by microscopy and the percentages of the positive from each district studied

Introduction The HIV pandemic in South Africa, now in its third decade, remains one of the biggest challenges this country has to confront. This occurs together with poverty, joblessness and other socio-economic challenges that the departments of health and social development strive to eradicate (Posel, Kahn and Walker, 2007). The HIV pandemic appears to have debatably generated more challenges to science and medicine than any other single disease. The virus has taken a huge toll in human suffering and has had an astounding socio-economic impact on healthcare in South Africa, and throughout the world, with conceivably more significant challenges occurring in third-world countries than in developed countries (Goudge, Gilson, Russel, Gumede and Mills, 2009). It is also believed that the incidence of HIV has also crafted a devastating burden and inimitable challenge to audiological health-service delivery in South Africa (Swanepoel, 2006; Khoza-Shangase, Mupawose, and Mlangeni. 2009). This challenge can partially be surmounted by ensuring that relevant amount of research is conducted to establish the physiological effects of HIV/ AIDS and its drug treatments on those who are infected.

udiological health-service delivery in South Africa (Swanepoel, 2006; Khoza-Shangase, Mupawose, and Mlangeni. 2009). This challenge can partially be surmounted by ensuring that relevant amount of research is conducted to establish the physiological effects of HIV/ AIDS and its drug treatments on those who are infected. Globally, research has shown numerous associations between HIV/AIDS and auditory performance in adults, including sensorineural hearing loss possibly linked with antiretroviral therapy (ART). However, the acquired evidence is predominantly from the developed world and research from developing countries remains limited. The known effects of HIV/AIDS on the auditory system are based mainly on cross-sectional studies and case reports conducted internationally in industrialised countries, with very scant information emanating from third-world countries where the presentation of the virus and its treatments may be different. Although much research has been conducted internationally, replication of such studies in the developing world is still required. The current study investigates specifically the auditory manifestations of AIDS in adults.

from third-world countries where the presentation of the virus and its treatments may be different. Although much research has been conducted internationally, replication of such studies in the developing world is still required. The current study investigates specifically the auditory manifestations of AIDS in adults. There are suggestions that hearing changes may be one of the presentations at any stage of HIV disease (Birchall, Wight, French, Cockbain and Smith, 1992; Noffsinger and Friedman, 1996; Campanini, Marani, Mastroianni, Cancellieri and Vicini, 2005; Khoza-Shangase, 2010). The documented prevalence and incidence of hearing loss in cases of HIV/ AIDS varies extensively, but this discrepancy may be credited to the vastly diverse samples, variable research methodologies, as well as different participant inclusion criteria used in the published studies. In general, the incidence of otologic manifestations of HIV/AIDS has been described to be reasonably small (Abemayor and Calcaterra, 1983; Kohan, Rothstein and Cohen, 1988; Lalwani and Sooy, 1992; Gold and Tami, 1998; Chandrasekhar, Connelly, Brahmbhatt, Chetan, Shah, Kloser and Baredes, 2000; Khoza and Ross, 2002). However, Flower (1991) reported on the high prevalence of hearing loss in patients with HIV/ AIDS when he declared that about 75% of adults with AIDS illness and 50% of persons with AIDS-related complex will present with clinical auditory-system abnormalities. Other researchers, such as Sooy (1987), have indicated that hearing impairment occurs in up to half of patients with AIDS. Khoza and Ross (2002) reported findings from South Africa, where hearing loss was found in 23% of their study sample of persons with HIV/ AIDS. The different results reported by these studies could well be due to methodological differences as opposed to differences in actual incidence of hearing loss (Khoza-Shangase, 2010). The most obvious difference among these studies is that while some focused on general HIV/ AIDS (with samples that included patients in all stages of HIV disease); others were more specific in their sampling.

ethodological differences as opposed to differences in actual incidence of hearing loss (Khoza-Shangase, 2010). The most obvious difference among these studies is that while some focused on general HIV/ AIDS (with samples that included patients in all stages of HIV disease); others were more specific in their sampling. Consistent with the reports that state minor auditory manifestations of HIV/ AIDS, are claims that this small incidence rate is exclusive to adults, but may be vastly different in children. These studies have resolved that there is an extremely low incidence (or no incidence) of otologic disease in adults with HIV infection, with possibly much higher incidence in paediatric patients (Khoza-Shangase, 2010). The high occurrence of conductive hearing loss in paediatric patients, globally, has been ascribed in part to the high incidence of serous otitis media, which may occur in up to 80% of cases (Smith and Canalis, 1989). Rosenberg, Schneider and Cohen (1985) examined medical records of adults with AIDS and discovered that while 71% of the patients had symptoms localised in the head and neck, none had otologic signs and symptoms. Similar findings were reported by Marcusen and Sooy (1985) who found no otologic abnormalities in their sample. Lalwani and Sooy (1992) maintain that otologic manifestations associated with HIV/ AIDS do occur but are less prevalent than other head and neck conditions.

ead and neck, none had otologic signs and symptoms. Similar findings were reported by Marcusen and Sooy (1985) who found no otologic abnormalities in their sample. Lalwani and Sooy (1992) maintain that otologic manifestations associated with HIV/ AIDS do occur but are less prevalent than other head and neck conditions. With regard to comparing the occurrence of auditory manifestations to other head and neck effects of HIV/AIDS, there is significant variation in the reports. For instance, Rosenberg et al. (1985) reported a total lack of otologic symptoms in patients who presented with head and neck signs and symptoms, while Booth (1997) mentioned an upsurge in the number of people living with HIV/ AIDS who presented with otologic symptoms. Lalwani and Sooy (1992) maintained that ear-related manifestations of HIV/ AIDS occur to a lesser degree when compared to other head and neck conditions. It should be noted that none of these studies utilized an audiological test battery method which would facilitate definitive conclusions about the auditory symptoms. Chandrasekhar et al. (2000) claimed that as many as one-third of patients with HIV may have significant otologic complaints or difficulties, and they maintained that this is a significant percentage that may be higher than figures reported in the literature on otolaryngologic and infectious diseases. Research that has incorporated audiological assessments, such as Sooy's (1987) research, illustrate abnormal audiologic findings greater than 25 dB HL on pure-tone testing in 49% of the total sample tested, while Birchall et al. (1992) stated a regular presence of hearing loss on pure-tone testing in 39% of a small sample of patients. Not so long ago, Prasad, Bhojwani, Shenoy and Prasad (2006) reported that in their study, although 80% of HIV infections presented with otolaryngologic symptoms, otologic manifestations were seen in only 20% of the cases seen, with 13% of these symptoms linked to chronic suppurative otitis media.

ll sample of patients. Not so long ago, Prasad, Bhojwani, Shenoy and Prasad (2006) reported that in their study, although 80% of HIV infections presented with otolaryngologic symptoms, otologic manifestations were seen in only 20% of the cases seen, with 13% of these symptoms linked to chronic suppurative otitis media. A review of the literature on auditory findings implies variability as far as nature, degree, and configuration of the hearing losses diagnosed in patients with HIV/ AIDS. The documented hearing impairment may be conductive, sensorineural, or central; the severity of the loss may range from mild to severe; the onset of the loss may be sudden or gradual; and the hearing loss may be stable or may fluctuate (Timon and Walsh, 1989; Lalwani and Sooy, 1992; Bankaitis; 1996; Friedman and Noffsinger, 1998; Chandrasekhar et al., 2000; Khoza and Ross, 2002; Khoza-Shangase, 2010). Khoza and Ross (2002) also argue that the hearing loss may also be profound in nature.

or gradual; and the hearing loss may be stable or may fluctuate (Timon and Walsh, 1989; Lalwani and Sooy, 1992; Bankaitis; 1996; Friedman and Noffsinger, 1998; Chandrasekhar et al., 2000; Khoza and Ross, 2002; Khoza-Shangase, 2010). Khoza and Ross (2002) also argue that the hearing loss may also be profound in nature. Friedman and Noffsinger (1998), Gold and Tami (1998) and Lalwani and Sooy (1992) have all stated that the most common otologic problems reported in this population are serous otitis media and recurrent acute otitis media, which are primarily due to eustachian tube dysfunction; suggesting that one would anticipate the conductive type of hearing loss to be more common (Khoza-Shangase, 2010). Nevertheless, that finding is not substantiated in the literature. The rate of occurrence of sensorineural hearing loss (SNHL) is reported by Gold and Tami (1998) to range between 20% and 50%, based on reviews of studies by Bankaitis and Keith (1995), Lalwani and Sooy (1992) and Tami and Lee (1994). Similarly, Khoza and Ross (2002) found higher prevalence of SNHL in a sample of adults with HIV infection, where a definite increase in the number of occurrences of SNHL from stage 1 (asymptomatic HIV infection) to stage 3 (clinical AIDS) was also established in adults who were not on ART.

992) and Tami and Lee (1994). Similarly, Khoza and Ross (2002) found higher prevalence of SNHL in a sample of adults with HIV infection, where a definite increase in the number of occurrences of SNHL from stage 1 (asymptomatic HIV infection) to stage 3 (clinical AIDS) was also established in adults who were not on ART. Very limited published literature exists with regards the degree of hearing loss in patients with HIV/AIDS. Sooy (1987) reported abnormal audiologic results of thresholds worse than 25 dB HL, with a sloping configuration on pure-tone audiometry of between 30 and 50 dB HL at 8000 Hz. A third of the patients in Sooy's study had moderate to severe hearing loss at three or more test frequencies, in at least one ear. Chandrasekhar et al. (2000), however indicated that all audiometric data in their study (i.e. pure tone, speech discrimination, and otoacoustic emissions), which was collapsed across CDC group, otologic complaint and age, there was no statistically significant effect on the ear. However, when data for right and left ear were combined, a statistically significant effect for the high frequencies (4000–8000 Hz) was found; with these high frequencies being significantly elevated relative to lower frequencies. Likewise, previously reported results have revealed that the audiometric data trends suggest deteriorating hearing loss in high frequencies. For example, Marcusen and Sooy (1985) found that some of their patients presented with a hearing loss on pure-tone testing involving 8000 Hz.

evated relative to lower frequencies. Likewise, previously reported results have revealed that the audiometric data trends suggest deteriorating hearing loss in high frequencies. For example, Marcusen and Sooy (1985) found that some of their patients presented with a hearing loss on pure-tone testing involving 8000 Hz. Chandrasekhar et al. (2000), Gold and Tami (1998) and Lalwani and Sooy (1992) state that such hearing loss steadily worsens with an increase in frequency, with high frequencies at a moderate degree of severity. Mata, Yebra Bango, Tutor de Ureta, Villarreal and Garcia (2000) reported that high-frequency sensorineural hearing loss was a common finding among the 30 patients studied. However, Khoza and Ross (2002) claim that the configuration of the hearing loss in HIV disease may not be frequency-range-specific, but may involve all frequencies. Again, these variations in reports may be attributed to the differences in the samples studied.

ng loss was a common finding among the 30 patients studied. However, Khoza and Ross (2002) claim that the configuration of the hearing loss in HIV disease may not be frequency-range-specific, but may involve all frequencies. Again, these variations in reports may be attributed to the differences in the samples studied. The description of hearing impairment in HIV/ AIDS varies even when one considers type of onset and symmetry of hearing loss (Khoza-Shangase, 2010). Several studies have reported on sudden SNHL in patients with HIV or AIDS (Real, Thomas and Gerwins, 1987; Timon and Walsh, 1989; Solanellas, Soldado and Lozano, 1996; Khoza and Ross, 2002). Smith and Canalis (1989) found that the otologic symptoms can include unilateral or bilateral SNHL, which can occasionally be of a sudden onset in nature, but that this loss is commonly rapidly progressive in nature. Khoza and Ross (2002) found the sudden onset of the hearing loss to be more prevalent in participants with more severe SNHL than in cases of conductive or mixed hearing losses. Chandrasekhar et al. (2000) evaluated 50 patients and reported that of the 29% with hearing loss, 3% presented with sudden onset and 21% with gradual onset, with the remainder presenting with intermittent onset of the symptoms. Comparison of these studies suggests a greater tendency towards bilateral gradual onset of hearing loss rather than sudden onset (Khoza-Shangase, 2010).

ed that of the 29% with hearing loss, 3% presented with sudden onset and 21% with gradual onset, with the remainder presenting with intermittent onset of the symptoms. Comparison of these studies suggests a greater tendency towards bilateral gradual onset of hearing loss rather than sudden onset (Khoza-Shangase, 2010). Numerous clinical and medically oriented studies have exhibited the occurrence of hearing loss and other auditory manifestations in HIV/AIDS. According to the literature, auditory abnormalities associated with HIV/AIDS and its treatments have been reported in persons with varying degrees of HIV infection, among both symptomatic and asymptomatic patients, as well as in patients on ART (Khoza-Shangase, 2010). Indications exist which indicate that the HIV-effects on the auditory system can be direct as well as indirect (Larsen, 1998); however, this distinction is not always clear and consistent (Khoza-Shangase, 2010). Early reports in the literature showed that HIV infection might directly affect the auditory function due to the fact that the virus is neurotropic and commonly manifests itself neurologically (McArthur, 1987), which may be what Kallail, Downs and Schertz (2008) refer to as HIV/AIDS being the primary cause of auditory system disorders. Reportedly, these direct causes possibly give rise to central auditory pathology found in this population (Bankaitis; 1996; Lalwani and Sooy, 1992). More commonly, though, reports in the literature have focused on the indirect effects of the virus on the ear. It is believed that indirect causes that result in hearing loss arise from opportunistic infections, which require suppressive therapy, thereby leading to ototoxicity (Lalwani and Sooy, 1992; Bankaitis; 1996; Bankaitis and Schountz, 1998), and which Kallail et al. (2008) refer to as iatrogenic sources (Khoza-Shangase, 2010).

believed that indirect causes that result in hearing loss arise from opportunistic infections, which require suppressive therapy, thereby leading to ototoxicity (Lalwani and Sooy, 1992; Bankaitis; 1996; Bankaitis and Schountz, 1998), and which Kallail et al. (2008) refer to as iatrogenic sources (Khoza-Shangase, 2010). Larsen's (1998) more generalised delineation of ‘direct’ versus ‘indirect’ effects of HIV/ AIDS can be applied to the causes of auditory manifestations. Based on this distinction, it would seem that, generally, the majority of auditory manifestations can be attributed to indirect effects in the form of opportunistic infections (e.g. SNHL due to cytomegalovirus, otosyphilis, meningitis, encephalitis, otitis media, and tuberculosis of the ear), and neoplasms, with some direct effects including central hearing loss due to primary CNS lymphomas (Friedmann and Arnold, 1993), necrosis of the vestibulocochlear nerve due to herpes simplex and herpes zoster, and SNHL due to cryptococcal (Kwartler, Linthicum, Jahn and Hawke, 1991) and aseptic meningitis, as well as neurosyphilis (Little, Gardner, Acker and Land, 1995) and cytomegalovirus (Meynard, El Amrani, Meyohas, Fligny, Gozlan, Rozenbaum, Roullet and Frottier, 1997). Lastly, the side effects of HIV/AIDS treatment can also be viewed as indirect effects (Stern, Lin and Lucente, 1990; Lalwani and Sooy, 1992; Friedmann and Arnold, 1993; Gold and Tami, 1998; Larsen, 1998; Chandrasekhar et al., 2000; Khoza-Shangase, 2010).

Meyohas, Fligny, Gozlan, Rozenbaum, Roullet and Frottier, 1997). Lastly, the side effects of HIV/AIDS treatment can also be viewed as indirect effects (Stern, Lin and Lucente, 1990; Lalwani and Sooy, 1992; Friedmann and Arnold, 1993; Gold and Tami, 1998; Larsen, 1998; Chandrasekhar et al., 2000; Khoza-Shangase, 2010). A review of the literature with regard to causes of hearing loss in cases of HIV/ AIDS confirms the substantial role that opportunistic infections play in the development of auditory manifestations. Khoza and Ross (2002) stated that opportunistic infections in the form of intracranial events (such as encephalitis and meningitis), and syphilis and herpes contributed to the causes of hearing loss. Moreover, auditory manifestations in this population could be due to the fact that serous otitis media and associated conductive hearing loss have been found to be more common, presenting as one of the opportunistic infections (Gold and Tami, 1998; Khoza-Shangase, 2010).

erpes contributed to the causes of hearing loss. Moreover, auditory manifestations in this population could be due to the fact that serous otitis media and associated conductive hearing loss have been found to be more common, presenting as one of the opportunistic infections (Gold and Tami, 1998; Khoza-Shangase, 2010). Literature states that conductive hearing loss in this population is commonly caused by otitis media, which may be due to eustachian tube dysfunction (Gold and Tami, 1998). Various factors have been associated with this eustachian tube dysfunction and middle ear effusions including decreased cell-mediated immunity, recurrent viral infections, non-malignant lymphoid hyperplasia of the adenoids, nasopharyngeal tumours, sinusitis, and allergic autoimmune reaction to HIV (Stern, Lin, and Lucente, 1990; Lalwani and Sooy, 1992; Friedmann and Arnold, 1993; Gold and Tami, 1998; Khoza-Shangase, 2010). Chandrasekhar et al. (2000) claim that otitis media, which is reportedly exceptionally scarce in adults with ‘normal’ health, affects up to 23% of HIV-infected patients.

immune reaction to HIV (Stern, Lin, and Lucente, 1990; Lalwani and Sooy, 1992; Friedmann and Arnold, 1993; Gold and Tami, 1998; Khoza-Shangase, 2010). Chandrasekhar et al. (2000) claim that otitis media, which is reportedly exceptionally scarce in adults with ‘normal’ health, affects up to 23% of HIV-infected patients. A large body of evidence exists in the literature regarding causes of SNHL in HIV/AIDS (Real et al., 1987; Smith and Canalis, 1989; Birchall et al., 1992; Friedmann and Arnold, 1993). Larsen (1998) asserts that the precise cause of SNHL in people infected with HIV infection is undetermined, a claim corroborated by Lalwani and Sooy (1992) who argue that in up to half of HIV-infected people with hearing loss, no cause can be found. Friedmann and Arnold (1993) associate SNHL in HIV/ AIDS to involvement of the central nervous system (CNS) and of the sensory-end organs, which may be due to neurosyphilis and neoplasms. According to these authors, these conditions may be complicated by opportunistic infections, such as cryptococcus and cytomegalovirus, as well as the side effects of some drugs used to treat the opportunistic infections (Khoza-Shangase, 2010).

of the sensory-end organs, which may be due to neurosyphilis and neoplasms. According to these authors, these conditions may be complicated by opportunistic infections, such as cryptococcus and cytomegalovirus, as well as the side effects of some drugs used to treat the opportunistic infections (Khoza-Shangase, 2010). Because of all the diseases and infections that the population with HIV/ AIDS presents with, it is not startling to find patients with hearing loss attributable to ototoxicity, as this population undergoes treatment that often involves potentially ototoxic medications (Birchall et al., 1992; Khoza-Shangase, 2010). Bankaitis and Schountz (1998) report that the use of experimental ARVs with undetermined side effects contributes to this occurrence of hearing loss. Additionally, ototoxic drugs that are often utilized in the management of opportunistic infections (such as tuberculosis) may raise the possibility for drug-induced hearing loss in this population. The current study which aimed at determining the auditory manifestations of AIDS was therefore conducted in order to increase the evidence base that is more contextually relevant. Methodology Research Aim and Objectives Primary Aim To investigate the auditory status in a group of adults with AIDS prior to commencement of antiretroviral therapy in a hospital outpatient clinic in Gauteng, South Africa. Specific Objectives To estimate the prevalence of hearing loss and the presence of other otologic effects over and above hearing impairment (tinnitus, aural fullness, disequilibrium, and so forth)

Methodology Research Aim and Objectives Primary Aim To investigate the auditory status in a group of adults with AIDS prior to commencement of antiretroviral therapy in a hospital outpatient clinic in Gauteng, South Africa. Specific Objectives To estimate the prevalence of hearing loss and the presence of other otologic effects over and above hearing impairment (tinnitus, aural fullness, disequilibrium, and so forth) To assess the type, degree and configuration of the hearing loss To explore the type of hearing symptom onset (e.g. sudden or gradual/progressive onset) To document case history data such as signs and symptoms of each participating participant and to identify any associations between obtained signs and symptoms and hearing loss Design of the Study As an extensive literature search yielded a paucity of both South African and internationally published data on this topic, the study was exploratory. The design utilized was prospective and qualitative in nature (Devore, 1999). The aim was to investigate the auditory status in a group of adults with AIDS prior to commencement of antiretroviral therapy; hence all measures were taken before commencement of ART.

published data on this topic, the study was exploratory. The design utilized was prospective and qualitative in nature (Devore, 1999). The aim was to investigate the auditory status in a group of adults with AIDS prior to commencement of antiretroviral therapy; hence all measures were taken before commencement of ART. Participants A total sample of 150 participants comprised the study. The patients selected for this study were recruited from the Hospital's Adult HIV/AIDS clinic. Patients who attend this clinic have already been diagnosed with HIV/AIDS and are seen there for general medical management as well as antiretroviral treatment and monitoring. At the time of the study all patients with CD4+ counts below 200 cells/mm3 were assessed and counselled for ART at this clinic - and the current study targeted this group before they enrolled in the treatment. Participant selection criteria Due to the exploratory nature of the current study; and given the fact that little, if any, published research has been conducted on this aspect of HIV/AIDS in South Africa, the researcher believed that it was crucial to have a sample that was as closely representative of the general AIDS population as possible; while closely recording medical history that could have had an influence on the results. This medical history was obtained from both case history interviews and from medical records. To this end, criteria stipulated in Table 1 were observed. Table 1 Summary of participant Inclusion Criteria and Recorded history

Participant selection criteria Due to the exploratory nature of the current study; and given the fact that little, if any, published research has been conducted on this aspect of HIV/AIDS in South Africa, the researcher believed that it was crucial to have a sample that was as closely representative of the general AIDS population as possible; while closely recording medical history that could have had an influence on the results. This medical history was obtained from both case history interviews and from medical records. To this end, criteria stipulated in Table 1 were observed. Table 1 Summary of participant Inclusion Criteria and Recorded history Criterion Inclusion History recorded HIV/AIDS positive serology Yes √ History of ART √ Age between 18 and 50 years Yes Alert and oriented Yes Noise exposure √ Recent (less than 3 years) or current history of treatment for TB and radiotherapy √ Positive clinical or serological evidence of syphilis √ Middle ear pathology √ Medical ear related history √ Family history of hearing loss √ Recruitment and Sampling Procedure A nonprobability convenience sampling technique was utilized in the study since the sample was restricted to a part of the population that was readily available (Devore, 1999; Schiavetti and Metz, 2002), and true random sampling would have been difficult to achieve due to time, cost, and equipment limitations. Participants were approached at the clinic and asked to volunteer to participate in the study following an explanation of what the study entailed.

was readily available (Devore, 1999; Schiavetti and Metz, 2002), and true random sampling would have been difficult to achieve due to time, cost, and equipment limitations. Participants were approached at the clinic and asked to volunteer to participate in the study following an explanation of what the study entailed. Research procedures and materials Participants underwent case history interviews and medical record reviews, otoscopy and tympanometry, as well as conventional pure tone audiometry testing. Data were collected from assessing participants' dependant variables before administration of ART. Following infection control measures proposed by Kemp and Roeser (1998), all testing was conducted in a sound-proof booth. Case History: A case history form that targeted the signs and symptoms of auditory manifestations was utilized in order to gather all the important case history information, audiological data and some medical variables that could have exerted an impact on the results of the study. Otoscopy: The researcher evaluated the participants' ears for the presence of impacted wax; otitis externa; possible otitis media; perforated tympanic membranes; collapsed ear canals; presence of any growths and any other ear disorders (Friedman and Arnold, 1993). These otoscopic abnormalities are reported to have a significant effect on DPOAE and therefore needed to be documented before testing commenced (Hall, 2000). Tympanometry:

The researcher evaluated the participants' ears for the presence of impacted wax; otitis externa; possible otitis media; perforated tympanic membranes; collapsed ear canals; presence of any growths and any other ear disorders (Friedman and Arnold, 1993). These otoscopic abnormalities are reported to have a significant effect on DPOAE and therefore needed to be documented before testing commenced (Hall, 2000). Tympanometry: Tympanometry (through the use of the Inter-Acoustic AZ26 audiotympanometer) was utilized to assess the status and integrity of middle ear functioning. Standard single frequency tympanometry using an 85dB SPL tone set at 226Hz was done. Pure tone audiometry: Conventional (250Hz–8000Hz) pure tone audiometry was performed on all participants through the use of the Inter-Acoustic AC 40 diagnostic audiometer. The criteria used to define normal hearing, was that of pure tone thresholds of 25dBHL or lower across all frequencies, with the absence of an air-bone gap (Martin and Clark, 2003). Where pure tone air conduction and tympanometry were abnormal at any test frequencies, bone conduction testing was conducted to determine the type of hearing loss. Participants presenting with abnormal findings were referred to an Ear, Nose and Throat Specialist for assessment and management, and were subsequently offered appropriate audiological rehabilitation.

try were abnormal at any test frequencies, bone conduction testing was conducted to determine the type of hearing loss. Participants presenting with abnormal findings were referred to an Ear, Nose and Throat Specialist for assessment and management, and were subsequently offered appropriate audiological rehabilitation. Validity and Reliability Test reliability was controlled and maintained at a high level by standardizing test administration, ensuring proper equipment calibration, and controlling patient variables. For all audiological assessments precautionary measures advocated by Bess and Humes (1990) and Hall (2000) were followed in terms of proper maintenance and calibration of the equipment; optimizing testing environment; correct earphone and bone vibrator placement, and proper probe placement for tympanometry. All testing was conducted in a soundproof booth or sound-treated room with equipment that was calibrated on an annual basis, with biologic calibration conducted before every test session. All participants were tested by the same researcher using the same test procedure. Furthermore, all patients were tested in the mornings to reduce the effect that fatigue can have on patients' responses to behavioural audiometry testing.

on an annual basis, with biologic calibration conducted before every test session. All participants were tested by the same researcher using the same test procedure. Furthermore, all patients were tested in the mornings to reduce the effect that fatigue can have on patients' responses to behavioural audiometry testing. Data Analysis and Statistical Procedures Participants' results were descriptively analysed and classified as either normal or abnormal. Normal hearing was taken as normal tympanometric results in the presence of normal otoscopic findings with pure tone responses at or better than 25 dB HL, with hearing loss being thresholds greater than 25dB HL with air-bone gaps greater than 10dB (Bess and Humes, 1990; Silman and Silverman, 1991). This stage of analysis aimed at determining the prevalence of hearing loss in the sample evaluated. For all participants presenting with abnormal auditory function, findings were further categorized into type of hearing loss; severity or degree of the loss; and type of onset of the hearing loss. The hearing losses were classified into the three well-documented types of hearing losses (conductive - CHL; mixed - MHL; and sensorineural - SNHL) (Bess and Humes, 1990; Jacobson and Northern, 1990). SNHL was not further differentiated into cochlear (sensory) versus retrocochlear (neural), and this is acknowledged as another limitation of the current study.

the three well-documented types of hearing losses (conductive - CHL; mixed - MHL; and sensorineural - SNHL) (Bess and Humes, 1990; Jacobson and Northern, 1990). SNHL was not further differentiated into cochlear (sensory) versus retrocochlear (neural), and this is acknowledged as another limitation of the current study. The degree of hearing loss was determined using Silman and Silverman's (1991) classification of Magnitude of Hearing Impairment. This classification system, in line with Bess and Humes (1990), Gilbert, Smith and Stayner (2003), and Katz (1994) proposes that impaired hearing function begins at an average hearing level of 25 dB HL, and is categorized as seen in Table 2. Table 2 System of classification of hearing loss in terms of degree of loss (Silman and Silverman, 1991) used in the current study Average Hearing Level dB Description < 26 dB Normal range 26dB – 40 dB Mild hearing loss 41dB – 55 dB Moderate hearing loss 56dB – 70 dB Moderately severe hearing loss 71dB – 90 dB Severe hearing loss >91 dB Profound hearing loss Furthermore, in classifying the degree of the hearing loss, the researcher looked at the configuration of the loss and added categories depicting this change in degree of loss at frequency ranges (i.e. mild-moderate; severe-profound).

ely severe hearing loss 71dB – 90 dB Severe hearing loss >91 dB Profound hearing loss Furthermore, in classifying the degree of the hearing loss, the researcher looked at the configuration of the loss and added categories depicting this change in degree of loss at frequency ranges (i.e. mild-moderate; severe-profound). Configuration of hearing loss was established by describing the pure tone results as depicting flat, irregular, rising (low frequency), sloping (high frequency) configuration (Katz, 2002). Symmetry of hearing loss was also examined where the researcher established whether the hearing loss was unilateral or bilateral, and whether it was symmetrical or asymmetrical (Katz, 2002). The type of onset of hearing symptoms was also analysed where descriptors such as gradual, gradual/progressive, sudden, and sudden/progressive onset were used to define the manner in which the symptoms presented, based on patient reports. All significant case history factors and Ear, Nose and Throat Specialists' reports were recorded and analyzed along with the audiological results in order to obtain a comprehensive assessment and to ensure that participants presenting with hearing loss deemed (based on Ear, Nose and Throat Specialists' reports) to be opportunistic infection related were identified.

ose and Throat Specialists' reports were recorded and analyzed along with the audiological results in order to obtain a comprehensive assessment and to ensure that participants presenting with hearing loss deemed (based on Ear, Nose and Throat Specialists' reports) to be opportunistic infection related were identified. Ethical Consideration Prior to commencement of the study, permission to conduct the research project was sought from the University of the Witwatersrand Human Research Ethics Committee (Medical) which gave unconditional ethical clearance in the form of protocol number M041131. The researcher ensured that permission to conduct the study was obtained from the Hospital management and from the Heads of the Audiology and HIV/AIDS clinics at the research site. Written informed consent to participate in the study was obtained from all participants before the study was conducted with an assurance that confidentiality of all records would be maintained. Furthermore, to ensure anonymity, the researcher ensured that no personal or identifying information was included in the research report and research coding numbers instead of identifying information were used. The current study also reduced risks to the participants to a minimum by conforming to the ethical principles (South African Medical Research Council, 2003) and observing provisions of the Nuremberg Code of ethics (Newell and Burnard, 2006) during the study. Lastly, the hospital and participants were given the opportunity to request to see the research results if they were interested.

um by conforming to the ethical principles (South African Medical Research Council, 2003) and observing provisions of the Nuremberg Code of ethics (Newell and Burnard, 2006) during the study. Lastly, the hospital and participants were given the opportunity to request to see the research results if they were interested. Results and Discussion Description of Participants As depicted in Table 3, the sample included 53 (35%) males and 97 (65%) females between the ages of 20 and 46 years with a mean age of 33.9 years. The average CD4+ count was 124 cells/mm3 which was consistent with the CD4+ requirement that persons need to have before they can be enrolled in an ART programme in South Africa (i.e. CD4+ has to be below 200 cells/mm3). Table 3 Demographic and CD4 count data of participants (N = 150) FACTOR SUB-CATEGORY NO. Age (Years) Range Mean 20–46yrs 33.9yrs Gender Male Female 53 (35%) 97 (65%) Ethnic Group Black White Coloured Indian 141 (94%) 0 9 (6%) 0 CD4+ Count (cells/mm3) Mean Standard deviation 123.5133

Results and Discussion Description of Participants As depicted in Table 3, the sample included 53 (35%) males and 97 (65%) females between the ages of 20 and 46 years with a mean age of 33.9 years. The average CD4+ count was 124 cells/mm3 which was consistent with the CD4+ requirement that persons need to have before they can be enrolled in an ART programme in South Africa (i.e. CD4+ has to be below 200 cells/mm3). Table 3 Demographic and CD4 count data of participants (N = 150) FACTOR SUB-CATEGORY NO. Age (Years) Range Mean 20–46yrs 33.9yrs Gender Male Female 53 (35%) 97 (65%) Ethnic Group Black White Coloured Indian 141 (94%) 0 9 (6%) 0 CD4+ Count (cells/mm3) Mean Standard deviation 123.5133 9.00294 Close inspection of the demographic profile of the participants in the current study reveals similarities between the sample evaluated and the general South African population infected with HIV/AIDS with regard to age, gender and race (Dorrington et al., 2006). This therefore implies a strong similarity to the South African population infected with HIV/AIDS, suggesting that the current study was performed on a sample that was fairly representative of the South African situation, particularly persons attending public health clinics. Dorrington et al. (2006) assert that women continue to have the highest HIV prevalence rates in the country. Most recent antenatal clinic data show that the prevalence rates amongst women exceeds 30% and that of men just over 25% (Dorrington et al., 2006). As in the rest of sub-Saharan Africa, HIV has been noted to disproportionately affect more women than men (Shisana and Simbayi, 2005). This gender bias in the prevalence rates of the disease was also evident in the sample recruited for the present study, with a much higher number of participants in the study being female.

in the rest of sub-Saharan Africa, HIV has been noted to disproportionately affect more women than men (Shisana and Simbayi, 2005). This gender bias in the prevalence rates of the disease was also evident in the sample recruited for the present study, with a much higher number of participants in the study being female. The prevalence rate of hearing loss Of the total sample of 150 participants evaluated, 135 (90%) had normal hearing, and 15 (10%) presented with a clinical hearing loss (Figure 1). Over and above changes in audiological function, participants with clinical hearing loss in the current study also presented with associated symptoms of hearing loss in the form of tinnitus and dizziness (Figure 2). Ten (67%) of the participants presented with associated tinnitus, 4 (27%) experienced dizziness, and 4 (27%) had simultaneous experience of dizziness and tinnitus. The significantly lower prevalence of dizziness in comparison to tinnitus is reassuring since this sign is believed to be more debilitating to the patient and can significantly affect the patients' quality of life (Katz, 2002). Figure 1 The prevalence rate of hearing loss in the sample of patients with AIDS (N = 150) Figure 2 The occurrence of tinnitus and dizziness in the group of participants with clinical hearing loss (n=15)

The prevalence rate of hearing loss Of the total sample of 150 participants evaluated, 135 (90%) had normal hearing, and 15 (10%) presented with a clinical hearing loss (Figure 1). Over and above changes in audiological function, participants with clinical hearing loss in the current study also presented with associated symptoms of hearing loss in the form of tinnitus and dizziness (Figure 2). Ten (67%) of the participants presented with associated tinnitus, 4 (27%) experienced dizziness, and 4 (27%) had simultaneous experience of dizziness and tinnitus. The significantly lower prevalence of dizziness in comparison to tinnitus is reassuring since this sign is believed to be more debilitating to the patient and can significantly affect the patients' quality of life (Katz, 2002). Figure 1 The prevalence rate of hearing loss in the sample of patients with AIDS (N = 150) Figure 2 The occurrence of tinnitus and dizziness in the group of participants with clinical hearing loss (n=15) These findings are consistent with previous reports on auditory manifestations of HIV/AIDS. The 10% that presented with hearing loss in the current sample is considered to be high since this sample only consisted of participants in the AIDS stage and excluded the earlier stages of the disease. This sample also did not include the effects of medications on hearing function since participants had not enrolled in ART at the time of data collection. This high occurrence of hearing loss in the sample assessed underscores the need for more research into this population to enhance generalizability of results so that vital decisions can be made regarding the anticipated burden of disease. Given the prevalence and disease burden of undetected hearing impairment and the availability of effective treatments, it is important for audiologists to engage early in the assessment and management of this population before quality of life is severely compromised.

be made regarding the anticipated burden of disease. Given the prevalence and disease burden of undetected hearing impairment and the availability of effective treatments, it is important for audiologists to engage early in the assessment and management of this population before quality of life is severely compromised. Assessment of the type, degree, configuration, and symmetry of the hearing loss Types of hearing loss Of the 15 participants with clinical hearing loss, 11 (73%) had sensorineural hearing loss (SNHL); 4 (27%) had conductive hearing loss (CHL); and none had mixed hearing loss (MHL) (Figure 3). Figure 3 CHL = Conductive hearing loss; MHL = Mixed hearing loss; SNHL = Sensorineural hearing loss Types of hearing loss in the sub-sample of participants with clinical hearing loss (n =15)

Assessment of the type, degree, configuration, and symmetry of the hearing loss Types of hearing loss Of the 15 participants with clinical hearing loss, 11 (73%) had sensorineural hearing loss (SNHL); 4 (27%) had conductive hearing loss (CHL); and none had mixed hearing loss (MHL) (Figure 3). Figure 3 CHL = Conductive hearing loss; MHL = Mixed hearing loss; SNHL = Sensorineural hearing loss Types of hearing loss in the sub-sample of participants with clinical hearing loss (n =15) In reviewing the literature, the current findings provide support for the reports that claim the hearing loss seen in HIV/AIDS to be of any type (Chandrasekhar et al. 2000; Friedmann and Noffsinger, 1998; Khoza and Ross, 2002; Timon and Walsh, 1989). The limited occurrence of CHL was not a surprising finding particularly since this was consistent with previous findings (Khoza and Ross, 2002), of less occurrence of CHL in the AIDS stage even though otitis media has been reported to be most common in this population (Friedmann and Noffsinger, 1993; Gold and Tami, 1998; Lalwani and Sooy, 1992). The higher occurrence of SNHL in AIDS may be attributed to the progressive decline in patients' immunologic status which potentially places the patients at risk for being susceptible to the neurotrophic nature of the disease and to opportunistic infections, which have been found to cause hearing loss. Because of the permanent nature of SNHL when compared to CHL, findings from the current study highlight the need for increased awareness of this AIDS manifestation to ensure that rehabilitation in the form of diagnostic testing as well as amplification occurs. Moreover, counselling regarding communication enhancing strategies for both the patient and the family can be implemented early, thereby minimising the documented negative effects that a hearing impairment can have on an individual.

that rehabilitation in the form of diagnostic testing as well as amplification occurs. Moreover, counselling regarding communication enhancing strategies for both the patient and the family can be implemented early, thereby minimising the documented negative effects that a hearing impairment can have on an individual. Degree of hearing loss Following the classification of degree of hearing loss previously reported; it became evident that the hearing loss could occur in any degree of severity as depicted in Figure 4. However, the most prevalent degree of severity of hearing loss was the mild-moderate hearing loss followed by the severe degree of hearing impairment. Of the 15 participants, 9 (60%) presented with mild to moderate hearing loss while 3 (20%) presented with severe hearing loss. Figure 4 Key: Mod = Moderate; Sev = Severe; Prof = Profound; L = left; R = right; Bi = bilateral Degree and symmetry of hearing loss in the sub-sample of participants with clinical hearing loss (n = 15)

Degree of hearing loss Following the classification of degree of hearing loss previously reported; it became evident that the hearing loss could occur in any degree of severity as depicted in Figure 4. However, the most prevalent degree of severity of hearing loss was the mild-moderate hearing loss followed by the severe degree of hearing impairment. Of the 15 participants, 9 (60%) presented with mild to moderate hearing loss while 3 (20%) presented with severe hearing loss. Figure 4 Key: Mod = Moderate; Sev = Severe; Prof = Profound; L = left; R = right; Bi = bilateral Degree and symmetry of hearing loss in the sub-sample of participants with clinical hearing loss (n = 15) These results are consistent with those reported in the literature where severity of hearing loss has been described as ranging from mild to severe with little mention of profound hearing loss - as was found in the current study. Despite the absence of the profound degree of hearing impairment in this sample, the implications for the patients' quality of life are still significant; and the role of the audiologist with regard to early and prompt effective services to this population is highlighted. Unaided severe hearing loss can have an influence not only on the patient's everyday listening environment; but in this case, may also have an influence on the patient's ability to follow treatment protocols; and subsequently adherence to ART.

rd to early and prompt effective services to this population is highlighted. Unaided severe hearing loss can have an influence not only on the patient's everyday listening environment; but in this case, may also have an influence on the patient's ability to follow treatment protocols; and subsequently adherence to ART. Configuration of hearing loss No typical pattern of configuration of hearing loss could be established before antiretroviral treatment was instituted. In the total sub-sample (n = 15) of patients with hearing loss, 5 (33%) participants presented with a sloping/high frequency hearing loss while the remaining 67% presented with flat and/or irregular audiograms with an equal chance of involvement of all frequencies. These results are consistent with findings documented by Khoza and Ross (2002) on patients who were also not taking ART, where they found that all frequencies were affected equally or to varying degrees, depending on the possible cause of the hearing loss, and the hearing loss was not necessarily confined to high frequencies as previously reported. Several authors have reported a tendency towards sloping/high frequency hearing loss, and this finding was not the general trend observed in the current study. Findings from the current study were thought to vary due to the fact that the participants were treatment naïve, and so ototoxic hearing loss often presenting as high frequency hearing loss was not the main presenting manifestation.

hearing loss, and this finding was not the general trend observed in the current study. Findings from the current study were thought to vary due to the fact that the participants were treatment naïve, and so ototoxic hearing loss often presenting as high frequency hearing loss was not the main presenting manifestation. Symmetry of hearing loss Of the total sub-sample of 15 participants with abnormal hearing, 7 (47%) had unilateral hearing impairment while 8 (53%) presented with bilateral hearing loss (Figure 3 and Table 3). Although there appears to be a dearth of reported data in respect to symmetry of hearing loss in adults infected with HIV/AIDS, the current study demonstrated that the hearing loss can be unilateral or bilateral, and can also be bilaterally symmetrical or asymmetrical (Table 3). This finding, when combined with the degree of hearing loss most prevalent in this population, stresses the importance of prompt audiological assessment and management of patients with AIDS. These findings highlight the crucial need for communication rehabilitation services by audiologists to minimise or eliminate the impact that the hearing impairment may have on the individual's communicative skills.

pulation, stresses the importance of prompt audiological assessment and management of patients with AIDS. These findings highlight the crucial need for communication rehabilitation services by audiologists to minimise or eliminate the impact that the hearing impairment may have on the individual's communicative skills. Type of onset of hearing symptoms Of the entire sample with hearing loss, all the participants (100%) presented with gradual/progressive hearing loss with no participant presenting with sudden onset of symptoms. These results are inconsistent with earlier reports by Khoza and Ross (2002) which revealed that sudden onset was mostly experienced by participants who presented with severe to profound SNHL, while gradual onset was mostly found in participants who presented with conductive and/or mixed hearing losses. In the current study, regardless of the type or degree of hearing loss, all participants presented with gradual onset of hearing loss. This type of onset of hearing loss probably explains the late patient presentation of symptoms to their physicians as patients may learn to compensate as the hearing loss gradually deteriorates.

rrent study, regardless of the type or degree of hearing loss, all participants presented with gradual onset of hearing loss. This type of onset of hearing loss probably explains the late patient presentation of symptoms to their physicians as patients may learn to compensate as the hearing loss gradually deteriorates. Case history information Detailed analysis of the audiologic evaluation results together with the documented case history information where medical diagnoses had been confirmed by Ear, Nose and Throat Specialists (Tables 4 and 5) revealed the following: Patients who presented with SNHL had documented medical histories of meningitis; infections (syphilis and otosyphilis); and histories of ototoxic medication used in the treatment of TB and other opportunistic infections. Four participants had unknown causes of hearing loss. Patients who presented with CHL had a history of chronic suppurative otitis media, and otitis media with effusion. Table 4 Case history and medical information for participants with clinical hearing loss (n=15) Participant Age (yrs) Type of Hearing Loss Medical History Ear History Participant 44s 30 Bilateral severe conductive hearing loss Otitis media Perforated tympanic membrane; Chronic suppurative otitis media Participant 45s 41 L-mild-moderate sloping SNHL Unknown None Participant 53 24 R-mild moderate CHL Otitis media Otitis media with effusion Participant 54 32 Bilateral mild SNHL ? otosyphilis None Participant 61 46 L-Mild moderately severe SNHL Unknown History of otalgia Participant 80 39 R-mild-moderate CHL Otitis media Perforated tympanic membrane; Chronic

suppurative otitis media Participant 45s 41 L-mild-moderate sloping SNHL Unknown None Participant 53 24 R-mild moderate CHL Otitis media Otitis media with effusion Participant 54 32 Bilateral mild SNHL ? otosyphilis None Participant 61 46 L-Mild moderately severe SNHL Unknown History of otalgia Participant 80 39 R-mild-moderate CHL Otitis media Perforated tympanic membrane; Chronic suppurative otitis media Participant 83 33 Bilateral moderate severe SNHL Otosyphilis None Participant 88 46 Bilateral moderate severe SNHL Meningitis None Participant 89 41 L-moderate severe SNHL Unknown Hearing loss Participant 97 29 Bilateral mild moderate sloping SNHL TB treatment and syphilis treatment None Participant 102 20 L-severe profound SNHL Syphilis & Viral meningitis Hearing loss Participant 104 39 Bilateral mild moderate SNHL Unknown None Participant 2c 29 Bilateral sloping mild moderate SNHL Herpes None Participant 44c 30 Bilateral severe CHL Otitis media Perforated tympanic membrane; Chronic suppurative otitis media Participant 45c 41 L- mild moderate sloping SNHL TB treatment and noise exposure None Key: L = left; R = right Table 5 Summary of case history data and results for participants with clinical hearing loss (n=15) FACTOR SUB-CATEGORY NO. PERCENTAGE Gender Female Male 8 7 53 47 Age CD4+ Average Age Average CD4+ 33.9yrs (Range 20–46yrs) 123.5 (Range 2–265 cells/mm3) Not applicable Hearing Function Hearing loss 15 10 Type of Hearing Loss Conductive Hearing Loss Sensorineural Hearing Loss Mixed Hearing Loss 4 11 0 27 73 0 Type of onset of Hearing Loss Sudden Gradual 0 15 0 100 Symmetry of Hearing Loss Unilateral Bilateral 7 8 47 53 Possible aetiology of

Average CD4+ 33.9yrs (Range 20–46yrs) 123.5 (Range 2–265 cells/mm3) Not applicable Hearing Function Hearing loss 15 10 Type of Hearing Loss Conductive Hearing Loss Sensorineural Hearing Loss Mixed Hearing Loss 4 11 0 27 73 0 Type of onset of Hearing Loss Sudden Gradual 0 15 0 100 Symmetry of Hearing Loss Unilateral Bilateral 7 8 47 53 Possible aetiology of Hearing Loss * Meningitis Oto/syphilis Otitis Media Herpes TB Treatment Unknown Noise exposure 2 4 4 1 2 4 1 13% 27% 27% 7% 13% 27% 7% Degree of Hearing Loss (n=15) Mild Mild-moderate Moderate Moderate-severe Severe Severe-profound Profound 1 8 1 1 3 1 0 7 53 7 7 20 7 0 Tinnitus Present Absent 10 5 67 33 Vertigo Present Absent 4 11 27 73 Tinnitus & Vertigo Present 4 27 * % Scores do not add up to 100% as some participants presented with more than one possible aetiological factor. Possible causes of hearing loss found in the current study are consistent with literature reviewed. These findings were not surprising as these conditions are commonly seen in persons living with HIV/AIDS where immunological status has been severely compromised (Khoza and Ross, 2002; Larsen, 1998), and in up to 50% of HIV-infected people with hearing loss, no cause can be identified (Lalwani and Sooy, 1992). The fact that some of these causes are treatable causes which means reversible hearing loss once again highlights the importance of ensuring that patients with AIDS get audiologically monitored so that auditory manifestations can be identified early and Ear, Nose and Throat specialist management can be instituted to prevent the symptoms from becoming permanent.

table causes which means reversible hearing loss once again highlights the importance of ensuring that patients with AIDS get audiologically monitored so that auditory manifestations can be identified early and Ear, Nose and Throat specialist management can be instituted to prevent the symptoms from becoming permanent. Conclusions Limitations to the current study were present, and they included the fact that there was limited control over confounding variables such as previous exposure to ototoxic drugs. Furthermore, due to the sample size and the fact that the data were collected in one hospital in Gauteng, South Africa, the researcher's ability to generalize the results from the sample studied to the total population of adults with AIDS in South Africa is limited. From the findings of the current study it is clear, however, that there is great heterogeneity in presentation of the auditory manifestations of AIDS. With the exception of Khoza and Ross (2002), studies reviewed were mostly from developed countries where the presentation as well as the treatment of HIV/ AIDS may be different to that in developing countries. Hence, more investigation is required in this population, which underpins the rationale for intensified efforts into audiological research into HIV/ AIDS.

studies reviewed were mostly from developed countries where the presentation as well as the treatment of HIV/ AIDS may be different to that in developing countries. Hence, more investigation is required in this population, which underpins the rationale for intensified efforts into audiological research into HIV/ AIDS. Audiological research into HIV/ AIDS may clearly demonstrate the potential role of the audiologist in both the assessment and treatment of patients AIDS. Most importantly, research conducted in specific contexts where the disease has varied effects as well as varied treatments may yield findings that are more relevant and evidence-based. Research conducted within the South African context; a context considered to be the epicentre of the African HIV pandemic; and a context that is acknowledged to present unique challenges of which political, social, economic, health, equipment, and personnel problems are deemed non-exhaustive; is crucial. Such research can directly inform the assessment and management protocols of this population, thereby impacting positively on their quality of life. These protocols can only be implemented if the audiological manifestations of AIDS and its treatments within a specific context have been clearly characterised.

s crucial. Such research can directly inform the assessment and management protocols of this population, thereby impacting positively on their quality of life. These protocols can only be implemented if the audiological manifestations of AIDS and its treatments within a specific context have been clearly characterised. Characterisation of the burden of disease has significant implications for developing countries where financial constraints guide the prioritisation of resources. For example, if the correct prevalence of audiologic manifestations of AIDS could be established, appropriate budget planning could be employed, which would have a direct influence on resource management for this population. Furthermore, when budgets are allocated for comprehensive HIV/ AIDS management, audiological services could be included in the programmes. Recommendations may need to be made for audiological services in terms of the provision of sensitive and objective equipment (such as measures of otoacoustic emissions [OAEs]) as well as the appointment of audiologists at HIV/AIDS clinics in state institutions.

audiological services could be included in the programmes. Recommendations may need to be made for audiological services in terms of the provision of sensitive and objective equipment (such as measures of otoacoustic emissions [OAEs]) as well as the appointment of audiologists at HIV/AIDS clinics in state institutions. While there are some clear auditory manifestations that were found in the current study, there is still a small evidence base. Findings regarding the prevalence as well as nature of auditory dysfunction in adults with HIV/ AIDS need to be replicated in other communities and populations in Africa, especially since sub-Saharan Africa has such high HIV incidence. Because of the vast variance in contexts, the differences in the patient populations studied, the wide range of methodological approaches employed, and so on — findings from international studies cannot easily be generalised to the African context. Furthermore, certain issues such as time of ART initiation, use of complimentary or alternative medicine in the African context, and how these may impact auditory function have not received attention. Hence, more context-relevant, current and updated research which utilises standardised and sensitive research instruments for ease of comparability of the findings is clearly needed to address this situation. Such research should also include more controlled studies to establish definitive links between HIV/ AIDS and its drug treatments with auditory function. Research should also include continuous reviews of prevalence and incidence data, as the nature of the disease and its manifestations may be changing. These research efforts would greatly enhance the development of a more consolidated body of evidence that can then be used to contribute to policy-making and programme design and implementation, while at the same time improving the efficacy of clinical practice.

re of the disease and its manifestations may be changing. These research efforts would greatly enhance the development of a more consolidated body of evidence that can then be used to contribute to policy-making and programme design and implementation, while at the same time improving the efficacy of clinical practice. Acknowledgements The author wishes to acknowledge the funding support from Carnegie Foundation for her continued research in this field.

Introduction Tuberculosis (TB) remains a significant public health problem worldwide. There were an estimated 9.2 million new cases and 1.7 million deaths from TB in 2008 (Global Tuberculosis Control Report 2008). Although genito-urinary disease is common, isolated ovarian TB is rare (Nebhani et al, 2004). We report here a case in a young girl treated in Antananarivo, Madagascar. The clinical features and diagnosis of ovarian TB are discussed, with a review of the literature. Case Report A nulliparous woman, aged 17 years, presented to hospital with a 2 month-history of pelvic pain. This was associated with a low-grade fever, weakness, and anorexia. She also reported a weight loss of 4kg in 6 months, with a BMI of just 15. She had received the Bacille Calmette-Guerin (BCG) vaccination at birth and there was no history of contact with tuberculosis. Menarche was at age 12, with regular cycles, however her last menstrual period was over four months ago. Vaginal examination revealed a right lateral uterine mass tender to palpation. Blood tests showed a moderate anaemia with a haemoglobin of 10g/dL, and an erythrocyte sedimentation rate of 90mm. Tumour markers were measured, and the level of CA-125 was 450 units/mL (15 times the upper limit). HIV serology was negative.

Vaginal examination revealed a right lateral uterine mass tender to palpation. Blood tests showed a moderate anaemia with a haemoglobin of 10g/dL, and an erythrocyte sedimentation rate of 90mm. Tumour markers were measured, and the level of CA-125 was 450 units/mL (15 times the upper limit). HIV serology was negative. Plain radiography of chest and abdomen was normal. Pelvic ultrasound demonstrated a heterogenous right adnexal mass of 50 × 45mm and a small amount of ascites in the sac of Douglas. The initial diagnosis was ovarian carcinoma, and we proceeded to laparotomy. This revealed a discrete cystic mass of the right ovary which was fully excised (Figure 1). The rest of the peritoneal cavity was completely unremarkable. Figure 1 Intraoperative image of right ovarian mass measuring 60×45mm. Postoperative histopathological examination showed giant cell proliferation with central caseous necrosis (Figure 2). There was no sign of malignancy, and the diagnosis was revised to ovarian tuberculosis. No other focus of TB was found, including pulmonary and urinary disease. Antituberculosis treatment was commenced, and continued for eight months as per current guidelines in Madagascar. Recovery was marked by complete resolution of the pelvic pain, a weight gain of 2 kg in 2 months, normalisation of her menstrual cycles, and a decrease in the CA-125 level. Figure 2 Histology slide showing caseous necrosis and giant cell proliferation, confirming ovarian tuberculosis (haematoxylin and eosin X100)

Postoperative histopathological examination showed giant cell proliferation with central caseous necrosis (Figure 2). There was no sign of malignancy, and the diagnosis was revised to ovarian tuberculosis. No other focus of TB was found, including pulmonary and urinary disease. Antituberculosis treatment was commenced, and continued for eight months as per current guidelines in Madagascar. Recovery was marked by complete resolution of the pelvic pain, a weight gain of 2 kg in 2 months, normalisation of her menstrual cycles, and a decrease in the CA-125 level. Figure 2 Histology slide showing caseous necrosis and giant cell proliferation, confirming ovarian tuberculosis (haematoxylin and eosin X100) Discussion Genito-urinary tuberculosis is the second most frequent location for extra-pulmonary tuberculosis, after the lymphatic system (Watfa and Michel, 2005). This site can represent up to 19% of gynaecological admissions in some developing countries (Sfar et al, 1990). The endometrium and fallopian tubes are almost always affected by the disease (Agarwal and Gupta, 1993). The ovaries were involved in 62.5% of cases in one study (Agarwal and Gupta, 1993). However, isolated ovarian TB with no other organ involvement as in this case, is rarely reported in the literature. It classically affects young women aged 20–30 years who are living in endemic zones. However, with increased immigration, travel, and the re-emergence of tuberculosis worldwide, reports from Western countries are also found (Falk et al., 1980; Pesut and Stojsic, 2007, Straughn and Robertson,, 2000).

Discussion Genito-urinary tuberculosis is the second most frequent location for extra-pulmonary tuberculosis, after the lymphatic system (Watfa and Michel, 2005). This site can represent up to 19% of gynaecological admissions in some developing countries (Sfar et al, 1990). The endometrium and fallopian tubes are almost always affected by the disease (Agarwal and Gupta, 1993). The ovaries were involved in 62.5% of cases in one study (Agarwal and Gupta, 1993). However, isolated ovarian TB with no other organ involvement as in this case, is rarely reported in the literature. It classically affects young women aged 20–30 years who are living in endemic zones. However, with increased immigration, travel, and the re-emergence of tuberculosis worldwide, reports from Western countries are also found (Falk et al., 1980; Pesut and Stojsic, 2007, Straughn and Robertson,, 2000). Pulmonary TB can be described prior to the ovarian disease, however this is not obligatory, as demonstrated by our case. Presenting symptoms include infertility, pelvic pain, abdmino-pelvic masses, ascites, weight loss and menstrual problems such as amenorrhoea and dysmenorrhoea (Nebhani et al, 2004). However, the patient can also be asymptomatic, which is estimated to account for at least 11% of cases in the population (Varma, 1991). Pre-operative tests which may aid the diagnosis include a positive Mantoux (tuberculin) test, and staining for acid-fast bacilli in either ascitic or pleural fluid. However, these can be negative despite extensive disease (Jana and Dhali, 2007).

Pulmonary TB can be described prior to the ovarian disease, however this is not obligatory, as demonstrated by our case. Presenting symptoms include infertility, pelvic pain, abdmino-pelvic masses, ascites, weight loss and menstrual problems such as amenorrhoea and dysmenorrhoea (Nebhani et al, 2004). However, the patient can also be asymptomatic, which is estimated to account for at least 11% of cases in the population (Varma, 1991). Pre-operative tests which may aid the diagnosis include a positive Mantoux (tuberculin) test, and staining for acid-fast bacilli in either ascitic or pleural fluid. However, these can be negative despite extensive disease (Jana and Dhali, 2007). Ca-125 is an antigenic determinant which is expressed in most nonmucinous epithelial ovarian carcinomas, and is raised in more than 80% of cases (Nebhani et al 2004, Straughn and Robertson, 2000). It is very useful in postmenopausal women (Pesut and Stojsic 2007), where the positive predictive value for malignancy is nearly 95%. However, in premenopausal women, it can be elevated by benign conditions such as endometriosis, fibroids, and pelvic inflammatory disease, and indeed tuberculosis (Straughn and Robertson, 2000). In the case of ovarian TB, its level rarely rises above 500U/ml (450U/ml in this case). (Nebhani et al., 2004, Lantheaume et al 2003). Simsek et al.(1997) have shown that decreasing levels of CA-125 correlate with the resolution of the disease on antituberculous treatment. They suggest that serial measurements should be used to determine treatment efficacy.

rely rises above 500U/ml (450U/ml in this case). (Nebhani et al., 2004, Lantheaume et al 2003). Simsek et al.(1997) have shown that decreasing levels of CA-125 correlate with the resolution of the disease on antituberculous treatment. They suggest that serial measurements should be used to determine treatment efficacy. Imaging has low specificity, with both an ovarian malignancy and a tuberculous abscess having similar appearances on ultrasound, competurised tomography, and magnetic resonance imaging (Lantheaume et al 2003). Both can be heterogenous masses, which can infiltrate omentum and neighbouring organs. Ascites and lympadenopathy are both frequently present, further confusing the diagnosis (Nebhani et al 2004). Ultrasound-guided transvaginal or transabdomenal biopsies may be used for preoperative diagnosis (Caspi et al 2000). Laparoscopy has been a great advance as it allows the diagnosis of tuberculosis in more than 97% of cases whilst avoiding laparotomy (Nebhani et al 2004, Caspi et al 2000). Nevertheless, in cases with high suspicion of malignancy, laparotomy is often the first choice to avoid tumour seeding along port tracts. However, even at open operation, it may be difficult to distinguish between the two diagnoses as the macroscopic appearance of pelvic tuberculosis can be similar to the carcinomatosis of extraovarian carcinoma (Straughn and Robertson,, 2000).

parotomy is often the first choice to avoid tumour seeding along port tracts. However, even at open operation, it may be difficult to distinguish between the two diagnoses as the macroscopic appearance of pelvic tuberculosis can be similar to the carcinomatosis of extraovarian carcinoma (Straughn and Robertson,, 2000). Intraoperative frozen section of tissue specimens can be very helpful in oncological surgery (Straughn and Robertson,, 2000). Although histological demonstration of TB can be difficult, the lack of malignant cells may indicate an alternative diagnosis. This would be recommended if the resources are available. Treatment for genital TB is medical (Global Tuberculosis Control Report 2008, Nebhani et al 2004). The national programme of TB control in Madagascar recommends a regime of eight months treatment, with quadruple therapy (rifampicin, isoniazid, ethambutol, pyrazinamide) for the first two months, followed by 6 months of isoniazid and thiacetazone. Although most cases resolve with this regime, the long-term prognosis for patients' fertility is poor. One study estimated that pelvic TB was responsible for more than 39% of cases of tubulo-ovarian infertility (Nebhani et al 2004). Early diagnosis and the prevention of tuberculosis, including BCG immunisation campaigns, are important in order to avoid this devastating outcome.

prognosis for patients' fertility is poor. One study estimated that pelvic TB was responsible for more than 39% of cases of tubulo-ovarian infertility (Nebhani et al 2004). Early diagnosis and the prevention of tuberculosis, including BCG immunisation campaigns, are important in order to avoid this devastating outcome. Conclusion Isolated ovarian tuberculosis is rare. Its presentation can mimic that of an ovarian malignancy, including an ovarian mass, ascites and a rise in CA-125 level. It should be kept in mind as a differential diagnosis, both in developing and developed countries. Acknowledgements KM is funded by a NIHR Academic Clinical Fellowship.

Introduction In 1984, Rao et al reported that renal lesions were found in HIV infected patients. They further described a glomerulopathy, which was characterised by heavy proteinuria, biochemical features of nephrotic syndrome, renal impairment that rapidly progressed to end stage renal disease (Rao et al., 1984). Males and young adults are more commonly affected (Szczech et al., 2004), but nephropathy has been demonstrated in all subsets of HIV infected patients regardless of age, sex, race, and mode of HIV acquisition (Nuermberger, 2007). Various presentations have been reported in these patients, varying from asymptomatic proteinuria to End Stage Renal Disease (ESRD) with different features of uraemia (Nochy et al., 1993; Winston and Klotman 1996; Seney et al., 1990; Weiner et al., 2002). Urinary abnormalities including oliguria, haematuria, and proteinuria has been reported as presentations in HIV patients with impaired renal function (Moro and Sidhartha 2006; Ijoma, 1996). Extremities oedema and hypertension are not common in these patients but some studies have documented them as clinical presentations of renal impairment in HIV patients especially in patients without HIV associated nephropathy (i.e. non-HIVAN) and acute renal failure (Franceschini et al., 2004; Ruldolph, 2003). Uraemia with its various presentations occur in these patients especially in those with severe renal functional impairment (Iglesias et al., 2006).

l impairment in HIV patients especially in patients without HIV associated nephropathy (i.e. non-HIVAN) and acute renal failure (Franceschini et al., 2004; Ruldolph, 2003). Uraemia with its various presentations occur in these patients especially in those with severe renal functional impairment (Iglesias et al., 2006). There are few studies on the clinical features of HIV patients with renal impairment. This study aims to highlight the spectrum of clinical presentations of HIV infected patients with renal disease.

l impairment in HIV patients especially in patients without HIV associated nephropathy (i.e. non-HIVAN) and acute renal failure (Franceschini et al., 2004; Ruldolph, 2003). Uraemia with its various presentations occur in these patients especially in those with severe renal functional impairment (Iglesias et al., 2006). There are few studies on the clinical features of HIV patients with renal impairment. This study aims to highlight the spectrum of clinical presentations of HIV infected patients with renal disease. Subjects and Method This is a cross sectional study. Three hundred and eighty three HIV seropositive patients presenting at University of Benin Teaching Hospital (out/inpatients) from 1st January 2007 to 30th June 2007 were randomly selected, and screened for renal function impairment (RFI) using glomerular filtration rate (GFR) which was calculated by using six variable of Modification of Diet in Renal Disease (MDRD) equation. and urine protein excretion using spot urine protein creatinine ratio (PCR). Two hundred and four (53.3%) patients among the total screened had RFI detected by GFR < 60ml/min/1.73m2 or PCR ≥ 200. They were stratified into mild (GFR ≥ 60ml/min/1.73m2 but PCR ≥ 200), moderate (GFR 30 to 59ml/min/1.73m2) and severe (GFR < 30ml/min/1.73ml/min/1.73m2) RFI. Eighty two patients (40.2%), 77 patients (37.7%) and 45 patients (22.2%) had mild, moderate and severe RFI respectively. Forty patients were recruited from each stratum by simple random sampling as subjects for the study. Forty patients were also recruited as control by simple random sampling from those patients with normal renal functions detected by GFR >60ml/min/1.73m2 and PCR < 200.

2%) had mild, moderate and severe RFI respectively. Forty patients were recruited from each stratum by simple random sampling as subjects for the study. Forty patients were also recruited as control by simple random sampling from those patients with normal renal functions detected by GFR >60ml/min/1.73m2 and PCR < 200. Ethical clearance was obtained from the ethical committee of the hospital. The study was explained to the subjects and control. Consent was obtained from each subject and control before the study. The data obtained from subjects and control was documented. The data was analysed using statistical package SPSS Vs 15.0. The frequency distributions of demographic variables were computed. A one way analysis of variance (ANOVA) was used to test the comparative analysis of clinical presentations as they vary with the severity of RFI. P value < 0.05 was considered significant. Results Two hundred and four (204) of the three hundred and eighty three (383) HIV infected patients screened constituting 53.3% had renal function impairment (RFI). Male constitute 61.7% of patients with renal impairment. The distribution of various severity of RFI is shown in Table 1 below. Table 1 Distribution of severity of RFI

Results Two hundred and four (204) of the three hundred and eighty three (383) HIV infected patients screened constituting 53.3% had renal function impairment (RFI). Male constitute 61.7% of patients with renal impairment. The distribution of various severity of RFI is shown in Table 1 below. Table 1 Distribution of severity of RFI Severity of RFI Number Percent Mild 82 40.2 Moderate 77 37.7 Severe 45 22.2 Total 204 100 Age distribution Figure 1 shows the mean age distribution of patients with various severities of RFI and control. Patients with mild RFI were younger with mean age of 35.6±8.3years but patients with moderate and severe RFI had mean age of 36.0±9.9 and 36.3±8.3 years respectively. There was no statistically significant difference between them (p = 0.95). Figure 1 Mean age of subjects with renal impairment and control groups Control − GFR ≥60ml/min/1.73m2 & PCR < 200mg/g ; Mild − GFR ≥60ml/min/1.73m2 and PCR ≥200mg/g Moderate − GFR 30 − 59ml/min/1.73m2; Severe − GFR <30ml/min/1.73m2

Severity of RFI Number Percent Mild 82 40.2 Moderate 77 37.7 Severe 45 22.2 Total 204 100 Age distribution Figure 1 shows the mean age distribution of patients with various severities of RFI and control. Patients with mild RFI were younger with mean age of 35.6±8.3years but patients with moderate and severe RFI had mean age of 36.0±9.9 and 36.3±8.3 years respectively. There was no statistically significant difference between them (p = 0.95). Figure 1 Mean age of subjects with renal impairment and control groups Control − GFR ≥60ml/min/1.73m2 & PCR < 200mg/g ; Mild − GFR ≥60ml/min/1.73m2 and PCR ≥200mg/g Moderate − GFR 30 − 59ml/min/1.73m2; Severe − GFR <30ml/min/1.73m2 Clinical features Table 2 shows the major complaints of the subjects and control. Easy fatigability was the commonest symptoms occurring in 19 (47.5%), 12 (30.0%), 15 (37.5%) and 9 (22.5%) of control, mild RFI, moderate RFI and severe RFI subjects respectively. This was not statistically significant (P = 0.568). Nausea and vomiting were commonest in patients with severe RFI occurring in 10(25.0%) and 16(40.0%) of these patients respectively but they were not statistically significant. Oliguria, facial and body swelling also occurred more in patients with RFI especially those with severe renal impairment. The difference was statistically significant (p = 0.046, 0.041, and 0.033 respectively). Pruritus was a commoner symptom in control but the difference was not statistically significant (P = 0.323). Table 2 Symptoms in groups of subjects and control

Clinical features Table 2 shows the major complaints of the subjects and control. Easy fatigability was the commonest symptoms occurring in 19 (47.5%), 12 (30.0%), 15 (37.5%) and 9 (22.5%) of control, mild RFI, moderate RFI and severe RFI subjects respectively. This was not statistically significant (P = 0.568). Nausea and vomiting were commonest in patients with severe RFI occurring in 10(25.0%) and 16(40.0%) of these patients respectively but they were not statistically significant. Oliguria, facial and body swelling also occurred more in patients with RFI especially those with severe renal impairment. The difference was statistically significant (p = 0.046, 0.041, and 0.033 respectively). Pruritus was a commoner symptom in control but the difference was not statistically significant (P = 0.323). Table 2 Symptoms in groups of subjects and control SYMPTOMS CONTROL N = 40 (%) MILD N=40(%) MODERATE N = 40 (%) SEVERE N=40 (%) P VALUE Easy fatiguability 19 (47.5) 12 (30.0) 15 (37.5) 9 (22.5) 0.568 Weakness of the body 13 (32.5) 9 (22.5) 10 (25.0) 12 (30.0) 0.089 Nausea 9 (22.5) 8 (20.0) 6 (15.0) 10 (25.0) 0.093 Vomiting 5 (12.5) 2 (5.0) 6 (15.0) 16 (40.0) 0.067 Pruritus 12 (30.0) 5 (12.5) 7 (17.5) 2 (5.0) 0.323 Leg swelling 2 (5.0) 3 (7.5) 1 (2.5) 15 (37.5) 0.056 Polyuria 5 (12.5) 3 (7.5) 3 (7.5) 3 (7.5) 0.125 Nocturia 4 (10.0) 4 (10.0) 2 (5.0) 4 (10.0) 0.098 Oliguria 1 (2.5) 1 (2.5) 3 (7.5) 7 (17.5) 0.046* Facial swelling 1 (2.5) 0 (0.0) 1 (2.5) 8 (20.0) 0.041* Abdominal swelling 0 (0.0) 2 (5.0) 0 (0.0) 5 (12.5) 0.033* Loss of

g swelling 2 (5.0) 3 (7.5) 1 (2.5) 15 (37.5) 0.056 Polyuria 5 (12.5) 3 (7.5) 3 (7.5) 3 (7.5) 0.125 Nocturia 4 (10.0) 4 (10.0) 2 (5.0) 4 (10.0) 0.098 Oliguria 1 (2.5) 1 (2.5) 3 (7.5) 7 (17.5) 0.046* Facial swelling 1 (2.5) 0 (0.0) 1 (2.5) 8 (20.0) 0.041* Abdominal swelling 0 (0.0) 2 (5.0) 0 (0.0) 5 (12.5) 0.033* Loss of consciousness 4 (10.0) 1 (2.5) 0 (0.0) 2 (5.0) 0.254 Haematuria 3 (7.5) 0 (0.0) 1 (2.5) 1 (2.5) 0.098 The major clinical findings in patients with various stages of RFI and control were detailed in Table 3. Pallor was the commonest signs occurring in 13 (32.5%), 20(50.0%), 14(35.0%) and 25(62.5%) of control and patients with mild, moderate, and severe RFI respectively. This was not statistically significant (P= 0.459). Impaired conscious state, fluffy hair, hepatomegaly, splenomegaly, and jaundice were commoner in patients with severe RFI but the differences were not statistically significant. Ascites, facial puffiness and pedal oedema were commoner in patients with RFI especially those with severe RFI. The differences were statistically significant (P = 0.048, 0.019, and 0.008 respectively). The mean systolic and diastolic blood pressure, and body mass index (BMI) were within normal range in both control and subjects. Table 3 signs in groups of subjects

consciousness 4 (10.0) 1 (2.5) 0 (0.0) 2 (5.0) 0.254 Haematuria 3 (7.5) 0 (0.0) 1 (2.5) 1 (2.5) 0.098 The major clinical findings in patients with various stages of RFI and control were detailed in Table 3. Pallor was the commonest signs occurring in 13 (32.5%), 20(50.0%), 14(35.0%) and 25(62.5%) of control and patients with mild, moderate, and severe RFI respectively. This was not statistically significant (P= 0.459). Impaired conscious state, fluffy hair, hepatomegaly, splenomegaly, and jaundice were commoner in patients with severe RFI but the differences were not statistically significant. Ascites, facial puffiness and pedal oedema were commoner in patients with RFI especially those with severe RFI. The differences were statistically significant (P = 0.048, 0.019, and 0.008 respectively). The mean systolic and diastolic blood pressure, and body mass index (BMI) were within normal range in both control and subjects. Table 3 signs in groups of subjects SIGNS CONTROL N = 40 (%) MILD N = 40 (%) MODERATE N = 40 (%) SEVERE N = 40 (%) P VALUE Pallor 13 (32.5) 20 (50.0) 14 (35.0) 25 (62.5) 0.459 Impaired consciousness 0 (0.0) 8 (20.0) 8 (20.0) 14 (35.0) 0.051 Fever 1 (2.5) 5 (12.5) 7 (17.5) 6 (15.0) 0.086 Fluffy hair 3 (7.5) 7 (17.5) 1 (2.5) 7 (17.5) 0.061 Hepatomegaly 1 (2.5) 2 (10.0) 1 (2.5) 8 (20.0) 0.056 Ascites 0 (0.0) 3 (7.5) 0 (0.0) 6 (15.0) 0.048* Splenomegaly 0 (0.0) 1 (2.5) 0 (0.0) 5 (12.5) 0.056 Facial puffiness 0 (0.0) 0 (0.0) 2(5.0) 3 (7.5) 0.019* Jaundice 0 (0.0) 0 (0.0) 1 (2.5) 1 (2.5) 0.054 Asterexis 1 (2.5) 0 (0.0) 0 (0.0) 1 (2.5) 0.128 Pedal oedema 0 (0.0) 1(2.5) 2 (5.0) 3 (7.5) 0.008* Mean systolic BP (mmHg) 115.33±17.17 109.64±17.08 112.11±11.23 118.00±19.34 0.912 Mean diastolic BP (mmHg) 72.33±14.31 74.19±12.21 68.15±10.15 72.52±21.15 0.578 Mean BMI 20.13±4.31 23.9±6.9 22.7±8.8 20.04±4.4 0.652 Discussion Renal disorder is a common manifestation in HIV infection. The occurrence rate of 53.3% HIV patients with renal impairment reported in this study is high when compared to other studies (Jones et al., 2007, Ham et al., 2006). However, Agaba in North Central Nigeria reported a prevalence rate of 52%, which compares with this study (Agaba et al., 2003). The mean age of about 36 years and predominant male sex in this study is consistent with other studies (Szczech et al., 2004). Renal diseases presenting as either acute or chronic renal impairment are characterised by varying degrees of clinical features. This depends on the degree of severity of impairment in renal function. In addition, human immunodeficiency virus infection has many and diverse clinical presentations. Thus there is overlap of clinical features attributable to both HIV infection and RFI, such features include easy fatiguability, weakness of the body, nausea, vomiting, pruritus and impaired conscious state (Ijoma, 1996; Onyemelukwe and Musa, 2002; Oyediran and Akinkugbe, 1970). This compares with this study, which showed no statistical significant difference in these symptoms between subjects and control.

ch features include easy fatiguability, weakness of the body, nausea, vomiting, pruritus and impaired conscious state (Ijoma, 1996; Onyemelukwe and Musa, 2002; Oyediran and Akinkugbe, 1970). This compares with this study, which showed no statistical significant difference in these symptoms between subjects and control. Body swelling manifesting as pedal oedema, facial puffiness and occasionally ascites are characteristics features of renal impairment, degree and distribution depends on the type and severity of the renal impairment with facial puffiness as one of the earliest presentations of impaired renal function. Various studies reported body swelling as an uncommon presentation in HIV patients with renal disease, especially in HIV associated nephropathy (HIVAN) (D'Agati and Appel, 1997). This contrast with this study where oedema is a common presentation in HIV infected patients with renal impairment especially those with severe renal function impairment. This may have resulted from added burden of malnutrition prevalent in our environment especially in HIV patients, also hypoalbuminaemia, and vascular abnormality are common in patients with impaired renal function. These promote interstitial fluid exudation and thus the oedema formation. In addition, this study included HIV patients with various causes and type of renal impairment and not just HIVAN. This compares with the studies of Agaba et al and Ijoma that reported oedema as one of the clinical presentations in HIV patients with renal disease.

rstitial fluid exudation and thus the oedema formation. In addition, this study included HIV patients with various causes and type of renal impairment and not just HIVAN. This compares with the studies of Agaba et al and Ijoma that reported oedema as one of the clinical presentations in HIV patients with renal disease. Urinary symptoms, which include oliguria, polyuria, nocturia, and haematuria, have been reported as common clinical presentations in renal impairment depending on the type/cause of renal disorder (Moro and Sidhartha, 2006; Ijoma, 1996; D'Agati and Appel, 1997). In this study, oliguria was the only urinary symptoms significantly commoner in patients with RFI. This is consistent with study by Ijoma at Enugu Nigeria (Ijoma, 1996). HIV is associated with various other abnormalities independent of renal function that affects frequency and characteristics of urine. These include urinary tract infection, biochemical and renal tubular abnormalities. Hypertension is a common presentation in both acute and chronic renal impairment in non-HIV patients but has been reported as uncommon in HIV related renal disorder. The mean diastolic and systolic blood pressure in this study is not elevated. The reason for the absence of hypertension is not known.

Urinary symptoms, which include oliguria, polyuria, nocturia, and haematuria, have been reported as common clinical presentations in renal impairment depending on the type/cause of renal disorder (Moro and Sidhartha, 2006; Ijoma, 1996; D'Agati and Appel, 1997). In this study, oliguria was the only urinary symptoms significantly commoner in patients with RFI. This is consistent with study by Ijoma at Enugu Nigeria (Ijoma, 1996). HIV is associated with various other abnormalities independent of renal function that affects frequency and characteristics of urine. These include urinary tract infection, biochemical and renal tubular abnormalities. Hypertension is a common presentation in both acute and chronic renal impairment in non-HIV patients but has been reported as uncommon in HIV related renal disorder. The mean diastolic and systolic blood pressure in this study is not elevated. The reason for the absence of hypertension is not known. Anaemia, which present as pallor is a common presentation in HIV infection and renal impairment and this was clearly shown in this study. The causes of anaemia in both clinical conditions are multifactorial. The co-existence of HIV infection and renal impairment worsens the burden of anaemia in these patients in terms of morbidity and mortality (Onyemelukwe and Musa, 2002; Horwich et al., 2002).

renal impairment and this was clearly shown in this study. The causes of anaemia in both clinical conditions are multifactorial. The co-existence of HIV infection and renal impairment worsens the burden of anaemia in these patients in terms of morbidity and mortality (Onyemelukwe and Musa, 2002; Horwich et al., 2002). Other clinical presentations such as jaundice, hepatomegaly, and splenomegaly may have resulted from other non-renal HIV related or unrelated clinical conditions. Also it is noted that most of the clinical presentations mentioned above were common in patients with severe RFI. This is consistent with observations in non HIV patients whose clinical features vary with severity of RFI. Furthermore severity of RFI has an inverse relationship with the level of CD4 cell count, thus severe RFI may be coexistent with severe immunosupression with antecedent exposure to renal related risk factors like infections, electrolyte abnormality, and drugs (Okafor et al., 2008). This study acknowledged some limitations which included absence of renal scan, the cross section nature of the study and inability to associate the clinical presentations with various risk factors like low CD4 cell count, drugs, co infections etc contributing to RFI. Thus patients with shrunken kidneys, and transient proteinuria may have been included as patients. However these may not have influenced the result of the study as the patients in this category will have been categorised as normal or mild RFI with unremarkable difference in their clinical presentation.

ributing to RFI. Thus patients with shrunken kidneys, and transient proteinuria may have been included as patients. However these may not have influenced the result of the study as the patients in this category will have been categorised as normal or mild RFI with unremarkable difference in their clinical presentation. In conclusion, renal disease is common in HIV infected patients and clinical presentations are many occurring more in severe renal impairment. However, few are specific, thus, use of clinical presentations as the only tool for diagnosis of renal impairment in HIV patients are not reliable. This makes a detailed assessment of renal function in all HIV patients at presentation imperative.

Introduction Praziquantel (PZQ) is efficacious against Schistosoma mansoni (Sleigh et al., 1985). Presently in Uganda, one of schistosomiasis endemic countries in sub Saharan Africa, PZQ is the drug of choice in controlling morbidity due to schistosomiasis because the mean cost of treatment per dose per person in Uganda is about $0.3 (Mott, 1982; Doenhoff et al., 2002). In spite of its relative low cost, Uganda Ministry of Health budget is still unable to procure adequate PZQ for short period mass chemotherapy. Previous studies in the seventies showed that in Uganda, the efficacy of PZQ was evaluated after six months to determine the cure proportion (Odongo-Aginya and Mugisha, 1987). Nevertheless in other tropical countries, few studies evaluated the efficacy of PZQ at the interval of one year (Stelma et al., 1995; Correa-Oliveira, et al., 2000; Davis, 1993). At present, studies in different endemic settings using the single oral dose regiment of 40 (mg/kg wt) of PZQ against S. mansoni, and mixed infections, the efficacy of PZQ stands at 60-90 % (Cioli and Pica- Mattoccia, 2000). Extensive use of PZQ in Uganda and elsewhere in the tropics has been linked to the development of parasite resistance to the drug. This is evidenced by reduced cure proportion in humans treated with PZQ in the Richard Toll area of Senegal (Stelma et al., 1995). However, factors like intensity of infections and high transmission of infections have been known to influence schistosomicidal activity of PZQ (Gryseels, 1994). In spite this fact, repeated treatment with PZQ has been shown to improve cure proportion as observed in West Nile districts of Uganda areas of high transmission and intensity (Ongom and Bradley, 1972).

s and high transmission of infections have been known to influence schistosomicidal activity of PZQ (Gryseels, 1994). In spite this fact, repeated treatment with PZQ has been shown to improve cure proportion as observed in West Nile districts of Uganda areas of high transmission and intensity (Ongom and Bradley, 1972). Consequently it was deemed necessary to establish a treatment regimen for schistosomiasis endemic foci of Uganda. This study investigated specifically the effect of PZQ on cure proportion as measured by S. mansoni egg reduction in the cohorts 18 months after treatment in Kigungu-fishing village, a community with high exposure risk to S. mansoni infection in order to establish morbidity reduction, hence, the patients benefit from treatment with PZQ and cost effective treatment regimen that could be adopted in Uganda.

y S. mansoni egg reduction in the cohorts 18 months after treatment in Kigungu-fishing village, a community with high exposure risk to S. mansoni infection in order to establish morbidity reduction, hence, the patients benefit from treatment with PZQ and cost effective treatment regimen that could be adopted in Uganda. Patients and Methods Study sample The participants were registered in the three studies using their study code numbers, names, sex, age, home locations and the names of head of the families. Nine hundred and forty five residents of Kigungu fishing village were registered in this study in January 2004. Their stool specimens were examined microscopically for S.mansoni eggs. Out of the 945 participants examined 448 (47.4%) of them were stool positive for S. mansoni, and 495 (52.6%) participants were stool negative for S. mansoni. Treatment with PZQ at 40mg/kg was administered to the 448 S. mansoni positive participants. While the S.mansoni stools negative participants were treated for other ailments where necessary. The two categories of participants were requested to report for review in July 2004. Six months later, 901 out of 945 (95.5%) cohorts were reviewed. These consisted of 433 out of 448 (96.7 %) treated S. mansoni positive cohorts. On re-examination of their stool specimens, 40 cohorts still had S.mansoni egg. They were again treated but were left out of the study. Similarly 468 out 495 (94.5%) who were S.mansoni negative at baseline were reviewed. Ninety two of them were found with eggs of S. mansoni in their stool. They were treated with PZQ 40mg/kg body weight and left out of the study. Twelve months later 625 out of 769 (81.3%) of the cohorts came back for the third evaluation. Among the 310 cohorts who were stool negative after PZQ clearance at baseline 80 became positive for S. mansoni while 230 were still stool negative for S. mansoni. Meanwhile among the 315 cohorts who were S.mansoni negative at baseline, 102 Patients became infected with S. mansoni while 213 cohorts continued to be stool negative for S. mansoni (see flow chart below).

er PZQ clearance at baseline 80 became positive for S. mansoni while 230 were still stool negative for S. mansoni. Meanwhile among the 315 cohorts who were S.mansoni negative at baseline, 102 Patients became infected with S. mansoni while 213 cohorts continued to be stool negative for S. mansoni (see flow chart below). Study area This study was conducted in Kigungu fishing village, situated along Lake Victoria in Entebbe peninsula. This village is located to the extreme end of the peninsula, at latitude 35° to 38° East and 03° to 07° North. Kigungu is about 15 kilometers from Entebbe Town Municipality and about half a kilometer from the Entebbe international airport. This fishing village was selected because previously studies on S. mansoni and other soil-transmitted helminths showed that the village is a focus for these parasites (Odongo-Aginya and Mugisha, 1987). The population of Kigungu is estimated to be 6,000 people with nearly an equal sex 1:1 ratio. They are mainly fishermen and women. Besides fishing, they do a little subsistence farming mostly for food crops. Their water exposure is high, hence, the source of infection and reinfection.

(Odongo-Aginya and Mugisha, 1987). The population of Kigungu is estimated to be 6,000 people with nearly an equal sex 1:1 ratio. They are mainly fishermen and women. Besides fishing, they do a little subsistence farming mostly for food crops. Their water exposure is high, hence, the source of infection and reinfection. Procedure of the study Informed consent was obtained from all the participants. One thousand Residents who had consented to participate in the study and children between 5 and 18 years old who had been granted permission to participate in the study by their parents/guardians and have not taken antischistosomal treatment six months prior to the baseline study were recruited. This explains the reason for the 55 people who were excluded from the base line registration. Those giving their consent and were literate, were asked to sign an official form showing acceptance. Meanwhile the illiterate patients used thumb prints on official form showing acceptance. Patients unwilling to participate in the study were not penalised in any way and normal clinical services and treatments including antischistosomal therapy were not conditioned to the patient's participation in this research.

ance. Meanwhile the illiterate patients used thumb prints on official form showing acceptance. Patients unwilling to participate in the study were not penalised in any way and normal clinical services and treatments including antischistosomal therapy were not conditioned to the patient's participation in this research. Residents reported for the study at our outreach clinic in Kigungu primary school between 9 a.m and 12 p.m every project workday. On each working day, thirty consecutive patients in a row, and who met the inclusion criteria, were registered into the study. This was to allow the laboratory technologist to examine the specimens in the afternoon and deliver the stool results the following day in the morning for treatment. The patients to be recruited were interviewed and examined by a physician and a nurse during initial screening. The physicians examined the patients clinically with special attention to condition of the abdominal organs commonly affected by S. mansoni worm. Anaemia and fever were also clinically noted. Patients with body temperature greater than 37.5°C had blood smear test for malaria parasites done. All patients infected with S. mansoni were treated at the study site with praziquantel (from Medochemie Ltd.Limassol-Cyprus Europe) at 40 mg per kg body weight. Illnesses, other than schistosomiasis, detected during examination were appropriately treated or referred to other health facilities. Patient's privacy was duly respected.

infected with S. mansoni were treated at the study site with praziquantel (from Medochemie Ltd.Limassol-Cyprus Europe) at 40 mg per kg body weight. Illnesses, other than schistosomiasis, detected during examination were appropriately treated or referred to other health facilities. Patient's privacy was duly respected. Determination of intensity of intestinal worms A stool container labelled with individual identification was given to each patient to return with about 5–10 gram of stool specimen. Eggs in the stool were quantified using modified Kato-Katz method (Odongo-Aginya, et al., 1997; Mahdi, et al., 1999). Essentially each stool specimen was initially strained through a stainless steel sieve 250µm mesh size to remove artefacts. The strained stool was then used to fill a hole in a template measuring 41.7 milligram of stool. Three separate aliquots of such measured weights were delivered on three separate slides from each stool specimen. About 10µl drop of compound stain consisting of eosin 5% in 10% formalin and nigrosin 7.5% in 10% formalin was added to stool smear on each slide. The stain was stirred in the stool smear on the slide. A wettable cellophane cover slip cut 32 × 41 mm pre soaked in 50% glycerine was placed on the stained stool smear and pressed down. The excess stain from the smear on slide was blotted out on an absorbent paper before the prepared slide was read immediately using objective × 10. The arithmetic mean of the eggs counted in three slides was recorded as the count in 41.7 milligram of stool. To convert the mean egg count into egg per gram of faeces a factor of 24 was multiplied by the mean of the eggs counted i.e. number of eggs (n) × 1000 mg/ 41.7 mg = 24 × n = eggs per gram faeces. Intensities of infections were classified as follows: low 1–100 eggs per gram, medium 101–400 and high ≥ 401eggs per gram of stool (Sleigh et al., 1985).

into egg per gram of faeces a factor of 24 was multiplied by the mean of the eggs counted i.e. number of eggs (n) × 1000 mg/ 41.7 mg = 24 × n = eggs per gram faeces. Intensities of infections were classified as follows: low 1–100 eggs per gram, medium 101–400 and high ≥ 401eggs per gram of stool (Sleigh et al., 1985). Data management and statistical analysis Data were double entered using Microsoft Excel and crosschecked by different individuals. The arithmetic mean egg counts of the individuals were categorised according to infection intensities as follows 0–100; 101–200; 201–300; 301–400; and ≥ 401. Similarly age group was also categorised into five groups 5–10; 11–20; 21–30; 31–40 and ≥ 41. The cure proportion was calculated from individuals who had no S. mansoni egg in their stool after treatment divided by the total number of individuals who had S. mansoni egg in their stool before treatment multiply by 100%. The percentage of egg count reduction was calculated as the geometric mean egg count after treatment/ by geometric mean egg count before treatment multiplied by 100%. The T-test was used to compare the intensity of infections between S. mansoni amongst 80 patients who were reinfected 18 months later and 102 patients who were S. mansoni negative but became S. mansoni positive 18 months later at 95% level of confidence while the Chi χ2 was used to show the force of infection which was evaluated by egg count reduction and the cure proportion after 6 months and 18 months against force of reinfection in six months and 18 months after treatment at 95% level of confidence.

ame S. mansoni positive 18 months later at 95% level of confidence while the Chi χ2 was used to show the force of infection which was evaluated by egg count reduction and the cure proportion after 6 months and 18 months against force of reinfection in six months and 18 months after treatment at 95% level of confidence. Results A very small proportion (2.4%) of the 433 patients who were S. mansoni positive at the baseline survey continued to excrete eggs of S. mansoni in their faeces. Meanwhile among the 468 negative patients at the baseline (2.5%) of them were infected (Figure 1). Comparison of eggs intensity excreted by the 80 cohorts who were positive at base line, cleared their infections after six months but got re-infected twelve months later are shone in (Table 1).Meanwhile Comparison of Intensity of S. mansoni amongst 80 patients who were reinfected 18 months later and 102 patients who were S. mansoni negative but became S. mansoni positive 18 months later by Student's T-test did not find any significant difference This implies that the benefit of treatment with PZQ continues to be felt in the community even after 18 months (Table 2). Figure 1 Flow chart showing the follow up of patients from first recruitment up to 18 months Table 1 Comparison of Intensity of S. mansoni amongst 80 cohorts who were reinfected 18 months but had cleared the infections after six months.

Results A very small proportion (2.4%) of the 433 patients who were S. mansoni positive at the baseline survey continued to excrete eggs of S. mansoni in their faeces. Meanwhile among the 468 negative patients at the baseline (2.5%) of them were infected (Figure 1). Comparison of eggs intensity excreted by the 80 cohorts who were positive at base line, cleared their infections after six months but got re-infected twelve months later are shone in (Table 1).Meanwhile Comparison of Intensity of S. mansoni amongst 80 patients who were reinfected 18 months later and 102 patients who were S. mansoni negative but became S. mansoni positive 18 months later by Student's T-test did not find any significant difference This implies that the benefit of treatment with PZQ continues to be felt in the community even after 18 months (Table 2). Figure 1 Flow chart showing the follow up of patients from first recruitment up to 18 months Table 1 Comparison of Intensity of S. mansoni amongst 80 cohorts who were reinfected 18 months but had cleared the infections after six months. BASE LINE INFECTION OF THE 80 COHORTS INFECTION AFTER 18 MONTHS OF SAME 80 COHORTS Age group No of cohorts / % of infected before and after 18 months in age groups Eggs excreted at base line Geometric Mean count Eggs excreted after 18 months Geometric Mean count 5–10 9 (11.25) 2632 463.86 2139 133.77 11–20 47 (58.75) 18812 285.05 9680 151.97 21–30 13 (16.25) 9184 317.11 2662 151.74 31–40 7 (8.75) 2200 284.20 1924 156.43 ≥ 41 4 (5.0) 1079 260.08 396 138.94 TOTAL 80 (100) 33897 285.05 16801 151.96 NB The Table 1: showed that there was 50.4 percent reduction in eggs excretion from the 80 cohorts studied at base line and 18 months later.

812 285.05 9680 151.97 21–30 13 (16.25) 9184 317.11 2662 151.74 31–40 7 (8.75) 2200 284.20 1924 156.43 ≥ 41 4 (5.0) 1079 260.08 396 138.94 TOTAL 80 (100) 33897 285.05 16801 151.96 NB The Table 1: showed that there was 50.4 percent reduction in eggs excretion from the 80 cohorts studied at base line and 18 months later. Table 2 Comparison of Intensity of S. mansoni amongst 80 patients who were reinfected 18 months later and 102 patients who were S. mansoni negative but became S. mansoni positive 18 months later. Re-infection with S. mansoni 18 months later N=80 Infection with S. mansoni 18months amongst 102 patients who were initially S.mansoni negative Age group N Mean eggs Std N Mean eggs Std T test (p-value) 5–10 9 133.77 202.8 23 79.4 119.9 0.555 11–20 47 151.97 173.2 47 60.9 85.6 0.031* 21–30 13 151.74 232.4 17 206.6 244.2 0.265 31–40 7 156.43 48.7 9 131 251.3 0.286 ≥ 41 4 138.94 28.9 6 20 6.9 0.427 TOTAL 80 151.96 152.09 102 105.03 179.86 0.586 Legend: Std = standard deviation. N= 80 the total number individuals re- infected with S. mansoni after the second treatment and the 102 individuals who were negative initially but became infected at the end of the 18 months. * The only age group which showed significant T test. ∑=Sum of mean

Re-infection with S. mansoni 18 months later N=80 Infection with S. mansoni 18months amongst 102 patients who were initially S.mansoni negative Age group N Mean eggs Std N Mean eggs Std T test (p-value) 5–10 9 133.77 202.8 23 79.4 119.9 0.555 11–20 47 151.97 173.2 47 60.9 85.6 0.031* 21–30 13 151.74 232.4 17 206.6 244.2 0.265 31–40 7 156.43 48.7 9 131 251.3 0.286 ≥ 41 4 138.94 28.9 6 20 6.9 0.427 TOTAL 80 151.96 152.09 102 105.03 179.86 0.586 Legend: Std = standard deviation. N= 80 the total number individuals re- infected with S. mansoni after the second treatment and the 102 individuals who were negative initially but became infected at the end of the 18 months. * The only age group which showed significant T test. ∑=Sum of mean Comparing the difference between those who remained negative after treatment with those who were infected in the baseline study the force of clearance of PZQ showed significant difference (P= 0.001). Similarly the force of clearing the infection of PZQ after 18 was also significant (P=0.001). While the re-infection rate was significant (P=0.001) after six months there was no significant difference in the re-infection rate after 18 months (P= 0.766) (Table 2).

earance of PZQ showed significant difference (P= 0.001). Similarly the force of clearing the infection of PZQ after 18 was also significant (P=0.001). While the re-infection rate was significant (P=0.001) after six months there was no significant difference in the re-infection rate after 18 months (P= 0.766) (Table 2). Discussion Several studies of infections and reinfection with Schistosoma mansoni after treatments with PZQ of residents living in endemic areas in the Tropics have been shown to lead to reduction of prevalence and intensity to re-infection after treatment. The re-infection prevalence and intensity have been shown never to equal before treatment level (Correa-Oliveira, et al., 2000). This study showed that there was reduction in the percentage of infection from {47.4% (448 out of 945) to 25.8% (80 out 310)} and the percentage reduction of the sum of eggs excretion was 50.4% in post PZQ therapy. This was 18 months after the initial treatment with PZQ 40mg/kg/wt. (Stelma et al., 1995; van.Liehout, et al., 1999).

howed that there was reduction in the percentage of infection from {47.4% (448 out of 945) to 25.8% (80 out 310)} and the percentage reduction of the sum of eggs excretion was 50.4% in post PZQ therapy. This was 18 months after the initial treatment with PZQ 40mg/kg/wt. (Stelma et al., 1995; van.Liehout, et al., 1999). We deliberately set a period of 18 months in total with the first six months for the assessment of the success of baseline treatment. We subsequently followed the cohorts up for twelve months to allow the study residents exposures to reinfection. This was to find out the level of resistance and susceptibility developed after treatment (Mott, 1982). Follow up studies to establish the force of reinfection after treatment with PZQ after short interval has been documented (Mott, 1982; Davis, 1993). Nevertheless this short term administration of treatment is uneconomical for most countries in the Tropic. Therefore, this study was to establish a treatment regiment for economical reasons for Kigungu and eventually elsewhere. Most schistosomiasis control Programmes in the Tropics are based on a six monthly single dose administration of PZQ to large communities. Repeated treatment to re-infected individuals is recommended depending on the degree of transmission in areas (Cioli, 2000). In spite of the low cost of PZQ, most counties in Africa are unable to procure adequate PZQ for mass chemotherapy. Study of this kind helps to establish treatment regiments for communities in schistosomiasis endemic areas in the tropical countries with meagre budget for helminth control programmes (Sleigh et al., 1985; Mott, 1982).

he low cost of PZQ, most counties in Africa are unable to procure adequate PZQ for mass chemotherapy. Study of this kind helps to establish treatment regiments for communities in schistosomiasis endemic areas in the tropical countries with meagre budget for helminth control programmes (Sleigh et al., 1985; Mott, 1982). Mutapi in their study of changes in specific anti-egg antibody levels with PZQ followed their patients after 9 months (Mutapi, et al., 1998). In addition, Correa-Oliveira in their natural versus drug induced resistance in S. mansoni infections study followed their patients for five years at an interval of one year (Correa-Oliveira, et al., 2000). Our study demonstrating long term benefits of PZQ covered a period of 18 months. It is of importance to note here that Immunological responses due to dead S.mansoni adult worms and eggs antigens could be contributing to the resistance to the reinfections (Ana, et al.1995; Khalife, et al., 1986; Dunne, et al., 1992). Repeated treatment with PZQ within short intervals was also found to have no effect on the re-infection period in children in their first decade of life (Correa-Oliveira, et al., 2000). Nevertheless, the cure proportion of PZQ is greater if the treatment is repeated within a short period but the cost of the treatment remains relatively high (Correa-Oliveira, et al., 2000; Ongom and Bradley 1972).

fect on the re-infection period in children in their first decade of life (Correa-Oliveira, et al., 2000). Nevertheless, the cure proportion of PZQ is greater if the treatment is repeated within a short period but the cost of the treatment remains relatively high (Correa-Oliveira, et al., 2000; Ongom and Bradley 1972). On the other hand lower cure proportion of 18–39% was observed in very intense focus of S. mansoni infection in northern Senegal (Stelma et al., 1995; van.Leihout, et al., 1999). In our study, infections were lower for higher age groups ≥ 21 years indicating that there is relationship between S. mansoni infection and age (Tables 1 and 2). This is in line with phenomena commonly observed in endemic areas (Gryseels, 1994). Pre-treatment infections categorised according to the levels of intensity showed that 197 patients in Kigungu were in low intensity levels (1–100 epg), 145 of them were in the middle levels of intensity (101–400epg) and only 106 of them excreted high egg count greater than 401 epg (Sleigh et al., 1985). We observed that 213 cohorts remained S. mansoni negative through out the study. They confirmed to us that they have never been treated at any time for S. mansoni. The observation that 213 patients remained uninfected in all studies points out to an interesting situation in which some of the residents live all their lives in S. mansoni endemic areas but do not get the infection. This group of patients commonly known as endemic normal (putative resistance) was always stool negative for S. mansoni eggs. The putative resistant people of Kigungu have been found to be stool negative for S. mansoni before and after treatment (Gazzinelli, et al., 1985).

oni endemic areas but do not get the infection. This group of patients commonly known as endemic normal (putative resistance) was always stool negative for S. mansoni eggs. The putative resistant people of Kigungu have been found to be stool negative for S. mansoni before and after treatment (Gazzinelli, et al., 1985). Intensities of infection influence manifestation of clinical schistosomiasis (N' Goran, 2003). From our records before treatment, majority of patients with multiple clinical symptoms such as abdominal pains, diarrhoea and blood in stool were positive for S. mansoni (Table 4). There were a few male patients with haematuria in this study but they were all negative for S. haematobium. Many studies showed that S. mansoni is the predominant species of schistosome in Uganda (Ongom and Bradley 1972; Odongo-Aginya and Mugisha, 1987). However, Schwertz 1951 and Cridland 1957 reported visceral schistosomiasis in Lango and Acholi (Schwert, 1951; Cridland, 1957). The few patients with ascites detected in this study were all positive for S. mansoni. Ascites is an indicator of chronic schistosomiasis with enlarged liver and spleen palpable (Davis, 1993; Ongom and Bradley 1972). Histories of blood vomiting were also recorded but they were not severe oesophageal varices bleeding linked to S. mansoni. The latter two clinical symptoms are characteristic of chronic schistosomiasis (Mott, 1982; Correa-Oliveira, et al., 2000). Age and pre-treatment intensity were the main host- parasite factors, which were significantly associated with S. mansoni low cure proportion in this study. This study also adds evidence of association between PZQ cure proportion, age and pre-treatment intensity. This observation stresses the need of two doses of PZQ within a standardised period to reduced morbidity due to heavy schistosome infections and avoids environmental contamination with S.mansoni eggs in endemic areas (N' Goran, 2003)

evidence of association between PZQ cure proportion, age and pre-treatment intensity. This observation stresses the need of two doses of PZQ within a standardised period to reduced morbidity due to heavy schistosome infections and avoids environmental contamination with S.mansoni eggs in endemic areas (N' Goran, 2003) Table 4 Percentage of common clinical symptoms observed among the study participants. Clinical symptoms % Abdominal pain Diarrhoea Blood in stool Blood in urine Ascites Blood vomiting Asymptomatic Before treatment 44.5(421*) 21.1(199*) 13.3(126*) 0.8(8*) 0.4(4*) 0.6(6*) 13.8 (130*) After treatment 30.1(188*) 18.2(134*) 6.6(41*) 0 0 0 37(231) Legend: Clinical symptoms observed* before and after treatment. Before treatment, S. mansoni positive patients had one or more of these clinical symptoms (not shown). Nevertheless after treatment most of the asymptomatic and some symptomatic patients became S. mansoni negative. There were reductions of major clinical symptoms while the minor symptoms disappeared after treatment. The percentage clinical symptoms was the ratio of observed symptoms to total number of the patients before or after treatment {(N/945*100); (N/625*100)}.

matic and some symptomatic patients became S. mansoni negative. There were reductions of major clinical symptoms while the minor symptoms disappeared after treatment. The percentage clinical symptoms was the ratio of observed symptoms to total number of the patients before or after treatment {(N/945*100); (N/625*100)}. Our stool analysis was based on a single stool specimen from each patient. Nevertheless, three stool smears were prepared from each specimen to increase the accuracy of the egg count in each stool specimen (Ongom and Bradley 1972; Odongo-Aginya, et al., 1997). In most community-based studies, cure proportion have been estimated based on only one or two slides Kato/Katz reading usually derived from a single stool sample (Kathrine, et al., 1999). The multiple stool samples procedure is particularly relevant when the overall geometric mean egg count is low, because it increases the chances of estimating true cure proportion (Sleigh et al., 1985, Mott, 1982). Our findings raise very high hope about the long term benefit of morbidity reduction initiated by praziquantel treatment. Nevertheless, since praziquantel is used widely in Africa especially in Uganda for large-scale treatment of schistosomiasis, it is relevant to monitor praziquantel effectiveness and the development of the parasites resistance to the drugs according to the set time frame of a particular endemic focus (Mott, 1982; Stelma et al., 1995; Correa-Oliveira, et al., 2000).

also higher than rates reported from hospitals in developing countries such as Ghana (Newman, 2009) with 6.7% and Ethiopia (Gedebou, 1988) with 17%. There is need to strengthen infection control activities in Nigerian hospitals in order reduce the prevalence, mortality, morbidity, and cost of care associated with HAI. The prevalence rate of 5.9% for hospital-acquired enterococci infection recorded in this study is considered high in view of the fact that all the isolates were from clinically infected patients over a period of just 6 months. There is need for clinicians in our environment to be aware of the role enterococci plays in clinical infections especially of urinary tract and surgical site infections. Most of the hospitalized patients (79.7%) in the two hospitals were in the age group 20–50 years and all the enterococci infections occurred in this age group. Because this age group constitutes the work force of any society, it becomes imperative to be critically aware of danger of enterococci infections and the need for prompt diagnosis, treatment and prevention.

y in Africa especially in Uganda for large-scale treatment of schistosomiasis, it is relevant to monitor praziquantel effectiveness and the development of the parasites resistance to the drugs according to the set time frame of a particular endemic focus (Mott, 1982; Stelma et al., 1995; Correa-Oliveira, et al., 2000). Acknowledgement This consortium study received financial assistant from the European Union grant designation: SCHISTO-M-VAC for which we are indebted. We are very grateful for the assistance of all laboratory Technologists from Makerere Medical School Microbiology department for their assistances in various laboratory works. The invaluable work of the Local councillors Lukwago David and Sempala Edward (the late), in mobilising the residents of Kigungu fishing village to come and participate in the study deserve special thanks. Similar thanks go to the Headmaster, Mr.Sentongo Mustoffer and the entire staff of Kigungu Primary School for offering to use the school facilities and organising the pupils for the study. We thank them for their generosity and cooperation. To the pupils and others who registered voluntarily in this study, we thank them for the precious specimens. Table 3 Cure rates and reinfection rates

Acknowledgement This consortium study received financial assistant from the European Union grant designation: SCHISTO-M-VAC for which we are indebted. We are very grateful for the assistance of all laboratory Technologists from Makerere Medical School Microbiology department for their assistances in various laboratory works. The invaluable work of the Local councillors Lukwago David and Sempala Edward (the late), in mobilising the residents of Kigungu fishing village to come and participate in the study deserve special thanks. Similar thanks go to the Headmaster, Mr.Sentongo Mustoffer and the entire staff of Kigungu Primary School for offering to use the school facilities and organising the pupils for the study. We thank them for their generosity and cooperation. To the pupils and others who registered voluntarily in this study, we thank them for the precious specimens. Table 3 Cure rates and reinfection rates Cure rates after 6 months P value OR (CI) 393/433 92/468 0.001 4.63 (3.53 – 6.06) Cure rates after 18 months 210/310 102/376 0.001 2.20 (1.87 – 3.34) Re infection rate after 6 months 40/432 92/469 0.001 0.47 (0.31 – 0.71) Re infection rate after 18 months 80/310 102/376 0.766 0.95 (0.68 – 1.34) Legend: cure rate after 6 months and 18 months against force of re-infection in six months and 18 months after treatment In addition, the reductions of the clinical symptoms observed before and after treatment further demonstrate the evidence of long benefit of parazequantel in this community. In S. mansoni positive patients most patients had one or more clinical symptoms before treatment, (data not shown). Nevertheless after treatment, most of the asymptomatic and some with abdominal pains, diarrhoea and blood in stool became S. mansoni negative. These indicated that those persisting symptom could be of different causes other than those for S. mansoni. We detected mixed infection of S.mansoni with others intestinal parasites including soil transmitted helminths and protozoan infections. There were reductions of major clinical symptoms while the minor symptoms disappeared after treatment (Table 3).

Introduction Enterococci are hardy, facultatively anaerobic Gram positive cocci in pairs or short chains that can grow and survive in many environments (Murray, 1990). They are part of normal flora of intestine of humans and animals but may be responsible for serious infections. Of the over 20 Enterococcus species (Facklam et al, 2002), 2 species are particularly pathogenic to man; Enterococcus faecalis causes 85–90% of enterococci infections while Enterococcus faecium causes 5–10% (Lewis & Zervos, 1990; Gordon et al, 1992; Patterson et al, 1995). Other Enterococcus species known to cause human infections include E. avium, E. gallinarium, E. casseliflavus, E. dirans, E. raffinosus and E. mundtii

Enterococcus faecalis causes 85–90% of enterococci infections while Enterococcus faecium causes 5–10% (Lewis & Zervos, 1990; Gordon et al, 1992; Patterson et al, 1995). Other Enterococcus species known to cause human infections include E. avium, E. gallinarium, E. casseliflavus, E. dirans, E. raffinosus and E. mundtii Enterococci are among the most frequent causes of nosocomial infection, particularly in intensive care units (ICU) where they are selected by therapy with cephalosporin and other antibiotics to which they are resistant. They are transmitted from person to person primarily on the hand of hospital personnel, some of whom may carry the organism in their gastrointestinal tracts. Occasionally, enterococci are transmitted on medical devices. In patients, the most common sites of enterococci infections are the urinary tract, wound and biliary tract along with other species of bacteria where it may be difficult to define the pathogenic role of the enterococci (Murray, 1990). In neonates, meningitis and bacteraemia may occur and endocarditis may occur in adults. Enterococci infection is equally distributed between sexes (Gordon et al, 1992), although urinary tract infections are more common in healthy women than men and in elderly patients due to high incidence of urinary instrumentation.

. In neonates, meningitis and bacteraemia may occur and endocarditis may occur in adults. Enterococci infection is equally distributed between sexes (Gordon et al, 1992), although urinary tract infections are more common in healthy women than men and in elderly patients due to high incidence of urinary instrumentation. In the routine microbiology laboratory, enterococci are characterized by their morphologic appearance on Gram stain and on culture, and are distinguished from the non-group D streptococci by their ability to survive in the presence of 40% bile, ability to hydrolyze aesculin, growth in 6.5% NaCl and a positive pyrrolidonylarylamidase test (Facklam et al, 2002). The treatment of enterococci infection is usually problematic because they are usually resistant to β-lactam antibiotics and aminoglycosides, though synergistic action of a combination of these drugs may be effective. The glycopeptide, vancomycin was the drug of choice but resistance to this drug is now on the increase. Newer antibiotics, such as the combination of quinupristin and dalfopristin are currently used to treat vancomycin resistant enterococci infection (Arias & Murray, 2008).

combination of these drugs may be effective. The glycopeptide, vancomycin was the drug of choice but resistance to this drug is now on the increase. Newer antibiotics, such as the combination of quinupristin and dalfopristin are currently used to treat vancomycin resistant enterococci infection (Arias & Murray, 2008). In Nigeria, the role of enterococci in clinical infections has not been appreciated hence reports of enterococci infections are very few. A previous study in Ilorin (Taiwo et al, 2002) reported 2.8% of 642 bacteria wound isolates to be Streptococcus (Enterococcus) faecalis. Another study in Lagos reported that 11% of 35 vancomycin susceptible E. faecalis isolates obtained from different clinical specimens exhibited high-level resistance (HLR) to gentamicin (MIC ≥ 2,000 µg/ml) and 32% exhibited HLR to streptomycin (Iregbu et al, 2002). In recent times in the teaching hospital in Osogbo, we have isolated E. faecalis from blood stream and wound infections (Taiwo et al, 2008; Fadiora et al, 2009) amongst other pathogens, however, the prevalence and magnitude of enterococci infections in this environment is largely unknown. The objectives of this study are; to determine the prevalence of hospital acquired enterococci infection and the common Enterococcus species in primary-care hospitals in Osogbo, Nigeria and to determine their susceptibility profile to commonly prescribed antibiotics. This information is to serve as a pilot data for more extensive molecular epidemiology of enterococci infections that will be necessary for the formulation of control policy in this environment.

n primary-care hospitals in Osogbo, Nigeria and to determine their susceptibility profile to commonly prescribed antibiotics. This information is to serve as a pilot data for more extensive molecular epidemiology of enterococci infections that will be necessary for the formulation of control policy in this environment. Materials and Method Study area This research is a descriptive cross sectional study carried out over a 6 month period (January to June 2009) in two primary-care hospitals and the bacteriology laboratory of the Ladoke Akintola University of Technology, Osogbo Southwestern Nigeria. The two hospitals offer general medical and surgical as well as gynaecologic and obstetric services, and were selected because of their relatively high patient patronage and consent of their hospital management board. Subjects The subjects were patients who developed clinical evidence of infections at least 48 hours after hospital admission. One hundred and eighteen patients were studied. Informed consent was obtained from each subject and the ethical approval of the two hospital management was obtained before the conduct of the study. Demographic and clinical data were collected from each patient into a designed form.

st 48 hours after hospital admission. One hundred and eighteen patients were studied. Informed consent was obtained from each subject and the ethical approval of the two hospital management was obtained before the conduct of the study. Demographic and clinical data were collected from each patient into a designed form. Laboratory procedure Appropriate clinical specimens (blood, urine, wound swabs, sputum and stool) were collected from each subject as applicable and transported to the bacteriology laboratory for processing, aerobic cultures and isolation on Sheep Blood agar/other appropriate culture media, and biochemical identification of enterococci and other bacterial pathogens according to recommended techniques and procedures (Cheesbrough, 2000). Isolation/identification of enterococci Enterococci were identified on Sheep Blood agar plate as non-haemolytic 0.5–1mm size streptococci-like colonies; on MacConkey agar as small dark-red magenta colonies and on CLED agar as small yellow colonies from fermentation of lactose (Cheesbrough, 2000). The colonies were confirmed as enterococci with Gram stain positivity, negative catalase test, positive bile-aesculin (bile insolubility) test, growth in 6.5% NaCl broth and as Enterococcus species by specific sugar (glucose, lactose, mannitol, sorbitol and arabinose) fermentation reactions (Facklam & Collins, 1989; Facklam et al, 1989).

onfirmed as enterococci with Gram stain positivity, negative catalase test, positive bile-aesculin (bile insolubility) test, growth in 6.5% NaCl broth and as Enterococcus species by specific sugar (glucose, lactose, mannitol, sorbitol and arabinose) fermentation reactions (Facklam & Collins, 1989; Facklam et al, 1989). Antibiotic susceptibility test The susceptibility of each enterococci isolate to oxacillin and vancomycin was determined using Clinical and Laboratory Standards Institute (CLSI) disk diffusion method (CLSI, 2007) on Mueller-Hinton agar (supplemented with 2% NaCl) with 1µg oxacillin and 30µg vancomycin disks and incubating at 35°C for 24 hours. Oxacillin zone diameter (ZD) of inhibition ≥14mm defined oxacillin susceptibility in enterococci while vancomycin ZD ≥17mm defined vancomycin susceptibility (CLSI, 2007). Susceptibility of each isolate to other antibiotics (ampicillin 10µg, erythromycin 15µg, gentamicin 10µg, cotrimoxazole 25µg, tetracycline 10µg, ceftazidime 30µg and ciprofloxacin 5µg) was performed using the disk diffusion method of Bauer et al (1966). ZD for susceptibility to these antibiotics in enterococci were; ampicillin ≥17mm, erythromycin ≥23mm, gentamicin ≥15mm, cotrimoxazole ≥16mm, tetracycline ≥19mm, ceftazidime ≥18mm and ciprofloxacin ≥21mm (CLSI, 2007). Staphylococcus aureus ATCC 25923 serve as negative and E. faecalis ATCC 51299 as positive control strains.

for susceptibility to these antibiotics in enterococci were; ampicillin ≥17mm, erythromycin ≥23mm, gentamicin ≥15mm, cotrimoxazole ≥16mm, tetracycline ≥19mm, ceftazidime ≥18mm and ciprofloxacin ≥21mm (CLSI, 2007). Staphylococcus aureus ATCC 25923 serve as negative and E. faecalis ATCC 51299 as positive control strains. Data entry and Statistical analysis All data (demographic and clinical) were entered into Window Vista 2007 laptop computer with GraphPad statistical software. Frequency tables were generated and relationship between variables tested with Chi square or Fisher's Exact test with significant value set at P<0.05. Results Over the 6 months period of study, there were 525 hospital admissions in the two primary-care hospitals in Osogbo, Nigeria. A total of 118 patients who developed clinical evidence of infection 48 hours after hospitalization were enrolled into the study. The hospital-acquired infection (HAI) rate in the two hospitals was 22.5%. Table 2 show the age and sex distribution of the eligible patients. The age group 20–29years constitute the largest proportion (42.4%) followed by age group 30–39 years (23.7%) and others as shown in Table 2. Table 3 shows the prevalence of enterococci infection of 5.9% (7 of 118) among the patients, with surgical site and urinary tract infections being the most prevalent with 18.8% and 2.6% rates respectively. Table 2 Prevalence of hospital acquired enterococci infection in two primary- care hospitals in Osogbo, Nigeria

Results Over the 6 months period of study, there were 525 hospital admissions in the two primary-care hospitals in Osogbo, Nigeria. A total of 118 patients who developed clinical evidence of infection 48 hours after hospitalization were enrolled into the study. The hospital-acquired infection (HAI) rate in the two hospitals was 22.5%. Table 2 show the age and sex distribution of the eligible patients. The age group 20–29years constitute the largest proportion (42.4%) followed by age group 30–39 years (23.7%) and others as shown in Table 2. Table 3 shows the prevalence of enterococci infection of 5.9% (7 of 118) among the patients, with surgical site and urinary tract infections being the most prevalent with 18.8% and 2.6% rates respectively. Table 2 Prevalence of hospital acquired enterococci infection in two primary- care hospitals in Osogbo, Nigeria Clinical diseases Specimen Number of patients (%) Enterococci isolates (%) Gastro-enteritis Stool 18 (15.3) - Blood stream infection Blood 20 (16.9) - Respiratory tract infection Sputum 9 (7.6) - Surgical site infection Swab/biopsy 32 (27.1) 6 Urinary tract infection Urine 39 (33.1) 1 Total 118 (100) 7 (5.9) Table 3 Enterococci species identified by sugar fermentation reactions in two primary–care hospitals in Osogbo Species No of strain Sugar reaction (% positive)

Clinical diseases Specimen Number of patients (%) Enterococci isolates (%) Gastro-enteritis Stool 18 (15.3) - Blood stream infection Blood 20 (16.9) - Respiratory tract infection Sputum 9 (7.6) - Surgical site infection Swab/biopsy 32 (27.1) 6 Urinary tract infection Urine 39 (33.1) 1 Total 118 (100) 7 (5.9) Table 3 Enterococci species identified by sugar fermentation reactions in two primary–care hospitals in Osogbo Species No of strain Sugar reaction (% positive) Glucose Lactose Mannitol Sorbitol Arabinose E. faecalis 6 + (100) + (100) + (100) + (100) − (0) E. faecium 1 − (0) + (100) + (100) − (0) + (100) Of the 118 patients with clinical infection, 58 (49.2%) were cultured positive for bacterial pathogens (with one microbial pathogen isolated in each case) while 60 (50.8%) were bacteriologically sterile. Enterococci were isolated in 7 (5.9%) patients and 2 species were identified using the sugar fermentation test described by Facklam & Collins (1989); E. faecalis in 6 and E. faecium in 1 (Table 3). Other non-enterococci bacteria recovered were Staphylococcus aureus 10 (17.2%), Staphylococcus epidermidis 2 (3.4%), Streptococcus pneumoniae 1 (1.7%), Klebsiella spp 18 (31.0%), Serratia spp 1 (1.7%), Escherichia coli 7 (12.1%) and Pseudomonas spp 12 (20.7%). The six E. faecalis were isolated from cases of SSI (5) and UTI (1) while the only E. faecium was isolated from a case of UTI (Table 4). Table 4 Bacterial isolates from hospital acquired infections in two primary-care hospitals in Osogbo, Nigeria

Glucose Lactose Mannitol Sorbitol Arabinose E. faecalis 6 + (100) + (100) + (100) + (100) − (0) E. faecium 1 − (0) + (100) + (100) − (0) + (100) Of the 118 patients with clinical infection, 58 (49.2%) were cultured positive for bacterial pathogens (with one microbial pathogen isolated in each case) while 60 (50.8%) were bacteriologically sterile. Enterococci were isolated in 7 (5.9%) patients and 2 species were identified using the sugar fermentation test described by Facklam & Collins (1989); E. faecalis in 6 and E. faecium in 1 (Table 3). Other non-enterococci bacteria recovered were Staphylococcus aureus 10 (17.2%), Staphylococcus epidermidis 2 (3.4%), Streptococcus pneumoniae 1 (1.7%), Klebsiella spp 18 (31.0%), Serratia spp 1 (1.7%), Escherichia coli 7 (12.1%) and Pseudomonas spp 12 (20.7%). The six E. faecalis were isolated from cases of SSI (5) and UTI (1) while the only E. faecium was isolated from a case of UTI (Table 4). Table 4 Bacterial isolates from hospital acquired infections in two primary-care hospitals in Osogbo, Nigeria Isolate Number Percentage Gram positive Enterococcus faecalis 6 10.4 Enterococcus faecium 1 1.7 Staphylococcus aureus 10 17.2 Staphylococcus epidermidis 2 3.4 Streptococcus pneumoniae 1 1.7 Gram negative Klebsiella spp 18 31.0 Serratia spp 1 1.7 Escherichia coli 7 12.1 Pseudomonas spp 12 20.7

Table 4 Bacterial isolates from hospital acquired infections in two primary-care hospitals in Osogbo, Nigeria Isolate Number Percentage Gram positive Enterococcus faecalis 6 10.4 Enterococcus faecium 1 1.7 Staphylococcus aureus 10 17.2 Staphylococcus epidermidis 2 3.4 Streptococcus pneumoniae 1 1.7 Gram negative Klebsiella spp 18 31.0 Serratia spp 1 1.7 Escherichia coli 7 12.1 Pseudomonas spp 12 20.7 Total 58 100 Table 5 shows the resistant pattern of the Enterococcus species. All isolates were resistant to ampicillin and oxacillin while 3 (42.9%) including the only E. faecium were resistant to vancomycin (VRE). The isolates also showed multi-drug resistant patterns with the VRE showing resistant to wider range of antibiotics (7 antibiotics) than the vancomycin sensitive strains (4–5 antibiotics. Table 5 Resistance pattern of hospital-acquired enterococci isolates in Osogbo, Nigeria No of antibiotic Antibiotic Resistance Pattern No of isolate (%) 4 Amp, Oxa, Ceft, Gen 2 (28.6) 5 Amp, Oxa, Ceft, Gen, Cot 2 (28.6) 8 Amp, Oxa, Ceft, Gen, Cot, Cip, Ery, Van *3 (42.8) Total 7 (100) Amp=ampicillin, Oxa=oxacillin, Gen=Gentamicin, Ceft=Ceftazidime, Cot=Cotrimoxazole, Cip=Ciprofloxacin, Ery=Erythromycin, Van=Vancomycin * include the only E. faecium isolated

No of antibiotic Antibiotic Resistance Pattern No of isolate (%) 4 Amp, Oxa, Ceft, Gen 2 (28.6) 5 Amp, Oxa, Ceft, Gen, Cot 2 (28.6) 8 Amp, Oxa, Ceft, Gen, Cot, Cip, Ery, Van *3 (42.8) Total 7 (100) Amp=ampicillin, Oxa=oxacillin, Gen=Gentamicin, Ceft=Ceftazidime, Cot=Cotrimoxazole, Cip=Ciprofloxacin, Ery=Erythromycin, Van=Vancomycin * include the only E. faecium isolated Table 6 shows the susceptibility profile of other Gram-positive pathogens isolated. Most of the isolates were resistant to commonly used antibiotics in this environment. Table 7 shows antibiotic susceptibility of Gram-negative pathogens isolated in the study. Most of the isolates were also resistant to all antibiotics except E. coli which was sensitive to ciprofloxacin (85.7%) and Pseudomonas spp which was sensitive to ceftazidime (100%) and ciprofloxacin (58.3%). Table 6 % Resistance of other Gram positive bacteria isolates in two primary-care hospitals in Osogbo Isolate Antibiotics (%) Amp Oxa Gen Van Ery Cot Ceft Cip S. aureus (n=10) 10 (100) 5 (50) 10 (100) 2 (20) 6 (60) 10 (100) 10 (100) 5 (50) S. epidermidis (n=2) 2 (100) 2 (100) 2 (100) 0 2 (100) 2 (100) 2 (100) 2 (100) S. pneumoniae (n=1) 1 (100) 1 (100) 1 (100) 0 0 1 (100) 1 (100) 1 (100) Amp=Ampicillin, Oxa=Oxacillin, Gen=Gentamicin, Van=Vancomycin, Ery=Erythromycin, Cot=Cotrimoxazole, Ceft=Ceftazidime, Cip=Ciprofloxacin Table 7 % Resistance of Gram negative bacteria isolates in two primary-care hospitals in Osogbo, Nigeria Isolates Antibiotics (%)

Amp Oxa Gen Van Ery Cot Ceft Cip S. aureus (n=10) 10 (100) 5 (50) 10 (100) 2 (20) 6 (60) 10 (100) 10 (100) 5 (50) S. epidermidis (n=2) 2 (100) 2 (100) 2 (100) 0 2 (100) 2 (100) 2 (100) 2 (100) S. pneumoniae (n=1) 1 (100) 1 (100) 1 (100) 0 0 1 (100) 1 (100) 1 (100) Amp=Ampicillin, Oxa=Oxacillin, Gen=Gentamicin, Van=Vancomycin, Ery=Erythromycin, Cot=Cotrimoxazole, Ceft=Ceftazidime, Cip=Ciprofloxacin Table 7 % Resistance of Gram negative bacteria isolates in two primary-care hospitals in Osogbo, Nigeria Isolates Antibiotics (%) Amp Gen Tet Cot Ceft Cip Klebsiella spp (n=18) 18 (100) 12 (66.7) 18 (100) 18 (100) 18 (100) 13 (72.2) Pseudomonas sp (n=12) 12 (100) 12 (100) 12 (100) 12 (100) 0 5 (41.7) Escherichia coli (n=7) 7 (100) 5 (71.4) 5 (71.4) 7 (100) 7 (100) 1 (14.3) Serratia spp (n=1) 1 (100) 1 (100) 1 (100) 1 (100) 1 (100) 1 (100) Amp=Ampicillin, Gen=Gentamicin, Tet=Tetracycline, Cot=Cotrimoxazole, Ceft=Ceftazidime, Cip=Ciprofloxacin

72.2) Pseudomonas sp (n=12) 12 (100) 12 (100) 12 (100) 12 (100) 0 5 (41.7) Escherichia coli (n=7) 7 (100) 5 (71.4) 5 (71.4) 7 (100) 7 (100) 1 (14.3) Serratia spp (n=1) 1 (100) 1 (100) 1 (100) 1 (100) 1 (100) 1 (100) Amp=Ampicillin, Gen=Gentamicin, Tet=Tetracycline, Cot=Cotrimoxazole, Ceft=Ceftazidime, Cip=Ciprofloxacin Discussion Before now in our environment, enterococci have been largely regarded as commensal flora and generally disregarded when isolated from clinical specimens such as wound and urine. However, there are increasing reports that this opportunistic bacterium can become pathogenic when it colonizes ecological niche where it is not normally found with potential to become invasive. Since the beginning of the 21st century in the United States of America, enterococci have become major reservoir of antibiotic-resistant genes and VRE a major cause of nosocomial infections especially of the bloodstream, urinary tract and surgical sites (Cetinkaya et al, 2000). Also recently in our teaching and specialist hospitals, enterococci especially E. faecalis are increasingly isolated in pure cultures from patients with clinical evidence of infections (Taiwo et al, 2008; Fadiora et al, 2009). The aim of this study was to determine the prevalence of hospital-acquired enterococci infections in primary health care institutions in our environment.

rococci especially E. faecalis are increasingly isolated in pure cultures from patients with clinical evidence of infections (Taiwo et al, 2008; Fadiora et al, 2009). The aim of this study was to determine the prevalence of hospital-acquired enterococci infections in primary health care institutions in our environment. This study recorded hospital-acquired infection rate of 22.5% for the two hospitals. This rate is higher than what is reported in developed countries with rates of 5–10% (Meers et al, 1980; Moro et al, 1986; Mayon-White et al, 1988; Scheel & Stormark, 1999) and also higher than rates reported from hospitals in developing countries such as Ghana (Newman, 2009) with 6.7% and Ethiopia (Gedebou, 1988) with 17%. There is need to strengthen infection control activities in Nigerian hospitals in order reduce the prevalence, mortality, morbidity, and cost of care associated with HAI.

e two hospitals were in the age group 20–50 years and all the enterococci infections occurred in this age group. Because this age group constitutes the work force of any society, it becomes imperative to be critically aware of danger of enterococci infections and the need for prompt diagnosis, treatment and prevention. Identification of Enterococcus species in this study was done using the conventional physiologic test scheme described by Facklam & Collins (1989). This scheme has been shown to correlate well with miniaturized-dehydrated tests and DNA hybridization techniques of identifying enterococci (Facklam et al, 1989). The scheme allowed identification of two Enterococcus species in this study; 85.7% E. faecalis and 14.3% E. faecium which agrees with the trend reported worldwide where E. faecalis is said to be responsible for about 80 to 90% of all enterococcal infections and E. faecium accounts for most of the others (Gordon et al, 1992; Facklam et al, 2002). This scheme is appropriate for identification of enterococci in resource limited countries such as ours as it allowed the identification of E. faecium, the specie that has not before been reported in Nigeria from previous studies (Iregbu et al, 2002; Taiwo et al, 2002; Taiwo et al 2008; Fadiora et al; 2009).

al, 2002). This scheme is appropriate for identification of enterococci in resource limited countries such as ours as it allowed the identification of E. faecium, the specie that has not before been reported in Nigeria from previous studies (Iregbu et al, 2002; Taiwo et al, 2002; Taiwo et al 2008; Fadiora et al; 2009). The enterococci isolates were resistant to multiple antibiotics with 100% of the isolates resistant to ampicillin, oxacillin, ceftazidime and gentamicin; 71.4% resistant to cotrimoxazole and 42.9% resistant to erythromycin, ciprofloxacin and vancomycin. Three isolates including the only E. faecium isolate were resistant to all tested antibiotics including vancomycin. Some enterococci are known to be intrinsically resistant to β-lactam antibiotics as well as many aminoglycosides while some are known to have acquired multidrug resistance to tetracycline, erythromycin, chloramphenicol and fluoroquinolones. A previous Nigerian study (Iregbu et al, 2002) reported 100% susceptibility of E. faecalis to ampicillin and vancomycin but exhibited 11% and 34% high level resistance (HLR) to gentamicin and streptomycin respectively. This present study revealed resistance rate of 100% to ampicillin and gentamicin and 43% to vancomycin. VRE may have gradually emerged in Nigeria at the turn of the century.

ity of E. faecalis to ampicillin and vancomycin but exhibited 11% and 34% high level resistance (HLR) to gentamicin and streptomycin respectively. This present study revealed resistance rate of 100% to ampicillin and gentamicin and 43% to vancomycin. VRE may have gradually emerged in Nigeria at the turn of the century. The emergence of VRE strains at the turn of the 20th century has generated major concern among clinicians (Cetinkaya et al, 2000) particularly in the last two decades; virtually these strains have emerged in nosocomial infections of hospitalized patients in the USA. In this study, VRE form about 43% of all the enterococci isolates, a figure that is high when one considers the fact that vancomycin is not available for clinical use in Nigeria. The VRE isolates include two E. faecalis and the only E. faecium isolate and were resistant to all the eight antibiotics tested. This observation agrees with a recent study on wound infections in the teaching hospital in Osogbo, Nigeria which reported two E. faecalis isolates to be resistant to all antibiotics tested in that study (Fadiora et al, 2009). The clinical implication is that VRE in Nigeria may soon become a great threat unless proper control measures are initiated.

recent study on wound infections in the teaching hospital in Osogbo, Nigeria which reported two E. faecalis isolates to be resistant to all antibiotics tested in that study (Fadiora et al, 2009). The clinical implication is that VRE in Nigeria may soon become a great threat unless proper control measures are initiated. A major problem is the fact that enterococci harbor transferable genetic elements, which have an unusually broad host range and can be transferred to both Gram-negative and Gram-positive bacteria species by conjugation systems involving plasmids and transposons (Clewell and Dunny, 2002). The danger of such transmission to bacteria such as S. aureus and the enterobacteriaceae in our environment is apparent as some Nigerian studies have reported vancomycin resistance among clinical S. aureus isolates (Olayinka et al, 2005, Onolitola et al, 2007) without previous exposure to vancomycin. A cursory look at our study revealed that 50% and 20% of the S. aureus isolates are oxacillin and vancomycin resistant respectively. Transfer of vancomycin resistance (van) genes has been specifically reported in patients co-colonized with VRE and MRSA (Furuno et al, 2005). One limiting factor in our study is that we could not perform molecular analysis to detect the genes responsible for vancomycin resistance because of lack of facilities. Nevertheless, we believe that hospital acquired infections with VRE will become a significant health problem in this era of sophisticated medical and surgical procedures unless strategy of systematic surveillance and infection control is put in place.

responsible for vancomycin resistance because of lack of facilities. Nevertheless, we believe that hospital acquired infections with VRE will become a significant health problem in this era of sophisticated medical and surgical procedures unless strategy of systematic surveillance and infection control is put in place. Other Gram positive bacteria (S. aureus, S. epidermidis and S. pneumoniae) isolated from the patients demonstrated multiple resistances to all antibiotics except vancomycin. The Gram negative isolates (Klebsiella spp, Pseudomonas spp, E. coli and Serratia spp) were also multiply resistant and only ciprofloxacin appeared effective. These findings are in agreement with reports of recent studies in our environment on bacterial isolates of hospital environment (Taiwo et al, 2006), catheter associated urinary tract infection (Taiwo & Aderounmu, 2006), ear infection (Tobih et al, 2006), blood stream infections (Taiwo et al, 2008) and superficial wound infections (Fadiora et al, 2009). This high antibiotic resistance situation has arisen from poor antibiotic prescription policy and guidelines, with over-the-counter availability of most antibiotics including the fluoroquinolones in Nigeria. The need to regulate antibiotic consumption, prescription and usage is highly imperative in Nigeria. Multicentre studies are necessary to determine the national prevalence of enterococci infections in Nigeria and to study the evolution of vancomycin resistance strains, their distribution and spread in the country using molecular method.

te antibiotic consumption, prescription and usage is highly imperative in Nigeria. Multicentre studies are necessary to determine the national prevalence of enterococci infections in Nigeria and to study the evolution of vancomycin resistance strains, their distribution and spread in the country using molecular method. Acknowledgement The co-operation of the management team of the two hospitals, staff and patients and the kind assistance of Mr O. A. Adefioye in the laboratory aspect of the study is acknowledged. Table 1 Gender and age group distributions of hospitalized patients in two primary-care hospitals in Osogbo, Nigeria Gender Age Group(Years) Male Female Total (%) ≤ 10 _ _ _ 10–19 5 6 11 (9.3) 20–29 18 32 50 (42.4) 30–39 9 19 28 (23.7) 40–49 10 6 16 (13.6) 50–59 2 4 6 (5.1) 60–69 1 1 2 (1.7) 70–79 3 0 3 (2.5) 80–89 0 1 1 (0.8) 90–99 1 0 1 (0.8) > 100 _ _ _ Total 49 (41.5) 69 (58.5) 118 (100)

Introduction Lymphatic filariasis is a tropical disease caused by the parasitic filarid nematode worm Wuchereria bancrofti. About 128 million people in 73 countries are infected globally with an estimated 1.1 billion at risk of infection (Addiss and Brady, 2007). The disease is the second leading cause of permanent and long-term disability in the world, inflicting serious public health and socio-economic problem in endemic communities (Terranella et al., 2006). At least 40 million people are affected in Africa (Anosike et al., 2005). The disease is usually seen among the poorest of the poor, and for many years has had as very low public health rating in the priorities of most of the countries where it is prevalent. The visible manifestations of the disease are severe and disfiguring, it has been reported that one third of infected individuals present with overt clinical manifestations: lymphoedema and elephantiasis of the limbs, or genitals, hydrocoele, chyluria, or recurrent infections associated with damaged lymphatic (Sherchand et al., 2003). The acute attacks of adenolymphangitis (ADL) are characterized by fever, chills, local warmth and inflammation of the inguinal node. Patients are usually incapacitated for 4–7 days while the attack lasts. The swelling later becomes permanent in the form of lymphoedema and at times there is dysfunction of the genital lymphatic that leads to hydrocoeles (Person et al., 2006).

erized by fever, chills, local warmth and inflammation of the inguinal node. Patients are usually incapacitated for 4–7 days while the attack lasts. The swelling later becomes permanent in the form of lymphoedema and at times there is dysfunction of the genital lymphatic that leads to hydrocoeles (Person et al., 2006). Lymphatic filariasis is prevalent in Nigeria and widespread, Nigeria is believed to be the third most endemic country in the world after India and Indonesia (Eigege et al., 2002). Studies in Nigeria have reported prevalence rates ranging from 6% – 47% (Anosike et al., 2005, Eigege et al., 2002, Udoidung et al., 2008, Braide et al., 2003, Nwoke et al., 2006, Badaki and Akogun 2000, Mba and Njoku 2000, Omudu and Okafor 2007). A lot more epidemiological information is however needed on the distribution, clinical signs, burden and impact of the disease on individuals. Several researchers have also highlighted the dearth of sociocultural information on the local beliefs, perceptions and behaviours towards the disease (Addiss and Brady 2007). This paucity of sociocultural data is a common feature of lymphatic filariasis and other neglected tropical diseases. Gyapong et al. (1996) have argued that the lack of information and understanding of lymphatic filariasis sociocultural consequences have led to a gross underestimation of its impact in rural communities. Lack of the correct disease etiology may increase risk, impede progress of intervention and become obstacles to health seeking behaviour.

have argued that the lack of information and understanding of lymphatic filariasis sociocultural consequences have led to a gross underestimation of its impact in rural communities. Lack of the correct disease etiology may increase risk, impede progress of intervention and become obstacles to health seeking behaviour. The global effort to eliminate lymphatic filariasis as a public health problem focuses on Mass Drug Administration (MDA) following community diagnosis using the prevalence of hydrocoele and lymphoedema as rapid diagnostic features (Eigege et al., 2002, Nwoke et al., 2006, Gyapong et al., 1998). This paper presents findings from a comparative study using lymphatic filariasis-related clinical signs as rapid diagnostic feature and serological diagnosis using the immunochromatographic card test to detect circulating filarial antigen (CFA), and community perceptions and beliefs on these disease manifestations. The study intends to provide much needed epidemiological data that will guide in identifying high risk communities and develop evidence-based community education strategies.

munochromatographic card test to detect circulating filarial antigen (CFA), and community perceptions and beliefs on these disease manifestations. The study intends to provide much needed epidemiological data that will guide in identifying high risk communities and develop evidence-based community education strategies. Materials and Methods Description of Study Area Benue state is one of the 36 states in the Federal Republic of Nigeria, a tropical country on the west coast of Africa. The state derives its name from River Benue the second largest river in the country and is located in the central region of the country where it lies between latitude 7° 13 and 7° 49 longitude 8° 15 and 8° 42. The state covers an area of about 34, 059 square kilometers with a population of over 4.2 million people (National Population Commission, 2007). Majority of the inhabitants live in rural agricultural areas and engage in peasant agriculture, the state's reputation as the food basket of the nation is being seriously jeopardized by the socio-economic consequences of parasitic diseases. Ado Local Government Area (LGA) where the study was conducted is one of the twenty three (23) LGAs of the state with a population of 178.882 (National Population Commission, 2007). The Local Government is made up of five major districts, Agila, Igumale, Ulayi, Ijigbam and Utonkon. The LGA is located at the southern part of Benue State (Fiure 1) and has boundaries with Ebonyi and Enugu States. The inhabitants are mainly peasant farmers and live in small farming settlements. The Ado and Okpokwu rivers are the predominant rivers and provide breeding ground for vectors of some filarial diseases. The study was conducted from February – November 2009.

ate (Fiure 1) and has boundaries with Ebonyi and Enugu States. The inhabitants are mainly peasant farmers and live in small farming settlements. The Ado and Okpokwu rivers are the predominant rivers and provide breeding ground for vectors of some filarial diseases. The study was conducted from February – November 2009. Ethical Clearance This survey received ethical clearance from the State Ministry of Health and the proposal was approved by the Postgraduate Research Committee of the University of Nigeria, Nsukka. Informed consent was obtained from all the participants after the explanation of the procedures and the likely benefits of the study. After explaining the purpose of the study to the village chiefs and traditional leadership councils and obtaining their permission and consent, all participating adults (16 years of age and older) were asked to gather at the village Primary Health Care (PHC) Centre and randomly selected. Before clinical examination and testing could be carried, the objectives of the survey were explained in local language and each consenting individual provided demographic data. The participants were assigned identification numbers and their names, age, occupation and marital status were taken.

e and randomly selected. Before clinical examination and testing could be carried, the objectives of the survey were explained in local language and each consenting individual provided demographic data. The participants were assigned identification numbers and their names, age, occupation and marital status were taken. Search for Hydrocoele and Lymphoedema After obtaining demographic information (age, occupation, marital status, sex), participants were asked to partially disrobe and a local government health officer trained on the diagnosis of the various manifestations of filariasis including hydrocoele and lymphoedema. Hydrocoele was diagnosed based on the finding of a non-tender, soft, fluid-filled mass bigger than the size of an orange. Hydrocoele was distinguished from inguinal hernia (which would change with cough or straining, show an inguinal swelling at the internal ring) as described Eigege et al. (2005). Clinical examination also involved the search for lymphoedema which was easier to conduct than the search for hydrocoele. Participants were simply asked to lift up their clothing to expose their legs. Swollen limbs and leopard skin were observed and noted (Anosike et al., 2005; Eigege et al., 2002; Nwoke et al., 2006, Omudu and Okafor 2007). Dermal manifestations associated with Onchocerciasis like leopard skins, palpable nodules, onchocercal dermatitis (creeping eruption) and crawling sensation were also recorded during the clinical examination.

were observed and noted (Anosike et al., 2005; Eigege et al., 2002; Nwoke et al., 2006, Omudu and Okafor 2007). Dermal manifestations associated with Onchocerciasis like leopard skins, palpable nodules, onchocercal dermatitis (creeping eruption) and crawling sensation were also recorded during the clinical examination. Immunochromatographic card test procedure After collecting the demographic data, ICT testing was performed following kit instructions (NOW, ICT filariasis kits; Binax, Portland, ME, USA). The patient's left index finger was cleaned with methylated spirit and then punctured using a sterile lancet. The initial sample of blood was removed using a cotton swab, and sufficient fresh blood was then obtained to fill a 100-ql capillary tube. The blood was then transferred from the capillary tube to the pad on the ICT test kit card and then sealed. The results of each ICT test card were read after 15 mins. A positive result was when two pink lines appeared on the card's window, and a negative result was when a single line was seen. Test results with the individual's identification code were recorded on the patients' diagnostic data sheet.

it card and then sealed. The results of each ICT test card were read after 15 mins. A positive result was when two pink lines appeared on the card's window, and a negative result was when a single line was seen. Test results with the individual's identification code were recorded on the patients' diagnostic data sheet. Interviews and Questionnaire Administration Interviews, using semi-structured questionnaire were conducted with selected individuals from all the districts to gather descriptive information on villagers' knowledge and beliefs about the cause, mode of transmission and how to prevent the disease. Based on the descriptive information, a structured questionnaire was developed. It included 19 questions on villagers' awareness, knowledge beliefs and health seeking behaviour in relation to filariasis. Questionnaire was administered to both affected and unaffected residents of the community. A total of 103 volunteers comprising 15 persons with visible clinical signs of lymphatic filariasis and 88 persons without visible signs participated in the questionnaire aspect of the study. Data Analysis The quantitative epidemiological data was entered into Epi-info software (version 6.0: CDC Atlanta, USA) and was analysed in percentages using chi-square to test significance. The questionnaire data was analysed using the SPSS package.

Interviews and Questionnaire Administration Interviews, using semi-structured questionnaire were conducted with selected individuals from all the districts to gather descriptive information on villagers' knowledge and beliefs about the cause, mode of transmission and how to prevent the disease. Based on the descriptive information, a structured questionnaire was developed. It included 19 questions on villagers' awareness, knowledge beliefs and health seeking behaviour in relation to filariasis. Questionnaire was administered to both affected and unaffected residents of the community. A total of 103 volunteers comprising 15 persons with visible clinical signs of lymphatic filariasis and 88 persons without visible signs participated in the questionnaire aspect of the study. Data Analysis The quantitative epidemiological data was entered into Epi-info software (version 6.0: CDC Atlanta, USA) and was analysed in percentages using chi-square to test significance. The questionnaire data was analysed using the SPSS package. Results Two hundred and forty eight (248) persons from 7 communities situated in Ado LGA of Benue State, Nigeria, were clinically examined for manifestations of filariases and detection of circulating W. bancrofti using the ICT card tests. 81 (32.6%) out of the 248 persons were positive for circulating filarial antigen (CFA). Infection rates of CFA ranged from 41 (46.1%) in Uffia to 1(6.6%) in Ijigbam (Table 1). Distribution of community ICT prevalence showed a significant variation (X2 P < 0.05). The prevalence of clinical signs/symptoms in the communities also showed significant variations (X2 P < 0.05). Community hydrocoele prevalence ranged from 8 (9.0%) in Uffia to 1(6.6%) in Ijigbam, the overall hydrocoele prevalence was 21 (8.5%). While the overall lymphoedema prevalence was 16 (6.4%) and women accounted for 14 (87.5%) of persons with swollen limb. Clinical manifestations associated with Onchocerciasis were also prevalent in the communities. These include leopard skin which had overall prevalence of 19 (7.6%), palpable nodules 43 (17.3%), Dermatitis 105(42.3%) skin rashes and itching and/or crawling sensation 112(45.2%) (Table 1). While dermatitis and a crawling sensation appeared much earlier in life, hydrocoele, leopard skin and lymphoedema showed up from the age range of 40 and above (Table 2).

ll prevalence of 19 (7.6%), palpable nodules 43 (17.3%), Dermatitis 105(42.3%) skin rashes and itching and/or crawling sensation 112(45.2%) (Table 1). While dermatitis and a crawling sensation appeared much earlier in life, hydrocoele, leopard skin and lymphoedema showed up from the age range of 40 and above (Table 2). Table 1 Community prevalence of clinical manifestations of filariases and lymphatic filariasis antigen as detected by ICT card test. Communities Number examined Number with hydrocoele Number with lymphoedema Number with leopard skin ICT positive Number with palpable module Number with skin eruption Number with crawling sensation Number who had taken Ivermectin Uffia 89 8(9.0) 2(2.2) 4(4.5) 42(46.1) 13(14.6) 34(38.2) 31(34.8) 44(49.4) Apa 43 2(4.6) 4(9.3) 2(4.6) 14(32.5) 3(6.9) 2(48.8) 21(48.8) 23(53.4) Agila 25 1(4.0) 2(8.0) 3(12.0) 6(24.0) 5(20.0) 11(44.0) 11(44.0) 4(16.0) Ezza 38 4(10.5) 2(5.3) 3(7.9) 13(34.2) 7(44.7) 18(47.3) 15(39.5) 11(29.0) Igumale 20 3(15.0) 2(10.0) 4(20.0) 5(25.0) 5(25.0) 8(40.0) 4(70.0) 5(25.0) Ijigbam 15 1(6.6) 1(6.6) 1(6.6) 1(6.6) 6(40.0) 3(20.0) 9(60.0) 1(6.6) Ulayi 18 2(11.1) 3(16.6) 2(11.1) - 4(22.2) 10(55.5) 11(61.1) 8(44.9) Total 248 21(8.5) 16(6.4) 19(7.6) 81(32.6) 43(17.3) 105(42.3) 112(45.2) 106(42.7) Figures in parenthesis are percentages (%) Table 2 Age-related prevalence of clinical manifestations of filariasis and detection of lymphatic filariasis antigen using ICT card test.

Communities Number examined Number with hydrocoele Number with lymphoedema Number with leopard skin ICT positive Number with palpable module Number with skin eruption Number with crawling sensation Number who had taken Ivermectin Uffia 89 8(9.0) 2(2.2) 4(4.5) 42(46.1) 13(14.6) 34(38.2) 31(34.8) 44(49.4) Apa 43 2(4.6) 4(9.3) 2(4.6) 14(32.5) 3(6.9) 2(48.8) 21(48.8) 23(53.4) Agila 25 1(4.0) 2(8.0) 3(12.0) 6(24.0) 5(20.0) 11(44.0) 11(44.0) 4(16.0) Ezza 38 4(10.5) 2(5.3) 3(7.9) 13(34.2) 7(44.7) 18(47.3) 15(39.5) 11(29.0) Igumale 20 3(15.0) 2(10.0) 4(20.0) 5(25.0) 5(25.0) 8(40.0) 4(70.0) 5(25.0) Ijigbam 15 1(6.6) 1(6.6) 1(6.6) 1(6.6) 6(40.0) 3(20.0) 9(60.0) 1(6.6) Ulayi 18 2(11.1) 3(16.6) 2(11.1) - 4(22.2) 10(55.5) 11(61.1) 8(44.9) Total 248 21(8.5) 16(6.4) 19(7.6) 81(32.6) 43(17.3) 105(42.3) 112(45.2) 106(42.7) Figures in parenthesis are percentages (%) Table 2 Age-related prevalence of clinical manifestations of filariasis and detection of lymphatic filariasis antigen using ICT card test. Age group Number examined ICT positive Number with hydrocoele Number with lymphoedema No with leopard skin Number with palpable module Number with skin eruption Number with crawling sensation Number Ivermectin Treatment 1 – 10 3 −(0.0) −(0.0) −(0.0) −(0.0) −(0.0) 1(33.3) 1(33.3) 1(33.3) 11 – 20 12 4(33.3) −(0.0) −(0.0) −(0.0) −2(16.6) 2(16.6) 3(25.0) 4(33.3) 21 – 30 30 9(30.0) 1(3.3) 1(3.3) −(0.0) 6(20.0) 16(53.3) 20(66.6) 21(70.0) 31 – 40 73 28(38.3) 5(6.8) 2(2.7) 2(2.7) 13(17.8) 25(34.2) 20(27.3) 19(26.0) 41 – 50 71 21(29.5) 5 (7.0) 3(4.2) 9(12.6) 13(18.3) 34(47.8) 25(35.2) 20(28.1) 51 – 60 39 16(41.0) 7(17.9) 4(10.2) 6(15.3) 6(15.3) 18(46.1) 23(58.9) 27(69.2) 61 – 70 14 2(14.2) 1(7.1) 3(21.4) 1(7.1) 3(21.4) 6(42.8) 14(100.0) 8(57.1) 70 – above 6 1(16.6) 3(50.0) 3(50.0) 1(16.6) −(0.0) 3(50.0) 6(100.0) 6(100.0)

19(26.0) 41 – 50 71 21(29.5) 5 (7.0) 3(4.2) 9(12.6) 13(18.3) 34(47.8) 25(35.2) 20(28.1) 51 – 60 39 16(41.0) 7(17.9) 4(10.2) 6(15.3) 6(15.3) 18(46.1) 23(58.9) 27(69.2) 61 – 70 14 2(14.2) 1(7.1) 3(21.4) 1(7.1) 3(21.4) 6(42.8) 14(100.0) 8(57.1) 70 – above 6 1(16.6) 3(50.0) 3(50.0) 1(16.6) −(0.0) 3(50.0) 6(100.0) 6(100.0) Total 248 81(32.6) 21(8.5) 16(6.4) 19(7.6) 4.3(17.3) 105(42.2) 112(45.2) 106(42.7) Figures in parenthesis are percentages (%)

19(26.0) 41 – 50 71 21(29.5) 5 (7.0) 3(4.2) 9(12.6) 13(18.3) 34(47.8) 25(35.2) 20(28.1) 51 – 60 39 16(41.0) 7(17.9) 4(10.2) 6(15.3) 6(15.3) 18(46.1) 23(58.9) 27(69.2) 61 – 70 14 2(14.2) 1(7.1) 3(21.4) 1(7.1) 3(21.4) 6(42.8) 14(100.0) 8(57.1) 70 – above 6 1(16.6) 3(50.0) 3(50.0) 1(16.6) −(0.0) 3(50.0) 6(100.0) 6(100.0) Total 248 81(32.6) 21(8.5) 16(6.4) 19(7.6) 4.3(17.3) 105(42.2) 112(45.2) 106(42.7) Figures in parenthesis are percentages (%) Knowledge about the cause, transmission and prevention Only about 14 (15.9%) of unaffected respondents knew that lymphatic filariasis is caused through mosquito bites, this differ significantly from affected respondents 10 (66.6%) (X2, P < 0.05) (Table 4). Half of the unaffected respondents attributed the cause of the disease to stepping on charms. The other major responses on cause of the disease include lack of personal hygiene and sexual intercourse with infected persons. The respondents' perception of the cause of the disease is consistent with the beliefs on mode of transmission. Only 18 (20.4%) of unaffected respondents cited mosquito bite as route of transmission and this varied significantly from 8 (53.3%) affected respondents (X2 P < 0.05). 50 (56.8%) and 56 (63.6%) of unaffected respondents again attributed the mode of transmission of the disease to witchcraft and stepping on charms respectively (Table 5). While 14 (93.3%) of affected respondents knew that protection from mosquito bites can prevent filariasis, only 15 (17.0%) of unaffected respondents shared the same opinion. Other means of prevention of lymphatic filariasis as cited by 20 (22.7%) of unaffected respondents include avoidance of body contact and sexual intercourse with infected persons (Table 6). On the whole, affected respondents were more knowledgeable on the correct etiology of the disease than and unaffected respondents.

Other means of prevention of lymphatic filariasis as cited by 20 (22.7%) of unaffected respondents include avoidance of body contact and sexual intercourse with infected persons (Table 6). On the whole, affected respondents were more knowledgeable on the correct etiology of the disease than and unaffected respondents. Table 4 Respondents knowledge on causes of lymphatic filariasis Causes Affected n =15 Yes (%) Unaffected n = 88 Yes (%) Working in the sun 2 (13.3) 2 (2.3) Walking long distance 5 (33.3) 5 (5.7) Sexual intercourse 1 (6.6) 13 (14.7) Stepping on charm 5 (33.3) 44 (50.0) Contaminated food 2 (13.3) 13 (14.7) Lack of personal hygiene 8 (53.3) 43 (49.0) Fever - (0.0) 9 (10.2) Mosquito bites 10 (66.6) 14 (15.9) Table 5 Respondents beliefs on mode of transmission of lymphatic filariasis Mode of transmission Affected n =15 Yes (%) Unaffected n = 88 Yes (%) Sexual intercourse 1 (6.6) 18 (20.4) Body contact 4 (26.6) 17 (19.3) Witchcraft 3 (20.0) 50 (56.8) Food poisoning 1 (6.6) 29 (32.9) Mosquito bites 8 (53.3) 18 (20.4) Stepping on charm 2 (13.3) 56 (63.6) Inheritance 2 (13.3) 17 (19.3) Table 6 Respondents belief on the prevention of lymphatic filariasis

ed n = 88 Yes (%) Sexual intercourse 1 (6.6) 18 (20.4) Body contact 4 (26.6) 17 (19.3) Witchcraft 3 (20.0) 50 (56.8) Food poisoning 1 (6.6) 29 (32.9) Mosquito bites 8 (53.3) 18 (20.4) Stepping on charm 2 (13.3) 56 (63.6) Inheritance 2 (13.3) 17 (19.3) Table 6 Respondents belief on the prevention of lymphatic filariasis Preventive measures Affected n =15 Yes (%) Unaffected n =88 Yes (%) Avoid sexual intercourse with affected person 5 (33.3) 20 (22.7) Avoid body contact with affected person 1 (6.6) 20 (22.7) Sacrifice to appease gods 5 (33.3) 34 (38.6) Good personal hygiene 6 (40.0) 50 (56.8) Avoid mosquito bites 14 (93.3) 15 (17.0) Avoid eating with affected person 1 (6.6) 2 (2.3) Perceived socioeconomic and psychological implications of lymphatic filariasis Social and psychological impact of the disease include personal discomfort in public and inability to engage in income generating activities, both affected and unaffected respondents generally agreed on these consequences. However, there was a significant difference between affected and unaffected respondents in the belief that the disease manifestations affect sexual relation with spouse. While 60 (68.1%) of unaffected persons were of the opinion that the disease manifestations affected sexual relations with spouse. Only 4 (26.6%) of affected persons shared the same opinion (X2 P < 0.05) (Table 7). Lymphatic filariasis — related health expenditure seem to be underrated by unaffected persons, their estimate of monthly health seeking expenditure by patients were significantly lower that those of affected respondents. 9 (60.0%) of affected respondents estimated the monthly health seeking expenditure at between N2000 – N3000. only 25 (28.4%) of unaffected respondent corroborated this estimate (Table 8). Convenience and affordability were key factors influencing the choice of health provider by infected persons (Table 8). However unaffected respondents believed that the choice of health provider to patronize is influenced by family decision.

only 25 (28.4%) of unaffected respondent corroborated this estimate (Table 8). Convenience and affordability were key factors influencing the choice of health provider by infected persons (Table 8). However unaffected respondents believed that the choice of health provider to patronize is influenced by family decision. Table 7 Respondents perception on some socio-economic and psychological consequences of lymphatic filariasis Consequences Affected n =15 Yes (%) Unaffected n =88 Yes (%) Personally uncomfortable in public 14 (93.3) 77 (87.5) Affects work and income 14 (93.3) 83 (94.3) Affects sexual relation with spouse 4 (26.6) 60 (68.1) Hinder marriage prospects of other members of family 3 (20.0) 52 (59.1) Spouse desertion and divorce 3 (20.0) 39 (44.3) Suspicion of infidelity 2 (13.3) 30 (34.1) Table 8 Respondents perception of the monthly lymphatic filariasis related health expenditure and factors that influence choice of health providers.

0 (68.1) Hinder marriage prospects of other members of family 3 (20.0) 52 (59.1) Spouse desertion and divorce 3 (20.0) 39 (44.3) Suspicion of infidelity 2 (13.3) 30 (34.1) Table 8 Respondents perception of the monthly lymphatic filariasis related health expenditure and factors that influence choice of health providers. Expenditure Affected n =15 Yes (%) Unaffected n =88 Yes (%) Below N500 monthly 7 (38.8) 12 (13.6) Between N500 – N1000 5 (33.3) 20 (22.7) Between N1000 – N2000 8 (53.3) 17 (28.4) Factors influencing choice of health provider Convenience 10 (66.6) 27 (30.6) Affordability 8 (53.3) 61 (69.3) Family decision 5 (33.3) 40 (45.4) Providers reputation 8 (53.3) 31 (35.2) Confidentiality 2 (13.3) 15 (17.0) Discussion This present study shows that lymphatic filariasis is endemic in Ado LGA of Benue State, Nigeria; with an overall hydrocoele prevalence of 8.5%, lymphoedema prevalence of 6.4% and CFA prevalence of 32.6%. Lymphatic filariasis due to W. bancrofti infections is indeed a serious public health problem in this area. This prevalence rate is higher than that of earlier observation in other parts of Benue State by (Omudu and Okafor, 2007) and parts of Nigeria by (Anosike et al., 2005; Anosike, 1994) in Bauchi State; in Cross River State (Braide et al., 2003; Udiodung et al., 2008); in Plateau State (Eigege et al., 2002) and in Anambra State (Mba and Njoku, 2000). However, prevalence of lymphoedema, hydrocoele and CFA in this area is relatively low when compared with findings from the Niger Delta area (Udonsi, 1986), Taraba State (Badaki and Akogun, 2000) and Kogi State (Nwoke et al., 2006).

008); in Plateau State (Eigege et al., 2002) and in Anambra State (Mba and Njoku, 2000). However, prevalence of lymphoedema, hydrocoele and CFA in this area is relatively low when compared with findings from the Niger Delta area (Udonsi, 1986), Taraba State (Badaki and Akogun, 2000) and Kogi State (Nwoke et al., 2006). The high endemicity of lymphatic filariasis in these communities could be due to several factors, especially the local environmental conditions like the availability of numerous domestic and peri-domestic mosquito breeding sites and deteriorating sanitary conditions. The various activities of the local population such as rice farming, cassava processing, fishing and other outdoor related activities tend to increase man-mosquito contact rates in different communities. Lymphatic filariasis vectors have been reported to breed in pots used for cassava fermentation (Udonsi, 1986; Iwuala, 1979). The topography of the area also created conducive environment for breeding of other vectors of filariases, such as clear, highly-oxygenated and fast flowing rivers like Okpokwu and Ado provide breeding grounds for black flies, (vector of onchocerciasis). This accounted for the cases of leopard skins, ochocercal nodules and onchodermatitis encountered during clinical examinations.

for breeding of other vectors of filariases, such as clear, highly-oxygenated and fast flowing rivers like Okpokwu and Ado provide breeding grounds for black flies, (vector of onchocerciasis). This accounted for the cases of leopard skins, ochocercal nodules and onchodermatitis encountered during clinical examinations. Age-related infection rates observed in this study agree with previous findings (Anosike et al., 2005; Eigege et al., 2002; Nwoke et al., 2006; Omudu and Okafor, 2007), which showed that prevalence of hydrocoele, lymphoedema and CFA increased with age. Apart from immunological reasons, duration of exposure to vectors in middle age and farming age group may be the major reason (Anosike et al., 2005; Eigege et al., 2002; Gyapong et al., 1998). The use of clinical manifestations of lymphatic filariasis as rapid assessment procedures for community diagnosis has been suggested (Eigege et al., 2002; Nwoke et al., 2006; Gyapong et al., 1998; Mwobabia et al., 2000). Our findings support the recommendations for the use of community lymphoedema and hydrocoele prevalence as rapid community indicator for lymphatic filariasis endemicity. Available parasitological and serological methods are either too cumbersome or too expensive (Anosike et al., 2005; Eigege et al., 2002; Omudu and Okafor, 2007; Weil et al., 1997).

mendations for the use of community lymphoedema and hydrocoele prevalence as rapid community indicator for lymphatic filariasis endemicity. Available parasitological and serological methods are either too cumbersome or too expensive (Anosike et al., 2005; Eigege et al., 2002; Omudu and Okafor, 2007; Weil et al., 1997). On the communities' knowledge and beliefs in relation to lymphatic filariasis in the area, our findings revealed significant differences in lymphatic filariasis related knowledge between affected and unaffected respondents. This contrast with similar study in South India (Ramaiah et al., 1996) which reported that unaffected people, irrespective of their educational status, are more knowledgeable than the affected on the cause of filariasis. The general awareness of the cause transmission and prevention of the disease is poor; the role of mosquitoes in transmitting the parasitic agents of filariasis is poorly appreciated in many of the communities investigated. This study has demonstrated several other shortcomings in the communities' understanding of the disease. Our findings corroborate similar studies in Nigeria and elsewhere (Anosike et al., 2005; Braide et al., 2003; Ramaiah et al., 1996; Omudu and Okafor, 2008) and clearly underscored the importance of comprehensive community education to address identified gaps in perception and practices. The chronic manifestations of lymphatic filariasis can have significant, and often very negative social impacts. Clinical manifestations like lymphoedema of the limbs and external genitalia as seen in this area have a profoundly detrimental effect on the quality of life of affected individuals and their family members. The degree of social disability varies between cultural settings and prevailing community perceptions and practices, but the degree of stigmatization appear to be directly correlated with the severity of visible disease (Addiss and Brady, 2007); Person et al., 2006). There is significant evidence that patients experience stigma as a result of lymphatic filariasis in the communities investigated and this results from general community beliefs and perception on the causes and mode of transmission of the disease. While patients may experience withdrawal from social gathering, their family members could experience difficulty in finding desired spouses.

esult of lymphatic filariasis in the communities investigated and this results from general community beliefs and perception on the causes and mode of transmission of the disease. While patients may experience withdrawal from social gathering, their family members could experience difficulty in finding desired spouses. Similar findings have also been reported in Ghana (Ahorlu et al., 1999), Sri-Lanka (Wijesinghe et al., 2007), Haiti (Person et al., 2006), Dominican Island (Person et al., 2006) and has been a subject of reviews (Hartigan 1999; Okafor and Omudu, 2005).

esult of lymphatic filariasis in the communities investigated and this results from general community beliefs and perception on the causes and mode of transmission of the disease. While patients may experience withdrawal from social gathering, their family members could experience difficulty in finding desired spouses. Similar findings have also been reported in Ghana (Ahorlu et al., 1999), Sri-Lanka (Wijesinghe et al., 2007), Haiti (Person et al., 2006), Dominican Island (Person et al., 2006) and has been a subject of reviews (Hartigan 1999; Okafor and Omudu, 2005). Unaffected respondents seem to underestimate lymphatic filariasis related health expenditures; the disease is known to cause direct and indirect economic loss to individuals and families. Studies from India, Ghana and Haiti indicate treatment costs to patients range from US $0.25 to US$2.62 per episode, as much as two days wages (Ramaiah et al., 1996, 1998). The cost of treatment included direct costs of treatment, including self mediation, as well as travel, feeding and accommodation since in most instances, the health providers live outside the patients' community. Our study demonstrates the need for the development of health education programmes that will enable people to protect themselves against mosquito bites. As Nigeria commence her lymphatic filariasis elimination programmes, there is an urgent need to develop morbidity management activities that will alleviate the burden of patients. Studies of this nature are critical to delineating lymphatic filariasis communities and needs to be replicated in other parts of the country where the status of the disease is unknown.

elimination programmes, there is an urgent need to develop morbidity management activities that will alleviate the burden of patients. Studies of this nature are critical to delineating lymphatic filariasis communities and needs to be replicated in other parts of the country where the status of the disease is unknown. Acknowledgement The authors acknowledge Ado Local Government health officers for helping in interpretation during clinical examination and questionnaire administration. Table 3 Respondents perception of the most worrisome signs and symptoms of lymphatic filariasis Affected n =15 Yes (%) Unaffected n=88 Yes (%) Fever 5 (33.3) 14 (15.9) Chills 6 (40.0) 13 (14.7) Pains 13 (86.6) 31 (35.2) Swelling 15 (100.0) 50 (56.8) Functional impairment 10 (66.6) 24 (27.2) Appearance 11 (73.3) 37 (42.8) Physical discomfort 9 (60.0) 46 (52.3)

Introduction Human Immunodeficiency Virus (HIV) is an RNA virus that mainly targets the T-lymphocytes bearing CD4. Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS) is a major cause of infant and childhood morbidity, hospitalization and mortality, has devastated families and complicated the efforts to fight poverty, improve health and promotion of development (ANECCA, 2005). In 2010, about 33.3 million people, worldwide, were living with HIV including 2.5 million children with an annual death rate of 1.8 million due to AIDS. Nigeria accounted for about 3.5-4 million people living with HIV including 360,000 children, with 340,000 new infections out of which 170,000 were children. There were also 220,000 AIDS-related deaths (WHO/UNAIDS, 2010).

iving with HIV including 2.5 million children with an annual death rate of 1.8 million due to AIDS. Nigeria accounted for about 3.5-4 million people living with HIV including 360,000 children, with 340,000 new infections out of which 170,000 were children. There were also 220,000 AIDS-related deaths (WHO/UNAIDS, 2010). Hepatitis B Virus (HBV), on the other hand, is a hepatotropic virus that replicates predominantly in the hepatocytes, lymphocytes, spleen, kidney and pancreas with areas of highest prevalence being the sub-Saharan Africa and South-East Asia (Yazigi, Balistrere, 2007, WHO/Hepatitis B). It is also a major public health problem with increased risk of chronicity in children and subsequent increase in morbidity and mortality. Younger age at acquisition of infection is the single most important predictor of chronic carriage (Yazigi, Balistrere, 2007, Uleanya, Obidike, 2015). Worldwide, an estimated two billion people are infected, with 350-400 million of them remaining chronic carriers of HBV, a leading cause of chronic liver diseases and liver-related mortality (WHO/Hepatitis B, Puoti et al, 2008, Sharma et al, 2008, Rouet et al, 2008, Mphahlele et al, 2002, Kire, 1996). It has also been estimated that 15 - 25% of the chronic carriers will die of these sequelae (WHO/Hepatitis B). Consequently, worldwide estimated annual mortality directly related to hepatitis B liver disease and cancer is between 600,000-1 million (Puoti et al, 2008, Sharma et al, 2008, Mphahlele et al, 2002). Nigeria is hyper-endemic with endemicity rate as high as 37-46% (Emechebe et al, 2009).

ese sequelae (WHO/Hepatitis B). Consequently, worldwide estimated annual mortality directly related to hepatitis B liver disease and cancer is between 600,000-1 million (Puoti et al, 2008, Sharma et al, 2008, Mphahlele et al, 2002). Nigeria is hyper-endemic with endemicity rate as high as 37-46% (Emechebe et al, 2009). Hepatitis B virus co-infection is common among patients with HIV, because of shared routes and mechanisms of transmission (Thio et al, 2002, Levy et al, 2006). It has been estimated that about 6 to 15% of the 33.3 million people living with HIV infection are also chronically infected with HBV (Psevdos et al, 2010). The multidimensional immunosuppression caused by HIV can: compromise one’s ability to recover spontaneously from an acute HBV infection thus leading to a chronic infection; lower incidence of spontaneous loss of HB e antigen (HBeAg) or HBsAg and cause more rapid decline in hepatitis B surface antibody over time (Thio et al, 2002, Psevdos et al, 2010, Mgonda, 2004). Co-infection increases the risk for HBV-related liver disease progression and also the risk for antiretroviral drug related hepatotoxicity (Thio et al, 2005). This high risk of hepatotoxicity may then imply an increased risk of death from the liver disease in co-infected people (Thio et al, 2002). In viral hepatitis and drug induced hepatotoxicity, alanine transaminase is the most specific marker of hepatotoxicity (Baron et al 1994). Paradoxically, there is low serum alanine transferase concentration but high HBV DNA and HBe Ag levels found in the serum of co-infected persons (Puoti et al, 2008, Kellerman et al 2003, Thio et al, 2002). Such co-infected persons are at higher risk for a more rapid progression to chronic hepatitis, HBV-related cirrhosis and hepatic decompensation than are only HBV infected persons (Puoti et al, 2008, Kellerman et al, 2003). It has also been documented that the rate of liver-related deaths from progressive liver disease are highest among those with lowest CD4 count, which also increases after the introduction of HAART. It is noted that CD4 depletion alter the intrahepatic cytokine milieu contributing to increased fibrosis (Thio et al, 2002). Co-infection therefore poses a treatment challenge. With increasing access to HAART, it is expected that AIDS-related mortality will become reduced (Puoti et al, 2008, Rouet et al, 2008).

HAART. It is noted that CD4 depletion alter the intrahepatic cytokine milieu contributing to increased fibrosis (Thio et al, 2002). Co-infection therefore poses a treatment challenge. With increasing access to HAART, it is expected that AIDS-related mortality will become reduced (Puoti et al, 2008, Rouet et al, 2008). However, it is most likely that liver disease related to chronic HBV infection or drug-induced hepatotoxicity will increase and liver failure may emerge as a major cause of death whether amongst the treated or untreated co-infected patients. This study was, therefore, undertaken to highlight the burden of co-infection in our environment, determine the associated risk factors and compare the effect of co-infection as determined by derangement in liver enzyme and CD4 counts of co-infected children with that of surface antigen negative HIV infected children. Such information will be of help to health policy makers in generating guidelines for effective management and/or prevention of both infections. Methods This was a cross sectional study carried out among HIV positive children attending the paediatric antiretroviral (ARV) clinic of the University of Nigeria Teaching Hospital (UNTH), a tertiary medical institution in Enugu. The clinic provides care for about 240 HIV positive children from Enugu and its environs.

This study was, therefore, undertaken to highlight the burden of co-infection in our environment, determine the associated risk factors and compare the effect of co-infection as determined by derangement in liver enzyme and CD4 counts of co-infected children with that of surface antigen negative HIV infected children. Such information will be of help to health policy makers in generating guidelines for effective management and/or prevention of both infections. Methods This was a cross sectional study carried out among HIV positive children attending the paediatric antiretroviral (ARV) clinic of the University of Nigeria Teaching Hospital (UNTH), a tertiary medical institution in Enugu. The clinic provides care for about 240 HIV positive children from Enugu and its environs. A total of 140 Western Blot confirmed HIV positive children aged 18 months to 15 years were studied with HIV positive HBsAg negative children serving as controls. Exclusion criteria were those aged less than 18 months or older than 15 years, unconfirmed HIV status and those whose parent(s)/guardian did not consent.

Methods This was a cross sectional study carried out among HIV positive children attending the paediatric antiretroviral (ARV) clinic of the University of Nigeria Teaching Hospital (UNTH), a tertiary medical institution in Enugu. The clinic provides care for about 240 HIV positive children from Enugu and its environs. A total of 140 Western Blot confirmed HIV positive children aged 18 months to 15 years were studied with HIV positive HBsAg negative children serving as controls. Exclusion criteria were those aged less than 18 months or older than 15 years, unconfirmed HIV status and those whose parent(s)/guardian did not consent. Ethical clearance was given by the University of Nigeria Teaching Hospital Health Research and Ethics committee. Informed consent (both verbal and written) was obtained from the child and the parent(s) or guardian. They were duly educated on the need for, and benefits of the study. The specimen to be collected and how it was to be collected was explained to them before collection. A structured interviewer administered questionnaire was designed for this study. Information sought included bio-data, occupation and educational status of both parents/guardian for the determination of socioeconomic class, risk factors for HBV infection including previous history and frequency of blood transfusion, histories and instruments of scarification, tattooing, ear piercing, circumcision, use of contaminated needles and syringes for injection (either used or reused needle or syringe considered as contaminated), intravenous drug use, histories of sex (where necessary), and sharing of tooth brush were obtained. In this study, co-infection was defined by the presence of HBsAg positivity in HIV positive children.

contaminated needles and syringes for injection (either used or reused needle or syringe considered as contaminated), intravenous drug use, histories of sex (where necessary), and sharing of tooth brush were obtained. In this study, co-infection was defined by the presence of HBsAg positivity in HIV positive children. Patient’s baseline and recent CD4 count/CD4% and alanine aminotransferase results were retrieved from their folders. Assays for HBsAg was done using Abbott DETERMINE HBsAg test kits - an enzyme immunoassay (specificity of 99.85% and a sensitivity of 94.64%). The test was then read after a waiting interval of 15 minutes to 24 hours (as specified by the manufacturer). The data was analysed using SPSS version 15. Measures of central tendencies – mean and median were used to summarize quantitative and qualitative variables where applicable. Frequency tables were constructed as appropriate and analytical tests of significance were done using the Student t- test for continuous variables, Chi square and Fischer’s exact tests for non-continuous variables and p value at the level of < 0.05 was accepted as significant. Odds ratios for the risk factors were calculated. Socioeconomic class was determined using the method proposed by Oyedeji (Oyedeji, 1985). Results A total of 140 questionnaires were administered with a 100% response rate. Blood samples were collected and analysed from all 140 subjects for HBsAg. Eight and seven subjects had only baseline ALT and CD4 results respectively. These were confirmed HIV positives but were new to the clinic.

ular routes has been shown to be a possibility in chicken breeders if the reproductive organs of the males are infected with the virus or via transmission by semen as previously documented for turkeys (Samadieh and Bankowski, 1970). Hence, these birds could serve as reservoirs shedding the viruses into the environment. Additionally, the detection of antibodies to LPAIVs in this study corroborates earlier reports of occurrence of LPAI H5, H7 and H9 subtypes in poultry (Nguyen-Van-Tam et al., 2006; Cheng et al., 2010; Parker et al., 2012; Aiki-Raji et al., 2015). This finding is significant as it underscores the possibility of a future outbreak of HPAI in Oyo state especially considering previous reports (Swayne and Halvorson, 2003; Werner and Harder, 2006; Briand et al., 2010) of the possibility of a field LPAIV mutating into a highly pathogenic strain after circulating in susceptible poultry. Such mutation probably occurs after the viruses have moved from their natural wild bird host to poultry.

The data was analysed using SPSS version 15. Measures of central tendencies – mean and median were used to summarize quantitative and qualitative variables where applicable. Frequency tables were constructed as appropriate and analytical tests of significance were done using the Student t- test for continuous variables, Chi square and Fischer’s exact tests for non-continuous variables and p value at the level of < 0.05 was accepted as significant. Odds ratios for the risk factors were calculated. Socioeconomic class was determined using the method proposed by Oyedeji (Oyedeji, 1985). Results A total of 140 questionnaires were administered with a 100% response rate. Blood samples were collected and analysed from all 140 subjects for HBsAg. Eight and seven subjects had only baseline ALT and CD4 results respectively. These were confirmed HIV positives but were new to the clinic. The age and sex distribution of the study populations is as shown in Table 1. The mean age of subjects was 7.3 ± 3.6 years. There were 74 males and 66 females with a male to female ratio of 1.1: 1. The majority of the subjects 61 (43.6%) were in the age range of 6-10 years and the least represented age range was 11-15 years – 29 (20.7%). The socioeconomic class of the subjects is also as shown in Table 1. Most were in the lower class. Table 1 Socio-demographic characteristics of HIV positive Children.

The age and sex distribution of the study populations is as shown in Table 1. The mean age of subjects was 7.3 ± 3.6 years. There were 74 males and 66 females with a male to female ratio of 1.1: 1. The majority of the subjects 61 (43.6%) were in the age range of 6-10 years and the least represented age range was 11-15 years – 29 (20.7%). The socioeconomic class of the subjects is also as shown in Table 1. Most were in the lower class. Table 1 Socio-demographic characteristics of HIV positive Children. Age Range (years) HIV Positive N (%) 1-5 50 (35.7) 6-10 61 (43.6) 11-15 29 (20.7) Gender Male 74 (52.9) Female 66 (47.1) Social class Upper class 8 (5.7) Middle class 22 (15.7) Lower class 110 (78.6) *The lowest age in this study was 18 months Of the 140 children studied, 14 were positive for HBsAg, giving a prevalence rate of 10%. Ten (9.1%) of the HBsAg positive subjects were from the lower socioeconomic class, four (18.2%) from the middle class while none among the upper class was positive for HBsAg. Ten (13.5%) out of the 74 HIV positive males and 4 (6.1%) of the 66 HIV positive females were also positive for HBsAg. The prevalence of HBsAg increased with age among the subjects (Table 2). However, the highest prevalence was found among four children (13.8%) aged 11-15 years, (χ2 = 1.5, p = 0.47) Table 2 Age distribution of HBsAg among HIV positive children Age range (Years) HIV positive HBs HBs (+) HBs (-) N (%) N (%) 1-5 3 (6.0) 47 (94.0) 6 -10 7 (11.5) 54 (88.5) 11-15 4 (13.8) 25 (86.2) χ2 =1.5, p = 0.47

Of the 140 children studied, 14 were positive for HBsAg, giving a prevalence rate of 10%. Ten (9.1%) of the HBsAg positive subjects were from the lower socioeconomic class, four (18.2%) from the middle class while none among the upper class was positive for HBsAg. Ten (13.5%) out of the 74 HIV positive males and 4 (6.1%) of the 66 HIV positive females were also positive for HBsAg. The prevalence of HBsAg increased with age among the subjects (Table 2). However, the highest prevalence was found among four children (13.8%) aged 11-15 years, (χ2 = 1.5, p = 0.47) Table 2 Age distribution of HBsAg among HIV positive children Age range (Years) HIV positive HBs HBs (+) HBs (-) N (%) N (%) 1-5 3 (6.0) 47 (94.0) 6 -10 7 (11.5) 54 (88.5) 11-15 4 (13.8) 25 (86.2) χ2 =1.5, p = 0.47 None of the subjects was involved with intravenous drug injection though all had received intramuscular injections for one reason or the other as shown in Table 3. However, 19 (13.6%) used contaminated needle/syringes, 42 (30%) had past history of blood transfusion, 76 (54.3%) were circumcised, 33 (23.6%) had scarification marks, 3 (2.1%) had a history of tattoo/ritual marks, 65 (46.4%) had ear piercing, while 9 (6.4%) shared toothbrushes. One subject (0.7%) was involved in unprotected sex (a victim of sexual assault). The observed differences between the risk factors and HBsAg positivity were not statistically significant. However, the odds ratio of the risk of transmission of HBV through these risk factors is as shown in table 3. Table 3 Showing the relative risk of HBsAg positivity among HIV positive children

None of the subjects was involved with intravenous drug injection though all had received intramuscular injections for one reason or the other as shown in Table 3. However, 19 (13.6%) used contaminated needle/syringes, 42 (30%) had past history of blood transfusion, 76 (54.3%) were circumcised, 33 (23.6%) had scarification marks, 3 (2.1%) had a history of tattoo/ritual marks, 65 (46.4%) had ear piercing, while 9 (6.4%) shared toothbrushes. One subject (0.7%) was involved in unprotected sex (a victim of sexual assault). The observed differences between the risk factors and HBsAg positivity were not statistically significant. However, the odds ratio of the risk of transmission of HBV through these risk factors is as shown in table 3. Table 3 Showing the relative risk of HBsAg positivity among HIV positive children Risk factors Total Sample HBsAg positive N (%) χ2 p OR Blood transfusion Present 42 7 (16.7) 3.0 0.12 2.6 Absent 98 7 (7.1) Scarification marks Present 33 2 (6.1) 0.75 0.52 0.5 Absent 107 12 (11.2) Tattooing Present 3 0 (0.0) - - - Absent 137 14 (10.2) Circumcision Present 76 10 (13.2) 1.76 0.26 2.2 Absent 64 4 (6.3) Intravenous drug use Absent 140 14 (10.0) - - - Contaminated syringe/needle use Present 19 1 (5.3) 0.55 0.69 0.5 Absent 121 13 (10.7) Ear piercing Present 65 4 (6.2) 1.99 0.26 0.4 Absent 75 10 (13.3) Sharing of toothbrush Present 9 1 (11.1) 0.01 0.91 1.1 Absent 131 13 (9.9) Unprotected sex Present 1 0 (0.0) - - - Absent 139 14 (10.1) Among the 14 co-infected children, 7 (50.0%) had elevated baseline alanine amino-transaminase (ALT) above the upper limit of normal while 7 (50.0%) had baseline ALT within normal limits. Of the 126 HIV positive but HBsAg negative children, a total of 57 (45.2%) had elevated baseline ALT while most (69 [54.8%]) had baseline ALT within normal limits. Having been initiated on antiretroviral drugs for varying periods, among the co-infected children, 10 (76.9%) now had elevated ALT while three (23.1%) had normal ALT. Eighteen (15.1%) of the HBsAg negative HIV positive children had normal ALT while 101 (84.9%) had elevated ALT above the upper limit of normal. The mean of baseline ALT among the HBV co-infected subjects was 17.9 ±11.6 iu/l while it was 19.3 ± 17.6 iu/l among the surface antigen negative subjects (126) (t = 0.3, p = 0.76). The mean value of the most recent ALT of the co-infected children (13) was 24.7 ± 14.2 iu/l while that of the HBsAg negative children (119) was 28.6 ± 23.7 iu/l (t = 0.6, p = 0.56). The differences were not statistically significant (Table 4).

iu/l among the surface antigen negative subjects (126) (t = 0.3, p = 0.76). The mean value of the most recent ALT of the co-infected children (13) was 24.7 ± 14.2 iu/l while that of the HBsAg negative children (119) was 28.6 ± 23.7 iu/l (t = 0.6, p = 0.56). The differences were not statistically significant (Table 4). Table 4 Baseline and Recent ALT of HIV infected children Mean (SD) HBsAg status Positive Negative (N) (N) Baseline (ALT (iu/L) 17.9 (±11.6) 19.3 (±17.6) (14) (126) t = 0.3, p = 0.76 Recent ALT (iu/L) 24.7 (± 14.2) 28.6 (± 23.7) (13) (119) t = 0.6, P = 0.56 The median of baseline CD4 count of HBsAg negative children ≥ 6 years was 360 cells/mm3, with a mean of 458.3 ± 352.2 cells/mm3 while the co-infected children ≥ 6 years had a median baseline CD4 count of 230 cells/mm3 with a mean of 386.7 ± 419.8 cells/mm3 (t = 0.6,p = 0.60) (Table 5). The median of their most recent CD4 count (all having been on HAART for varying periods), of the children without HBV co-infection was 723 cells/mm3 with a mean of 741.2 ± 447.2 cells/mm3 while that of the co-infected was 682 cells/mm3 with a mean of 712.2 ± 610.5 cells/mm3 (t = 0.19, p = 0.85). Among the children ≤ 5 years, the baseline median CD4% of HBsAg positive children was 25% with a mean of 28.2 ± 13.0 while surface antigen negative children had baseline median CD4% of 15% with a mean of 16.3 ± 10.3. Having received HAART for varying periods, the median CD4% of the co-infected children was 21.8% while that of surface antigen negative children rose to 30.2%. Table 5 Median and Mean CD4 count/CD4% of HIV infected children

Mean (SD) HBsAg status Positive Negative (N) (N) Baseline (ALT (iu/L) 17.9 (±11.6) 19.3 (±17.6) (14) (126) t = 0.3, p = 0.76 Recent ALT (iu/L) 24.7 (± 14.2) 28.6 (± 23.7) (13) (119) t = 0.6, P = 0.56 The median of baseline CD4 count of HBsAg negative children ≥ 6 years was 360 cells/mm3, with a mean of 458.3 ± 352.2 cells/mm3 while the co-infected children ≥ 6 years had a median baseline CD4 count of 230 cells/mm3 with a mean of 386.7 ± 419.8 cells/mm3 (t = 0.6,p = 0.60) (Table 5). The median of their most recent CD4 count (all having been on HAART for varying periods), of the children without HBV co-infection was 723 cells/mm3 with a mean of 741.2 ± 447.2 cells/mm3 while that of the co-infected was 682 cells/mm3 with a mean of 712.2 ± 610.5 cells/mm3 (t = 0.19, p = 0.85). Among the children ≤ 5 years, the baseline median CD4% of HBsAg positive children was 25% with a mean of 28.2 ± 13.0 while surface antigen negative children had baseline median CD4% of 15% with a mean of 16.3 ± 10.3. Having received HAART for varying periods, the median CD4% of the co-infected children was 21.8% while that of surface antigen negative children rose to 30.2%. Table 5 Median and Mean CD4 count/CD4% of HIV infected children Baseline Median Recent Median (Mean ± SD) (Mean± SD) HBsAg HBsAg HBsAg HBsAg positive negative Positive negative CD4 count (cells/mm3) 230.50 (386.7 ± 419.8) 360 (458.3 ±352.2) 682 (712.2 ± 610.5) 723 (741.2± 447.2) Number of children 11 79 10 78 t = 0.6 p = 0.54 t = 0.19 p = 0.85 CD4% 25 (28.2 ±13.0) 15 (16.3 ± 10.3) 21.8 (20.8 ± 9.8) 30.2 (29.5 ± 10.7) Number of children 3 47 3 42 t = 1.9 P = 0.06 t = 1.4 P = 0.18 From the baseline CD4 count, 30 (33.3%) children ≥ 6 years studied did not have significant immunosuppression. Seventeen (18.9%) had mild immunosuppression, 16 (17.8%) had advanced immunosuppression and 27 (30%) had severe immunosuppression. Five (45.5%) of the co-infected subjects were severely immunosuppressed compared with 27.8% (22) of the non-co- infected subjects. However, there was no significant association between the levels of immunosuppression and HBsAg positivity (p = 0.64). Among the HIV positive HBsAg negative children, 34.2%, and 27.3% of the co-infected subjects had baseline CD4 count ≥ 500 cells/mm3 respectively. Having been on HAART for varying periods, their most recent CD4 reveals that 40% of the co-infected children still had CD4 count < 200 compared with 9% of the HIV positive HBsAg negative children (P = 0.027) (Table 6).

en, 34.2%, and 27.3% of the co-infected subjects had baseline CD4 count ≥ 500 cells/mm3 respectively. Having been on HAART for varying periods, their most recent CD4 reveals that 40% of the co-infected children still had CD4 count < 200 compared with 9% of the HIV positive HBsAg negative children (P = 0.027) (Table 6). Table 6 Immunologic classification of HIV positive children ≥ 6 years CD4 range Baseline CD4 Recent CD4 (Cells/mm3) HBsAg HBsAg HBsAg HBsAg negative positive negative positive N (%) N (%) N (%) N (%) <200 5 (45.5) 22 (27.8) 4 (40.0) 7 (9.0) 201-349 1 (9.1) 15 (19.0) 0 (0.0) 8 (10.3) 350-499 2 (18.2) 15 (19.0) 0 (0.0) 10 (12.8) ≥ 500 3 (27.3) 27 (34.2) 6 (60.0) 53 (67.9) Fischer’s exact test P=0.67 Fischer’s exact test P = 0.027 Among children ≤ 5 years, 44.7% (21) of the HIV positive HBsAg negative children had baseline CD4% < 15% while none of the co-infection children had a CD4% < 15%. However, 33.3% (1) of co-infected children had CD4% > 25% while 17% (8) of those without HBsAg had CD4% > 25% (Table 7). Table 7 Immunologic classification of HIV positive subjects ≤ 5 years. Baseline CD4% HBsAg status Positive Negative N (%) N (%) < 15 0 (0.0) 21 (44.7) 15 – 20 1 (33.3) 15 (31.9) 20-25 1 (33.3) 3 (6.4) > 25 1 (33.3) 8 (17.0) Fischer’s exact test P= 0.079

CD4 range Baseline CD4 Recent CD4 (Cells/mm3) HBsAg HBsAg HBsAg HBsAg negative positive negative positive N (%) N (%) N (%) N (%) <200 5 (45.5) 22 (27.8) 4 (40.0) 7 (9.0) 201-349 1 (9.1) 15 (19.0) 0 (0.0) 8 (10.3) 350-499 2 (18.2) 15 (19.0) 0 (0.0) 10 (12.8) ≥ 500 3 (27.3) 27 (34.2) 6 (60.0) 53 (67.9) Fischer’s exact test P=0.67 Fischer’s exact test P = 0.027 Among children ≤ 5 years, 44.7% (21) of the HIV positive HBsAg negative children had baseline CD4% < 15% while none of the co-infection children had a CD4% < 15%. However, 33.3% (1) of co-infected children had CD4% > 25% while 17% (8) of those without HBsAg had CD4% > 25% (Table 7). Table 7 Immunologic classification of HIV positive subjects ≤ 5 years. Baseline CD4% HBsAg status Positive Negative N (%) N (%) < 15 0 (0.0) 21 (44.7) 15 – 20 1 (33.3) 15 (31.9) 20-25 1 (33.3) 3 (6.4) > 25 1 (33.3) 8 (17.0) Fischer’s exact test P= 0.079 Discussion This study revealed a 10% prevalence rate of HBsAg among 140 HIV infected children, which is lower than the 19% and 12.1% among co-infected children in Maiduguri and Ivory Coast (Rouet et al 2008, Ashir et al, 2009) respectively. This difference may be due to higher prevalence of HIV and HBV infections in these areas compared to Enugu. It is higher than the seroprevalence of 7.7% in Benin (Sadoh et al, 2011) and 7.8% in Makurdi (Anigilaje, Olutola, 2013). It is also higher than the 1.2% in Dar-es-Salaam (Telatela et al, 2007). The role of universal HB immunization in Tanzania since 1999 may have accounted for this lower prevalence. Generally, this prevalence of 10% is higher than the estimated 8% for Africa (Puoti et al, 2008). This probably may be because Nigeria is hyper-endemic (Emechebe et al, 2009) and the fact that HIV infected children tends to have early exposure to HBV risk factors following repeated illnesses and hospitalizations. The large number of children co-infected with HBV can easily transmit the infection to other children, considering the fact that HBV is 100 times more infective than HIV (Mgonda, 2004). This makes the infection a major public health problem that requires enhanced measures of prevention and care in order to achieve eradication in sub-Saharan Africa. An increasing prevalence of HBsAg with age was observed among the HIV positive children in this study. Other studies have also noted a similar trend (Telatela et al, 2007, Emechebe et al, 2008). This pattern would suggest that there is a predominance of horizontal transmission of HBV infection among HIV infected children in our environment. Though the highest number of HBsAg positivity was found in those children aged 6 -10 years, the highest prevalence of HBsAg was found among those aged 11-15 years. The high prevalence of co-infected Nigerian children of this age may reflect their inability to lose HBsAg due to their impaired immunity (Levy et al, 2006). In this study also, zero prevalence was observed in the age group 18 months to 3 years, which implies that vertical transmission of HBV has remained low in Nigerian women.

prevalence of co-infected Nigerian children of this age may reflect their inability to lose HBsAg due to their impaired immunity (Levy et al, 2006). In this study also, zero prevalence was observed in the age group 18 months to 3 years, which implies that vertical transmission of HBV has remained low in Nigerian women. It may also be that the mothers were not co-infected with HBV, considering the fact that Ilboudo in Burkina Faso recorded that three co-infected mothers who were positive for HBeAg, HBsAg and HBV DNA vertically transmitted the virus to their children (Ilboudo et al, 2010). This finding is similar to that in Dar-es-salaam (Telatela et al, 2007). The social class of the children in this study was not significantly associated with HBsAg positivity. However, it was observed that the higher the social class, the lower the number of children positive to HBsAg. This is similar to the findings in Bangui (Komas et al, 2010), and in Enugu (Emechebe et al, 2008). This could be because people in the upper socioeconomic class are less likely to patronize medical quacks, or indulge in activities that may promote infection with HBV such as alternative medicine.

ren positive to HBsAg. This is similar to the findings in Bangui (Komas et al, 2010), and in Enugu (Emechebe et al, 2008). This could be because people in the upper socioeconomic class are less likely to patronize medical quacks, or indulge in activities that may promote infection with HBV such as alternative medicine. Though the risk factors in this study were not statistically significant, those transfused were about three times more likely to have HBV infection than those not transfused. It is worthy to note that blood transfusion may have contributed to this high prevalence rate considering the fact that infectious HBV can be present in blood without detectable HBsAg (Alexander, Kowdley, 2006). Al-Fawaz had earlier adduced that even an HBsAg free blood obtained by the very sensitive 3rd generation screening techniques can not completely safeguard against HBV (Al-Fawaz, Ramia, 1993). Also, it had been noted that history of blood transfusion was a sufficient risk factor for chronic hepatitis B infection, a major aetiological factor for primary hepatocellular carcinoma in Africa and Southeast Asia (Ndububa et al, 2001).

ning techniques can not completely safeguard against HBV (Al-Fawaz, Ramia, 1993). Also, it had been noted that history of blood transfusion was a sufficient risk factor for chronic hepatitis B infection, a major aetiological factor for primary hepatocellular carcinoma in Africa and Southeast Asia (Ndububa et al, 2001). Irrespective of age and sex, serum alanine (ALT) levels greater than the upper limits of normal (15 iu/l) is often considered abnormal. Though the difference in ALT between the co-infected and non-co-infected children was not statistically significant, it was higher in the study group when compared with normal values. It probably may imply in-apparent liver disease in these children. The observed higher values among HBsAg negative subjects could be as a result of the fact that seven of the subjects had moderate to severe hepatic derangement with values of 50-206 iu/l. It is also possible that the higher value is due to other causes of liver disease in HIV positive individuals considering that 50% of the co-infected children had elevated baseline ALT compared with 45.2% among HBsAg negative HIV infected children. This differed from findings in Maiduguri (Ashir et al, 2009), and in Dar-es-Salaam (Telatela et al, 2007). Having been on HAART for varying periods, the mean ALT increased among both the co-infected and non-co-infected children showing a worsening liver disease probably due to HAART.

sAg negative HIV infected children. This differed from findings in Maiduguri (Ashir et al, 2009), and in Dar-es-Salaam (Telatela et al, 2007). Having been on HAART for varying periods, the mean ALT increased among both the co-infected and non-co-infected children showing a worsening liver disease probably due to HAART. The immune status of the co-infected children as depicted by the baseline median CD4 count was lower than in those with HIV infection alone. They also had lower median CD4 value even after being on HAART for a variable period. The improvement in immune status of this children compared with the co-infected was statistically significant. This implies that HBV co-infection significantly impairs the immune system of HIV positive children and that immunologic failure is commoner among co-infected subjects. This finding is consistent with those of Anigilaje, et al, Rouet et al Thio et al and Psevdos et al. Conclusion Hepatitis B virus infection is common among HIV infected children and co-infection is associated with impaired immunity (lower baseline CD4 count), even more significantly after initiation of HAART. Hepatitis B Virus co-infection is also associated with liver enzyme derangement (liver disease) although the difference was not statistically significant and this is probably exacerbated by HAART. Conflict Interest Statement: The authors of this work hereby declares that there is no competing interest among them and that there are also no financial or professional affiliations with any group or company.

Introduction The relationship between people and dogs is unique. Among domesticated hunting of prey animals, dogs are capable of performing a wide variety of roles for human: sheep herding, sniffing out drugs and explosives, hunting of prey and security; breeding purposes and companionship (Pet). To be precise about when the friendship began, is very difficult but a reasonable guess suggest that it has been going strong for more than 14,000 years (Bradshaw 2012; Udell et al., 2008). Despite the fact that we live so closely with dogs, it is not entirely without any health risk. A high zoonotic risk is involved with the increasing number of people, rich and poor, keeping dogs for various purposes without much knowledge on the zoonoses (Omudu et al., 2010). Surveys of microbiota of the nasal cavities, tonsils, and pharynx of clinically healthy dogs have found many types of aerobic and facultative anaerobic bacteria (Weese, 2007) most of which are zoonotic and may escape into the air while the dog breaths. Greater numbers of organisms are routinely cultured from the rostral than from the caudal nasal cavity of dogs (Craig, 2011). Craig, 2011 reported that the same bacteria flora may not possibly be found in nasal cavity and pharynx in each animal because of marked individual variations, but the presence of a certain range of microbiota can be predicted (Craig, 2011). However, the antibiotic resistance reports of bacteria isolated from clinically healthy animals is continuously increasing (Coates et al., 2002; Davis et al., 2014; Henning et al., 2001; Manian, 2003). Since these resistance factors are transferable, antibiotic sensitive microbiota of human may acquire these resistance factors (Lee, 2003) after effective zoonotic transmission including direct contact with pets, contact with feces from pets, preparation of raw meat and bones for pet consumption, and the handling of commercial pet treats (Cherry et al., 2004; Health Canada, 2005).

sitive microbiota of human may acquire these resistance factors (Lee, 2003) after effective zoonotic transmission including direct contact with pets, contact with feces from pets, preparation of raw meat and bones for pet consumption, and the handling of commercial pet treats (Cherry et al., 2004; Health Canada, 2005). Currently, the behavioral habits of dog owners/lovers may expose them to pathogenic agents through either inhalation, ingestion, skin contact etc. The aerosolized transmission of disease can occur through both “droplet” and “airborne” means. Droplet transmission is defined as the transmission of diseases by expelled particles that are likely to settle into another surface quickly, typically within three feet (90 cm) of the source (Practical Guidelines for Infection Control in Health Care Facilities 2005). Thus, for example, for an infection to be caused by droplet transmission, a susceptible individual must be close enough to the source of the infection for the droplet (containing the infectious microorganism) to make contact with the susceptible individual’s respiratory tract, eyes, mouth, nasal passages, and so forth (Gralton et al., 2011). The increase in antibiotic resistance among many potential pathogenic bacteria poses a great threat to humans who inhale aerosolised bacteria from the dog’s nostril, mouth, or fur etc.

to make contact with the susceptible individual’s respiratory tract, eyes, mouth, nasal passages, and so forth (Gralton et al., 2011). The increase in antibiotic resistance among many potential pathogenic bacteria poses a great threat to humans who inhale aerosolised bacteria from the dog’s nostril, mouth, or fur etc. This study was carried out to determine the potential pathogenic bacteria in the nasal region of the upper respiratory tract of apparently healthy dogs, antibiotic resistance patterns of the isolated bacteria and the level of closeness (distance) between the dog owner/handler’s face and the body of the dog.

to make contact with the susceptible individual’s respiratory tract, eyes, mouth, nasal passages, and so forth (Gralton et al., 2011). The increase in antibiotic resistance among many potential pathogenic bacteria poses a great threat to humans who inhale aerosolised bacteria from the dog’s nostril, mouth, or fur etc. This study was carried out to determine the potential pathogenic bacteria in the nasal region of the upper respiratory tract of apparently healthy dogs, antibiotic resistance patterns of the isolated bacteria and the level of closeness (distance) between the dog owner/handler’s face and the body of the dog. Materials and Methods Collection of samples Nasal samples were collected from the vestibules of 173 dogs by using sterile swabs. These dogs were clinically healthy and were used as guards, pets, and for breeding and hunting purposes in urban and rural areas of Ibadan, Oyo state, Nigeria. None of the dogs had apparent bacterial infections or was receiving antimicrobial therapy at the time of sample collection. In an attempt to sample a representative portion of the population, samples were collected at irregular intervals. Also, close observation and structured questionnaire administration were done involving the human with closest interaction with the dog sampled. This was done to determine the actual closest human face to dog body distance when playing with the dog. Only one sample was collected per animal. Samples collected were transported in a cooler with ice packs for delivery to the Department of Veterinary Microbiology and Parasitology, Faculty of Veterinary Medicine, University of Ibadan, Ibadan, Nigeria, for bacteriological analysis.

ody distance when playing with the dog. Only one sample was collected per animal. Samples collected were transported in a cooler with ice packs for delivery to the Department of Veterinary Microbiology and Parasitology, Faculty of Veterinary Medicine, University of Ibadan, Ibadan, Nigeria, for bacteriological analysis. Isolation and identification of bacterial isolates Each nasal sample was inoculated unto nine milliliters of sterile tryptic soy broth (TSB) (OXOID, Basingstoke, UK) in universal bottles. The broth cultures were incubated at 37°C for 18 to 24 h. After incubation, a loopful of the TSB culture was inoculated onto MacConkey agar, 7% sheep blood agar, Eosin Methylene Blue (EMB) (LAB M, lancashire, UK). These inoculated media were incubated at 37°C for 18 to 24 h. Colonial morphology and Gram staining of all the isolates on the plates were carried out; all the isolates were selected for oxidase and catalase production. Coagulase test was carried out for all the Gram positive cocci in clusters. Haemolysis was observed and recorded. Other biochemical and sugar utilization tests were performed. Results of biochemical tests were interpreted using Cowan and Steel’s manual for the identification of medical bacteria 3rd edition (Barrow et al., 2003).

test was carried out for all the Gram positive cocci in clusters. Haemolysis was observed and recorded. Other biochemical and sugar utilization tests were performed. Results of biochemical tests were interpreted using Cowan and Steel’s manual for the identification of medical bacteria 3rd edition (Barrow et al., 2003). Antimicrobial Susceptibility Test The susceptibility of identified bacterial isolates to antimicrobial agents was determined by the standard Kirby-Bauer disk diffusion method. Susceptibility to the following antimicrobials was determined for sixty Gram positive bacterial isolates: amoxicillin (30μg), ampiclox (30μg), ciprofloxacin (10μg), rocephin (25μg), perfloxacin (10μg), zinnacef (30μg), erythromycin (10μg), gentamicin (10μg), septrin (30μg), streptomycin (10μg) while One hundred and sixty- five Gram negative bacterial isolates were tested for susceptibility to the following antibiotics: augmentin (30μg), ofloxacin (5μg), gentamycin (10μg) and ciprofloxacin (5μg), perfloxacin (10μg), streptomycin (10μg), amoxicillin (30μg), chloramphenicol (30μg)), septrin (30μg), sparfloxacin (10μg).

e hundred and sixty- five Gram negative bacterial isolates were tested for susceptibility to the following antibiotics: augmentin (30μg), ofloxacin (5μg), gentamycin (10μg) and ciprofloxacin (5μg), perfloxacin (10μg), streptomycin (10μg), amoxicillin (30μg), chloramphenicol (30μg)), septrin (30μg), sparfloxacin (10μg). Results A total of 222 bacterial isolates were obtained from 173 nasal swabs of apparently healthy dogs. The isolates included Escherichia coli 41 (18.5%) followed by Proteus species 38 (17.1%), Staphylococcus aureus 31 (14.0%), Klebsiella species 20 (9.0%), Acinetobacter species 20 (9.0%), coagulase negative Staphylococcus species 17 (7.7%), Pseudomonas species 15 (6.8%), Actinobacter species 15 (6.8%), Citrobacter species 13 (5.9%) and Streptococcus species 12 (5.4%) (Table 1, Chart 1). Staphylococcus aureus and Escherichia coli ranked highest in the isolation frequency for Gram positive and Gram negative bacteria respectively (Table 1). Table 1 Distribution of bacteria isolates Bacteria Frequency (%) Gram positive isolates 60 (27.0%) Staphylococcus aureus 31 (14.0%) Coagulase negative Staphylococcus species 17 (7.7%) Streptococcus species 12 (5.4%) Gram negative isolates 162 (73.0%) Escherichia coli 41 (18.5%) Proteus species 38 (17.1%) Klebsiella species 20 (9.0%) Acinetobacter species 20 (9.0%) Pseudomonas species 15 (6.8%) Actinobacter species 15 (6.8%) Citrobacter species 13 (5.9%) Total number of isolates 222 (100%) Chart 1 Percentage distribution of bacteria isolates

ive isolates 162 (73.0%) Escherichia coli 41 (18.5%) Proteus species 38 (17.1%) Klebsiella species 20 (9.0%) Acinetobacter species 20 (9.0%) Pseudomonas species 15 (6.8%) Actinobacter species 15 (6.8%) Citrobacter species 13 (5.9%) Total number of isolates 222 (100%) Chart 1 Percentage distribution of bacteria isolates The overall rate of Staphylococcus aureus (n= 31) displayed 26 (84%), 26 (84%), 21 (68%), 11 (35.5%), 8 (25.8%), 7 (22.6%), 5 (16.0%), 5 (16.0%), 2 (6.5%), 0 (0.0%) resistance to Ampiclox, Amoxicillin, Zinnacef, Gentamycin, Septrin, Streptomycin, Rocephin, Erythromycin, Perfloxacin and Ciprofloxacin respectively. Streptococcus species (n=12) showed 11 (92.0%), 11 (92.0%), 9 (75.0%), 8 (66.7%), 6 (50.0%), 6 (50.0%), 6 (50.0%), 3 (25.0%), 2 (16.7%) and 2 (16.7%) to Ampiclox, Amoxicillin, Zinnacef, Gentamycin, Rocephin, Septrin, Streptomycin, Erythromycin, Perfloxacin and Ciprofloxacin. Coagulase negative Staphylococcus species (n=17) had resistance 13 (77.0%), 14 (82.4%), 11 (65.0%), 6 (35.0%), 3 (17.6%), 3 (17.6%), 0 (0.0%), 0 (0.0%), 0 (0.0%) and 0 (0.0%) to Ampiclox, Amoxicillin, Zinnacef, Rocephin, Gentamycin, Septrin, Streptomycin, Perfloxacin, Ciprofloxacin and Erythromycin respectively (Table 2). Generally, more than 50% of the gram positive isolates (range: 68.3% - 85.0%) were resistance to Ampiclox, Zinnacef and Amoxicillin (Table 3, Chart 2) while one isolate of Staphylococcus aureus and two isolates of Streptococcus species were resistant to eight out of ten antibiotics used (Table 6). Table 2 Antibiotic resistance patterns for Gram positive isolates

The overall rate of Staphylococcus aureus (n= 31) displayed 26 (84%), 26 (84%), 21 (68%), 11 (35.5%), 8 (25.8%), 7 (22.6%), 5 (16.0%), 5 (16.0%), 2 (6.5%), 0 (0.0%) resistance to Ampiclox, Amoxicillin, Zinnacef, Gentamycin, Septrin, Streptomycin, Rocephin, Erythromycin, Perfloxacin and Ciprofloxacin respectively. Streptococcus species (n=12) showed 11 (92.0%), 11 (92.0%), 9 (75.0%), 8 (66.7%), 6 (50.0%), 6 (50.0%), 6 (50.0%), 3 (25.0%), 2 (16.7%) and 2 (16.7%) to Ampiclox, Amoxicillin, Zinnacef, Gentamycin, Rocephin, Septrin, Streptomycin, Erythromycin, Perfloxacin and Ciprofloxacin. Coagulase negative Staphylococcus species (n=17) had resistance 13 (77.0%), 14 (82.4%), 11 (65.0%), 6 (35.0%), 3 (17.6%), 3 (17.6%), 0 (0.0%), 0 (0.0%), 0 (0.0%) and 0 (0.0%) to Ampiclox, Amoxicillin, Zinnacef, Rocephin, Gentamycin, Septrin, Streptomycin, Perfloxacin, Ciprofloxacin and Erythromycin respectively (Table 2). Generally, more than 50% of the gram positive isolates (range: 68.3% - 85.0%) were resistance to Ampiclox, Zinnacef and Amoxicillin (Table 3, Chart 2) while one isolate of Staphylococcus aureus and two isolates of Streptococcus species were resistant to eight out of ten antibiotics used (Table 6). Table 2 Antibiotic resistance patterns for Gram positive isolates Antibiotic Organisms Staphylococcus aureus n= 31 Coagulase negative Staphylococcus spp. n= 17 Streptococcus spp. n= 12 TOTAL n= 60 Ampiclox Sensitivity 5(16.0%) 5(23.0%) 1(8.0%) 11(18.3%) Resistance 26(84.0%) 12(70.6%) 11(92.0%) 49(81.7%) Zinnacef Sensitivity 10(32.0%) 6(35.0%) 3(25.0%) 19(31.7%) Resistance 21(68.0%) 11(65.0%) 9(75.0%) 41(68.3%) Amoxicillin Sensitivity 5(16.0%) 3(17.6%) 1(8.0%) 9(15.0%) Resistance 26(84.0%) 14(82.4%) 11(92.0%) 51(85.0%) Rocephin Sensitivity 26(84.0%) 11(65.0%) 6(50.0%) 43(71.7%) Resistance 5(16.0%) 6(35.0%) 6(50.0%) 17(28.3%) Gentamycin Sensitivity 20(64.5%) 14(82.4%) 4(33.3%) 38(63.3%) Resistance 11(35.5%) 3(17.6%) 8(66.7%) 22(36.7%) Septrin Sensitivity 23(74.2%) 14(82.4%) 6(50.0%) 43(71.7%) Resistance 8(25.8%) 3(17.6%) 6(50.0%) 17(28.3%) Streptomycin Sensitivity 24(77.4%) 17(100.0%) 6(50.0%) 47(78.3%) Resistance 7(22.6%) 0(0.0%) 6(50.0%) 13(21.7%) Perfloxacin Sensitivity 29(93.5%) 17(100.0%) 10(83.3%) 56(93.3%) Resistance 2(6.5%) 0(0.0%) 2(16.7%) 4(6.7%) Ciprofloxacin Sensitivity 31(100.0%) 17(100.0%) 10(83.3%) 58(96.7%) Resistance 0(0.0%) 0(0.0%) 2(16.7%) 2(3.3%) Erythromycin Sensitivity 26(84.0%) 17(100.0%) 9(75.0%) 52(86.7%) Resistance 5(16.0%) 0(0.0%) 3(25.0%) 8(13.3%) Table 3 Antibiotic resistance patterns for Gram positive isolates

(6.5%) 0(0.0%) 2(16.7%) 4(6.7%) Ciprofloxacin Sensitivity 31(100.0%) 17(100.0%) 10(83.3%) 58(96.7%) Resistance 0(0.0%) 0(0.0%) 2(16.7%) 2(3.3%) Erythromycin Sensitivity 26(84.0%) 17(100.0%) 9(75.0%) 52(86.7%) Resistance 5(16.0%) 0(0.0%) 3(25.0%) 8(13.3%) Table 3 Antibiotic resistance patterns for Gram positive isolates Test drugs Amount (μg) Sensitive Resistant Ciprofloxacin 10 58(96.7%) 2(3.3%) Perfloxacin 10 56(93.3%) 4(6.7%) Erythromycin 10 52(86.7%) 8(13.3%) Streptomycin 30 47(78.3%) 13(21.7%) Rocephin 25 43(71.7%) 17(28.3%) Septrin 30 43(71.7%) 17(28.3%) Gentamycin 10 38(63.3%) 22(36.7%) Zinnacef 20 19(31.7%) 41(68.3%) Ampiclox 10 11(18.3%) 49(81.7%) Amoxicillin 30 9(15.0%) 51(85.0%) Chart 2 Antibiotic resistance patterns for Gram positive isolates Table 4 Antibiotic resistance patterns for Gram negative isolates

Test drugs Amount (μg) Sensitive Resistant Ciprofloxacin 10 58(96.7%) 2(3.3%) Perfloxacin 10 56(93.3%) 4(6.7%) Erythromycin 10 52(86.7%) 8(13.3%) Streptomycin 30 47(78.3%) 13(21.7%) Rocephin 25 43(71.7%) 17(28.3%) Septrin 30 43(71.7%) 17(28.3%) Gentamycin 10 38(63.3%) 22(36.7%) Zinnacef 20 19(31.7%) 41(68.3%) Ampiclox 10 11(18.3%) 49(81.7%) Amoxicillin 30 9(15.0%) 51(85.0%) Chart 2 Antibiotic resistance patterns for Gram positive isolates Table 4 Antibiotic resistance patterns for Gram negative isolates Antibiotics Organisms Escherichia coli N= 41 Proteus species N=38 Klebsiella species N=20 Acinetobact er species N=20 Pseudomonas species N=15 Actinobacter species N=15 Citrobacter species N=13 Total N= 162 Amoxacillin Sensitivity 19 (46.3%) 9 (23.7%) 14 (70.0%) 8 (40.0%) 6 (40.0%) 6 (40.0%) 4 (30.8%) 66 (40.7%) Resistance 22 (53.7%) 29 (76.3%) 6 (30.0%) 12 (60.0%) 9 (60.0%) 9 (60.0%) 9 (69.2%) 96 (59.3%) Augmentin Sensitivity 21 (51.2%) 9 (23.7%) 14 (70.0%) 9 (45.0%) 1 (6.7%) 5 (33.3%) 2 (15.4%) 61 (37.7%) Resistance 20 (48.8%) 29 (76.3%) 6 (30.0%) 11 (55%) 14 (93.3%) 10 (66.7%) 11 (84.6%) 101 (62.3%) Septrin Sensitivity 25 (61.0%) 9 (23.7%) 13 (65.0%) 10 (50%) 7 (46.7%) 10 (66.7%) 8 (61.5%) 82 (50.6%) Resistance 16 (39.0%) 29 76.3%) 7 (35.0%) 10 (50%) 8 (53.3%) 5 (33.3%) 5 (38.5%) 80 (49.4%) Chloramphenicol Sensitivity 32 (78.0%) 18 (47.4%) 13 (65.0%) 13 (65.0%) 10 (66.7%) 11 (73.3%) 9 (69.2%) 106 (65.4%) Resistance 9 (22.0%) 20 (52.6%) 7 (35.0%) 7 (35.0%) 5 (33.3%) 4 (26.7%) 4 (30.8%) 56 (34.6%) Sparfloxacin Sensitivity 35 (85.4%) 31 (81.6%) 17 (85.0%) 16 (80.0%) 15 (100.0%) 12 (80.0%) 10 (76.9%) 136 (84.0%) Resistance 6 (14.6%) 7 (18.4%) 3 (15.0%) 4 (20.0%) 0 (0.0%) 3 (20.0%) 3 (23.1%) 26 (16.0%) Ciprofloxacin Sensitivity 38 (92.7%) 32 (84.2%) 19 (95.0%) 17 (85.0%) 15 (100.0%) 14 (93.3%) 12 (92.3%) 147 (90.7%) Resistance 3 (7.3%) 6 (15.8%) 1 (5.0%) 3 (15.0%) 0 (0.0%) 1 (6.7%) 1 (7.7%) 15 (9.3%) Gentamycin Sensitivity 29 (70.7%) 21 (55.3%) 15 (75.0%) 11 (55.0%) 7 (46.7%) 11 (73.3%) 9 (69.2%) 103 (63.6%) Resistance 12 (29.3%) 17 (44.7%) 5 (25.0%) 9 (45.0%) 8 (53.3%) 4 (26.7%) 4 (30.8%) 59 (36.4%) Perfloxacin Sensitivity 37 (90.2%) 31 (81.6%) 15 (75.0%) 16 (80.0%) 13 (86.7%) 12 (80.0%) 10 (76.9%) 134 (82.7%) Resistance 4 (9.8%) 7 (18.4%) 5 (25.0%) 4 (20.0%) 2 (13.3%) 3 (20.0%) 3 (23.1%) 28 (17.3%) Ofloxacin Sensitivity 33 (80.5%) 26 (68.4%) 15 (75.0%) 16 (80.0%) 15 (100.0%) 12 (80.0%) 10 (76.9%) 127 (78.4%) Resistance 8 (19.5%) 12 (31.6%) 5 (25.0%) 4 (20.0%) 0 (0.0%) 3 (20.0%) 3 (23.1%) 35 (21.6%) Streptomycin Sensitivity 27 (65.9%) 20 (52.6%) 16 (80.0%) 11 (55%) 9 (60.0%) 10 (66.7%) 8 (61.5%) 101 (62.3%) Resistance 14 (34.1%) 18 (47.4%) 4 (20.0%) 9 (45%) 6 (40

.0%) 16 (80.0%) 15 (100.0%) 12 (80.0%) 10 (76.9%) 127 (78.4%) Resistance 8 (19.5%) 12 (31.6%) 5 (25.0%) 4 (20.0%) 0 (0.0%) 3 (20.0%) 3 (23.1%) 35 (21.6%) Streptomycin Sensitivity 27 (65.9%) 20 (52.6%) 16 (80.0%) 11 (55%) 9 (60.0%) 10 (66.7%) 8 (61.5%) 101 (62.3%) Resistance 14 (34.1%) 18 (47.4%) 4 (20.0%) 9 (45%) 6 (40 .0%) 5 (33.3%) 5 (38.5%) 61 (37.7%) Table 5 Antibiotic resistance patterns for Gram negative isolates Antibiotics Amount (μg) Sensitive Resistant Ciprofloxacin 5 147 (90.7%) 15 (9.3%) Sparfloxacin 10 136 (84.0%) 26 (16.0%) Perfloxacin 10 134 (82.7%) 28 (17.3%) Ofloxacin 5 127 (78.4%) 35 (21.6%) Chloramphenicol 30 106 (65.4%) 56 (34.6%) Gentamycin 10 103 (63.6%) 59 (36.4%) Streptomycin 10 101 (62.3%) 61 (37.7%) Septrin 30 82 (50.6%) 80 (49.4%) Amoxicillin 30 66 (40.7%) 96 (59.3%) Augmentin 30 61 (37.7%) 101 (62.3%) Table 6 Resistance patterns of Gram positive bacteria isolates. Resistant Pattern Staphylococcus aureus Coagulase Negative Staphylococcus species Streptococcus species AM 1 0 0 APX, AM 1 1 1 APX, AM, Z 6 1 0 APX, AM, CN 0 1 0 APX, AM, Z, R 2 1 0 APX, Z, CN, S 1 0 0 APX, AM, CN, R 0 1 0 APX, AM, Z, CN, 1 0 1 APX, AM, Z, SXT 3 0 0 APX, AM, Z, PEF 0 0 1 APX, AM, Z, CN, R 3 0 0 APX, AM, Z, SXT, E 0 1 0 APX, AM, Z, CN, SXT, S 0 1 0 APX, AM, Z, CN, SXT, S, E 1 0 0 APX, AM, Z, CN, R, SXT, S 0 0 2 APX, AM, Z, CN, R, SXT, S, E 1 0 2 LEGEND: PEF- Perfloxacin, E- Erythromycin, SXT- Streptomycin, S- Septrin, CN- Gentamycin, Z- Zinnacef, APX- Ampiclox, AM- Amoxacillin

Resistant Pattern Staphylococcus aureus Coagulase Negative Staphylococcus species Streptococcus species AM 1 0 0 APX, AM 1 1 1 APX, AM, Z 6 1 0 APX, AM, CN 0 1 0 APX, AM, Z, R 2 1 0 APX, Z, CN, S 1 0 0 APX, AM, CN, R 0 1 0 APX, AM, Z, CN, 1 0 1 APX, AM, Z, SXT 3 0 0 APX, AM, Z, PEF 0 0 1 APX, AM, Z, CN, R 3 0 0 APX, AM, Z, SXT, E 0 1 0 APX, AM, Z, CN, SXT, S 0 1 0 APX, AM, Z, CN, SXT, S, E 1 0 0 APX, AM, Z, CN, R, SXT, S 0 0 2 APX, AM, Z, CN, R, SXT, S, E 1 0 2 LEGEND: PEF- Perfloxacin, E- Erythromycin, SXT- Streptomycin, S- Septrin, CN- Gentamycin, Z- Zinnacef, APX- Ampiclox, AM- Amoxacillin Escherichia coli (n= 41) displayed 22 (53.7%), 20 (48.8%), 16 (39.0%), 14 (34.1%), 12 (29.3%), 9 (22.0%), 8 (19.5%), 6 (14.6%), 4 (9.8%) and 3 (7.3%) to Amoxacillin, Augmentin, Septrin, Streptomycin, Gentamycin, Chloramphenicol, Ofloxacin, Sparfloxacin, Perfloxacin and Ciprofloxacin respectively. Proteus species (n=38) had 29 (76.3%), 29 (76.3%), 29 (76.3%), 20 (52.6%), 18 (47.4%), 17 (44.7%), 12 (31.6%), 7 (18.4%), 7 (18.4%) and 6 (15.8%) resistance to Amoxacillin, Augmentin, Septrin, Chloramphenicol, Streptomycin, Gentamycin, Ofloxacin, Sparfloxacin, Perfloxacin and Ciprofloxacin respectively. Klebsiella species showed 7 (35.0%), 7 (35.0%), 6 (30.0%), 6 (30.0%), 5 (25.0%), 5 (25.0%), 5 (25.0%), 4 (20.0%), 3 (15.0%) and 1 (5.0%) resistance to Septrin, Chloramphenicol, Amoxacillin, Augmentin, Gentamycin, Perfloxacin, Ofloxacin, Streptomycin, Sparfloxacin and Ciprofloxacin respectively. Acinetobacter species (n=20 had resistance 12 (60.0%), 11 (55%), 10 (50%), 9 (45.0%), 9 (45.0%), 7 (35.0%), 4 (20.0%), 4 (20.0%), 4 (20.0%) and 3 (15.0%) to Amoxacillin, Augmentin, Septrin, Gentamycin, Streptomycin, Chloramphenicol, Sparfloxacin, Perfloxacin, Ofloxacin and Ciprofloxacin respectively. Pseudomonas species (n=15) showed 14 (93.3%), 9 (60.0%), 8 (53.3%), 8 (53.3%), 6(40.0%), 5 (33.3%), 2 (13.3%), 0 (0.0%), 0 (0.0%) and 0 (0.0%) resistance to Augmentin, Amoxacillin, Septrin, Gentamycin, Streptomycin, Chloramphenicol, Perfloxacin, Sparfloxacin, Ciprofloxacin and Ofloxacin respectively. Actinobacter species (n=15) had 10 (66.7%), 9 (60.0%), 5 (33.3%), 5 (33.3%), 4 (26.7%), 4 (26.7%), 3 (20.0%), 3 (20.0%), 3 (20.0%) and 1 (6.7%) resistance to Augmentin, Amoxacillin, Septrin, Streptomycin, Chloramphenicol,

treptomycin, Chloramphenicol, Perfloxacin, Sparfloxacin, Ciprofloxacin and Ofloxacin respectively. Actinobacter species (n=15) had 10 (66.7%), 9 (60.0%), 5 (33.3%), 5 (33.3%), 4 (26.7%), 4 (26.7%), 3 (20.0%), 3 (20.0%), 3 (20.0%) and 1 (6.7%) resistance to Augmentin, Amoxacillin, Septrin, Streptomycin, Chloramphenicol, Gentamycin, Sparfloxacin, Perfloxacin, Ofloxacin and Ciprofloxacin respectively. Citrobacter species (n=13) had resistance 11 (84.6%), 9 (69.2%), 5 (38.5%), 4 (30.8%), 5 (38.5%), 4 (30.8%), 3 (23.1%), 3 (23.1%), 3 (23.1%) and 1 (7.7%) to Augmentin, Amoxacillin, Septrin, Chloramphenicol, Streptomycin, Gentamycin, Sparfloxacin, Perfloxacin, Ofloxacin and Ciprofloxacin respectively (Table 4). Generally, more than 50% of the Gram negative isolates (range: 59.3% - 62.3%) were resistance to Amoxicillin and Augmentin (Table 5, Chart 3). One each of Escherichia coli, Klebsiella species, Pseudomonas species, Actinobacter species and Citrobacter species isolates, two Acinetobacter species isolates and four Proteus species isolates were resistance to ten different antibiotics (Table 7). Chart 3 Antibiotic resistance patterns for Gram negative isolates Table 7 Resistance patterns of Gram negative bacteria isolates

Gentamycin, Sparfloxacin, Perfloxacin, Ofloxacin and Ciprofloxacin respectively. Citrobacter species (n=13) had resistance 11 (84.6%), 9 (69.2%), 5 (38.5%), 4 (30.8%), 5 (38.5%), 4 (30.8%), 3 (23.1%), 3 (23.1%), 3 (23.1%) and 1 (7.7%) to Augmentin, Amoxacillin, Septrin, Chloramphenicol, Streptomycin, Gentamycin, Sparfloxacin, Perfloxacin, Ofloxacin and Ciprofloxacin respectively (Table 4). Generally, more than 50% of the Gram negative isolates (range: 59.3% - 62.3%) were resistance to Amoxicillin and Augmentin (Table 5, Chart 3). One each of Escherichia coli, Klebsiella species, Pseudomonas species, Actinobacter species and Citrobacter species isolates, two Acinetobacter species isolates and four Proteus species isolates were resistance to ten different antibiotics (Table 7). Chart 3 Antibiotic resistance patterns for Gram negative isolates Table 7 Resistance patterns of Gram negative bacteria isolates Resistant pattern E. coli Proteus species Klebsiella species Acinetobacter species Pseudomon as species Actinobacter species Citrobacter species AU 0 0 1 0 2 2 1 SXT 1 1 0 0 0 0 0 GN 1 0 0 0 0 0 0 CH 0 0 1 0 0 0 1 CPX 0 0 0 0 0 0 1 AM 1 0 0 0 0 0 0 S 0 1 0 1 0 0 0 AU, SXT 0 2 0 0 0 0 0 AU, AM 2 0 0 0 2 1 0 AU, GN 0 0 0 1 0 0 0 SXT, CH 0 0 1 0 0 0 0 SXT, S 1 0 0 0 0 0 0 S, OFX 1 0 0 0 0 0 0 AU, S 0 0 0 0 1 0 0 AU, AM, PEF 0 0 0 0 0 1 0 AU, AM, CH 0 1 0 0 0 1 0 SXT, OFX, S 0 0 0 1 0 0 0 AU, AM, SXT 2 1 0 1 2 0 0 AU, GN, CH 0 1 0 0 0 0 0 AU, AM, S 0 1 0 0 0 0 0 AU, AM, GN 2 0 1 0 0 0 0 AU, SXT, CH, 0 0 2 1 0 0 0 AM, SXT, CH 0 1 0 0 0 0 0 S, AM, GN 0 0 0 1 0 0 0 AU, AM, GN, S 0 0 0 1 1 0 0 AU, AM, CH, SP 1 0 0 0 0 0 0 AU, AM, GN 1 1 0 0 1 0 0 AU, SXT, S, CH 0 0 0 0 0 0 1 AU, AM, GN, OFX 0 0 0 0 0 0 1 AU, AM, SXT, CH 0 1 0 1 0 0 0 AM, SXT, OFX, PEF 0 0 1 0 0 0 0 SXT, GN, CH, OFX 0 1 0 0 0 0 0 AU, AM, SXT, S 1 2 0 0 0 0 0 AU, SXT, GN, CH 0 0 0 1 1 0 0 AU, AM, SXT, GN, CH 0 1 0 0 1 1 0 AU, AM, SXT, GN, S 1 1 0 0 1 0 0 AU, AM, SXT, S, CH 0 1 0 0 0 1 0 AU, AM, GN, OFX, PEF 0 1 0 0 0 0 1 AU, AM, SXT, S, OFX 1 0 0 0 0 0 0 AU, AM, SXT, CH, SP 0 1 0 0 0 0 0 AU, S, SXT, CH, OFX, 0 1 0 0 0 0 0 AU, AM, SXT, GN, S, 0 0 0 0 1 0 0 AU, AM, SXT, GN, CH 0 1 0 0 0 0 0 AM, SXT, S, CH, CPX 0 1 0 0 0 0 0 AU, AM, SXT, S, SP 1 0 0 0 0 0 1 AU, AM, GN, S, OFX, PEF 0 0 1 0 0 0 0 AU, AM, SXT, S, OFX, SP 0 1 0 0 0 0 0 AU, AM, SXT, GN, S, CH, 2 0 0 0 1 0 0 AU, AM, SXT, GN, S, OFX 0 0 0 0 0 0 1 AU, AM, GN, S, PEF, SP 0 0 0 1 0 0 0 AU, AM, SXT, S, CH, OFX 1 0 0 0 0 0 0 AU, AM, SXT, GN, S, CH 0 0 0 0 0 0 3 AU, AM, SXT, GN, S, OFX 0 0 0 0 0 1 0 AU, AM, SXT, GN, CH, OFX, PEF 0 1 0 0 0 0 0 AU, AM, SXT, GN, S, CH, OFX, PEF 1 0 0 0 0 0 0 AU, AM, SXT, GN, S, CH, OFX, PEF 0 0 1 0 0 0 0 AU, AM, SXT, GN, S, CH, OFX, CPX 0 0 1 0 0 0 0 AU, AM, SXT, GN, S, CH, OFX, SP, CPX 2 0 0 0 0 0 0 AU, AM, SXT, GN, S, CH, OFX, PEF, CPX 0 1 0 0 0 0 0 AU, AM, SXT, GN, S, CH, OFX, PEF, SP 1 0 2 0 0 0 1 AU, AM, SXT, GN, S, CH, OFX, PEF, SP, CPX 1 4 1 2 1 1 1 LEGEND:

0 0 0 0 AU, AM, SXT, GN, S, CH, OFX, PEF 0 0 1 0 0 0 0 AU, AM, SXT, GN, S, CH, OFX, CPX 0 0 1 0 0 0 0 AU, AM, SXT, GN, S, CH, OFX, SP, CPX 2 0 0 0 0 0 0 AU, AM, SXT, GN, S, CH, OFX, PEF, CPX 0 1 0 0 0 0 0 AU, AM, SXT, GN, S, CH, OFX, PEF, SP 1 0 2 0 0 0 1 AU, AM, SXT, GN, S, CH, OFX, PEF, SP, CPX 1 4 1 2 1 1 1 LEGEND: AU- augmentin (30μg), GN- gentamycin (10μg), CPX- ciprofloxacin (5μg), CH- chloramphenicol (30μg) PEF- perfloxacin (10μg), SXT- streptomycin (10μg), AM- amoxicillin (30μg), SP- Sparfloxacin (10μg) OFX- ofloxacin (5μg), S septrin (30μg) It was obtained from the questionnaire that 82% (123/150) of human had contacts, at different times, less than 50cm human face to dog body contact especially when playing with the dogs. 60 (40%) human contacts respondents belong to urban areas where dogs are majorly used as pet, security and breeding purpose while 63(42%) of human contacts belong to rural areas where dogs are majorly used for hunting purpose.

t times, less than 50cm human face to dog body contact especially when playing with the dogs. 60 (40%) human contacts respondents belong to urban areas where dogs are majorly used as pet, security and breeding purpose while 63(42%) of human contacts belong to rural areas where dogs are majorly used for hunting purpose. Discussion and Conclusion A total of 222 bacteria isolates were cultured from the 173 nasal swab samples, out of which 10 potentially pathogenic bacteria including 3 Gram positive bacteria (Staphylococcus aureus 14.0%, coagulase negative Staphylococcus species 7.7% and Streptococcus species 5.4%) and 7 Gram negative bacteria (Escherichia coli 18.5%, Proteus species 17.1%, Klebsiella species 9.0%, Acinetobacter species 9.0%, Pseudomonas species 6.8%, Actinobacter species 6.8% and Citrobacter species 5.9%) were identified. The most frequently isolated bacteria in decreasing order included Escherichia coli (18.5%), Proteus species (17.1%), Staphylococcus aureus (14.0%), Klebsiella species (9.0%), Acinetobacter species (9.0%), coagulase negative Staphylococcus species (7.7%), Pseudomonas species (6.8%), Actinobacter species (6.8%) and Citrobacter species (5.9%) (Table 1). This result is in close agreement with Bauer et al. 2003 who reported the presence of 17 different bacterial species of Enterobacteriaceae, Staphylococci species and Streptococci species, from nasal swabs of healthy dog. Most of these bacteria have been implicated in respiratory disease in dogs and human (Adaszek et al., 2009; Meyer et al., 2010; Lansing et al., 2002; Spaterna et al., 2012). The susceptible dogs may present varying clinical signs such as coughing, nasal discharge, sneezing, difficulty in breathing, fever, loss of appetite and lethargic behavior (Ayodhya et al., 2013). In this study, Bacillus species was disregarded because this might have been obtained from the surrounding soil, though it can also cause respiratory disease (Maden et al., 2000; Amin et al., 2015).

asal discharge, sneezing, difficulty in breathing, fever, loss of appetite and lethargic behavior (Ayodhya et al., 2013). In this study, Bacillus species was disregarded because this might have been obtained from the surrounding soil, though it can also cause respiratory disease (Maden et al., 2000; Amin et al., 2015). Less than 50% of Gram positive isolates (31.7%) were sensitive to second generation cephalosporin- cefuroxine (zinnacef). The next drugs include Ampiclox (18.3% for Gram positive and 40.7% for Gram negative isolates), Amoxicillin (15.0% for Gram positive) and Augmentin (37.7% for Gram negative isolates). Owing to these low sensitivities, the selection of Amoxicillin and Ampiclox among drugs of choice for first line therapy of dog with pneumonia is weakened (Lesley, 2010) since anyone of these isolates can cause respiratory disease. The susceptibility level was highest to ciprofloxacin for Gram positive 58 (96.7%) and Gram negative (90.7%) bacteria (table 3 and 5). Other fluoroquinolones such as Perfloxacin, Sparfloxacin and Ofloxacin had sensitivity ranging from 78.4% to 93.3% (Tables 3 and 5).

one of these isolates can cause respiratory disease. The susceptibility level was highest to ciprofloxacin for Gram positive 58 (96.7%) and Gram negative (90.7%) bacteria (table 3 and 5). Other fluoroquinolones such as Perfloxacin, Sparfloxacin and Ofloxacin had sensitivity ranging from 78.4% to 93.3% (Tables 3 and 5). A pathogen is multidrug resistant (MDR) when it is resistant to three or more antibiotics at any given time (Jan et al., 2004). The antibiotic susceptibility pattern suggested that the isolated bacteria have strains which possessed varying MDR genes. This finding was also reported by Davis et al., 2014 who reported the presence of multiple antibiotic resistances of Staphylococcus species isolated from healthy dog and cats. However, Staphylococcus aureus strains were 100% sensitive to Ciprofloxacin while coagulase negative Staphylococcus species strains were 100% sensitive to Ciprofloxacin, Perfloxacin, Streptomycin and Erythromycin (Table 2). MDR among normal flora of clinically healthy dogs calls for a great attention since most of them had no record of antibiotic therapy. The growing antibiotic resistance trend among bacteria in humans and animals in both diseased and clinically healthy state, instigates a need for continuous research to avert the impending danger of antibiotic resistance (CDC, 2010; Coates et al., 2002; FDA, 2000).

ince most of them had no record of antibiotic therapy. The growing antibiotic resistance trend among bacteria in humans and animals in both diseased and clinically healthy state, instigates a need for continuous research to avert the impending danger of antibiotic resistance (CDC, 2010; Coates et al., 2002; FDA, 2000). Interestingly, more than 80 percent of human contacts (82%) have had less than 50 cm face-to-face closeness with these dogs; thus possessing a great risk of acquiring MDR pathogens. These human contacts have high chance of inhaling aerosolized bacteria from the body surfaces such as skin, oral cavity, nasal cavity etc. (Henning et al., 2001) especially when those areas are disturbed by hand rubbing on the fur, excessive exercise etc. If this contaminated air is breathed in by man, within three feet (90 cm), there is risk of acquiring such pathogen (W.H.O., 2005). The presence of MDR bacteria among healthy dogs suggested that these dogs (companion animals) are reservoir of multidrug-resistant potentially pathogenic bacteria, which may be transferred to human, especially the closest human contacts and others who handle them in unabated endangering habit. Such carriage poses an underlying risk of infection and this should be considered during handling of healthy dogs by all in-contact humans such as dog owners, veterinary personnel and students etc. Although, the settling of these airborne bacteria from these dogs on the human respiratory epithelium may not result into infection or disease state, the exchange of antibiotic resistant genes (plasmid) should not be taken lightly (Lee, 2003). Transfer of antibiotic resistant plasmid in bacteria has been documented (Lansing et al., 2002). These can make an antibiotic susceptible bacterium to be resistant to same after acquiring resistant plasmid. It is of importance to implement strategies to reduce the rate of appearance and spread of resistance bacteria to allow new drug discovery to catch up with bacteria resistance development.

ansing et al., 2002). These can make an antibiotic susceptible bacterium to be resistant to same after acquiring resistant plasmid. It is of importance to implement strategies to reduce the rate of appearance and spread of resistance bacteria to allow new drug discovery to catch up with bacteria resistance development. Competing interest: The authors affirm that this study and its interpretations were not under any financial or otherwise competing interest. Acknowledgements: The authors appreciate the technical and moral contributions of Mrs O.O. Orioke and Mrs A. Iyanda of the Department of Veterinary Microbiology and Parasitology, Faculty of Veterinary Medicine, University of Ibadan, Ibadan, Oyo State, Nigeria.

Introduction Avian influenza viruses (AIV) continue to be a global problem because they are potential highly infectious, can rapidly spread and cause disease in domestic poultry, and some viruses may also infect other animal hosts, including humans (Feare, 2007). Globally, an enormous number of poultry have died from direct infection with AIV, and countless numbers of poultry flocks at risk have been depopulated as a measure to contain the virus and prevent its further spread (Feare, 2007). Apart from the severe economic losses it causes in commercial poultry, AIV can evolve rapidly and spillover into other species (Perdue and Swayne, 2005; Van Reeth, 2007). In addition, high pathogenic avian influenza (HPAI) viruses in domestic poultry are thought to have evolved from low pathogenic avian influenza (LPAI) viruses through mutations or re-combinations (Alexander, 2007). According to Swayne et al. (2011), LPAI is a reportable disease caused by H5, H7 and H9 subtype viruses that have become a major source of concern to the global poultry industry. Most LPAI viruses (LPAIVs) produce mild to moderate disease in commercial rearing settings, especially when complicated by secondary pathogens, immunosuppression, and stress factors in the environment. Drops in egg production have also been observed in chicken breeders and layers infected with these viruses (Lu et al., 2004; Pillai et al., 2009). Furthermore, outbreaks of HPAI that resulted from circulating LPAI H5, H7 and H9 viruses have been reported in poultry worldwide (Iqbal et al., 2009; Snoeck et al., 2011). Additionally, their transmission to humans has been described and this highlights their potential to cause zoonotic disease (Capua and Alexander, 2007a; Wang et al., 2009).

f HPAI that resulted from circulating LPAI H5, H7 and H9 viruses have been reported in poultry worldwide (Iqbal et al., 2009; Snoeck et al., 2011). Additionally, their transmission to humans has been described and this highlights their potential to cause zoonotic disease (Capua and Alexander, 2007a; Wang et al., 2009). Highly pathogenic avian influenza has extended from Asia to Europe and Africa since 2003, leading to the death or mass slaughter of millions of birds and significant economic losses. For instance, the 2003 outbreak of HPAI (H7N7 subtype) in the Netherlands, Belgium and Germany led to the destruction of over 33 million birds with the total cost estimated at €750 million while a 2004 outbreak of AI due to H7N3 subtype in Canada resulted in the destruction of 14 million birds and a loss of more than $300 million (Lupiani and Reddy, 2009). These outbreaks which also resulted in transmission of the virus to occupationally exposed people highlight the significance of global surveillance for AIV infections in the natural hosts. The method commonly used in serologic surveillance programs for AI is a test that detects antibodies directed against the cross-reactive nucleoprotein antigen shared by all influenza A viruses (OIE, 2012). In Nigeria where AI vaccine is not currently administered to poultry and where epidemiological and virological data are sparse, the benefits of surveillance may include estimation of the value of influenza prevention through demonstration of the local disease burden associated with influenza. In this way, surveillance can also help establish the epidemiological characteristics of influenza within the country which would inform the development of effective and sustainable prevention strategies (Breese, 2010). Hence, in Nigeria with a poultry population of about 170 million birds (FAOSTAT, 2014) that provides a source of livelihood for thousands of individuals, there is need for continuous surveillance for diseases such as AI which is of immense economic and public health significance. Therefore, in an attempt to further elucidate the epidemiology of AI in southwest Nigeria, we screened birds from commercial breeder and layer flocks in Oyo state for LPAIV antibodies and nucleic acid.

need for continuous surveillance for diseases such as AI which is of immense economic and public health significance. Therefore, in an attempt to further elucidate the epidemiology of AI in southwest Nigeria, we screened birds from commercial breeder and layer flocks in Oyo state for LPAIV antibodies and nucleic acid. Materials and methods Study design and area Purposive sampling method was used to focus the study on seven local government areas (LGAs) identified as areas of concentration of commercial poultry in Oyo state, namely Afijio, Lagelu, Oluyole, Ido, Egbeda, Akinyele and Ogbomoso South (Figure 1). With a preponderance of hatcheries, commercial breeder, layer, broiler and cockerel farms, backyard poultry, live-bird markets and feedmills, Oyo state is the nerve centre of poultry activities in southwest Nigeria which has previously been described as the hub of poultry production in the country (Owoade et al., 2004; Adene and Oguntade, 2006). Figure 1 Map showing the study area in Oyo state, Nigeria In addition, the poultry farmers/personnel were interviewed on AI vaccination practice in their farms. Samples were collected between March and October 2013 from 20 commercial poultry flocks located in these seven LGAs. The survey was targeted at breeder and layer flocks since these chicken types are regarded as the frontliners in poultry farming producing day-old chicks, table eggs and poultry meat for human use and consumption in the study area.

en March and October 2013 from 20 commercial poultry flocks located in these seven LGAs. The survey was targeted at breeder and layer flocks since these chicken types are regarded as the frontliners in poultry farming producing day-old chicks, table eggs and poultry meat for human use and consumption in the study area. Sample collection A total of 461 blood and cloacal swab samples were collected for this study. Of this, 180 were obtained from six breeder flocks located in Egbeda and Oluyole LGAs and 281 from 14 commercial layer flocks in the remaining 5 LGAs (Table 1). Two milliliters of blood collected from each bird via the brachial vein was allowed to clot at room temperature for about 3-4 hours. The separated sera were collected in labeled Eppendorf tubes and stored at -20°C until tested. Table 1 Prevalence of influenza A virus antibodies based on flock type

Sample collection A total of 461 blood and cloacal swab samples were collected for this study. Of this, 180 were obtained from six breeder flocks located in Egbeda and Oluyole LGAs and 281 from 14 commercial layer flocks in the remaining 5 LGAs (Table 1). Two milliliters of blood collected from each bird via the brachial vein was allowed to clot at room temperature for about 3-4 hours. The separated sera were collected in labeled Eppendorf tubes and stored at -20°C until tested. Table 1 Prevalence of influenza A virus antibodies based on flock type Flock type Location No. Positive/No. tested Prevalence (%) Breeders Egbeda 9/40 22.5 Oluyole 14/140 10.0 23/180 12.8 Layers Afijio 8/101 7.9 Lagelu 1/41 2.4 Ido 8/37 21.6 Akinyele 1/60 1.7 Ogbomoso south 8/42 19.0 26/281 9.3 Total 49/461 10.6 Cloacal swab samples were collected by inserting sterile cotton swab in the cloaca of each bird and swabbing it against the mucosal wall until some faecal materials was obtained. This was immediately transferred into virus transport medium containing antibiotics and fetal bovine serum in labeled cryovials. The swabs were then transported on ice to the laboratory where they were stored at -80 °C until processed for testing. None of the flocks showed clinical signs of AI at the time of sampling.

This was immediately transferred into virus transport medium containing antibiotics and fetal bovine serum in labeled cryovials. The swabs were then transported on ice to the laboratory where they were stored at -80 °C until processed for testing. None of the flocks showed clinical signs of AI at the time of sampling. Serologic testing The serum samples were tested for antibodies to influenza A virus with a commercial competitive enzyme-linked immunosorbent assay (ELISA) (Bionote Inc., Korea) following the manufacturer’s protocol. According to the manufacturer, the kit was developed to detect antibodies against LPAIV subtypes (H1-15, N1-6). Results were read at 450 nm using a microplate ELISA reader and positive samples (PI ≥ 50) were thereafter screened by haemagglutination inhibition (HI) assays for AIV subtype-specific antibodies using a panel of reference antigens comprising LPAI H5N2, H7N7 and H9N2 viruses, 4 haemagglutinating units of each antigen, and positive- and negative-control serum as recommended by the World Organization for Animal Health (OIE, 2014).

ned by haemagglutination inhibition (HI) assays for AIV subtype-specific antibodies using a panel of reference antigens comprising LPAI H5N2, H7N7 and H9N2 viruses, 4 haemagglutinating units of each antigen, and positive- and negative-control serum as recommended by the World Organization for Animal Health (OIE, 2014). Molecular testing Suspensions prepared from the collected cloacal swabs were centrifuged (4°C) at 1000 rpm for 10 minutes to obtain a clear supernatant. Extraction of each supernatant was done using the QIAmp viral RNA extraction kit (QIAGEN GmbH, Germany) according to the manufacturer’s manual. The extracts were amplified by conventional reverse transcriptase-polymerase chain reaction (RT-PCR) using the QIAGEN One-Step RT-PCR kit (Hilden, Germany) in a 50μl reaction volume containing 2μl of enzyme mixture (including Omniscript reverse transcriptase and hot-start Taq polymerase), 10μl of 1X RT-PCR buffer, 2μl dNTPs, 5μl of each primer, 0.25μl RNase inhibitor, 20.75μl nuclease-free water and 5μl of the extracted RNA. Each amplification run contained one negative and one positive control. The negative control was nuclease-free water while for the positive control, nucleic acid extracted from archived virus stock was used. Gene-specific primers designed for the AIV nucleoprotein: 5’ – AGC AGC ACA AAG AGC AAT GA - 3’ (forward) and 5’ – ACT CAT GTC AAA GGA GGG CAC GAT – 3’ (reverse) were used (Lu et al., 2013). The thermal profile was as follows: reverse transcription at 50°C for 30 min, a single cycle initial PCR activation step at 95°C for 15 min, followed by 35 cycles of 94°C for 1 min, 56°C for 30 sec and 72°C for 1 min. Final extension was done at 72°C for 10 min.

A GGG CAC GAT – 3’ (reverse) were used (Lu et al., 2013). The thermal profile was as follows: reverse transcription at 50°C for 30 min, a single cycle initial PCR activation step at 95°C for 15 min, followed by 35 cycles of 94°C for 1 min, 56°C for 30 sec and 72°C for 1 min. Final extension was done at 72°C for 10 min. Statistical analysis Data obtained were analysed with Fisher’s exact test (2-tailed) using Graph Pad prism version 5.0 (Graph Pad software, San Diego, CA, USA) at a 0.05. Results The responses obtained from interviewing the poultry farmers and personnel revealed that they did not vaccinate their birds against AI. Using the ELISA, an overall influenza A virus antibody prevalence of 10.6% (49/461) was obtained. A higher (12.8%) seroprevalence was obtained for breeder flocks compared to 9.3% for layers (Table 1). However, using the Fisher’s exact test (two-sided), there was no statistically significant difference between both flock types sampled. The HI assay to confirm the presence of AIV subtype-specific antibodies in the ELISA-positive sera revealed 22.0%, 2.0% and 78.0% prevalence of H5N2, H7N7 and H9N2 LPAIV antibodies, respectively (Table 2). Table 2 Prevalence of LPAIV antibodies by HI test

However, using the Fisher’s exact test (two-sided), there was no statistically significant difference between both flock types sampled. The HI assay to confirm the presence of AIV subtype-specific antibodies in the ELISA-positive sera revealed 22.0%, 2.0% and 78.0% prevalence of H5N2, H7N7 and H9N2 LPAIV antibodies, respectively (Table 2). Table 2 Prevalence of LPAIV antibodies by HI test No. positive by HI Flock type Location H5N2 H7N7 H9N2 Breeders Egbeda 2 0 7 Oluyole 1 1 12 Layers Afijio 2 0 7 Lagelu 1 0 1 Ido 3 0 5 Akinyele 0 0 1 Ogbomoso South 2 0 6 Total 11 (22.0%) 1 (2.0%) 39 (78.0%) Testing of the cloacal swabs by RT-PCR gave negative results as none yielded the expected band size of 750 bp following RT-PCR and agarose gel electrophoresis of the amplified products.

0 7 Oluyole 1 1 12 Layers Afijio 2 0 7 Lagelu 1 0 1 Ido 3 0 5 Akinyele 0 0 1 Ogbomoso South 2 0 6 Total 11 (22.0%) 1 (2.0%) 39 (78.0%) Testing of the cloacal swabs by RT-PCR gave negative results as none yielded the expected band size of 750 bp following RT-PCR and agarose gel electrophoresis of the amplified products. Discussion The importance of continuous surveillance for AI viruses in order to prevent outbreaks and possibly identify potential carriers and reservoirs of the virus that can be included in future surveillance programmes cannot be overemphasized (Coker et al., Considering that AI viruses have been reported to impact negatively on food supply and the economy through their effects on the poultry industry (Peiris et al., 2007), flock surveillance can help establish the epidemiological characteristics of AIV infection that would inform development of prevention and control strategies, especially in countries like Nigeria where vaccination against AI is currently prohibited. As part of routine screening for AIVs in southwest Nigeria (Aiki-Raji et al., 2015; Oluwayelu et al., 2015), serologic and molecular surveillance for LPAIVs was conducted in birds from commercial breeder and layer flocks in Oyo state. It has been reported that serologic screening provides insight into the infection history of an animal’s entire life (Couacy- Hymann et al., 2012) while RT-PCR detection of the shedding of viral RNA is often performed to monitor the current influenza status in birds (Wallerström et al., 2014).

ayer flocks in Oyo state. It has been reported that serologic screening provides insight into the infection history of an animal’s entire life (Couacy- Hymann et al., 2012) while RT-PCR detection of the shedding of viral RNA is often performed to monitor the current influenza status in birds (Wallerström et al., 2014). Our findings revealed a low prevalence (10.6%) of antibodies against influenza A virus in asymptomatic, unvaccinated commercial breeder and layer birds in Oyo state, southwest Nigeria. This finding is consistent with previous reports of low seroprevalence of AI in commercial chickens elsewhere: 20% in Bangladesh (Biswas et al., 2009), 11.7% in Grenada (Sabarinath et al., 2011) and 12.9% in Kano state, Nigeria (Wakawa et al., 2012). Since the birds were not vaccinated, the further detection of HI antibodies against LPAIV H5N2, H7N7 and H9N2 in them is an indication of response to natural infection with LPAIVs in the environment and suggests that these viruses were present in the sampled farms even in the absence of overt disease. It has been reported that antibodies to influenza A virus can be detected long after viral shedding has ceased (Spackman et al., 2009). Interestingly, HI antibodies against two LPAIV subtypes (H5N2 and H9N2) were detected in two layer (Afijio and Lagelu) flocks (Table 2) in this study. This is an indication that birds in these flocks were exposed to more than one LPAIV subtype at the same time, a situation that could potentially lead to genetic reassortment between the two viruses and possibility of emergence of novel virus strains with zoonotic tendencies as previously documented (Smolinski et al., 2004).

s an indication that birds in these flocks were exposed to more than one LPAIV subtype at the same time, a situation that could potentially lead to genetic reassortment between the two viruses and possibility of emergence of novel virus strains with zoonotic tendencies as previously documented (Smolinski et al., 2004). Other workers have shown that LPAIV infections may affect feed and water consumption sufficiently to cause distressed egg lay with a resultant decrease in or complete cessation of egg production in both breeder and layer birds (Pillai et al., 2009; Pantin-Jackwood et al., 2012). Moreover, LPAIVs have been associated with increased severity because of co-infection with other poultry viruses, thus they cause economic losses to the poultry industry and negatively impact food supply (Peiris et al., 2007; Samaha et al., 2015). Therefore, considering that the subclinical to mild illness caused by LPAIVs can result in serious disease outbreaks especially when complicated by concurrent infections and/or suboptimal environmental conditions (Seifi et al., 2010), the seropositive birds detected in this study could constitute an economic threat to the poultry industry in Oyo state, and possibly Nigeria if interstate spread occurs. Furthermore, environmental exposure to AI viruses by the cloacal and intraocular routes has been shown to be a possibility in chicken breeders if the reproductive organs of the males are infected with the virus or via transmission by semen as previously documented for turkeys (Samadieh and Bankowski, 1970). Hence, these birds could serve as reservoirs shedding the viruses into the environment.

ring previous reports (Swayne and Halvorson, 2003; Werner and Harder, 2006; Briand et al., 2010) of the possibility of a field LPAIV mutating into a highly pathogenic strain after circulating in susceptible poultry. Such mutation probably occurs after the viruses have moved from their natural wild bird host to poultry. The non-detection of AIV RNA by RT-PCR in the cloacal swabs collected from commercial breeder and layer birds in this study suggests that the birds were not actively shedding the virus at the time of sample collection. Since influenza virus is excreted continuously in faeces for only about 12 days in birds (Henaux and Samuel, 2011; Couacy-Hymann et al., 2012), it is possible that the period of virus shedding could have been missed in the birds sampled. It could also be due to too low titres of the virus in faeces which makes it undetectable by RT-PCR. Similar findings of absence of AIV RNA in seropositive birds have been reported previously (Obon et al., 2009; Molia et al., 2010; Sabarinath et al., 2011; Wakawa et al., 2012). Likewise, the evidence of LPAIV in commercial poultry in this study suggests that the birds could serve as carriers shedding the virus into the environment when immunity against AI is no longer sufficient, thereby playing a crucial role in the epidemiology of the disease and posing a potential public health risk especially to occupationally exposed humans in the study area. This possibility is highlighted by several reports which have implicated H5, H7 and H9 subtype AIVs in previous cases of human infections (Fouchier et al., 2004; Koopmans et al., 2004; Capua and Alexander, 2007b; Wang et al., 2009). Conclusions

health risk especially to occupationally exposed humans in the study area. This possibility is highlighted by several reports which have implicated H5, H7 and H9 subtype AIVs in previous cases of human infections (Fouchier et al., 2004; Koopmans et al., 2004; Capua and Alexander, 2007b; Wang et al., 2009). Conclusions The findings of this study show that LPAI H5N2, H7N7 and H9N2 viruses presently circulate without producing overt disease in commercial breeder and layer flocks in Oyo state, Nigeria. This highlights the possibility of a future outbreak of HPAI in the state. There is therefore a need for continuous active, flock-based serological, virological and molecular surveillance for AIV as part of early warning measure to achieve prompt detection of the virus and its sustainable control in Nigeria.

Introduction Household air pollution is a leading risk factor for morbidity and mortality in developing countries (Bruce et al., 2000; Lim et al., 2012; Rehfuess et al., 2009; Smith, 2000). Although outdoor air pollution is an important and growing concern in sub-Saharan Africa, it has been estimated that household air pollution (HAP) is actually a more significant environmental health threat (Lim et al., 2012). World-wide, exposure to high levels of HAP is a daily reality for approximately 3 billion users of solid cook-fuels (WHO, 2014). The World Health Organization (WHO) estimates that HAP leads to about 4 million deaths per year and is one of the major global environmental risk factors for reduced life expectancy (WHO, 2014). In Ghana, solid fuels are utilized by a large majority of the population (GSS/GHS/GNPHRL, 2015) and solid fuel use accounts for 2.2 percent of the national burden of disease according to WHO estimates (WHO, 2007). This health burden is largely borne by women and especially children. Among women and children, HAP is associated with the risk of poor birth outcomes (Amegah et al., 2013; Kim et al., 2011; Lakshmi et al., 2013), cardiovascular diseases (Brook, 2007; Miller et al., 2007) and acute lower respiratory infection (ALRI) (Perez-Padilla et al., 2010; Torres-Duque et al., 2008).

ially children. Among women and children, HAP is associated with the risk of poor birth outcomes (Amegah et al., 2013; Kim et al., 2011; Lakshmi et al., 2013), cardiovascular diseases (Brook, 2007; Miller et al., 2007) and acute lower respiratory infection (ALRI) (Perez-Padilla et al., 2010; Torres-Duque et al., 2008). In sub-Saharan Africa, the burden of ALRI is high (Walker et al., 2013) and contributes significantly to household expenditure or the health system (Sinha et al., 2012). Since HAP is one of the leading causes of ALRI, there is the need to develop interventions to reduce HAP in sub-Saharan Africa where the use of biomass as the main domestic energy is high (WHO, 2014). Meeting domestic energy while reducing the burden of ALRI may be a challenge and requires relevant local data that quantifies the magnitude of the current health burden and point to optimal solutions. In 2007, the Kintampo Health Research Centre (KHRC) and Columbia University started a collaboration to develop innovative approaches that will help reduce household air pollution, with the goal of preventing poor health outcomes among rural populations in Ghana. In this paper, we report the results of a large, population-based household survey carried out with the goal of characterizing household cooking practices and respiratory morbidity among infants.

will help reduce household air pollution, with the goal of preventing poor health outcomes among rural populations in Ghana. In this paper, we report the results of a large, population-based household survey carried out with the goal of characterizing household cooking practices and respiratory morbidity among infants. Method Study area The study was carried out in the Kintampo North Municipality and Kintampo South District of Ghana in 2007, with a total area of 7162 km2 and a resident population of approximately 140,000 (Owusu-Agyei et al., 2012). The study area is located within the forest-savannah transitional zone in Ghana where community members are predominantly subsistent farmers, with literacy rate of about 40%. Malaria and respiratory infections are among the top ten diseases reported at health facilities(KNMA, 2013). There is significant poor ill-health among new-borns and infants; low birth weight prevalence is about 10% (Asante et al., 2013), neonatal mortality is 32/1000 live-birth, and infant mortality is 64/1000 child years (Kirkwood et al., 2010a). Health care is basic in the study area and the first point of call for an acute illness is a local chemical shop (Asante et al., 2010). This region has been under regular surveillance for births, deaths and migration patterns since 2003 by the Kintampo Health Research Centre (KHRC) under the Kintampo Health and Demographic Surveillance System (KHDSS) (Owusu-Agyei et al., 2012). The KHDSS platform provides the strength of contacting all households in the study area with a high response rate (Owusu-Agyei et al., 2012).

and migration patterns since 2003 by the Kintampo Health Research Centre (KHRC) under the Kintampo Health and Demographic Surveillance System (KHDSS) (Owusu-Agyei et al., 2012). The KHDSS platform provides the strength of contacting all households in the study area with a high response rate (Owusu-Agyei et al., 2012). Data Collection The survey consisted of a short set of questions addressing childhood respiratory infections and household cooking practices that were administered to 12,333 households in the study area using the KHDSS. A more extensive questionnaire was later administered to a randomly selected sub-sample of 421 out of the 12,333 households to determine more details of household cooking practices. A trained fieldworker using a pretested questionnaire interviewed household heads or their representatives. A household was eligible for the survey if the respondent gave consent. Key variables included household cooking practices, including type of cook-stove used in cooking, type of fuel used for cooking, description of primary cooks and cooking area. We also assessed the presence of respiratory symptoms (cough, blocked nose, runny nose, fast breathing; or difficulty in breathing) among the youngest child in the household in the 2 weeks prior to the interview. Questions were also asked regarding family structure, decision-making and economic circumstances. The time for each interview was about 45 minutes.

tory symptoms (cough, blocked nose, runny nose, fast breathing; or difficulty in breathing) among the youngest child in the household in the 2 weeks prior to the interview. Questions were also asked regarding family structure, decision-making and economic circumstances. The time for each interview was about 45 minutes. Data Management and Analysis Answered questionnaires were checked for completeness and consistency and sent to the KHRC computer laboratory for double entry into a password-protected database in Microsoft FoxPro version 9.0. Clean dataset was analyzed using Stata version 11.0 (Stata Corp, TX). Simple proportions and means were used to describe categorical and continuous data respectively. Socioeconomic status was defined using household assets such as household architecture or social amenities. Principal component analysis was applied to these variables and the first component was used as described others (Houweling et al., 2003; Howe et al., 2012; Vyas and Kumaranayake, 2006) The scores for the first component of wealth index were further classified into terciles as most poor, less poor and least poor.

ties. Principal component analysis was applied to these variables and the first component was used as described others (Houweling et al., 2003; Howe et al., 2012; Vyas and Kumaranayake, 2006) The scores for the first component of wealth index were further classified into terciles as most poor, less poor and least poor. Ethical Consideration Scientific and ethical approvals for the study were obtained from the Kintampo Health Research Centre Scientific Review Committee, the Kintampo Health Research Centre Institution’s Ethics Committee (FWA00011103) and the Institutional Review Board of Columbia University. All participants were individually consented for voluntary participation in the study. The consenting process involved explaining the purpose of the study, confidentiality procedures, risks, benefits and freedom to opt out of the study at any time. Consent was indicated by a signature or thumbprint and a witness was used where respondent was illiterate. Completed survey forms have been kept safely under lock and key in KHRC and could be accessed only by named study investigators. Results Twelve thousand, three hundred and thirty-three households were approached and interviewed with 100% response rate. As shown in Table 1, majority of household heads/respondents were males (61.5%) and with no education (54.1%). Most (67%) of households were located in rural areas. Table 1 Characteristics of all households contacted (N= 12,333).

Results Twelve thousand, three hundred and thirty-three households were approached and interviewed with 100% response rate. As shown in Table 1, majority of household heads/respondents were males (61.5%) and with no education (54.1%). Most (67%) of households were located in rural areas. Table 1 Characteristics of all households contacted (N= 12,333). n % Gender of household head Male 7585 61.5 Female 4339 35.2 Missing 409 3.3 Age of Household head in 2007 20-39 4605 37.3 40-59 4782 38.8 60+ 2449 19.9 Missing 497 4.0 Educational attainment of house head No education 6676 54.1 Primary 1003 8.1 Middle/JSS 2527 20.5 Secondary+ 999 8.1 Missing 1128 9.2 Household size 1 1314 10.6 2-5 2597 21.1 6-9 1988 16.1 10+ 6025 48.9 Missing 409 3.3 Area of stay Rural 8265 67.0 Urban 4068 33.0 Household wealth terciles Most poor 3980 32.3 Less poor 3942 32.0 Least poor 4002 32.4 Missing 409 3.3 Cigarettes smoking in the last month Yes 1687 13.7 No 10646 86.3 Commercial cooking Yes 1765 14.3 No 10568 85.7 The prevalence of smoking among household members was 13.7% within the one month period prior to the survey. Approximately fifty-seven percent (7006/12333) of households had at least one child less than five years of age. Fifty-two percent of children were males [Table 2]. Table 2 Characteristics of households and characteristics of children assessed for respiratory morbidity Total At least one respiratory sign. n (%) Symptoms of acute lower respiratory infection. n (%)

n % Gender of household head Male 7585 61.5 Female 4339 35.2 Missing 409 3.3 Age of Household head in 2007 20-39 4605 37.3 40-59 4782 38.8 60+ 2449 19.9 Missing 497 4.0 Educational attainment of house head No education 6676 54.1 Primary 1003 8.1 Middle/JSS 2527 20.5 Secondary+ 999 8.1 Missing 1128 9.2 Household size 1 1314 10.6 2-5 2597 21.1 6-9 1988 16.1 10+ 6025 48.9 Missing 409 3.3 Area of stay Rural 8265 67.0 Urban 4068 33.0 Household wealth terciles Most poor 3980 32.3 Less poor 3942 32.0 Least poor 4002 32.4 Missing 409 3.3 Cigarettes smoking in the last month Yes 1687 13.7 No 10646 86.3 Commercial cooking Yes 1765 14.3 No 10568 85.7 The prevalence of smoking among household members was 13.7% within the one month period prior to the survey. Approximately fifty-seven percent (7006/12333) of households had at least one child less than five years of age. Fifty-two percent of children were males [Table 2]. Table 2 Characteristics of households and characteristics of children assessed for respiratory morbidity Total At least one respiratory sign. n (%) Symptoms of acute lower respiratory infection. n (%) N=7006 N=2413 N=4593 N=957 N=6049 n (%) Yes n(%) No n(%) Yes n(%) No n(%) Gender of household head Male 4624 (66.0) 1589 (65.8) 3035 (66.1) 609 (63.6) 4015 (66.4) Female 2176 (31.1) 748 (31.0) 1428 (31.1) 311 (32.5) 1865 (30.8) Missing 206 (2.9) 76 (3.2) 130 (2.8) 37 (2.9) 169 (2.8) Age of Household head at time of survey 20-39 3050 (43.5) 1077 (44.6) 1973 (43.0) 439 (45.9) 2611 (43.2) 40-59 2592 (37.0) 884 (36.6) 1708 (37.2) 331 (34.6) 2261 (37.4) 60+ 1118 (16.0) 361 (15.0) 757 (16.5) 143 (14.9) 975 (16.1) Missing 246 (3.5) 91 (3.8) 155 (3.6) 44 (4.6) 201 (3.3) Educational attainment of house head No education 3810 (54.4) 1301 (53.9) 2509 (54.6) 497 (51.9) 3313 (54.8) Primary 587 (8.4) 185 (7.7) 402 (8.7) 66 (6.9) 521 (8.6) Middle/JSSa 1400 (20.0) 475 (19.7) 925 (20.1) 204 (21.3) 1196 (19.7) Secondary+ 523 (7.4) 197 (8.2) 326 (7.1) 90 (9.4) 433 (7.2) Missing 686 (9.8) 255 (10.5) 431 (9.4) 100 (10.5) 586 (9.7) Household size 1 620 (8.8) 255 (10.6) 365 (7.9) 113 (11.8) 507 (8.4) 2-5 1395 (19.9) 490 (20.3) 905 (19.7) 216 (22.6) 1179 (19.5) 6-9 1074 (15.3) 381 (15.8) 693 (15.1) 124 (13.0) 950 (15.7) 10+ 3711 (53.0) 1211 (50.2) 2500 (54.4) 467 (48.8) 3244 (53.6) Missing 206 (2.9) 76 (3.1) 130 (2.8) 37 (3.8) 169 (2.8) Area of residence Rural 4855 (69.3) 1577 (65.3) 3278 (71.4) 558 (58.3) 4289 (71.0) Urban 2151 (30.7) 836 (34.7) 1315 (28.6) 399 (41.7) 1752 (29.0) Cigarettes smoking in the last month Yes 1042 (14.9) 376 (15.6) 666 (14.5) 139 (14.5) 903 (14.9) No 5964 (85.1) 2037 (84.4) 3927 (85.5) 818 (85.5) 5146 (85.1) Household wealth terciles Most poor 2137 (30.5) 755 (31.3) 1382 (30.1) 283 (29.6) 1854 (30.7) Less poor 2381 (34.0) 824 (34.1) 1557 (33.9) 322 (33.6) 2059 (34.0) Least poor 2282 (32.6) 758 (31.4) 1524 (33.2) 315 (32.9) 1967 (32.5) Missing 206 (2.9) 76 (3.2) 130 (2.8) 37 (3.9) 169 (2.8) Commercial cooking in household Yes 1082 (15.4) 420 (17.4) 662 (14.4) 775 (81.0) 5149 (85.1) No 5924 (84.6) 1993 (82.6) 3931 (85.6) 182 (19.0) 900 (14.9) Sex of youngest child Male 3627 (51.8) 1300 (53.9) 2327 (50.7) 3100 (51.3) 527 (55.1) Female 3345 (47.7) 1102 (45.7) 2243 (48.8) 2917 (48.2) 428 (44.7) Missing 34 (0.5) 11 (0.4) 23 (0.5) 32 (0.53) 2 (0.21) Age of child 0

7.4) 662 (14.4) 775 (81.0) 5149 (85.1) No 5924 (84.6) 1993 (82.6) 3931 (85.6) 182 (19.0) 900 (14.9) Sex of youngest child Male 3627 (51.8) 1300 (53.9) 2327 (50.7) 3100 (51.3) 527 (55.1) Female 3345 (47.7) 1102 (45.7) 2243 (48.8) 2917 (48.2) 428 (44.7) Missing 34 (0.5) 11 (0.4) 23 (0.5) 32 (0.53) 2 (0.21) Age of child 0 1626 (23.2) 594 (24.6) 1032 (22.5) 251 (26.2) 1375 (22.7) 1 1851 (26.4) 696 (28.8) 1155 (25.1) 287 (30.0) 1564 (25.9) 2 1857 (26.5) 588 (24.4) 1269 (27.6) 214 (22.4) 1643 (27.2) 3 841 (12.0) 281 (11.6) 560 (12.2) 98 (10.2) 743 (12.2) 4 831 (11.9) 254 (10.6) 577 (12.6) 107 (11.2) 724 (12.0) Primary cooking fuel LPG/ Electricity 16 (0.2) 8 (0.3) 8 (0.2) 4 (0.4) 12 (0.2) Biomass fuel (Wood/ Charcoal) 6957 (99.3) 2390 (99.1) 4567 (99.4) 945 (98.8) 6012 (99.4) Other 33 (0.5) 15 (0.6) 18 (0.4) 8 (0.8) 25 (0.4) Primary cooking area Open with no roof and walls 4071 (58.1) 1360 (56.3) 2711 (59.0) 556 (58.1) 3515 (58.1) Open with roof only 433 (6.2) 154 (6.4) 279 (6.1) 62 (6.5) 371 (6.1) Partially enclosed with walls and roof 1260 (18.0) 484 (20.1) 776 (16.9) 187 (19.5) 1073 (17.8) Fully enclosed 1227 (17.5) 413 (17.1) 814 (17.7) 151 (15.8) 1076 (17.8) No primary cooking area 15 (0.2) 2 (0.1) 13 (0.3) 1 (0.1) 14 (0.2) Stove for burning wood Open mokyiab 5764 (82.3) 1975 (81.9) 3789 (82.5) 748 (78.2) 5016 (82.9) Clay mokyiab 197 (2.8) 69 (2.9) 128 (2.8) 35 (3.7) 162 (2.7) Metal mokyiab 131 (1.9) 23 (0.9) 108 (2.3) 10 (1.0) 121 (2.0) Other 29 (0.4) 12 (0.5) 17 (0.4) 7 (0.7) 22 (0.4) Do not use wood 885 (12.6) 334 (13.8) 551 (12.0) 157 (16.4) 728 (12.0) Way in obtaining wood Gather from the field only 1287 (18.4) 449 (18.6) 838 (18.3) 721 (75.3) 5059 (83.6) Gather from the field and purchase from market 120 (1.7) 54 (2.2) 66 (1.4) 15 (1.6) 75 (1.3) Purchase from the market only 2455 (35.0) 950 (39.4) 1505 (32.8) 63 (6.6) 190 (3.1) Do not gather wood 3144 (44.9) 960 (39.8) 2184 (47.5) 158 (16.5) 725 (12.0) a JSS - Junior Secondary School

) 838 (18.3) 721 (75.3) 5059 (83.6) Gather from the field and purchase from market 120 (1.7) 54 (2.2) 66 (1.4) 15 (1.6) 75 (1.3) Purchase from the market only 2455 (35.0) 950 (39.4) 1505 (32.8) 63 (6.6) 190 (3.1) Do not gather wood 3144 (44.9) 960 (39.8) 2184 (47.5) 158 (16.5) 725 (12.0) a JSS - Junior Secondary School b Mokyia - A local stove Prevalence of at Least One Respiratory Symptom and Potential Risk Factors At least one respiratory symptom (cough, blocked nose, fast breathing, or chest in drawing) was reported in 34.4% (n=2413, 95% CI 33.3 - 35.5) of children <5 years of age (N=7006). A majority (51.9%) of children with at least one respiratory symptom lived in households with a male head, with those in the rural areas going up to 65.3%. Also 99.3% of male-headed households used biomass fuel (wood or charcoal) as the main primary fuel (Table 2). Blocked nose was the commonest symptom reported (29.6%, 95% CI: 28.4 – 30.6) (Figure 1). Seventeen percent (413/2413) of children who were reported to have had at least one respiratory symptom sought medical treatment. Figure 1 Bar chart of Prevalence of respiratory morbidity among children in the last two weeks prior to the survey (with confidence intervals).

Prevalence of at Least One Respiratory Symptom and Potential Risk Factors At least one respiratory symptom (cough, blocked nose, fast breathing, or chest in drawing) was reported in 34.4% (n=2413, 95% CI 33.3 - 35.5) of children <5 years of age (N=7006). A majority (51.9%) of children with at least one respiratory symptom lived in households with a male head, with those in the rural areas going up to 65.3%. Also 99.3% of male-headed households used biomass fuel (wood or charcoal) as the main primary fuel (Table 2). Blocked nose was the commonest symptom reported (29.6%, 95% CI: 28.4 – 30.6) (Figure 1). Seventeen percent (413/2413) of children who were reported to have had at least one respiratory symptom sought medical treatment. Figure 1 Bar chart of Prevalence of respiratory morbidity among children in the last two weeks prior to the survey (with confidence intervals). Prevalence of ALRI Symptoms and Potential Risk Factors The prevalence of ALRI symptoms (cough with fast breathing and/or difficulty in breathing) was 13.7% (n= 957, 95% CI: 12.8–15.5%). A majority of children with ALRI symptoms lived in households with household heads who had no education (51.9%) and also in households that used biomass fuel (98.8%) as the main primary fuel (Table 2).

ce of ALRI symptoms (cough with fast breathing and/or difficulty in breathing) was 13.7% (n= 957, 95% CI: 12.8–15.5%). A majority of children with ALRI symptoms lived in households with household heads who had no education (51.9%) and also in households that used biomass fuel (98.8%) as the main primary fuel (Table 2). Cooking Practices In all households contacted, a majority (77.8%, 95% CI 77.7 - 78.5%) of households used wood as their primary fuel (Table 3). Majority of respondents who used wood as their primary fuel obtained them by gathering wood from their neighborhood (95.6%, 9177/9595) and used a 3-stone local stove for cooking (94.9%, 9101/9595). Charcoal use was less common (Table 3). LPG or electricity use was rare (Table 3). A majority of respondents primarily cooked in open areas with no roofs and walls (56.7%). Partially enclosed areas were the commonest (38.0%) secondary cooking area (Figure 2). None of the cooking practice variables was significantly associated with respiratory symptoms. Table 3 Cooking practices among all households N=12,333

Cooking Practices In all households contacted, a majority (77.8%, 95% CI 77.7 - 78.5%) of households used wood as their primary fuel (Table 3). Majority of respondents who used wood as their primary fuel obtained them by gathering wood from their neighborhood (95.6%, 9177/9595) and used a 3-stone local stove for cooking (94.9%, 9101/9595). Charcoal use was less common (Table 3). LPG or electricity use was rare (Table 3). A majority of respondents primarily cooked in open areas with no roofs and walls (56.7%). Partially enclosed areas were the commonest (38.0%) secondary cooking area (Figure 2). None of the cooking practice variables was significantly associated with respiratory symptoms. Table 3 Cooking practices among all households N=12,333 N % Primary cooking fuel LPG/ Electricity 63 0.5 Charcoal 2364 19.2 Wood 9595 77.8 Other 311 2.5 Primary cooking area Open with no roof and walls 6996 56.7 Open with roof only 748 6.1 Partially enclosed with walls and roof 2208 17.9 Fully enclosed 2101 17.0 No primary cooking area 280 2.3 Stove for burning wood Open mokyia 9691 78.6 Clay mokyia 319 2.6 Metal mokyia 214 1.7 Other 41 0.3 Do not use wood 2068 16.8 Way in obtaining wood* Gather 9660 78.3 Gather and purchase 150 1.2 Purchase 455 3.7 Do not use wood 2068 16.8 * Includes respondents who use wood as their primary or secondary source of fuel.

.3 Stove for burning wood Open mokyia 9691 78.6 Clay mokyia 319 2.6 Metal mokyia 214 1.7 Other 41 0.3 Do not use wood 2068 16.8 Way in obtaining wood* Gather 9660 78.3 Gather and purchase 150 1.2 Purchase 455 3.7 Do not use wood 2068 16.8 * Includes respondents who use wood as their primary or secondary source of fuel. Figure 2 Proportion of secondary cooking areas among all households that had a secondary cooking area (N= 4825). Numerator (n) for each secondary cooking area is as follows: Open with no roof and walls =1027, Open with roof only =348, partially enclosed with walls and roof= 1833, fully enclosed=1617 Legend: Bar chart of the prevalence of respiratory symptoms among children less than five years old (N=7006) in the Kintampo area, 2007. Numerator (n) for each symptom is as follows: cough =1775, blocked nose/runny nose=2070, fast breathing =944, chest in-drawing =692 In a randomly selected subset of respondents, females were the persons who mostly gathered firewood from the fields (90.8%, 296/326) and did the cooking (94.8%, 384/406) for the household; 58.7% (247/421) of households cooked on their farms outside their homes. In the same subset of households, 74% (313/421), 47.7% (201/421), and 93.6% (394/421) of households cooked breakfast, lunch, or supper respectively. The median time for cooking was 2 hours and 20 minutes (range 0 – 5 hours, 30 minutes).

household; 58.7% (247/421) of households cooked on their farms outside their homes. In the same subset of households, 74% (313/421), 47.7% (201/421), and 93.6% (394/421) of households cooked breakfast, lunch, or supper respectively. The median time for cooking was 2 hours and 20 minutes (range 0 – 5 hours, 30 minutes). Awareness of Cook-Stoves and Health Risk of Smoke In the randomly selected subset of households, a majority (92.4%, n=389/421) of respondents were not aware of improved cookstoves. However, most respondents were aware of health hazards associated with smoke. About 97.4% (410/421) were aware that tobacco could lead to health hazard and cook smoke could lead to childhood pneumonia (96.7%, n=407/421) or lung disease in adults (96.4%, n=406/421). Other sources of smoke identified by household members were those from mosquito coils (39.7%, 167/421) as well as from paraffin or kerosene lantern (92.6%, 390/421). Household Decision-Making and Sources of Information In all households, fathers/husbands were the primary decision makers for households in health seeking (64.3%, 7924/12,333) or for major item purchases (67.2%, 8286/12,333). Mothers/wives made primary decisions in 25.4% (3135/12,333) of health-seeking in households and 23.1% (2850/12,333) for major items purchases. The local radio was the commonest primary source of agricultural (55.2%) or health (51.4%) information (Table 4). Other sources included agricultural officers or health officers. Table 4 Source of information among households (N=12,333)

Household Decision-Making and Sources of Information In all households, fathers/husbands were the primary decision makers for households in health seeking (64.3%, 7924/12,333) or for major item purchases (67.2%, 8286/12,333). Mothers/wives made primary decisions in 25.4% (3135/12,333) of health-seeking in households and 23.1% (2850/12,333) for major items purchases. The local radio was the commonest primary source of agricultural (55.2%) or health (51.4%) information (Table 4). Other sources included agricultural officers or health officers. Table 4 Source of information among households (N=12,333) Agricultural information Health information Source N % N % Radio 6,813 55.2 6,338 51.4 Nearby neighbors 1,275 10.3 302 2.5 Agricultural officer 877 7.1 26 0.2 Community elders 810 6.6 313 2.5 Other 384 3.1 80 0.7 Other community members 330 2.7 210 1.7 Health officer 47 0.4 4,285 34.7 Extended family 26 0.2 12 0.1 Friend elsewhere 24.0 0.2 8 0.1 NK 1,747 14.2 759 6.2 Discussion A survey was conducted in a predominantly rural area of Ghana to determine household respiratory morbidity and cooking practices among young children. The prevalence of at least one respiratory symptom (cough, blocked nose, fast breathing, or chest in drawing) was very high among children who were less than five years of age at the time of the survey. Cough was one of the commonest symptoms reported. The reported cough could be pathological or non-pathological (Bonney et al., 2012). Though it is likely that a proportion of the symptoms reported may not be pathological, the prevalence of runny nose suggests a high probability of an upper respiratory tract infection (URTI). URTI may result from common viruses such as Influenza Sp that sometimes occur as outbreaks but are usually eliminated within a short period (i.e. within periods less than 6 months) (Iskander et al., 2013). It could have been possible an URTI outbreak occurred at the time of this survey, but this was unlikely as there was no report of such an outbreak by health authorities in our surveyed area in the six-month period of our survey. The prevalence of respiratory symptoms determined in this survey is similar to that obtained in other surveys in rural areas of Kenya (Feikin et al., 2011), and in industrialized countries where air pollution is high (Kumar et al., 2007; Ranzi et al., 2014).

s in our surveyed area in the six-month period of our survey. The prevalence of respiratory symptoms determined in this survey is similar to that obtained in other surveys in rural areas of Kenya (Feikin et al., 2011), and in industrialized countries where air pollution is high (Kumar et al., 2007; Ranzi et al., 2014). The prevalence of ALRI symptoms (13.7%), though lower than that of URTI represents a significant health burden if they truly reflect underlying ALRI prevalence. In studies conducted in southern Ghana, the prevalence of ALRI symptoms assessed by trained community health personnel was <10% (Chinbuah et al., 2013). In our study, ALRI symptoms were reported by mothers or caregivers and not by health professionals or trained community volunteers. It is therefore likely that the prevalence determined in this survey could have been under-estimated since mild to moderate breathlessness or chest in-drawing may go unnoticed by the mother as was found in surveys in other parts of Ghana (Denno et al., 1994) and in Ethiopia (Muhe, 1996).

r trained community volunteers. It is therefore likely that the prevalence determined in this survey could have been under-estimated since mild to moderate breathlessness or chest in-drawing may go unnoticed by the mother as was found in surveys in other parts of Ghana (Denno et al., 1994) and in Ethiopia (Muhe, 1996). The use of firewood for cooking was very high in the study area and similar to other rural areas of Ghana and developing countries (Bonjour et al., 2013; GSS/GHS/GNPHRL, 2015). Uncontrolled, unventilated firewood combustion leads to air pollution, which is associated with high disease burden including respiratory diseases (Thacher et al., 2013; Wong et al., 2013). In this survey, no associations were sought between use of firewood and respiratory symptoms. This is due to the fact that few households (0.5%) used clean cook stoves or fuels (electricity or LPG) as their primary cooking fuel and we lacked exposure monitoring at the time of the survey to test for exposure-response among those families using wood for cooking. Respiratory symptoms were higher among children living in urban areas. It is likely that the children living in urban areas are exposed to other risk factors of respiratory signs such as smoke from vehicles.

monitoring at the time of the survey to test for exposure-response among those families using wood for cooking. Respiratory symptoms were higher among children living in urban areas. It is likely that the children living in urban areas are exposed to other risk factors of respiratory signs such as smoke from vehicles. A majority of households primary cooks were women of childbearing age. We assume that, if pregnant, the foetus of these primary cooks were likely to be exposed to environmental pollutants such as carbon monoxide that may lead to poor health outcomes (Naeher et al., 2000). The GRAPHS study (Jack et al., 2015) currently under way in Ghana is seeking to evaluate the potential impact of use of improved cook-stoves on low birth weight and infant respiratory morbidity and will be able to provide additional data to help explain this assumption being made in the manuscript. Other cohort studies will be required to investigate the potential impact of using improved cook-stoves on other infant health outcomes such as motor and neurodevelopment and infant anthropometry growth.

morbidity and will be able to provide additional data to help explain this assumption being made in the manuscript. Other cohort studies will be required to investigate the potential impact of using improved cook-stoves on other infant health outcomes such as motor and neurodevelopment and infant anthropometry growth. In many developing sub-Saharan countries, government policies recognize the need to provide improved cook-stoves as means of maintaining trees and plantations to halt climate change (Kgathi and Zhou, 1995). In Ghana, pilot programmes are being implemented by providing free LPG stoves to some rural communities. There is the need to take advantage of such programmes to assess the health benefits of improved cook-stoves on non-communicable diseases such as cardiovascular diseases. In planning such intervention studies, there is the need to consider the social context within which the study will be carried out. In the current study, knowledge of improved cook-stoves is documented to be limited. Community members will therefore need to be educated on the use of the chosen improved cook-stove to ensure compliance through an appropriate medium. Community durbars and community worker discussion of topical issues in the study were found to be the best ways of deploying information. Even though, interventions of this nature may be targeted at women and their children, household decision makers will need to be consulted to ensure acceptance and compliance to the improved cook-stove as majority of households decisions are made by male household heads.

nd to be the best ways of deploying information. Even though, interventions of this nature may be targeted at women and their children, household decision makers will need to be consulted to ensure acceptance and compliance to the improved cook-stove as majority of households decisions are made by male household heads. Study Limitation A cross-sectional survey was conducted and therefore temporal relationship between use of cook-stoves and respiratory symptoms cannot be assessed. Secondly the symptoms such as breathlessness was not counted but was dependent on the subjective observations of the mother or caregiver of the child. Studies will however require counting and observations by a trained health professional or community worker as was used in other studies in Ghana (Kirkwood et al., 2010b) and the RESPIRE study (Smith et al., 2011). Conclusion Symptoms of ALRI reported by caregivers are high in the Kintampo area of Ghana where use of biomass fuel use is also high. There is the need to plan interventions to use improved cook-stoves and to test the health benefits of such interventions. Acknowledgement The authors are grateful to the staff of Kintampo Health Research Centre and to Columbia University for supporting this study; the community leadership and community members, local political leaders and local government agencies and Ghana Health Service. The Kintampo Health Research Centre is a member-centre of the INDEPTH Network.

Acknowledgement The authors are grateful to the staff of Kintampo Health Research Centre and to Columbia University for supporting this study; the community leadership and community members, local political leaders and local government agencies and Ghana Health Service. The Kintampo Health Research Centre is a member-centre of the INDEPTH Network. Authors’ contribution KP, DJ, SOA and PK conceived the idea. All authors contributed to the study implementation. KP and EN analyzed the data. KP wrote the first draft of the paper and the final version was reviewed and approved by all authors. SOA, the Director of the Kintampo Health Research Centre and PK, the Director of Columbia Climate and Health Program, Department of Environmental Health Sciences, Mailman School of Public Health reviewed and approved the paper on behalf of the participating institutions. Competing Interest: All authors declared that they have no competing interest.

Introduction Avian Influenza (AI) is an infectious disease of birds caused by influenza A viruses. Migratory waterfowls - most notably wild ducks - are the natural reservoirs of all influenza A viruses (Hinshaw and Webster, 1982; Webster et al., 1992; Stallknecht and Brown 2007). There are 16 main subtypes of influenza A viruses, of which strains within the H5 and H7 subtypes cause Highly Pathogenic Avian Influenza (HPAI), which is highly contagious and rapidly fatal resulting in nearly 100% mortality in infected domestic flocks (Center for Infectious Diseases Research and Policy, 2007).

Introduction Avian Influenza (AI) is an infectious disease of birds caused by influenza A viruses. Migratory waterfowls - most notably wild ducks - are the natural reservoirs of all influenza A viruses (Hinshaw and Webster, 1982; Webster et al., 1992; Stallknecht and Brown 2007). There are 16 main subtypes of influenza A viruses, of which strains within the H5 and H7 subtypes cause Highly Pathogenic Avian Influenza (HPAI), which is highly contagious and rapidly fatal resulting in nearly 100% mortality in infected domestic flocks (Center for Infectious Diseases Research and Policy, 2007). Recently, avian influenza has acquired world-wide attention because the H5N1 virus had traversed interclass barriers (Perkins and Swayne, 2003) and had been transmitted from birds to mammals (cats, swine, and humans). Substantial number of documented cases in humans are associated with severe disease and several fatalities (Klempner and Shapiro, 2004; Webster, 2006). There are several further lines of evidence suggesting that the H5N1 virus has acquired increased pathogenic potency for several mammalian species. Chickens and turkeys are particularly susceptible to epidemics; direct or indirect contact of domestic flocks with wild waterfowl has been implicated as a frequent cause (Easterday, et al 1997; Stalknecht and Brown, 2007). Birds that survive infection often excrete virus for up to 10 days, orally and in faeces, thus facilitating further spread (Olsen et al., 2005; Swayne and Beck, 2005). Suspicions that birds may be carrying highly pathogenic virus along their migratory routes were underscored following the detection of outbreaks in wild and domestic birds in the Russian Federation and adjacent parts of Kazakhstan, so also Turkey, Romania, and Croatia (OIE, 2005, WHO, Global Alert and Response).

2005). Suspicions that birds may be carrying highly pathogenic virus along their migratory routes were underscored following the detection of outbreaks in wild and domestic birds in the Russian Federation and adjacent parts of Kazakhstan, so also Turkey, Romania, and Croatia (OIE, 2005, WHO, Global Alert and Response). Most avian influenza viruses affecting humans have caused mild respiratory symptoms or conjunctivitis, with one important exception: the H5N1 strain. The H5N1 strain has caused severe disease throughout the world with high fatality rates starting from 1997, till date. Studies comparing virus samples overtime show that H5N1 has become progressively more pathogenic for mammals, and is now hardier than in the past, surviving several days longer in the environment (Olsen et al., 2005; Beck, 2005; EC, 1992 amended 2004). In 2004, H5N1 caused fatal disease in naturally infected large felines (tigers and leopards) and experimentally infected domestic cats-species not previously considered susceptible to disease caused by any influenza A virus. Several mutations in the virus have been detected during 2005, but the significance of these mutations in terms of virulence and transmissibility in humans is not fully understood.

opards) and experimentally infected domestic cats-species not previously considered susceptible to disease caused by any influenza A virus. Several mutations in the virus have been detected during 2005, but the significance of these mutations in terms of virulence and transmissibility in humans is not fully understood. In April 2007, despite a ban on importation of poultry and poultry products from HPAI H5N1 affected countries (mostly Southeast Asian countries) and several biosecurity measures enforced by the Government of Ghana, the first outbreak of H5N1 was reported in Ghana by VSD at a small – scale poultry farm at Kakasunanka, near Michel Camp in the Tema Metropolis (April 24, 2007). Subsequently, outbreaks of AI were reported in Sunyani in the Brong Ahafo region (May 15, 2007) and Aflao in the Volta region in the same year (June 13, 2007). To date, the possible reasons for the emergence and spread of the disease in the country have not been determined. The disease is associated with enormous economic loss and the cost of veterinary interventions and public education on prevention and control of the disease was estimated at 2 million US dollars of which came from donor partners including USAID and FAO (VSD Annual Report, 2009). Unlike chickens, some domestic ducks are known to be resistant to the viruses and can be asymptomatic carriers of the viruses, thus acting as a “silent reservoir” that perpetuates transmission (Swayne and Beck, 2005 and European Commission (EC), 1992 amended 2004).

The disease is associated with enormous economic loss and the cost of veterinary interventions and public education on prevention and control of the disease was estimated at 2 million US dollars of which came from donor partners including USAID and FAO (VSD Annual Report, 2009). Unlike chickens, some domestic ducks are known to be resistant to the viruses and can be asymptomatic carriers of the viruses, thus acting as a “silent reservoir” that perpetuates transmission (Swayne and Beck, 2005 and European Commission (EC), 1992 amended 2004). The experience of a second outbreak of HPAI in Togo in 2008 after the 2007 outbreak has shown the ability of H5N1 virus to persist discreetly among traditional farms (scavenging poultry) where chicken mortality is common and usually go unreported. Although the role of ducks and related species in the viral circulation of avian influenza was reported in many countries including Nigeria in the 2008 (Alice et al., 2008), the status of avian influenza virus infections in domestic ducks in Ghana especially that associated with the Sunyani Municipality which shares border with La Cote d’Ivoire, a country which reported AI outbreaks in 2006 has not been determined, hence this study.

tries including Nigeria in the 2008 (Alice et al., 2008), the status of avian influenza virus infections in domestic ducks in Ghana especially that associated with the Sunyani Municipality which shares border with La Cote d’Ivoire, a country which reported AI outbreaks in 2006 has not been determined, hence this study. Method Study Area The study was conducted in the Sunyani Municipality, one of the twenty-two districts of the region which is the capital of the Brong Ahajo region. It lies between latitude 70 20’N and 70 05’N and longitude 20 30’W and 20 10 W and shares boundaries with Sunyani West District to the north, Dormaa District to the west, and Asutifi District to the South and Tano North District to the East. The Municipality has a total land area of 829.3 square kilometers (320.1 square miles). Sunyani Municipality (Fig 1) had an estimated ducks population of 25,036 (according to the Statistics, Research and Information Directorate data, MOFA, 2005) before the 2007 H5N1 outbreak. Many of these ducks were destroyed during the 2007 HPAI outbreaks. Presently, fourteen ducks farmers in the Municipality who abandoned the trade because of the outbreaks in 2007 have become operational again. The population of ducks keepers in the Municipality is estimated at 107. Duck population in the area was estimated at 5,000 after collecting census data from the various active ducks rearing sites in the Municipality. Figure 1 Source: Epidemiology Unit, VSD, Accra Ghana, 2011. Map showing Sampling Sites in the Sunyani Municipality, July 2009- October, 2010.

Sunyani Municipality (Fig 1) had an estimated ducks population of 25,036 (according to the Statistics, Research and Information Directorate data, MOFA, 2005) before the 2007 H5N1 outbreak. Many of these ducks were destroyed during the 2007 HPAI outbreaks. Presently, fourteen ducks farmers in the Municipality who abandoned the trade because of the outbreaks in 2007 have become operational again. The population of ducks keepers in the Municipality is estimated at 107. Duck population in the area was estimated at 5,000 after collecting census data from the various active ducks rearing sites in the Municipality. Figure 1 Source: Epidemiology Unit, VSD, Accra Ghana, 2011. Map showing Sampling Sites in the Sunyani Municipality, July 2009- October, 2010. Study Design A descriptive cross-sectional study using active surveillance approach was carried out as indicated in the scheme below. It entailed simultaneous collection of cloacal swabs and feather tissues from three hundred and eighty-four domestic ducks, and the administration of a structured questionnaire to 17 ducks owners/workers on issues of husbandry practices and bio-security at their farms or premises (households). The study was carried out from July 2009 to August 2010.

tion of cloacal swabs and feather tissues from three hundred and eighty-four domestic ducks, and the administration of a structured questionnaire to 17 ducks owners/workers on issues of husbandry practices and bio-security at their farms or premises (households). The study was carried out from July 2009 to August 2010. Data Collection Technique Data was collected with structured questionnaires (Appendix II). The questionnaire was developed and pre-tested in Berekum, an adjacent district before it was administered to 17 persons who were either owners or workers of the ducks sampled as well as live birds vendors at the Sunyani live birds market whose birds were sampled. Questionnaire administration was done simultaneously with sample collection. Sample Size Determination The sample size was calculated by the Epi info version 3.4.1 at 95% confidence level, absolute precision of 5% and assuming 0.5 prevalence of Avian Influenza among domestic ducks in Sunyani: where N= sample size, z= risk of Type 1 error=1.96 at 95% confidence level p= prevalence of AI = 0.5 (arbitrary proxy) d= absolute precision = 5% = 0.05 Total number of domestic ducks sampled = 384 Sample size = 384 cloacal swabs plus 142 feather tissues = 526

Sample Size Determination The sample size was calculated by the Epi info version 3.4.1 at 95% confidence level, absolute precision of 5% and assuming 0.5 prevalence of Avian Influenza among domestic ducks in Sunyani: where N= sample size, z= risk of Type 1 error=1.96 at 95% confidence level p= prevalence of AI = 0.5 (arbitrary proxy) d= absolute precision = 5% = 0.05 Total number of domestic ducks sampled = 384 Sample size = 384 cloacal swabs plus 142 feather tissues = 526 Sampling Method Thirteen (13) epidemiological units (epi units) or communities in the Sunyani Municipality were identified for the study. Simple random sampling method was used to select a community at a time for ducks to be identified in households and farms within that community for sample collection. This procedure was repeated till the desired sample size for the study was attained from nine (9) sites in six communities. Ethical Considerations and Consent Approval for the study was duly obtained from the Scientific Technical Committee of Noguchi Memorial Institute for Medical Research (NMIMR). A written informed consent was obtained from the Brong Ahafo Regional Veterinary Officer, Commercial and Backyard ducks owners, and vendors at the Live Birds Market in the selected communities to carry out the study.

ned from the Scientific Technical Committee of Noguchi Memorial Institute for Medical Research (NMIMR). A written informed consent was obtained from the Brong Ahafo Regional Veterinary Officer, Commercial and Backyard ducks owners, and vendors at the Live Birds Market in the selected communities to carry out the study. Sample Collection and Processing 384 Cloacal swabs and 142 feather tissues (calamuses) were obtained from backyard ducks, a commercial farm and a live birds market. All field samples collected were pooled separately by type (cloacal swabs separate from feather tissues). Two cloacal swabs from two (2) different birds from the same site were placed into a 2.0 ml tube while ten (10) feather calamuses with similar characteristics were pooled into a 25 ml vacutainer tube with both tubes containing 2 ml and 5 ml of viral transport medium (VTM) which contained 2.5% Veal Infusion Broth (SIGMA), 0.5% Bovine Serum Albumin (SIGMA), 100 μg/ml Gentamycin sulphate (SIGMA) and 2 μg/ml Fungizone (Amphotericin B) solution. The vacutainer tubes containing these samples were properly labelled with information that described a unique identification number and dates of collection. They were placed on ice in “cold boxes” from the field and transported to the Sunyani Veterinary office where they were stored at -70 °C. RNA extraction and RRT-PCR was carried out in the Virology Department of NMIMR in Accra.

elled with information that described a unique identification number and dates of collection. They were placed on ice in “cold boxes” from the field and transported to the Sunyani Veterinary office where they were stored at -70 °C. RNA extraction and RRT-PCR was carried out in the Virology Department of NMIMR in Accra. Preparation for RNA Extraction The 526 samples (384 cloacal swabs and 142 feather tissues) collected from the field were pooled into 43 eppendof f tubes according to the farm/household where samples were obtained from. Thirty one (31) of these pooled samples were cloacal swabs and 12 were feather specimens. Pooling of the samples was done to ensure optimization of the use of reagents. RNA Extraction Single stranded viral RNA was extracted using the QIAamp® Viral RNA Mini Kit commercially available from QIAGEN (Qiagen, Hilden, Germany). The viral RNA mini spin procedure as recommended by the manufacturer was used and the manufacturer’s instructions were followed. Real-Time Reverse Transcription-PCR PLATE SETUP The RT-PCR plate setup for both protocols; Spackman et al, 2002 and CDC protocol (WHO, 2009) was the same and as follows: the Negative and Positive Controls were distantly placed (A1 and H12 wells of the plates respectively) to avoid contamination. The field samples were placed systematically from the A4 well to C9 well of the PCR plate.

tup for both protocols; Spackman et al, 2002 and CDC protocol (WHO, 2009) was the same and as follows: the Negative and Positive Controls were distantly placed (A1 and H12 wells of the plates respectively) to avoid contamination. The field samples were placed systematically from the A4 well to C9 well of the PCR plate. Spackman et al Protocol The Qiagen one-step RT-PCR kit was used with a 20ul reaction mixture under the following conditions: 0.8 ul of kit supplied enzyme mixture (including RT and hot-start Taq polymerase), 10 pmol of each primer, 0.3 uM probe, 400 uM (each) dNTPs, 3.75mM MgCl2 and 6.5U of RNase inhibitor (Promega, Madison, Wisconsin). Reverse transcription was achieved at 50°C for 30 minutes .Taq polymerase activation was at 90°C for 15 minutes. A two-step PCR cycling protocol was then used for the matrix gene primer and probe set as follows 45 cycles of 94°C for 0 seconds for denaturation and 60°C for 20 seconds annealing. Real-time RT-PCR was performed with Applied Biosystems Incorporated (ABI) 7300 system thermocycler and software.

s at 90°C for 15 minutes. A two-step PCR cycling protocol was then used for the matrix gene primer and probe set as follows 45 cycles of 94°C for 0 seconds for denaturation and 60°C for 20 seconds annealing. Real-time RT-PCR was performed with Applied Biosystems Incorporated (ABI) 7300 system thermocycler and software. Confirmation of Results Using Real-Time Reverse Transcription-PCR, CDC Protocol (WHO, 2009) The negative results obtained using the Spackman protocol were confirmed as described by the Centres for Disease Control and Prevention, Atlanta USA in their protocol CDC Real-time RTPCR (rRT-PCR) Protocol (Table 1) for Detection and Characterization of Influenza (version 2009) using reagents from the Invitrogen One-Step Superscript III RT-PCR kit. Primers designed and supplied by the CDC were used at a concentration of 20 pmol each in a 25 ul reaction mix with 0.5 ul kit supplied enzyme, 0.25 uM probe (designed by the CDC) and 12.5 ul of kit supplied 2X reaction mix, a buffer containing 0.4 mM of each dNTP, 2.4 mM MgSO4. Cycling conditions for all primer sets (Table 2) was 50°C for 30 minutes for the reverse transcriptase step, a Taq polymerase activation step of 95°C for 2 minutes and 45cycles of 95°C for 15 seconds and 55°C for 30seconds denoting denaturing and annealing steps (Table 3, 4 and 5). Flourescent data was collected during the annealing step at 55°C. Real-time RT-PCR was performed with the Applied Biosysytems Incorporated 7300 system thermocycler and software. Table 1 PCR Master Mix Formula (Spackman et al., 2002)

Confirmation of Results Using Real-Time Reverse Transcription-PCR, CDC Protocol (WHO, 2009) The negative results obtained using the Spackman protocol were confirmed as described by the Centres for Disease Control and Prevention, Atlanta USA in their protocol CDC Real-time RTPCR (rRT-PCR) Protocol (Table 1) for Detection and Characterization of Influenza (version 2009) using reagents from the Invitrogen One-Step Superscript III RT-PCR kit. Primers designed and supplied by the CDC were used at a concentration of 20 pmol each in a 25 ul reaction mix with 0.5 ul kit supplied enzyme, 0.25 uM probe (designed by the CDC) and 12.5 ul of kit supplied 2X reaction mix, a buffer containing 0.4 mM of each dNTP, 2.4 mM MgSO4. Cycling conditions for all primer sets (Table 2) was 50°C for 30 minutes for the reverse transcriptase step, a Taq polymerase activation step of 95°C for 2 minutes and 45cycles of 95°C for 15 seconds and 55°C for 30seconds denoting denaturing and annealing steps (Table 3, 4 and 5). Flourescent data was collected during the annealing step at 55°C. Real-time RT-PCR was performed with the Applied Biosysytems Incorporated 7300 system thermocycler and software. Table 1 PCR Master Mix Formula (Spackman et al., 2002) MASTER MIX VOLUME (μl) VOLUME/TUBE TOTAL Water 3.1 3.1 155 5 x Buffer of Qiagen kit 4 4 200 MgCl (Promega) 1 1 50 dNTPs 0.8 0.8 40 M+64(FAM-TAMRA) probe 0.3 0.3 15 M+25 Primer 0.5 0.5 25 M+124 Primer 0.5 0.5 25 ROX dye working dilution (1:100) 1 1 50 Qiagen one-step Enzyme Mix 0.8 0.8 40 12 600 Template Volume 8 Number of tubes: 50 Reaction Volume 20 Table 2 PCR Primer and Hydrolysis Probe Sequence for AI Virus Detection (Spackman)

NTPs 0.8 0.8 40 M+64(FAM-TAMRA) probe 0.3 0.3 15 M+25 Primer 0.5 0.5 25 M+124 Primer 0.5 0.5 25 ROX dye working dilution (1:100) 1 1 50 Qiagen one-step Enzyme Mix 0.8 0.8 40 12 600 Template Volume 8 Number of tubes: 50 Reaction Volume 20 Table 2 PCR Primer and Hydrolysis Probe Sequence for AI Virus Detection (Spackman) Specificity Primer/probe Sequence "(5’-3’) Influenza Matrix AM + 25 AGA TGA GTC TTC TAA CCG AGG TCG M- 124 TGC AAA AAC ATC TTC AAG TCT CTG M+64 FAM-TCA GGC CCC CTC AAA GCC GA-TAMRA FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetralrhodamine. Source: Spackman et al 2002 Table 3 Amplification Cycling (Spackman) RT Reaction Starting Denaturation Annealing 50°C 94°C 94°C 60°C 30 min 15 min 0 sec 29 sec 45 cycles Table 4 PCR Master Mix Formula (CDC Protocol) MASTER MIX VOLUME (μl) VOLUME/TUBE TOTAL Nuclease Free Water 5.0 5.0 230 2 x Reaction Mix 12.5 12.5 575 Probe (FAM-TAMRA) 0.5 0.5 23 Forward Primer 0.5 0.5 23 Reverse Primer 0.5 0.5 23 25 x RT-PCR Enzyme 1.0 1.0 46 Total 20 920 Template Volume 5.0 5.0 Number of tubes: 46 Reaction Volume 25 Table 5 Amplification cycling (CDC Protocol) RT Reaction Starting Denaturation Denaturing Annealing 50°C 95°C 95°C 55°C 30 min 2 min 15 sec 30 sec

MASTER MIX VOLUME (μl) VOLUME/TUBE TOTAL Nuclease Free Water 5.0 5.0 230 2 x Reaction Mix 12.5 12.5 575 Probe (FAM-TAMRA) 0.5 0.5 23 Forward Primer 0.5 0.5 23 Reverse Primer 0.5 0.5 23 25 x RT-PCR Enzyme 1.0 1.0 46 Total 20 920 Template Volume 5.0 5.0 Number of tubes: 46 Reaction Volume 25 Table 5 Amplification cycling (CDC Protocol) RT Reaction Starting Denaturation Denaturing Annealing 50°C 95°C 95°C 55°C 30 min 2 min 15 sec 30 sec 45 cycles Data Analysis/Processing Both quantitative and qualitative data obtained from the questionnaires and data from the RRT-PCR testing was double entered into Epidata Software (2007 version) and coded accordingly. This was then exported to SPSS Software version 17.0 for analysis. Data was analyzed into percentages and tables based on adherence or non-adherence of each farm premises to the 19 independent variables or biosecurity practices (Tables 11a-11c) investigated in this study. Test of association (chi-square) was also performed on certain variables (age and sex) to determine the significance. Results Descriptive Characteristics of Birds Sampled Apart from being raised for meat and eggs, some ducks in the area were also kept as pets or for ornamental value. It was observed that the ducks in the area were mostly hybrids from the Anas platyrhynchos domesticus family, particularly the Mallards. Eighty four percent (323/384) of the ducks sampled were hybrids; the remaining 16% (61/384) were made up of thorough Mallard breeds (Aylesbury, peking and pennine).

ornamental value. It was observed that the ducks in the area were mostly hybrids from the Anas platyrhynchos domesticus family, particularly the Mallards. Eighty four percent (323/384) of the ducks sampled were hybrids; the remaining 16% (61/384) were made up of thorough Mallard breeds (Aylesbury, peking and pennine). Out of the 526 samples collected (Table 6), 58.6% (308/526) were from backyard holdings, 34% (179/526) from a commercial farm and 7.4% (39/526) from a live birds market (LBM). Table 6 Details of samples collected from ducks in the Sunyani Municipality (July 2009 - October 2010). Farm Number Farm Size Quantity of Samples Collected Location of Farm Cloacal swabs Feather samples 1* 1,113 119 60 Dumasua 2 25 24 7 New Dormaa 3 12 12 7 New Dormaa 4 23 21 7 New Dormaa 5 241 57 20 Abesim 6** 38 30 10 New Dormaa 7* 44 32 7 Sunyani LBM 8 78 46 17 Odomase 9 75 43 7 Adantia Total 1,649 384 142 * Farm numbers 1 and 7 are; a Commercial Farm and a Live Birds Market (LBM) respectively. The remaining seven are backyard holdings. ** The only duck farm sampled that experienced outbreak in 2007. Seventy six percent (292/384) of the ducks were females (ducks) and 24% (92/384) were males (drakes) (Table 7). Adult ducks (> 1 year old) represented 79.9% (307/384) of the sampled population while growers or ducklings (< 1 year old) formed only 20.1% (77/384) of the same population (Table 8). The number of ducks of age greater than 1 year compared to those less than 1 year was found to be statistically significant at p<0.0001. The same was for male and female ducks sampled (Table 9 and 10).

pled population while growers or ducklings (< 1 year old) formed only 20.1% (77/384) of the same population (Table 8). The number of ducks of age greater than 1 year compared to those less than 1 year was found to be statistically significant at p<0.0001. The same was for male and female ducks sampled (Table 9 and 10). Table 7 Sex Distribution of Ducks by Farm/Household in Sunyani Municipality (July 2009 - August 2010). Farm Number Male Female Total Number % Number % Number % 1 25 20 100 80 125 100 2 5 27.8 13 72.2 18 100 3 3 25 9 75 12 100 4 6 28.6 15 71.4 21 100 5 16 28.1 41 71.9 57 100 6 8 26.7 22 73.3 30 100 7 11 34.4 21 65.6 32 100 8 10 21.7 36 78.3 46 100 9 8 18.6 35 81.4 43 100 Total 92 24 292 76 384 100 The above Table shows the number of males and female ducks in each of the nine sites studied. The calculated Pearson Chi-square was 5.195 with a p < 0.0001. The difference was found to be statistically significant at 99.9% CL in all the nine sites. Table 8 Age Grouping of Ducks Sampled in the Sunyani Municipality, (July 2009 - August 2010).

Farm Number Male Female Total Number % Number % Number % 1 25 20 100 80 125 100 2 5 27.8 13 72.2 18 100 3 3 25 9 75 12 100 4 6 28.6 15 71.4 21 100 5 16 28.1 41 71.9 57 100 6 8 26.7 22 73.3 30 100 7 11 34.4 21 65.6 32 100 8 10 21.7 36 78.3 46 100 9 8 18.6 35 81.4 43 100 Total 92 24 292 76 384 100 The above Table shows the number of males and female ducks in each of the nine sites studied. The calculated Pearson Chi-square was 5.195 with a p < 0.0001. The difference was found to be statistically significant at 99.9% CL in all the nine sites. Table 8 Age Grouping of Ducks Sampled in the Sunyani Municipality, (July 2009 - August 2010). Farm Number Ducks greater than one year old Ducks less than 1 year old Total Number % Number % Number % 1 94 75.2 31 24.8 125 100.0 2 5 27.8 13 72.2 18 100.0 3 12 100.0 0 0.0 12 100.0 4 15 71.4 6 28.6 21 100.0 5 54 94.7 3 5.3 57 100.0 6 30 100.0 0 0.0 30 100.0 7 26 81.3 6 18.8 32 100.0 8 40 87.0 6 13.0 46 100.0 9 31 72.1 12 27.9 43 100.0 Total 307 79.9 77 20.1 384 100.0 The above Table shows the number of adult ducks (age > 1 year) and that of ducklings (age < 1 year) in each of the nine sites studied. The calculated Pearson Chi-square was 54.501 with a p < 0.0001. This was found to be statistically significant at 99.9% CL in all nine cases. Table 9 Results of RRT-PCR Tests for AI conducted at the NMIMR Virology Department using both Spackman et al., 2002 and CDC Protocols (WHO, 2009), between October 2010 and April 2011 respectively

ercial fetchers go around scavenging for water. This discourages the copious use of water among the butchers for pig slaughtering and cleaning. The water supply facilities in Lagos and Ogun were functional although the bore-hole facility was majorly run on locally generated power. This made supply occasionally erratic. Biological Intrusions These are humans and animals which were observed on the slaughter area without direct involvement and benefit to the slaughter processes. (Figure 2 & 4). In Oyo state, the second area of the abattoir which is an in-door area also serves as the sales point. This arrangement therefore makes this area often occupied with people who are only interested in buying the products. They intrude into the slaughter area with their wares and containers for collection of their purchase. Observed animal intrusions include house flies, stray dogs, free range pigs and chickens (Table 1). Table 1 Biological intrusions on Oyo, Ogun and Lagos state pig abattoirs Intruder State Human Chicken Dogs Cattle Egrets Stray pigs Lizards House flies Oyo Yes(+++) Yes(++) Yes(+) No Yes(+) Yes(+) Yes(+++) Ogun Yes(+++) Yes(+) Yes(+) Yes(+++) No Yes(+) Yes(+++) Lagos Yes(+) No No Yes(++) No Yes(+) Yes(+++) + Occasional, ++ Frequent, +++Always.

Farm Number Ducks greater than one year old Ducks less than 1 year old Total Number % Number % Number % 1 94 75.2 31 24.8 125 100.0 2 5 27.8 13 72.2 18 100.0 3 12 100.0 0 0.0 12 100.0 4 15 71.4 6 28.6 21 100.0 5 54 94.7 3 5.3 57 100.0 6 30 100.0 0 0.0 30 100.0 7 26 81.3 6 18.8 32 100.0 8 40 87.0 6 13.0 46 100.0 9 31 72.1 12 27.9 43 100.0 Total 307 79.9 77 20.1 384 100.0 The above Table shows the number of adult ducks (age > 1 year) and that of ducklings (age < 1 year) in each of the nine sites studied. The calculated Pearson Chi-square was 54.501 with a p < 0.0001. This was found to be statistically significant at 99.9% CL in all nine cases. Table 9 Results of RRT-PCR Tests for AI conducted at the NMIMR Virology Department using both Spackman et al., 2002 and CDC Protocols (WHO, 2009), between October 2010 and April 2011 respectively Sample Target Gene Cycle Threshold (Ct) Positive Control Flu-A Matrix Gene (Qiagen) 27.50/20.50 Negative Control ” Undetected Commercial Ducks Samples ” Undetected Backyard Ducks Samples ” Undetected Live Birds Market Samples ” Undetected “Undetected”= Negative results. The 27.50 cycle threshold value recorded for the “Positive Control” is a Positive result. The cut-off point for this test was ≤ 35 ct value. The male to female ratio in breeding pens was found to be 5-8 females (ducks) to a male (drake).

Sample Target Gene Cycle Threshold (Ct) Positive Control Flu-A Matrix Gene (Qiagen) 27.50/20.50 Negative Control ” Undetected Commercial Ducks Samples ” Undetected Backyard Ducks Samples ” Undetected Live Birds Market Samples ” Undetected “Undetected”= Negative results. The 27.50 cycle threshold value recorded for the “Positive Control” is a Positive result. The cut-off point for this test was ≤ 35 ct value. The male to female ratio in breeding pens was found to be 5-8 females (ducks) to a male (drake). Sources of Parent Stock Sources of ducks in all the sites were identified. The commercial farm acquired parent stock from a farm in Dormaa Ahenkro in 2005 (Anonymous). Our findings also indicated that 71.4% (5/7) of the backyard holdings acquired their birds from live birds markets near and far. We also found that various species of birds in the live birds market investigated came from sources within and outside the municipality. Birds were purchased by customers for different purposes such as consumption (household, and restaurant), offering in ceremonies/gifts and religious festivals, and for replacement stock for farmers. The study further showed that transportation and management of birds by market vendors involved poor biosecurity practices. Collectors (intermediary men) and vendors did not separate birds according to species and sources of birds. Also, birds were mixed in cages during transportation and at the market place.

Sources of Parent Stock Sources of ducks in all the sites were identified. The commercial farm acquired parent stock from a farm in Dormaa Ahenkro in 2005 (Anonymous). Our findings also indicated that 71.4% (5/7) of the backyard holdings acquired their birds from live birds markets near and far. We also found that various species of birds in the live birds market investigated came from sources within and outside the municipality. Birds were purchased by customers for different purposes such as consumption (household, and restaurant), offering in ceremonies/gifts and religious festivals, and for replacement stock for farmers. The study further showed that transportation and management of birds by market vendors involved poor biosecurity practices. Collectors (intermediary men) and vendors did not separate birds according to species and sources of birds. Also, birds were mixed in cages during transportation and at the market place. Molecular Investigation for Avian Influenza Two different RT-PCR protocols (Spackman et al., 2002 and the CDC Real Time PCR protocol) were applied on 526 ducks samples (384 cloacal swabs and 142 feather tissues) to determine the presence of AI virus in ducks. All the samples were negative for AI virus (Table 9, Fig 2, 3). This included ducks sampled from the only duck farm in the Sunyani Municipality which was affected by the 2007 HPAI H5N1 outbreaks. This farm was closed down as a result of the outbreak in May 2007 and became operational three years ago with current population of 38 ducks (Table 8).

ve for AI virus (Table 9, Fig 2, 3). This included ducks sampled from the only duck farm in the Sunyani Municipality which was affected by the 2007 HPAI H5N1 outbreaks. This farm was closed down as a result of the outbreak in May 2007 and became operational three years ago with current population of 38 ducks (Table 8). Figure 2 Real-Time PCR (Spackman et al 2002) Amplification Plot for AI Virus Detection in the 43 Pooled samples, April 12, 2011 “Undetected’ - Negative results. The 27.50 cycle threshold value recorded for the “Positive Control” is a Positive result. The cut-off point for this test was < 35 ct value. Figure 2 shows RRT-PCR amplification plot for Flu A Matrix Gene analysis for the 43 pooled samples analyzed using the Spackman et al. (2002) Protocol. The Positive Control (light green line above baseline indicated with an arrow) increased exponentially with a cycle threshold (CT) value of 27.50. The Negative Control and the 43 pooled samples were undetected by the system’s software. The cut off point for the test was a threshold < 35 CT value. The RRT-PCR lasted for less than three hours with 45 cycles. Figure 3: displays the RRT-PCR amplification plot for Flu A Matrix Gene analysis for the 43 pooled samples analyzed using the CDC Protocol. The Positive Control (light green thin line above baseline indicated with an arrow) increased exponentially with a CT value of 20.50. The Negative Control and the 43 pooled samples were undetected by the system’s software. The cut off point was a threshold < 35 CT value. The test lasted for less than three hours with 45 cycles.

ive Control (light green thin line above baseline indicated with an arrow) increased exponentially with a CT value of 20.50. The Negative Control and the 43 pooled samples were undetected by the system’s software. The cut off point was a threshold < 35 CT value. The test lasted for less than three hours with 45 cycles. Figure 3 Real-Time PCR (CDC Protocol) Amplification Plot for AI Virus Detection in the 43 Pooled Samples, October 17, 2010

ive Control (light green thin line above baseline indicated with an arrow) increased exponentially with a CT value of 20.50. The Negative Control and the 43 pooled samples were undetected by the system’s software. The cut off point was a threshold < 35 CT value. The test lasted for less than three hours with 45 cycles. Figure 3 Real-Time PCR (CDC Protocol) Amplification Plot for AI Virus Detection in the 43 Pooled Samples, October 17, 2010 Discussion This study was conducted to increase the body of knowledge on the role of waterfowls (domestic ducks) as potential reservoirs of AI viruses and also to ascertain the profile of AI in domesticated ducks in the Sunyani Municipality area after the H5N1 outbreaks in May 2007. Avian influenza virus was isolated from outbreaks in parts of the Municipality particularly the New Dormaa area during this period (May, 2007). Also, because waterfowls (ducks) have the unique characteristic of being asymptomatic reservoir of the AI virus, the need to verify whether or not the virus was still in circulation is expedient. The results obtained from the present study showed no evidence of the presence of AI virus in the five hundred and twenty six (526) AI samples collected from domestic ducks in nine farms in the Sunyani Municipality. These samples were tested using two different RT-PCR protocols; Spackman et al., (2002) and CDC protocol (WHO, 2009). Hence, the fact that two different “tried and tested” RRT-PCR protocols were applied in this study and both tested negative for the virus in all the samples probably confirmed the validity of the results obtained and therefore suggest that there is currently no circulation of the virus in the area. Similar studies conducted in migratory and resident birds in Argentine, Bolivia and Caribbean countries, identified H1, H3, H4, H10 and H13 subtypes (Douglas et al., 2007; Spackman et al., 2007a; Pereda et al., 2008; Ghersi et al., 2009; Alvarez et al., 2010). In Mexico, results of similar research have been incorporated into activities performed by sanitary officials at poultry farms as part of the campaign for early detection and prevention of HPAI outbreaks (Villarreal-Chavez and Rivera-Cruz, 2003).

, 2007a; Pereda et al., 2008; Ghersi et al., 2009; Alvarez et al., 2010). In Mexico, results of similar research have been incorporated into activities performed by sanitary officials at poultry farms as part of the campaign for early detection and prevention of HPAI outbreaks (Villarreal-Chavez and Rivera-Cruz, 2003). A similar study carried out in commercial, backyard and live birds market in the Tema Metropolis of Ghana where there have been outbreaks (May, 2007) from May 2009 to September 2010 also yielded negative results (Danso et al., 2010). This could be connected with destruction of the poultry in the only duck farm where there was an outbreak in 2007 and the subsequent containment measures put in place. However, evidence gathered by the present study pointed to deterioration of farm management practices particularly biosecurity, and if left unattended to and reintroduction of AI virus occurs, the effect may be more disastrous to the poultry industry than the 2007 outbreaks, with possible human incursions. In this study, we found out that biosecurity practices which had to do with the use of disinfectants (disinfection of premises 11.1%, personnel disinfection 22.2%, use of footbath 0% and disinfection of vehicles 0%) were considered expensive hence were either not practiced or were less practiced compared to those that were not disinfectant dependent. This was also observed by Danso and others (2010) in their findings.

(disinfection of premises 11.1%, personnel disinfection 22.2%, use of footbath 0% and disinfection of vehicles 0%) were considered expensive hence were either not practiced or were less practiced compared to those that were not disinfectant dependent. This was also observed by Danso and others (2010) in their findings. Activity such as delivery of water and feed to birds could act as a source of contamination which could enhance the spread of poultry diseases when being administered manually without proper hygienic practices. According to Adak G.K et al. (1995) and Rodriguez et al, (2001), contaminated food and water are believed to be major sources of infection in warm-blooded animals including poultry. However, our study revealed an awfully low patronage (11.1%) for cleaning and disinfection of premises. Also, visitors to these premises did not put on protective gears to avoid been infected by sick birds or to ensure they did not introduce pathogens to farm premises. Again, the study showed that none of the sites investigated practised this very important biosecurity measure (donning of PPEs by visitors). Another equally important biosecurity measure was the disinfection of vehicles to and from the farm premises which was never observed in any of the sites. Though there was no available data on the biosecurity situation in the area prior to this study, it was clear that many farms (89%) did not still adhere to strict biosecurity and farm management practices.

asure was the disinfection of vehicles to and from the farm premises which was never observed in any of the sites. Though there was no available data on the biosecurity situation in the area prior to this study, it was clear that many farms (89%) did not still adhere to strict biosecurity and farm management practices. At the live birds market, vendors did not adhere to any of the requirements of bio-security except for daily sweeping. In this study, the difference in sex amongst the ducks investigated was found to be statistically significant with a p-value of 0.0001 at 95% confidence level, and the accepted male/female ratio of ducks in breeding according to Koney (1998) is 1 male to 5-8 females. However, the male/female ratio in this study was found to be 1:3 which suggested that there could be in-fighting among males in mating females since there was deviation from the norm (1 male to 5-8 females). This difference could then encourage males (those in free-range) to migrate to other territories in search of females hence favoring the spread of diseases amongst birds. However, because high mortality rates were recorded in two backyard holdings and the live birds market (Table 10) which were characterised by non-adherence to biosecurity practices, we suspected possible deficiencies in biosecurity to be the cause of the deaths. Study Limitation/Constraint An equal number of feather tissue and cloacal swabs could not be collected due to shortage of Viral Transport Medium (VTM). Also, viral isolation method which is a gold standard test for AI diagnosis was not applied.

However, because high mortality rates were recorded in two backyard holdings and the live birds market (Table 10) which were characterised by non-adherence to biosecurity practices, we suspected possible deficiencies in biosecurity to be the cause of the deaths. Study Limitation/Constraint An equal number of feather tissue and cloacal swabs could not be collected due to shortage of Viral Transport Medium (VTM). Also, viral isolation method which is a gold standard test for AI diagnosis was not applied. Conclusions There was no evidence of AI virus in domestic ducks in the Sunyani Municipality. Hence domestic ducks in the Municipality are not acting as reservoir of AI viruses. However, adherence to strict farm management and biosecurity practices was not observed by 89% of the sites investigated. Generally, ducks farmers in the Municipality had little knowledge about biosecurity practices. Also, deep burying of dead birds and burning were the most common methods of disposal of dead birds practiced though burying was most preferred (77.8% of the sites). Daily sweeping of the premises was the major sanitation practice in the LBM involved in this study.

Introduction Pre-slaughter animal welfare and meat hygiene is a concern worldwide (FAO, 1992) with most developed countries having humane slaughter laws that ensure that food animals are killed quickly, painless and without suffering in other ways. In Nigeria, there are laws on animal welfare and abattoir operations but compliance to these laws are not fully enforced. This situation leads to excessive pre-slaughter stress and poor hygiene conditions of the slaughter areas. Sources of pre-slaughter stress may range from physical, such as high ambient temperature, vibration and changes in acceleration during transportation, confinement, noise, and crowding; to psychological such as the breakdown of social groupings and mixing with unfamiliar animals, unfamiliar or noxious smells and novel environment (Warriss, 2000). Animals could also suffer from pre-slaughter stresses arising from bruises, injuries, starvation, tiredness, and loading and unloading onto vehicles. Lawrie (2006) reported that with higher levels of stress poorer meat quality is eminent, quite apart from being inhumane. Besides stress, genotype, transportation, lairage time, season of the year, environmental conditions and many other factors will affect pork quality (Küchenmeister, 2005).

d unloading onto vehicles. Lawrie (2006) reported that with higher levels of stress poorer meat quality is eminent, quite apart from being inhumane. Besides stress, genotype, transportation, lairage time, season of the year, environmental conditions and many other factors will affect pork quality (Küchenmeister, 2005). Slaughter methods vary with geographic location depending on the technology available and/or adopted and may be influenced by cultural or religious orientation of the people. Quality of equipment and training of abattoir personnel impacts to a large extent on the quality of pork products. With proper stunning methods and equipment, the animal is expected to be unconscious and with no sensitivity to pains. Therefore, well trained personnel and right choice of stunning equipment should be used to avoid unnecessary stress and distress to animals (Adzitey, 2011). Hygienic practices during pig slaughtering including proper effluent disposal are of utmost importance as these have effect on the health of the public through the wholesomeness of pork products and the direct effects of effluents on the surrounding. In Nigeria, many abattoirs dispose their effluents directly into streams and rivers without any form of treatment (Adelegan, 2002). Such is the situation in several private and government abattoirs in most parts of the country (Osibanjo and Adie, 2007). This research seeks to investigate and create a better understanding of the current pig slaughtering practices and the animal welfare and hygiene situation in the process in Southwestern Nigeria.

Hygienic practices during pig slaughtering including proper effluent disposal are of utmost importance as these have effect on the health of the public through the wholesomeness of pork products and the direct effects of effluents on the surrounding. In Nigeria, many abattoirs dispose their effluents directly into streams and rivers without any form of treatment (Adelegan, 2002). Such is the situation in several private and government abattoirs in most parts of the country (Osibanjo and Adie, 2007). This research seeks to investigate and create a better understanding of the current pig slaughtering practices and the animal welfare and hygiene situation in the process in Southwestern Nigeria. Materials and Methods Study Area The study was conducted on government designated abattoirs for pig slaughtering in Oyo, Ogun and Lagos State which are major pig producing states in Southwestern Nigeria. The three government-owned abattoirs serve as designated areas for commercial scale pig slaughtering. These are Bodija Municipal (Ibadan, Oyo State, Latitude 7.4208050, Longitude 3.9237550), Oke aro (Akute, Ogun State Latitude. 6.6896910, Longitude 3.3325720) and Oko oba (Agege, Lagos State, Latitude 6.6646080, Longitude.3.6914060) abattoirs.

ed abattoirs serve as designated areas for commercial scale pig slaughtering. These are Bodija Municipal (Ibadan, Oyo State, Latitude 7.4208050, Longitude 3.9237550), Oke aro (Akute, Ogun State Latitude. 6.6896910, Longitude 3.3325720) and Oko oba (Agege, Lagos State, Latitude 6.6646080, Longitude.3.6914060) abattoirs. Observational study The observational study design required a 2-week continuous period of observation of activities on each abattoir. The slaughtering of a total of 2,450 pigs was observed in order to assess normal slaughter operations. Information on different aspects including pre-slaughter handling, stunning, slaughter method, cleaning and splitting of carcass and abattoir facilities were obtained by the authors’ observation and data capture on digital camera. Questionnaires and Focus Group Interviews Information on the demography and training of the personnel were obtained by structured questionnaires administered to 58 workers that were consistent atthe abattoirs. Focus group interviews were conducted for twelve and six representatives of the abattoir workers and health officials (veterinarians and animal health officers) respectively. This was done after getting the informed consent of the abattoir authorities and the individuals involved. Statistical Analysis Statistical analysis was done by using descriptive statistics.

Questionnaires and Focus Group Interviews Information on the demography and training of the personnel were obtained by structured questionnaires administered to 58 workers that were consistent atthe abattoirs. Focus group interviews were conducted for twelve and six representatives of the abattoir workers and health officials (veterinarians and animal health officers) respectively. This was done after getting the informed consent of the abattoir authorities and the individuals involved. Statistical Analysis Statistical analysis was done by using descriptive statistics. Results The findings of this study based on the observational study, questionnaires and focus group interviews reveal peculiarities, a number of animal welfare concerns and situations with public health implications in pork processing in Southwestern Nigeria. Observational Study Abattoir Location, Facilities and Practices

Statistical Analysis Statistical analysis was done by using descriptive statistics. Results The findings of this study based on the observational study, questionnaires and focus group interviews reveal peculiarities, a number of animal welfare concerns and situations with public health implications in pork processing in Southwestern Nigeria. Observational Study Abattoir Location, Facilities and Practices The design and facilities available in the three abattoir locations supports manual method of pig slaughtering. This makes the process labor intensive, time consuming and usually with low output/capita. Different segments of the pork processing area were poorly demarcated often with no clear cut clean or dirty area. The slaughter areas are usually divided vaguely into two segments: the first is meant for activities such as mechanical stunning, slaughtering, and flaying. The second area serves for further cleaning, washing and evisceration of carcass. Approximately 60-80% of processing activities starting from stunning to sales of pork are done on bare cemented floors (Figure 1-8). There are certain observed variations in the three abattoirs. In Oyo state, the abattoir is located in the popular Bodija market in Ibadan, adjacent to the cattle slaughter area. The first area of the abattoir which is an outdoor segment serves basically for mechanical stunning, slaughter, boiling of water with firewood and flaying while the second area which is an indoor segment is designated for further cleaning of the carcass usually with razor blades after the initial flaying with knifes. The carcasses are washed with water and eviscerated. They are placed on tables where transactions between the butchers and customers who buy for consumption or further distribution takes place. The separated viscera is cleaned out with water, inverted for proper rinsing and parboiled before display for sale. The parboiling practice is generally accepted in the three locations as it is believed to make the visceral keep longer and remain compact. The visceral content and other waste produced are gathered and disposed manually with buckets on nearby dung hills. The processing of carcasses is done simultaneously and the average processing time per animal per butcher is estimated to be 30 minutes.

it is believed to make the visceral keep longer and remain compact. The visceral content and other waste produced are gathered and disposed manually with buckets on nearby dung hills. The processing of carcasses is done simultaneously and the average processing time per animal per butcher is estimated to be 30 minutes. Figure 1 Lagos state abattoir: Means of transportation (Bus) and restraint method (Snout to hind limb) Figure 2 Oyo state abattoir: Indoor flaying and viscera cleaning area, predominantly female butchers, biological intrusions (Humans and Stray dog), floor and table carcass processing, poor hygiene, water supplied in buckets. Figure 3 Lagos state abattoir: Simultaneous slaughtering, jugular slit without nuchal cut, no personnel uniform/protective clothing, flaying on cemented floors with hot water sourced from drums. Figure 4 Lagos state abattoir: Biological intrusions (farmer/supplier, buyer, onlookers), restraint with rope and identification by shaving, weight evaluation (with snout to hock restraint), inconsistent use of protective gears, floor slaughteringwith no clear cut slaughter clean or dirty area. Figure 5 Ogun state Abattoir: Mechanical stunning, Nuchal ligament slit, floor processing. Figure 6 Ogun state abattoir: Farm structures of adjacent pig farming village, Biological intrusions (cattle egrets, house flies), effluent flows directly into adjacent stream, floor slaughtering. Figure 7 Ogun state abattoir: Water source, no uniform/protective clothing, flaying and cleaning, floor processing, poor slaughter area boundaries.

Figure 6 Ogun state abattoir: Farm structures of adjacent pig farming village, Biological intrusions (cattle egrets, house flies), effluent flows directly into adjacent stream, floor slaughtering. Figure 7 Ogun state abattoir: Water source, no uniform/protective clothing, flaying and cleaning, floor processing, poor slaughter area boundaries. Figure 8 Oyo state abattoir: Predominantly female abattoir workers with their babies, Floor slaughtering, Water supplied in buckets. In Ogun State, the abattoir is located close to Okearo pig village. The pig village scheme which is government owned is an expansive area that accommodates over 1,000 independent pig farmers. The pigs slaughtered on the abattoir facilities are almost entirely those raised within the farming village. The slaughter area is located by a stream that runs through the farm. The farm and abattoir effluents run directly into the drainage. The slaughter area is poorly demarcated. Stunning and slaughtering is done in a segment which is not clearly demarcated from the rest of the slaughter area. Several pigs are slaughtered concurrently with the average processing time per animal per butcher estimated to be about 20 minutes. Sales are based on processed weight.

laughter area is poorly demarcated. Stunning and slaughtering is done in a segment which is not clearly demarcated from the rest of the slaughter area. Several pigs are slaughtered concurrently with the average processing time per animal per butcher estimated to be about 20 minutes. Sales are based on processed weight. The Lagos abattoir is located at Oko-oba in Agege area of Lagos State. It has a better layout and appears more organized and managed than the two other locations. It is also located beside a flowing stream that also drains the close-by cattle slaughter area. There are three fairly demarcated segments on the slaughter area and a fourth adjacent separate area is for pork display and sales. The first area is basically for mechanical stunning, slaughtering, boiling of water, flaying and evisceration. The second area which is adjacent to the first is for cleaning of the viscera and the third area which is opposite the first is for washing the carcass and cutting to sizes. The pork is then transferred to a fourth area where tables are available for buyers to transact. Sales are based on dressed weight basis. The average processing time per animal per butcher was estimated to be 15 minutes. Pre-Slaughter Handling The pre-slaughter practices observed included transportation, loading and off-loading, restraint methods, identification and lairaging.

The Lagos abattoir is located at Oko-oba in Agege area of Lagos State. It has a better layout and appears more organized and managed than the two other locations. It is also located beside a flowing stream that also drains the close-by cattle slaughter area. There are three fairly demarcated segments on the slaughter area and a fourth adjacent separate area is for pork display and sales. The first area is basically for mechanical stunning, slaughtering, boiling of water, flaying and evisceration. The second area which is adjacent to the first is for cleaning of the viscera and the third area which is opposite the first is for washing the carcass and cutting to sizes. The pork is then transferred to a fourth area where tables are available for buyers to transact. Sales are based on dressed weight basis. The average processing time per animal per butcher was estimated to be 15 minutes. Pre-Slaughter Handling The pre-slaughter practices observed included transportation, loading and off-loading, restraint methods, identification and lairaging. Transportation to Abattoir, Restraint Methods and Identification In Oyo and Lagos states, the pigs are transported to the abattoir from different areas of the state and other neighboring states. The butchers usually go in search of pigs and buy from farms, but a few farmers prefer to bring their produce to the abattoir for sales. They usually transport them with trucks, buses, cars and occasionally bikes. The pigs are loaded on and off-loaded the vehicles manually and are restrained with ropes or the barricades formed by the walls of the vehicle. Those transported in buses and cars are usually restrained with short ropes applied to tie their snout to one hind limb above the hock joint (Figure 1). In Ogun state, the pigs are not usually transported with vehicles since they mostly originate from the nearby farming village. They are herded on foot to the abattoir. The butchers have a similar identification system. This is done by scrapping off the bristles of the pigs with sharp blades to form signs unique to each individual.

e, the pigs are not usually transported with vehicles since they mostly originate from the nearby farming village. They are herded on foot to the abattoir. The butchers have a similar identification system. This is done by scrapping off the bristles of the pigs with sharp blades to form signs unique to each individual. Lairage Facilities Lairage facilities available in Oyo and Lagos abattoirs are grossly inadequate. The facilities in Oyo state are two small rooms (12ft2each) attached to the in-door slaughter house with make shift feeding and watering facilities. The Lagos state lairage is made up of cubicles with concrete floor full of crevices, concrete and wooden walls and galvanized roof. In Ogun state, there are no lairages as the pigs move to the slaughter area directly from the farm houses after purchase. Slaughter Processes The slaughter processes observed are fully manual, laborious and time consuming. The observed processes includes: stunning, slaughter practices, flaying, evisceration and splitting of carcass, processing of viscera and distribution of pork.

Lairage Facilities Lairage facilities available in Oyo and Lagos abattoirs are grossly inadequate. The facilities in Oyo state are two small rooms (12ft2each) attached to the in-door slaughter house with make shift feeding and watering facilities. The Lagos state lairage is made up of cubicles with concrete floor full of crevices, concrete and wooden walls and galvanized roof. In Ogun state, there are no lairages as the pigs move to the slaughter area directly from the farm houses after purchase. Slaughter Processes The slaughter processes observed are fully manual, laborious and time consuming. The observed processes includes: stunning, slaughter practices, flaying, evisceration and splitting of carcass, processing of viscera and distribution of pork. Stunning and Slaughter Practices The stunning practices adopted in the three locations are similar. Physical stunning is employed. Usually, a heavy metal or wooden material is used to apply a sharp force on the frontal bone area of the pigs, then a jugular or nuchal ligament slit is done. The stunning method is inhumane and often ineffective as many pigs are observed to show post slaughter wriggles as signs of consciousness and pains. The slaughtering practices vary slightly. In Ogun state, a peculiar slaughter practice in the abattoir after physical stunning is the making of a deep dorso-ventral cut on the neck region to severe the nuchal ligament as against jugular slit in the other abattoirs (Figure 5). This immobilizes the pigs and is believed to reduce post-slaughter wriggles which occasionally follow the jugular slit. Jugular slit exclusively is practiced in Lagos and Oyo abattoirs (Figure 3).

rso-ventral cut on the neck region to severe the nuchal ligament as against jugular slit in the other abattoirs (Figure 5). This immobilizes the pigs and is believed to reduce post-slaughter wriggles which occasionally follow the jugular slit. Jugular slit exclusively is practiced in Lagos and Oyo abattoirs (Figure 3). Flaying, Washing, Evisceration, Splitting of Carcass and Processing of Visceras Flaying is done using hot water and knives or cans. The carcass is placed on the cemented floor and hot water boiled often with fire wood is poured over it. The carcass occasionally may be dipped in the drums of boiling water. Knives or used metal cans are then used to flay to remove bristles and epidermal coatings of the skin (Figure 7 & 8). The heads are removed, further boiled and flayed. Evisceration is done after majority of the bristles have been removed and the carcass washed with water. This is done by making a slit from the pubis to the sternum along the medial line and a further anterior cut through the sternal cartilages to expose the abdominal and thoracic cavity. The thoracic organs are removed as a pluck starting from the trachea and kept separate. The intestines are removed with caution to prevent burst of the gall bladder which is carefully discarded. The viscera is transferred to certain butchers that are specialized in their cleaning.. In Oyo state, majority of the intestinal content are cleaned out before placing in water filled buckets. The intestines are cut into segments and carefully inverted and washed in buckets to minimize water use. In Lagos and Ogun states, there are designated running taps for this purpose. Water is run into the lumen of the intestine from the anterior portion to flush out the content and the viscera is then inverted. It is a generally accepted practice to parboil the viscera after cleaning to prolong its shelf life. The carcasses are split into various sizes and are set for distribution.

or this purpose. Water is run into the lumen of the intestine from the anterior portion to flush out the content and the viscera is then inverted. It is a generally accepted practice to parboil the viscera after cleaning to prolong its shelf life. The carcasses are split into various sizes and are set for distribution. Sales and Distribution of Processed Pork The numberof pigs slaughtered per day in all the abattoirs variesand isdetermined by demand as there are no functional facilities for freeze preservation of pork. The average number of pigs slaughtered per day in Oyo, Ogun and Lagos state abattoir was 22, 80 and 105 respectively. In Oyo state, almost all the pigs slaughtered are supplied to individual meat traders and consumers who come to the abattoir. In Ogun and Lagos states, there are corporate and bulk buyers in addition to the regular individual buyers. Water Source Bore-hole with associated facilities is available in the three abattoirs. The facility in the Oyo state abattoir was not functional at the time of the study and therefore creating a need for commercial water fetchers to supply water. The quality of water supplied could not be ascertained as the commercial fetchers go around scavenging for water. This discourages the copious use of water among the butchers for pig slaughtering and cleaning. The water supply facilities in Lagos and Ogun were functional although the bore-hole facility was majorly run on locally generated power. This made supply occasionally erratic.

Table 1 Biological intrusions on Oyo, Ogun and Lagos state pig abattoirs Intruder State Human Chicken Dogs Cattle Egrets Stray pigs Lizards House flies Oyo Yes(+++) Yes(++) Yes(+) No Yes(+) Yes(+) Yes(+++) Ogun Yes(+++) Yes(+) Yes(+) Yes(+++) No Yes(+) Yes(+++) Lagos Yes(+) No No Yes(++) No Yes(+) Yes(+++) + Occasional, ++ Frequent, +++Always. In Ogun state, there are also similar human intrusions as there are no limits set for customers. Cattle egrets were observed to be abundant on the fringes of the nearby stream and occasionally came over to the slaughter area to feed (Figure 6). House flies were abundant and stray chicken were also observed. These animals are observed to feed on the nearby stream and stray around thereby serving as a potential source of transmission of food borne diseases. In Lagos state, Human intrusions are minimal as there is a fourth area clearly set for sales. Cattle egrets were also abundant on the fringes of the stream which occasionally strayed into the slaughter area to feed. House flies and Lizards were also observed.

In Ogun state, there are also similar human intrusions as there are no limits set for customers. Cattle egrets were observed to be abundant on the fringes of the nearby stream and occasionally came over to the slaughter area to feed (Figure 6). House flies were abundant and stray chicken were also observed. These animals are observed to feed on the nearby stream and stray around thereby serving as a potential source of transmission of food borne diseases. In Lagos state, Human intrusions are minimal as there is a fourth area clearly set for sales. Cattle egrets were also abundant on the fringes of the stream which occasionally strayed into the slaughter area to feed. House flies and Lizards were also observed. Questionnaires The findings on the demography and training of abattoir workers on animal welfare, pork processing and hygiene from the questionnaires shows that most of the workers in Oyo state are female (92.86%) which is in contrast to the predominantly male workers in Lagos (75%) and Ogun state (80%). People with various religious affiliations and sentiments are involved in pig slaughtering in the three locations although with a higher proportion being Christians. Most of the workers were between 20 and 60 years of age (Table 2). There is currently no form of formal training or prequalification for abattoir workers involved in pork processing and inadequate periodic enlightenment trainings (Table 3). Table 2 Demographic characteristics of abattoir workers in Oyo, Ogun and Lagos states

Questionnaires The findings on the demography and training of abattoir workers on animal welfare, pork processing and hygiene from the questionnaires shows that most of the workers in Oyo state are female (92.86%) which is in contrast to the predominantly male workers in Lagos (75%) and Ogun state (80%). People with various religious affiliations and sentiments are involved in pig slaughtering in the three locations although with a higher proportion being Christians. Most of the workers were between 20 and 60 years of age (Table 2). There is currently no form of formal training or prequalification for abattoir workers involved in pork processing and inadequate periodic enlightenment trainings (Table 3). Table 2 Demographic characteristics of abattoir workers in Oyo, Ogun and Lagos states Oyo Ogun Lagos Sex Male 1 (7.14%) 16 (80%) 18 (75%) Female 13 (92.86%) 4 (20%) 6 (25%) Total 14 20 24 Religion Christianity 8 (57.14%) 13 (65%) 17 (70.83%) Islam 5 (35.71%) 7 (35%) 7 (29.17%) Traditional 1 (7.14%) 0 (0%) 0 (0%) Non Total 14 20 24 Age(Years) 21-30 1 (7.14%) 4 (20%) 3 (12.5%) 31-40 3 (21.4%) 12 (60%) 9 (37.5%) 41-50 6 (42.9%) 3 (15%) 7 (29.2%) 51-60 3 (21.4%) 1 (5%) 4 (16.7%) 61-70 1 (7.14%) 0 (0%) 1 (4.2%) Total 14 20 24 Table 3 Formal training of abattoir workers on animal welfare, pork processing and hygiene in Southwestern Nigeria

al 14 20 24 Age(Years) 21-30 1 (7.14%) 4 (20%) 3 (12.5%) 31-40 3 (21.4%) 12 (60%) 9 (37.5%) 41-50 6 (42.9%) 3 (15%) 7 (29.2%) 51-60 3 (21.4%) 1 (5%) 4 (16.7%) 61-70 1 (7.14%) 0 (0%) 1 (4.2%) Total 14 20 24 Table 3 Formal training of abattoir workers on animal welfare, pork processing and hygiene in Southwestern Nigeria Oyo Ogun Lagos Formal training on proper animal handling and welfare Non Non Non Formal training on pork processing Non Non Non Periodic enlightenment training on public health Non Yes Yes The use of uniforms, coveralls and protective gears Non Non Non Focus Group Interviews The representatives of the focus groups were interviewed after due informed consent on the limitations to optimal functioning of the abattoirs. The first group comprised of four representatives of the abattoir workers on each of the three locations. They highlighted the different factors that were limitations to their optimal productivity, responsible for the poor state of facilities and poor hygiene of the abattoirs. These included: Long period of neglect of the facilities and inadequate intervention of the government in the running of the abattoirs. Poor state of basic amenities such as waste disposal facilities, electricity and water supply. No compensation for owners of condemned carcass: The abattoir workers believe it was not fair for them to bear the loss of condemned carcass as they would have paid the farmers or suppliers fully. High cost of transportation of the animals to the abattoir.

Poor state of basic amenities such as waste disposal facilities, electricity and water supply. No compensation for owners of condemned carcass: The abattoir workers believe it was not fair for them to bear the loss of condemned carcass as they would have paid the farmers or suppliers fully. High cost of transportation of the animals to the abattoir. The second group comprised of two health officials (veterinarians and animal health officers) on each of the abattoirs. They discussed limitations to their optimal performance as it relates to the meat inspection and abattoir hygiene. They highlighted the recent interest of the government in upgrading the abattoirs and future plans to build more modern facilities for pig slaughtering. They also highlighted limitations they encounter in the line of their duty to include: Inadequate number of staff relative to animal traffic on the abattoirs. Poor state of available facilities due to long period of neglect which makes enforcement of standard abattoir practices difficult. Lack of modern abattoir equipment. Poor compliance of abattoir workers to laid down rules on individual and public health due to weak system of enforcing compliance. General apprehension of abattoir workers to partial or total condemnation of carcasses.

Poor state of available facilities due to long period of neglect which makes enforcement of standard abattoir practices difficult. Lack of modern abattoir equipment. Poor compliance of abattoir workers to laid down rules on individual and public health due to weak system of enforcing compliance. General apprehension of abattoir workers to partial or total condemnation of carcasses. Discussion This study highlights pig slaughtering activities in Southwestern Nigeria, its peculiarities and inadequacies which are of animal welfare concerns and public health implications. The slaughter facilities available were observed to be inadequate, poorly managed and the personnel were never formally trained. The systems adopted and currently in operation were therefore arrived at by the use of individual intuitions, apprenticeship and also by trial and error practices.

public health implications. The slaughter facilities available were observed to be inadequate, poorly managed and the personnel were never formally trained. The systems adopted and currently in operation were therefore arrived at by the use of individual intuitions, apprenticeship and also by trial and error practices. The practices that are of animal welfare concerns identified include inadequate transportation facilities and practices, inhumane restraint practices which are sometimes applied over prolonged transport periods, poor lairage facilities which usually subject pigs to prolonged fast periods and the brute means of physical stunning. These observations amount to excessive stress and poor animal welfare. These practices are a reflection of the orientation of pig handlers on animal welfare in Southwestern Nigeria. Poor animal welfare on the farm, through the production stage of the pigs may be a significant contributor to sub-optimal output from pig husbandry in Nigeria as high stress level predisposes to myriads of disease conditions (Thomson and Friendship, 2012). These cruel practices observed with pigs have also been reported in other livestock in Nigeria (Adeyemo et al., 2009).

stage of the pigs may be a significant contributor to sub-optimal output from pig husbandry in Nigeria as high stress level predisposes to myriads of disease conditions (Thomson and Friendship, 2012). These cruel practices observed with pigs have also been reported in other livestock in Nigeria (Adeyemo et al., 2009). The practices that are of public health implications observed include widespread embrace of floor pig slaughtering, inadequate water supply and sub-optimal water use, improper demarcation of slaughter areas, excessive biological intrusions, poor environmental hygiene, poor waste disposal and the failure of abattoir workers to use protective clothing which increases the risk of meat contamination and exposes workers to injuries and infection. Floor animal slaughtering which is widely practiced in Southwestern Nigeria (Figure 1-8) is also practiced in abattoirs in Northern Nigeria and other parts of Africa as flaying, evisceration and splitting of carcass are carried out on unhygienic floors (Lawan et al., 2013; Fearon et al., 2014). This practice may lead to a higher microbial load of meat and meat products, thereby constituting a source of high risk to the public (Warriss, 2000 Adzitey et al., 2010). There is therefore need for urgent intervention by the government who owns most of the abattoirs and relevant stakeholders to reduce the public health effects of these unhygienic practices.

ial load of meat and meat products, thereby constituting a source of high risk to the public (Warriss, 2000 Adzitey et al., 2010). There is therefore need for urgent intervention by the government who owns most of the abattoirs and relevant stakeholders to reduce the public health effects of these unhygienic practices. Inadequate water supply to the abattoirs encourages improper cleaning during pig slaughtering. The practice of scavenging for water observed in Oyo state abattoir (Figure 2 & 8) is of significant public health implication as it increases the potentials of water borne disease agents being transferred to meat products. This finding is similar to that of Lawan et al., (2013) who observed that in four abattoirs surveyed in northern Nigeria, there was no regular supply of portable water and electricity. Water was usually obtained from truck pushers, who sold water from unidentified sources for carcass washing. Occasionally the butchers sourced water from nearby streams. The meat inspectors in the study of Lawan et al.,(2013) adduced this deplorable state to government’s insensitivity and lack of concern towards general management of abattoirs.

from truck pushers, who sold water from unidentified sources for carcass washing. Occasionally the butchers sourced water from nearby streams. The meat inspectors in the study of Lawan et al.,(2013) adduced this deplorable state to government’s insensitivity and lack of concern towards general management of abattoirs. The ownership of most abattoirs in Nigeria belongs to the government. The three tiers of government (federal, state and local government) participate in meat inspection, however the local government authorities (LGA) are allowed legally to own slaughter sites and abattoirs within their boundaries, subject to the approval of the supervising veterinary division (Adeyemo 2002). The three locations in Southwestern Nigeria have veterinary officers and animal health officers assigned to each abattoir but the number was grossly inadequate compared with the slaughter population. The government officials stated that the inadequacy of the abattoir facilities hampers their thoroughness in meat inspection. They stressed the need for government to put stricter measures in place in order to enforce compliance to the judgment of meat inspectors. The butchers often resist partial or total condemnation of diseased carcasses in order to avoid bearing the financial losses of pigs purchased.

heir thoroughness in meat inspection. They stressed the need for government to put stricter measures in place in order to enforce compliance to the judgment of meat inspectors. The butchers often resist partial or total condemnation of diseased carcasses in order to avoid bearing the financial losses of pigs purchased. Excessive biological intrusions appear to be a major source of risk in pig slaughtering in South-western Nigeria. There are no clearly demarcated limits of access to external humans and also poor control of stray animals. This is similar to reports from the northern part of Nigeria where movement in and out of the abattoir by human and animals were uncontrolled. Stray dogs and hawkers were allowed access to the abattoir premises (Ogbaje et al., 2012). These biological intrusions are of public health implications as they may be involved in transmission of food borne diseases or zoonotic agents to the pork products.. Scavenging animals such as dogs, cattle egrets, lizards, chicken, pigs and house flies were observed in varying frequencies on the three surveyed locations. These should be prevented to improve the wholesomeness of pork products.

involved in transmission of food borne diseases or zoonotic agents to the pork products.. Scavenging animals such as dogs, cattle egrets, lizards, chicken, pigs and house flies were observed in varying frequencies on the three surveyed locations. These should be prevented to improve the wholesomeness of pork products. Poor hygiene and deficient waste disposal are common problems observed in the three abattoir locations. This was arrogated by the abattoir workers to poor provision of basic facilities by the government. The urban location of these abattoirs and the direct release of untreated waste into the environment and water bodies pose a direct public health threat. This practice of poor waste disposal is similar to the findings in other locations in Nigeria (Bello and Oyedemi 2009; Lawan, 2013). Some of the peri-abattoir water bodies in which waste is released into also serve the dual purpose of drinking water for the butchers and others working in the abattoir, and for dressing of the carcasses to be sold for human consumption (Adeyemo, 2002). Livestock waste contamination is known to increase the level of nitrates in ground water, which causes methaemoglobinemia or “blue baby syndrome” (Meadows, 1995).

se of drinking water for the butchers and others working in the abattoir, and for dressing of the carcasses to be sold for human consumption (Adeyemo, 2002). Livestock waste contamination is known to increase the level of nitrates in ground water, which causes methaemoglobinemia or “blue baby syndrome” (Meadows, 1995). The personnel in all the locations were observed to place little premium on personal protection as protective gears were not worn. This exposes the abattoir workers to injuries and risk of contraction of zoonotic diseases. There are a number of endemic bacterial, viral and parasitic diseases of livestock in Nigeria that are of zoonotic importance which can be readily acquired from meat and handling of food animals (Adeyemo, 2002). Aworh et al. (2013)] reported a high sero-prevalence of human brucellosis among abattoir workers in the two busiest abattoirs in Abuja, Nigeria. Similar findings have been documented from studies done in South west Nigeria, Tanzania and Egypt (Cadmus et al., 2006; Swai et al., 2009 and El Kholy et al., 2009). Among the various categories of abattoir workers that were screened by Aworh et al., (2013), butchers had the highest seropositivity rate. The missing factor of personnel education and enlightenment on hygiene and biosecurity in an abattoir system which is labor driven greatly contributes to the high incidence of zoonotic diseases among abattoir workers. Proper training of abattoir workers and enforcement of safe practices by the government will greatly reduce this alarming occurrence.

education and enlightenment on hygiene and biosecurity in an abattoir system which is labor driven greatly contributes to the high incidence of zoonotic diseases among abattoir workers. Proper training of abattoir workers and enforcement of safe practices by the government will greatly reduce this alarming occurrence. Pig slaughtering in Oyo state abattoir is almost an exclusive preserve for female butchers (92.86%). Many of the women were observed to be of child bearing age and some had babies strapped to their back as they slaughtered pigs This practice exposes these children to infections. Prejudice in relation to pig slaughtering is fading as people with various religious sentiments are observed to be involved in the processing in the three locations studied. The pig slaughtering practices, personnel training and facilities in Southwestern Nigeria re observed to be grossly deficient. There is therefore urgent need for the intervention of government and relevant stakeholders in the area of personnel training on best practices, provision of modern facilities and basic amenities in the abattoirs in order to ensure the wholesomeness of pork products and protection of public health.

sease ensued thereafter. Among the 318 human cases, 280 deaths (88% mortality) occurred, 38 serologically confirmed survivors were recorded, and the etiologic agent was identified to be Ebola virus, later renamed strain Mayinga, the prototype virus, after a Nurse Mayinga N’seka who died in the DRC outbreak (WHO, 1978). Nomenclature of Ebola virus Ebola virus belongs to the Order Mononegavirales; Family Filoviridae; Genus Ebolavirus. By serology and genetic analyses, the distinct five virulent species are: Zaire ebolavirus ZEBOV (identified in Zaire in 1976, now renamed Ebola virus, EBOV), Sudan ebolavirus SEBOV (identified in Sudan in1976, now renamed Sudan virus, SUDV), Reston ebolavirus REBOV (transmitted by Macaca fascicularis from Philippines, identified in 1989 in Reston USA, and in Sienna Italy in 1992, now renamed Reston virus, RESTV), Cote d’ Ivoire ebolavirus CIEBOV (identified in Tai forest, CI in 1994, now renamed Tai Forest ebolavirus, TAFV), and Bundibugyo ebolavirus BEBOV (identified in Uganda in 2007, now renamed Bundibugyo virus, BDBV) (Kuhn et al., 2013).

Introduction Ebola virus is the etiological agent of Ebola virus disease (EVD), an acutely fatal haemorrhagic disease, that is characterized by high fever, body aches, joint pains, vomiting, diarrhea, multiorgan liquefaction, delirium, coma and death within two weeks of infection. Ebola virus has not been as successful in penetrating the human species because of its virulence and ferocity in devouring the host. “While HIV/AIDS is a silent stalker, Ebola is a violent, hot, and bloody predator”.

s, vomiting, diarrhea, multiorgan liquefaction, delirium, coma and death within two weeks of infection. Ebola virus has not been as successful in penetrating the human species because of its virulence and ferocity in devouring the host. “While HIV/AIDS is a silent stalker, Ebola is a violent, hot, and bloody predator”. In accordance with one of the accepted methods of naming newly discovered viruses, Ebola Virus (EBOV) is named after a river in Yambuku area in the Democractic Republic of Congo (formerly Zaire) where the first outbreak occurred in 1976. There was a simultaneous outbreak in Sudan the same year. Literature has it that the index case was Mabalo Lokela, a 44 year-old male teacher at the Yambuku catholic mission school who fell ill after extensive travels in the northern Equateur Province of Zaire (now the Democratic Republic of Congo, DRC), having bought pieces of bushmeat (fresh and smoked antelope and monkey) as delicacies on his way back from the journey. At Yambuku, he took ill, and was treated for presumptive malaria at the Yambuku hospital. However, a week later, he had uncontrolled vomiting, bloody diarrhea, bleeding from the nostrils, mouth, and rectum, and died on 8th September 1976, about 14 days after the onset of symptoms. An outbreak of the disease ensued thereafter. Among the 318 human cases, 280 deaths (88% mortality) occurred, 38 serologically confirmed survivors were recorded, and the etiologic agent was identified to be Ebola virus, later renamed strain Mayinga, the prototype virus, after a Nurse Mayinga N’seka who died in the DRC outbreak (WHO, 1978).

in 1989 in Reston USA, and in Sienna Italy in 1992, now renamed Reston virus, RESTV), Cote d’ Ivoire ebolavirus CIEBOV (identified in Tai forest, CI in 1994, now renamed Tai Forest ebolavirus, TAFV), and Bundibugyo ebolavirus BEBOV (identified in Uganda in 2007, now renamed Bundibugyo virus, BDBV) (Kuhn et al., 2013). Morphology of Ebola virus Morphologically, EBOV is a helical, enveloped RNA virus. Under the electron microscope, the virion is pleomorphic, long, sometimes branched, twisted, filamentous, thread-like, shaped like a “6”, “U”, a circle, or better still like a “hockey stick”. It is approximately 800-1400nm in length and 60-80 nm in diameter (Fig. 1). The genome (consisting of 7 genes) is typically approximately 19 kb (19,000 bp) in length of molecular weight 4.2x106 Dalton. The linear, non-segmented, negative-sense, single-stranded RNA is encoded in a 3’ to 5’ direction in an overlapping reading frame (Figs 1 and 2). The seven sequentially arranged genes are transcribed into 8 major mRNAs (7 structural proteins and 1 nonstructural protein). The proteins expressed by the ebola viruses are: nucleoprotein (NP) which encapsulates the RNA genome, glycoprotein (GP), RNA-dependent RNA polymerase (L), and four structural proteins, namely, minor matrix proteinVP24, transcription activator (VP30), Polymerase cofactor (VP35), and VP40 (Fig 1). The Viral Matrix Protein VP40, and VP24 form the internal viral membranes while the surface of the viral envelope are spiked with arrays of glycoprotein GP trimers which are 10nm long and 10 nm apart (Feldmann et al., 1994).

trix proteinVP24, transcription activator (VP30), Polymerase cofactor (VP35), and VP40 (Fig 1). The Viral Matrix Protein VP40, and VP24 form the internal viral membranes while the surface of the viral envelope are spiked with arrays of glycoprotein GP trimers which are 10nm long and 10 nm apart (Feldmann et al., 1994). Figure 1 Morphology of the ebola virion (Adapted from: http://www.ncbi.nlm.nih.gov). Figure 2 Schematic diagram and genomic arrangement of the Ebolavirus. Courtesy: http://www.ncbi.nlm.nih.gov

trix proteinVP24, transcription activator (VP30), Polymerase cofactor (VP35), and VP40 (Fig 1). The Viral Matrix Protein VP40, and VP24 form the internal viral membranes while the surface of the viral envelope are spiked with arrays of glycoprotein GP trimers which are 10nm long and 10 nm apart (Feldmann et al., 1994). Figure 1 Morphology of the ebola virion (Adapted from: http://www.ncbi.nlm.nih.gov). Figure 2 Schematic diagram and genomic arrangement of the Ebolavirus. Courtesy: http://www.ncbi.nlm.nih.gov Due to RNA editing, transcription of the GP gene results in the synthesis of several GP gene - specific mRNAs coding for viral glycoproteins including non-structural glycoprotein sGP and virion surface Transmembrane glycoprotein GP (Volchkov et al., 1998). Both glycoproteins are synthesized as a precursor molecule that is proteolytically cleaved by furin (a cellular protease) during intracellular processing (Volchkov et al., 1998). The sGP forms dimers (Falzarano et al., 2006) whereas the cleaved carboxy- terminal fragment, termed delta peptide, is a monomer (Volchkova et al., 1999). Viral envelope surface spikes are formed as a trimer of GP1,2, made up of two subunits, GP1 and GP2 linked by a disulfide bond (Jeffers et al., 2002). GP1 is known as the virus ligand which mediate virus attachment to the host cells’ receptor whereas GP2 is involved in membrane fusion (Alazard – Dany et al., 2006). GP 1, 2 is a type 1 glycoprotein containing multiple N- and O-linked glycans and the majority of O-glycans are grouped in a region termed mucin-like domain (Lee et al., 2008), while the GP trimers are the target for neutralizing and protective antibodies (Feldmann et al., 2001).

ne fusion (Alazard – Dany et al., 2006). GP 1, 2 is a type 1 glycoprotein containing multiple N- and O-linked glycans and the majority of O-glycans are grouped in a region termed mucin-like domain (Lee et al., 2008), while the GP trimers are the target for neutralizing and protective antibodies (Feldmann et al., 2001). EVD is a zoonotic disease, one that can be transmitted from animals to humans or from humans to animals. Majorly, Humans, bats, antelope, Deer, Pig, Chimpanzee, Gorilla, and Monkey are the culprits. In Africa, the major EVD outbreaks have occurred in these countries and years: the Democratic Republic of Congo (1976, 1977, 1995, 2007, 2008, 2012), Congo Brazzaville (2001, 2002, 2003) Sudan (1976, 1979, 2004), Cote d’Ivoire (1994), Gabon (1994, 1996, 1997, 2001), South Africa (1996), Uganda (2000, 2007, 2011, 2012), the 2013-2015 West Africa outbreaks in Guinea, Liberia, Sierra Leone, Mali, Nigeria, Spain, Senegal, USA, Spain, and UK (WHO, 2012; Carlos et al., 2014; Torpiano et al., 2014) resulting in a total reported case counts of 22,560 and confirmed mortality of 9,019 as at Jan 29 2015 (WHO, 2015). The epidemic is usually considered complete after an interval of at least twice the maximum incubation period (42 days) after the death or recovery of the last confirmed case. However, fresh cases were reported in Liberia and Sierra Leone in June 2015, month after the epidemic was declared over.

an 29 2015 (WHO, 2015). The epidemic is usually considered complete after an interval of at least twice the maximum incubation period (42 days) after the death or recovery of the last confirmed case. However, fresh cases were reported in Liberia and Sierra Leone in June 2015, month after the epidemic was declared over. The Concept of SWOT SWOT is an acronym for Strengths, Weaknesses, Opportunities, and Threats. A SWOT analysis is usually represented as a grid. It is a business or strategic planning technique used to summarise the key components of the strategic environments. The technique is credited to Albert Humphrey at Stanford University between the 1960s and 70s, who led a research project that involved 5,000 interviews, funded by the fortune 500 and took 9 years to develop (Turner, 2002).

strategic planning technique used to summarise the key components of the strategic environments. The technique is credited to Albert Humphrey at Stanford University between the 1960s and 70s, who led a research project that involved 5,000 interviews, funded by the fortune 500 and took 9 years to develop (Turner, 2002). SWOT is a widely used framework for organizing and using data and information gained from situation analysis. The technique enables a group or individual to move from everyday problems or traditional strategies to a fresh perspective. It generally is a framework for identifying and analyzing the internal and external factors that can have an impact on the viability of a project, product, place, organism, or person. The SWOT concept was originally developed for business and industry, but it is equally useful in other areas, and even personal growth. SWOT is an assessment technique with a long track record of effectiveness. The strengths of this concept are its simplicity and application to a variety of levels of operation (Kotler, 1997). The benefits of SWOT include; identification of areas of strength and weakness, provision of comprehensive overview for contingency planning, and development of a “plan of action” to act on in a snapshot. SWOT analysis might be used to explore possibilities for new efforts or solutions to problems, to make decisions about the best path for an initiative, and is an excellent way to organize derived information from studies or surveys. The objectives of this innovative review was to educate all and sundry by adapting the SWOT concept to: identifying the biological strengths of ebola virus, determining the weaknesses of ebola virus, identifying the opportunities harnessed by ebola virus, determining the threats to ebola virus, present gaps where scientists and researchers could key-in for productive researches, and to encourage scientists, researchers, industry and the academia to explore this paradigm shift for holistic knowledge at combating recalcitrant, emerging, reemerging and potentially pandemic infectious pathogens and diseases.

rus, present gaps where scientists and researchers could key-in for productive researches, and to encourage scientists, researchers, industry and the academia to explore this paradigm shift for holistic knowledge at combating recalcitrant, emerging, reemerging and potentially pandemic infectious pathogens and diseases. Methods Databases used in this study included: Medline, PubMed, Embase, Web of Science, BIOSIS Previews and Google search engines. Text word searches (including “wildcards” to capture term variations, e.g., SWOT analysis, Ebola virus, Pathogenesis, diagnosis, zoonotic disease, Ebola virus disease, biological strengths of Ebola virus, weaknesses of Ebola virus, Opportunities harnessed by Ebola virus, Threats to Ebola virus, viral hemorrhagic fever, Disseminated Intravascular Coagulation) were conducted using keywords pertaining to Ebola (bats, monkeys, deers, bushmeat trade), bat-associated zoonoses and the urban environment (rural, endemicity, epidemicity, urban, city, cities, metropol). Groups of key words were combined using Boolean operators. Medline and Embasse were searched using Medical Subject Headings (natural reservoir, fruit bats, zoonoses, Ebola virus disease, urban health, and human Ebola outbreaks) in various combinations. Papers in languages other than English and French were excluded. The literature search was conducted between August 2014, and March 2015. A total of 150 papers were identified for initial consideration. To ensure that the review was focused on the most up to-date researches, all papers published prior to 1976, and in languages other than English and French were excluded (n=12). Remaining papers (n=138) were organized for inclusion and reviewed according to the amount of information they contributed regarding the ecology, the strengths, weaknesses, opportunities and threats of Ebola virus pathogen associated with animals and humans. Papers with significant ecologic content (e.g., reviews, observational studies, modeling studies, large case series, studies focused exclusively on pathogenesis, pathogen genetics, treatment methodology, etc.) were retained (n= 140) and all other papers (e.g. case studies, short communications) were also included considering the importance of the virus (n= 10). Finally, papers not focused on the epicenter of ebola virus outbreak and monkeys/bats in urban centers (e.g., studies focused on other haemorrhagic fever viruses species, etc.) were excluded (n=15).

ther papers (e.g. case studies, short communications) were also included considering the importance of the virus (n= 10). Finally, papers not focused on the epicenter of ebola virus outbreak and monkeys/bats in urban centers (e.g., studies focused on other haemorrhagic fever viruses species, etc.) were excluded (n=15). Additional sources (n=5) were added through citation searching and to fill specific information gaps such as, biological strengths of Ebola virus. A total of 138 papers were reviewed in detail (Fig 3). Data from these papers were extracted and synthesized based on the methodology for narrative synthesis described by Arai et al. (2007). The goal of narrative synthesis is to identify common themes across research regarding a particular subject that then can be used to identify commonalities and critical differences among included papers. Figure 3 Flow diagram of Literature searches and the systematic review Results and Discussion STRENGTHS OF EBOLA VIRUS Strengths are the characteristics of Ebola virus that gives it advantages over the host and other diseases. It comprises the positive tangible and intangible attributes, internal to Ebola. Essentially, the biological strengths and attributes that facilitate the pathogenicity, establishment, and spread of the Ebola virus include the followings:

eristics of Ebola virus that gives it advantages over the host and other diseases. It comprises the positive tangible and intangible attributes, internal to Ebola. Essentially, the biological strengths and attributes that facilitate the pathogenicity, establishment, and spread of the Ebola virus include the followings: Ebola virus is an RNA virus with inherent capability to mutate, reassort and recombine to generate mutant or reassortant virulent strains: Typical of RNA coded viruses, ebola virus mutates rapidly, both within a person during the progression of a disease and in the reservoir among the local human population, at a rate of 2.0x10-3 substitutions per site per year. This is likely to represent rapid adaptation to human hosts as the virus is repeatedly passed from human to human, and may pose serious challenges to the development of anti ebola vaccines (Biek et al., 2006). Bayesian phylogenetic analyses incorporating sequences derived from both human outbreaks and bats suggest that ‘spillover’ (Fig 4) occurs from bats to generate outbreaks in humans (Biek et al., 2006). Figure 4 Ebola transmission Dynamics (Olival et al., 2014).

tional study carried out in Bangladesh reported that reproductive tract infections (RTI), including bacterial vaginosis, are a major public-health problem among sexually active women. Among RTI investigated, bacterial vaginosis was responsible for 40 -50% of vaginal infections in sexually -active women (Spiegel, 1991). Bacterial vaginosis (BV) resolves spontaneously in up to one-third of non-pregnant and one-half of pregnant women (Klebanoff et al., 2004). Treatment is indicated for relief of symptoms in women with symptomatic infection and to prevent postoperative infection in those with symptomatic and asymptomatic infection prior to abortion or hysterectomy or any post vaginal surgical procedure. Treatment of BV may also reduce the risk of acquiring sexually transmitted diseases (STDs), including human immunodeficiency virus (HIV) (Schwebke et al., 2004). For this reason, some experts support the concept of treating all women with BV regardless of presence or absence of symptoms.

Ebola virus is an RNA virus with inherent capability to mutate, reassort and recombine to generate mutant or reassortant virulent strains: Typical of RNA coded viruses, ebola virus mutates rapidly, both within a person during the progression of a disease and in the reservoir among the local human population, at a rate of 2.0x10-3 substitutions per site per year. This is likely to represent rapid adaptation to human hosts as the virus is repeatedly passed from human to human, and may pose serious challenges to the development of anti ebola vaccines (Biek et al., 2006). Bayesian phylogenetic analyses incorporating sequences derived from both human outbreaks and bats suggest that ‘spillover’ (Fig 4) occurs from bats to generate outbreaks in humans (Biek et al., 2006). Figure 4 Ebola transmission Dynamics (Olival et al., 2014). The 2012 outbreak of EVD in the Democratic Republic of Congo (DRC) was caused by the Bundibugyo ebolavirus. However, the cause of the 2014 Guinea outbreak has been confirmed by full-genome sequencing (Baize et al., 2014) to be an outlier strain (Gue’cke’dou-C05) of Zaire ebolavirus, the species from which the genus takes its name. Within species, divergence is rarely more than 4% (Gatherer, 2014). Recent study has shown that within EBOV, the species involved in the Guinea outbreak, the greatest divergence was 3% between the 1994 Gabon strain and the Guinea strain Gue’cke’dou-C05 (Baize et al., 2014).

species from which the genus takes its name. Within species, divergence is rarely more than 4% (Gatherer, 2014). Recent study has shown that within EBOV, the species involved in the Guinea outbreak, the greatest divergence was 3% between the 1994 Gabon strain and the Guinea strain Gue’cke’dou-C05 (Baize et al., 2014). The fact that the Guinea outbreak strain is an outlier within EBOV suggests that it is not an introduction of a central African strain into West Africa but has been present in bat populations in Guinea without previously infecting humans (Baize et al., 2014). Although it is yet to be proven if survivors of ebola virus disease possesses immunological memory to sustain immunity or to mop up subsequent reinfection, it is suffice to state that the susceptible host may not have developed immunity to fight the new mutant strains, with tendencies to elicit a pandemic.

al., 2014). Although it is yet to be proven if survivors of ebola virus disease possesses immunological memory to sustain immunity or to mop up subsequent reinfection, it is suffice to state that the susceptible host may not have developed immunity to fight the new mutant strains, with tendencies to elicit a pandemic. Ebola virus has a broad cellular tropism: Cellular tropism is the affinity or propensity of a virus to preferentially establish interaction and attachment in a particular cell line when compared to the other cell types. While some viruses are only capable of interacting and replicating in a single cell line, others are efficient in replicating in multiple types of cell. The target cell range for EBOV infection is considerably broad. In-situ hybridisation and electron microscopic analyses of tissues from patients with fatal disease or from experimentally infected non-human primates show that monocytes, macrophages, dendritic cells, endothelial cells, fibroblasts, hepatocytes, adrenal cortical cells, and several types of epithelial cells were permissive of infection and lend support to replication of ebola viruses (Feldmann and Geisbert, 2011).

se or from experimentally infected non-human primates show that monocytes, macrophages, dendritic cells, endothelial cells, fibroblasts, hepatocytes, adrenal cortical cells, and several types of epithelial cells were permissive of infection and lend support to replication of ebola viruses (Feldmann and Geisbert, 2011). Natural Reservoir of ebola virus is unconfirmed but fruit bats, arthropods, and plants are hypothesized: The natural reservoir of a pathogen is the natural host that harbors the pathogen and in which it replicates without any clinical manifestation of the disease. The exact origin, locations, and natural habitat (known as the “natural reservoir”) of Ebola virus remain unknown. Very diverse taxa have been suggested as potential reservoirs for filoviruses over the years, including bats, rodents, arthropods, and plants (Germain, 1977; Arata and Johnson, 1977; Leirs et al., 1999; Swanepoel et al., 1996). The epidemiology and ecology of Ebola virus including identification of its natural reservoir hosts, remains a formidable challenge for public health and scientific communities. However, fruit bats of the Pteropodidae family are considered to be the natural host of the Ebola virus. Evidence of asymptomatic infection by Ebola virus was found in some species of fruit bat that are widely spread in Africa, including Eidolon helvum, Hypsignathus monstrosus (the hammer-headed fruit bat) Myonycteris torquata (the little-collared bat) and Epomops franqueti (Franquet’s epaulettes fruit bat) that migrate long distances (>2,500 km), indicating that these animals may be acting as a reservoir for the virus and could explain multiple remote epidemic clusters (Thomas et al., 1983). The first direct evidence from field studies that bats were reservoir hosts for Ebolavirus was reported by (Leroy et al., 2005) and research has since been growing to understand the role that bats play in the maintenance, transmission, and evolution of filoviruses. It was hypothesized that lower animals such as palm civets pick up infectious ebola viruses from ejected remnants and saliva contaminated fruit pellets from fruit bats and culled indigestible insect parts by insectivorous bats. The discovery of EBOV sequences in fruit bats near the locations of human outbreaks implies that EVD is a zoonosis, transmitted from a reservoir in bats.

ck up infectious ebola viruses from ejected remnants and saliva contaminated fruit pellets from fruit bats and culled indigestible insect parts by insectivorous bats. The discovery of EBOV sequences in fruit bats near the locations of human outbreaks implies that EVD is a zoonosis, transmitted from a reservoir in bats. Human Ebola outbreaks between 2001 and 2005 in Gabon and the DRC were linked to concurrent outbreaks that devastated local gorillas (Gorilla gorilla) and chimpanzee (Pan troglodytes) populations, making it unlikely that these non-human primates were the natural reservoirs but accidental or dead end hosts (Groseth et al., 2007). Ebola virus primarily targets and selectively destroys the immune system: Ebola virus targets the immune cells by infecting and replicating in the dendritic cells, monocytes, macrophages, fibroblast reticular cells, and lymph nodes using the same method as HIV, to spread through the human body (Weingartl et al., 2013) before multiorgan involvement. Failure of early T-cell activation and extensive apoptosis of blood leukocytes lead to rapid systemic spread and death (Geisbert et al., 2003). Although EBOV does not replicate in lymphocytes, large numbers of these cells undergo apoptosis (Baize et al., 1999).

dy (Weingartl et al., 2013) before multiorgan involvement. Failure of early T-cell activation and extensive apoptosis of blood leukocytes lead to rapid systemic spread and death (Geisbert et al., 2003). Although EBOV does not replicate in lymphocytes, large numbers of these cells undergo apoptosis (Baize et al., 1999). Recent studies indicate that Natural Killer (NK) cells, CD4+, and CD8+ lymphocytes are the principally infected target cells (Hotchkiss and Karl, 2003). Studies in macaques show that major features of illness are caused by effects of viral replication on macrophages and dendritic cells. Infected macrophages produce massive proinflammatory cytokines, chemokines and tissue factor, attracting additional target cells and inducing vasodilatation, disintegration of endothelial barrier, increased vascular permeability and disseminated intravascular coagulation. However, the macrophages cannot restrict viral replication, possibly because of suppression of interferon responses. Infected dendritic cells also secrete proinflammatory mediators, but cannot initiate antigen specific responses. In consequence, the virus disseminates to these and other cell types throughout the body, causing multifocal necrosis and a syndrome resembling refractory hypotension, Disseminated Intravascular Coagulopathy (DIC) and septic shock (Bray and Mahanty, 2003). The massive apoptosis of “bystander” NK and T cells further impairs the immune functions (Reed et al., 2004). However, in a study, treatment of infected macaques with a tissue-factor inhibitor reduced both inflammation and viral replication and improved survival (Bray and Geisbert, 2005). Therefore, these findings suggest that modifying host responses would be an effective therapeutic strategy against ebola.

eed et al., 2004). However, in a study, treatment of infected macaques with a tissue-factor inhibitor reduced both inflammation and viral replication and improved survival (Bray and Geisbert, 2005). Therefore, these findings suggest that modifying host responses would be an effective therapeutic strategy against ebola. Ebola viruses possess accessory proteins that inhibit the host’ immune responses: The VP35 protein is a Type 1 Interferon (IFN) antagonist that combats the host INF response. VP35 protein blocks IFN gene transcription and production by virus-infected cells. It prevents the cellular recognition of dsRNA that normally leads to phosphorylation of IRF-3 and its translocation to the nucleus. These activities are in concert with another viral protein VP24 which blocks responses to exogenous IFN (Basler and Palese, 2004). In effect, these inhibitory capacities impair antiviral defenses mediated by type I and II IFN needed to activate NK cells and upregulation of Major Histocompatibility Complex (MHC) for effective antimicrobial functions, thereby enhancing the replicative ability of the virus.

to exogenous IFN (Basler and Palese, 2004). In effect, these inhibitory capacities impair antiviral defenses mediated by type I and II IFN needed to activate NK cells and upregulation of Major Histocompatibility Complex (MHC) for effective antimicrobial functions, thereby enhancing the replicative ability of the virus. Secreted glycoprotein (sGP), a truncated soluble protein that triggers immune activation and increased vascular permeability is uniquely associated with Ebola virus only: Following infection, Ebola virus encoded glycoproteins (non-structural sGP) are uniquely released from infected cells in soluble forms into the host milieu. During EBOV infection significant amounts of both the virions and soluble glycoproteins, including shed GP and secreted sGP, are released from virus-infected cells into the extracellular environment. The soluble shed GP resembles virion GP 1,2, and has been shown to bind and sequester virus neutralizing antibodies directed against the surface GP (Dolnik et al., 2004). In addition, secretory glycoprotein (sGP) acts as decoys for immune cells and forms a dimeric protein that interferes with the signaling of neutrophils, which allows the virus to evade the immune system by inhibiting early steps of neutrophil activation. Contrary to other viruses’s glycoproteins, EBOV shed GP as a soluble mediator is able to activate non-infected immune cells, making it distinct from a number of other viruses whose surface glycoproteins have been shown to act as ligands for Toll-Like Receptor 4 (TLR4) recognition but that either do not shed their surface glycoproteins into the extracellular environment or do not spread systemically and thus only cause local inflammatory disorders (Xie et al., 2008; Bukreyev et al., 2008). The release of shed GP contributes to increased vascular permeability, DIC, disregulated inflammation and impairment of cell (hepatocytes and renal cells) functions and organ failure (Escudo-Perez et al., 2014) Therefore, as the shed GP has been found to possess a strong antibody-neutralizing role, it is conceivable that neutralizing antibodies targeting the shed GP could help to alleviate the systemic shock-like syndrome in EBOV infection.

(hepatocytes and renal cells) functions and organ failure (Escudo-Perez et al., 2014) Therefore, as the shed GP has been found to possess a strong antibody-neutralizing role, it is conceivable that neutralizing antibodies targeting the shed GP could help to alleviate the systemic shock-like syndrome in EBOV infection. Ability to effectively cross the species barrier and establish productive infection in Humans, Non Human primates, and other mammals: The species barriers separating nonhuman animal species from humans represent a major challenge for effective exposure to, infection by, and subsequent spread of zoonotic pathogens among humans (Kuiken et al., 2006). These species barriers can be divided into three largely complementary sets accordingly. First, the interspecies barrier which determines the nature and level of human exposure to zoonotic pathogens. Second, the intrahuman barrier which determines the ability of zoonotic pathogens to productively infect a human host and effectively cope with the immune response. Third, the interhuman barrier which determines the ability of zoonotic pathogens to efficiently transmit among humans, causing outbreaks, epidemics, or pandemics. Zoonotic pathogens may cross one or more of these sets of barriers, more or less efficiently. Only pathogens that cross the three barriers have the potential to sustainably establish in the human population (Gortazar et al, 2014). Identifying the factors that allow ebola virus to cross each of these three sets of barriers is essential to mitigate burdens of EVD. Notably, ebola virions are able to infect a broad range of primate cells, perhaps because the heavily glycosylated surface glycoprotein (GP) can bind to a variety of target molecules, including cell surface lectins (Takada et al., 2004). In addition, intensive domestic animal breeding facilitates viral mixing and increased targets for spillover from wild viruses (Taylor et al., 2001). Dogs and pigs are so far the only domestic animals identified that can be infected with Ebola Zaire. However, in Africa, infection has been documented through the handling of infected chimpanzees, gorillas, fruit bats, monkeys, forest antelope and porcupines found ill or dead in the forest.

ruses (Taylor et al., 2001). Dogs and pigs are so far the only domestic animals identified that can be infected with Ebola Zaire. However, in Africa, infection has been documented through the handling of infected chimpanzees, gorillas, fruit bats, monkeys, forest antelope and porcupines found ill or dead in the forest. Multiple portals of virion entry: Ebola virus enters the susceptible hosts through mucous membranes of the mouth, nose, eyes, skin cuts, abrasions, and open wound. Healthcare associated transmission occurs by reuse of needles and syringes, exposure to infectious tissues, excretions, and hospital wastes. Aerosol transmission has been proven in non human Primate but highly probable in humans. Transmission is enhanced by direct intimate person to person contact: Through contacts with infected individuals, EVD debilitated, dead, or funeral apparels of dead individuals, the virus spreads to families and friends who come in close contact with infectious secretions when caring for infected persons. Ebola virus attacks every part of the human body: Subsequent upon infection and replication, massive progeny viruses disseminate to every part of the body killing a huge amount of tissues, except the skeletal muscle and bones, converting it into a digested mass of virus particle. If the bones and skeletal muscles are spared during ebola infection, these sites could be researched and harnessed for generation of anti ebola medications.

disseminate to every part of the body killing a huge amount of tissues, except the skeletal muscle and bones, converting it into a digested mass of virus particle. If the bones and skeletal muscles are spared during ebola infection, these sites could be researched and harnessed for generation of anti ebola medications. The virus can survive in liquid or dried material for a number of days: Ebola virus is stable at room temperature or at 4°C for several days and indefinitely at -70°C. It is moderately thermolabile and can be inactivated by heating for 30-60 minutes at 60°C, and boiling for 5 minutes. Ebola virus is highly infectious at very low doses and has a short incubation period: About 1-10 pfu of the virus is sufficient for infection. The time interval from infection with the virus to the onset of symptoms, is between 2 - 21 days, but more often within 4-9 days. Ebola induces severe hemorrhage and DIC on infection: The bleeding phase typically begins within five to seven days after the first onset of symptoms. EVD patients expresses bleeding under the skin, the eye, and the orifices (nostrils, mouth, ears, genitals, rectum). The symptoms of hemorrhagic diathesis: bloody diarrhea, haematemesis, conjunctival injection, gingival bleeding, epitasis and bleeding at the site of injection (Fig 6) are consistent with DIC, while skin manifestations include petaechiae and puerperal (morbiliform skin rash) (Muyembe-Tamfum et al., 2012). Figure 5 Potential hosts of ebola virus disease (Courtesy AALAS Learning Library).

Ebola induces severe hemorrhage and DIC on infection: The bleeding phase typically begins within five to seven days after the first onset of symptoms. EVD patients expresses bleeding under the skin, the eye, and the orifices (nostrils, mouth, ears, genitals, rectum). The symptoms of hemorrhagic diathesis: bloody diarrhea, haematemesis, conjunctival injection, gingival bleeding, epitasis and bleeding at the site of injection (Fig 6) are consistent with DIC, while skin manifestations include petaechiae and puerperal (morbiliform skin rash) (Muyembe-Tamfum et al., 2012). Figure 5 Potential hosts of ebola virus disease (Courtesy AALAS Learning Library). Figure 6 Haemorrrhagic manifestations in Ebola patients: oral bleeding; skin, conjunctiva and diathesis at transfusion (Muyembe-Tamfum et al., 2012)..

Ebola induces severe hemorrhage and DIC on infection: The bleeding phase typically begins within five to seven days after the first onset of symptoms. EVD patients expresses bleeding under the skin, the eye, and the orifices (nostrils, mouth, ears, genitals, rectum). The symptoms of hemorrhagic diathesis: bloody diarrhea, haematemesis, conjunctival injection, gingival bleeding, epitasis and bleeding at the site of injection (Fig 6) are consistent with DIC, while skin manifestations include petaechiae and puerperal (morbiliform skin rash) (Muyembe-Tamfum et al., 2012). Figure 5 Potential hosts of ebola virus disease (Courtesy AALAS Learning Library). Figure 6 Haemorrrhagic manifestations in Ebola patients: oral bleeding; skin, conjunctiva and diathesis at transfusion (Muyembe-Tamfum et al., 2012).. Ebola elicits complications and Induces multi-system collapse: Ebolavirus damages many kinds of tissue in the body, either by direct infection of cells or by the body’s extreme inflammatory response. Cytokine release causes clots in the bloodstream, thereby blocking the blood flow to organs such as the liver and kidneys. Red blood cells are lysed when moving through small clot-filled vessels. As cells in the liver are destroyed, the blood loses its normal ability to clot, exacerbating any internal or external hemorrhaging. A breakdown of the adrenal glands leads to dangerously low blood pressure and a decreased ability to produce steroid hormones. The body’s connective tissues are attacked and the extracellular matrices are broken down, as are the cells that line body cavities and surfaces, leading to accumulation of fluid in the brain. The resultant convulsions can cause patients to spew and spread infectious blood and bodily fluids. People who die from Ebola succumb to very low blood pressure, multiple organ failure, delirium, coma and the shock of severe infection (ECDC, 2014).

y cavities and surfaces, leading to accumulation of fluid in the brain. The resultant convulsions can cause patients to spew and spread infectious blood and bodily fluids. People who die from Ebola succumb to very low blood pressure, multiple organ failure, delirium, coma and the shock of severe infection (ECDC, 2014). Absence of carrier state and high mortality: There is no carrier state. The case fatality rate (cfr) is between 60 – 90%. Previous outbreak had a cfr of 88% while the recent outbreaks were also above 50%. Ebola death (“crashing out”) ensues as early as 2 days after expression of symptoms and in many cases, death occurs between 7-16 days in 60-90% of cases, and recovery is usually painful (Muyembe-Tamfum et al., 2012). Confounding pattern of signs, symptoms and ill defined clinical course: Variability of clinical presentations complicate early detection and management. EVD mimicks the presentation of other common diseases such as malaria, typhoid fever, and dysentery. Non-specific prodrome typically lasts less than 1 week. In ten to 12 days after the onset of disease, the sustained fever may break, with improvement and eventual recovery of the patient. However, between 1-2 weeks after onset of symptoms, hemorrhage and death occurs (Roddy et al., 2012).

typhoid fever, and dysentery. Non-specific prodrome typically lasts less than 1 week. In ten to 12 days after the onset of disease, the sustained fever may break, with improvement and eventual recovery of the patient. However, between 1-2 weeks after onset of symptoms, hemorrhage and death occurs (Roddy et al., 2012). WEAKNESSES OF EBOLA VIRUS: These are the characteristics that places the virus at a disadvantage relative to the host and the environment. The virus weaknesses detract it from its ability to attain the core goal, and influence its infectivity, growth, and establishment. Essentially, the internal attributes of Ebola virus that present as “Achilles’ heel” and targets for its elimination are:

virus at a disadvantage relative to the host and the environment. The virus weaknesses detract it from its ability to attain the core goal, and influence its infectivity, growth, and establishment. Essentially, the internal attributes of Ebola virus that present as “Achilles’ heel” and targets for its elimination are: Ebola virus transmission and persistence is severely limited by its virulence: Virulence is defined as the infectivity of a pathogen in a population and severity of the disease in the individual host. The basic model of virulence evolution is called the Trade-Off Model which states that disease virulence and transmission are negatively correlated. Higher virulence causes a disease to proliferate in a host, increasing host incapacitation and therefore potentially decreasing transmission potential. The high mortality associated with EVD represents an auto limitation for the virus expansion; this explains why most of the outbreaks began and ended in rural areas. It is infective only in the symptomatic stage and diffusion during the incubation stages is not reported by the literature. Ebola virus is ferociously pathogenic. It devours the patient rapidly, burns out the infected locality, thereby limiting its succession and sustenance during an outbreak. As long as contact with the case is cut off, transmission can be broken and the disease can be brought under control. This is where quarantine is important.

irus is ferociously pathogenic. It devours the patient rapidly, burns out the infected locality, thereby limiting its succession and sustenance during an outbreak. As long as contact with the case is cut off, transmission can be broken and the disease can be brought under control. This is where quarantine is important. Ebola virus essentially requires host encoded protein Niemann-Pick C1 (NPC1) for host’s cell entry: A cholesterol transporter protein NPC1 is essential for Ebola virion entry and replication (Carette et al., 2012). The virus use the protein to gain entry into host cells, essentially tricking the cell into thinking that the virus is cholesterol. In a study, mice that were heterozygous for NPC1 were shown to be protected from lethal challenge with mouse-adapted Ebola virus. In another study, small molecules were shown to inhibit Ebola virus infection by preventing viral envelope glycoprotein (GP) from binding to NPC1 (Alexandra, 2011). Hence NPC1 was shown to be critical to entry of this virus, because it mediates infection by direct binding to the viral GP (Cote et al., 2012). When cells from Niemann Pick Type C patients lacking this transporter were exposed to Ebola virus in the laboratory, the cells survived and appeared impervious to the virus, further indicating that Ebola relies on NPC1 for cell entry. Therefore, mutations in the NPC1 gene in human were conjectured as a possible mode of effecting resistant in some individuals to this virus (Babin et al., 2014).

xposed to Ebola virus in the laboratory, the cells survived and appeared impervious to the virus, further indicating that Ebola relies on NPC1 for cell entry. Therefore, mutations in the NPC1 gene in human were conjectured as a possible mode of effecting resistant in some individuals to this virus (Babin et al., 2014). Ebola virus essentially requires host encoded proteins (TIM-1) for cell’ entry: T-Cell immunoglobulin and mucin domain1 (TIM-1, aka HAVCR1) was shown to bind to the receptor binding domain of the EBOV glycoprotein, to increase the receptivity of Vero cells in vitro. Silencing its effect with siRNA prevented infection of Vero cells. TIM-1 is expressed in tissues known to be seriously impacted by EBOV lysis (the trachea, cornea and conjunctiva). A monoclonal antibody against the IgGV domain of TIM-1, ARD5, blocked EBOV binding and infection. These studies suggests that TIM-1 may be potential therapeutic targets for an Ebola drug as a basis for a rapid field diagnostic assay (Kondratowicz et al., 2011).

acted by EBOV lysis (the trachea, cornea and conjunctiva). A monoclonal antibody against the IgGV domain of TIM-1, ARD5, blocked EBOV binding and infection. These studies suggests that TIM-1 may be potential therapeutic targets for an Ebola drug as a basis for a rapid field diagnostic assay (Kondratowicz et al., 2011). Relative abundance of Ebolavirus Nucleoprotein than the other virion components: The filovirus Nucleoprotein consists of739 or 695 amino acid residues, with a conserved hydrophobic N-terminus and a variable hydrophilic C-terminal (Niikura et al., 2001; Sanchez et al., 2007). The NP plays an important role in the replication of the viral genome, it is essential for formation of the nucleocapsid (Watanabe et al., 2006) and the C-terminal half has strong antigenicity (Saijo et al., 2001). The conformational and linear epitopes of nucleoprotein have been identified for several Ebola viruses (Ikegami et al., 2003) from which recombinants can be developed. Therefore the filovirus nucleoprotein may be an ideal target antigen because of its strong antigenicity and abundance in filovirus particles (Niikura et al., 2003; Bharat et al., 2012) for serological detection without handling live viruses.

Ebola viruses (Ikegami et al., 2003) from which recombinants can be developed. Therefore the filovirus nucleoprotein may be an ideal target antigen because of its strong antigenicity and abundance in filovirus particles (Niikura et al., 2003; Bharat et al., 2012) for serological detection without handling live viruses. OPPORTUNITIES FOR EBOLA VIRUS: These are the chances to make greater gains in the environment i.e external attractive factors that represent the reason for Ebola to exist, infect and spread. Opportunities arise when the virus can take benefit of conditions in its environment to infect and execute strategies that enables it to become established and spread. The various host and environmental factors harnessed for infection by Ebola virus are: In an outbreak setting, the virus can be transmitted in several ways after the first case-patient: EBOV disease is spread through infectious bodily fluids, including blood, excrement, saliva, breast milk, vaginal fluids, semen, and vomits by direct bodily contact with available viable infectious viruses in fomites, contaminated objects, reuse of needles or syringes and reinsertion into multi-dose vials of medicine. The virus can persist in semen for up to 61 days after the onset of illness and transmission through semen occurs up to seven weeks after clinical recovery (WHO, 2014) which could elicit infection and disease through sexual intercourse.

ts, reuse of needles or syringes and reinsertion into multi-dose vials of medicine. The virus can persist in semen for up to 61 days after the onset of illness and transmission through semen occurs up to seven weeks after clinical recovery (WHO, 2014) which could elicit infection and disease through sexual intercourse. Lack of infection control practices in African health-care facilities and paucity of health infrastructures, especially in the endemic zones: Owing to the dearth of funding, medical equipment and accessories, patients are often cared for without the use of a mask, gown, or gloves, leading to exposure of health care providers (CDC, 2014) in the affected areas. In addition, disinfection of equipment are usually absent and slow to become available in numerous health facilities (Bah, 2014). As a measure, it is important to empower the local hospitals to shift reliance on distant facilities and also establish linkages /communication with International responders such as the CDC and the Medecins Sans Frontieres. African governments should establish effective and functional public health facilities with the appropriate infrastructure to cope with emergency situations and emergency preparedness. Social mobilization and awareness campaigns should also be strengthened to dispel dangerous rumors that encourage spread of dangerous infectious diseases.

should establish effective and functional public health facilities with the appropriate infrastructure to cope with emergency situations and emergency preparedness. Social mobilization and awareness campaigns should also be strengthened to dispel dangerous rumors that encourage spread of dangerous infectious diseases. Permissiveness of circulating Monocytes, Macrophages and dendritic cells in virus mobilization and dissemination: Ebola virus attacks immune cells. These cells seem to have pivotal roles in dissemination of the virus as it spreads from the initial infection site via monocytes, macrophages, and dendritic cells to regional lymph nodes, probably through the lymphatic system, and to the liver and spleen through the blood (Fig 7). Monocytes, macrophages, and dendritic cells infected with Ebola virus migrate out of the spleen and lymph nodes to other tissues, thus disseminating the virus to other tissues and organs in the course of the taxis (Schnittler and Feldmann, 1998). Figure 7 Pathogenesis of ebola virus disease (Hotchkiss and Karl, 2003).

Permissiveness of circulating Monocytes, Macrophages and dendritic cells in virus mobilization and dissemination: Ebola virus attacks immune cells. These cells seem to have pivotal roles in dissemination of the virus as it spreads from the initial infection site via monocytes, macrophages, and dendritic cells to regional lymph nodes, probably through the lymphatic system, and to the liver and spleen through the blood (Fig 7). Monocytes, macrophages, and dendritic cells infected with Ebola virus migrate out of the spleen and lymph nodes to other tissues, thus disseminating the virus to other tissues and organs in the course of the taxis (Schnittler and Feldmann, 1998). Figure 7 Pathogenesis of ebola virus disease (Hotchkiss and Karl, 2003). Diagnosis can be difficult as the symptoms can be confused with common diseases: Early signs of ebola infection are non specific and mimicks malaria, dengue, lassa, and typhoid fevers. EVD is characterized by sudden onset of fever (>39°C), chills, malaise, exhaustion, sore throat, hiccups, rashes, headache, and joint/muscle aches. This is followed by diarrhea, vomiting, and stomach pains. Late signs are severe hemorrhage. The delay in clinical diagnosis and epidemic alert represent a major issue because it exacerbates the spread of the virus. Other variables that hinder the adequate management of outbreaks include training of health workers who fail to immediately identify the disease, especially in areas where EVD has never been observed, either because of nonspecific clinical symptoms or lack of experience (CDC, 2014).

exacerbates the spread of the virus. Other variables that hinder the adequate management of outbreaks include training of health workers who fail to immediately identify the disease, especially in areas where EVD has never been observed, either because of nonspecific clinical symptoms or lack of experience (CDC, 2014). Collection, consumption and trade of wild games (bushmeat): In areas where Non Human Primates were rare or absent, as in Kikwit (DRC) in 1995, Mweka (DRC) in 2007, Gulu (Uganda) in 2000 and Yambio (Sudan) in 2004, the hunting and eating of fruit bats were suggested to have resulted in the primary transmission of Ebola virus to humans. The index case of several outbreaks have been more or less directly associated with hunting or consumption of bush meat such as monkeys, antelopes, and bats (Leroy et al., 2009; Olson et al., 2012). About 13000 lbs of bushmeat were estimated to be imported into the UK annually. Therefore, the trade and consumption of ‘bushmeat’ should be highly regulated as these could serve as sources of outbreak in the respective destinations.

sh meat such as monkeys, antelopes, and bats (Leroy et al., 2009; Olson et al., 2012). About 13000 lbs of bushmeat were estimated to be imported into the UK annually. Therefore, the trade and consumption of ‘bushmeat’ should be highly regulated as these could serve as sources of outbreak in the respective destinations. Pertubation and drastic changes in forest ecosystems present opportunities for Ebola virus: The reduction in biological diversity by deforestation can trigger the invasion and spread of opportunistic species, while the dry season attracts rodents to peridomestic environments, heralding the emergence of diseases through increased contact with local community. Human population in Africa has doubled in 27 years while native animal habitats have been destroyed or fragmented, and wild animal food sources made less diverse. When analysing the risk factors for primary transmission of EBOV from a broad anthropological point of view, it was noticeable that the increase in Ebola outbreak since 1994 was frequently associated with drastic changes in forest ecosystems in tropical Africa. The perturbation of these ecosystems due to extensive deforestation and human activities in the depth of the forests may have promoted direct or indirect contact between humans and a natural reservoir of the virus. EBOV infection has therefore been related to human economic activities like hunting (young hunters were infected by a chimpanzee in the forest near Mayibout, Gabon in 1996), farming (the infection of charcoal makers in the forest near Kikwit, DRC in 1995), and gold mining (in Minkebe Forest, Gabon in 1994). For instance, the forest region that borders Sierra Leone, Liberia, and Cote d’Ivoire was decimated by clear-cut logging, leaving the “Guinea Forest Region” largely deforested. Consequently, the region has found itself home to tens of thousands of refugees fleeing regional war and conflicts, adding to both the ecologic and economic burden. It is speculated that prolonged dry season, extreme deforestation, proportion of Ebola virus–infected bats and/or the frequency of human contact are some of the triggers of ebola virus outbreaks (Bausch and Schwarz, 2014).

of refugees fleeing regional war and conflicts, adding to both the ecologic and economic burden. It is speculated that prolonged dry season, extreme deforestation, proportion of Ebola virus–infected bats and/or the frequency of human contact are some of the triggers of ebola virus outbreaks (Bausch and Schwarz, 2014). Use of dogs in hunting predisposes man and animals to inter-species contact: Human settlements provide fertile grounds for interspecies transmission between wild /farm animals, rodents, dogs, cats, and insects. It is estimated that approximately 350,000 wild caught animals are traded around the world annually, adding to the risk of potentially zoonotic infections crossing the species barrier into humans. Once established in humans, the diseases could be maintained indefinitely. In 2009, a survey in Gabon found a greater than 30% seroprevalence for EBOV in dogs during the 2001–2002 outbreak (Barrette et al., 1989). An individual or animal who was likely infected from the suspected zoonotic reservoir on a hunting expedition in the forest would then cause secondary infections that may be amplified back in their local villages. Wild animals held or bred in captivity as companion animals, agricultural, research, or zoological parks, have long fascinated human societies. This implies that companion monkeys on long distance trucks, use of dogs for hunting as it is practiced in some African settings should therefore be discouraged. If domestic animals do indeed prove to play a role in the transmission through viral shedding of the Ebola strains to man, it may be necessary to develop veterinary vaccines.

t companion monkeys on long distance trucks, use of dogs for hunting as it is practiced in some African settings should therefore be discouraged. If domestic animals do indeed prove to play a role in the transmission through viral shedding of the Ebola strains to man, it may be necessary to develop veterinary vaccines. Poverty, Malnutrition, crowding, social disorder, mobility and political instability: The effect of a stalled economy and government is 3-fold. First, poverty drives people to expand their range of activities to stay alive, plunging deeper into the forest to expand the geographic as well as species range of hunted game, to find wood to make charcoal, and deeper into mines to extract minerals, thereby increasing their risk of exposure to Ebola virus and other zoonotic pathogens in these remote areas. While the devastating effects of the civil wars in Liberia and Sierra Leone are evident and well documented, Guinea is in a state of stalled or even retrograde development. Guinea is ranked 178 (behind Liberia, 174 and Sierra Leone, 177) out of 187 countries on the UNDP Human Development Index. More than half of Guineans live below the national poverty line and about 20% live in extreme poverty. The indirect impact of the recent outbreak on the Guinean economy (and any other country affected) had been extensive, with the transport, tourism and entertainment sectors badly affected as people avoided crowded situations. Fewer workers have reported for work, thereby eventually having global implications, given that Guinea has one-half of the world’s supply of bauxite, as well as significant iron, diamond and gold deposits (Bah et al., 2014b).

nsport, tourism and entertainment sectors badly affected as people avoided crowded situations. Fewer workers have reported for work, thereby eventually having global implications, given that Guinea has one-half of the world’s supply of bauxite, as well as significant iron, diamond and gold deposits (Bah et al., 2014b). Emotional and behavioral disposition of susceptible host by direct contact with the body or funeral apparels of deceased EVD patient: Burial ceremonies in which mourners have direct contact with the body of the deceased can play a role in the transmission of Ebola. In the community setting, new infections related to the ministration of funeral rites, which involve ritual cleansing of the cadaver and removal of hair, finger nails, toe nails and clothing before burial, constitute great risks. People visiting or taking care of infected persons in their homes or in hospitals also risk being exposed to Ebola infections (CDC, 2014). In the Guinean outbreak, the first death in the capital city of Conakry (population >2 million) was a businessman who had travelled from Dabola in central Guinea. He became ill on 17 March 2014 and died the following day. He was suspected to have contracted EVD in Dabola through contact with a visitor from Gue’cke’dou who also subsequently died from suspected EVD. The businessman’s cadaver was taken from Conakry to Watagala, his home village (near Dinguiraye, north of Dabola). His four siblings who lived in Conakry, and who travelled with the body, and four other mourners at his funeral were all tested positive for EBOV (Guinea News, 2014). Thereafter, the total number of suspected cases presenting in the capital had risen (WHO, 2014b). Burial customs involving open viewing of the deceased potentially exposes the mourners to the virus and may amplify the spread of the virus. Learning to pay respect to the deceased person without touching or even seeing them would help reduce the spread of ebola.

cases presenting in the capital had risen (WHO, 2014b). Burial customs involving open viewing of the deceased potentially exposes the mourners to the virus and may amplify the spread of the virus. Learning to pay respect to the deceased person without touching or even seeing them would help reduce the spread of ebola. Demographics: changes in the age, race, gender, culture of residents in area of outbreak: It has been shown, in several epidemics, that affected people perceived the illness as divine punishment or evil spell, and even, sometimes, denied the disease itself (Epelboin, 2012). Hesitations about diagnosis and the diversity of explanatory anthropological models result in the delay of the alert and difficulties in the implementation of measures against epidemics (Milleliri et al., 2004). Before appropriate measures are taken, which may take several weeks or months, the deceased persons are transported to their home community where people sometimes come from far away to attend the funeral and then go back home infected. The always frightening and often contradictory messages– and rumors– prompt patients to avoid going to the hospital due to fear of isolation and because of the lack of effective treatments. Even, violent protests– with loss of life, involving sometimes the medical staff– have been reported in some outbreaks. It becomes impossible to identify the cases, confirm diagnosis, protect and monitor contacts (Formenty et al., 2003). For instance it was beamed on GPBO Television and confirmed during the recent 2014 outbreak in Liberia, that some residents carried dead ebola victims on their heads, dead bodies littered the streets (Fig 9), violent protesters prevented health workers from intervening, with a consequential exponential increase in the number of cases after the protest (Adu unpublished correspondence).

the recent 2014 outbreak in Liberia, that some residents carried dead ebola victims on their heads, dead bodies littered the streets (Fig 9), violent protesters prevented health workers from intervening, with a consequential exponential increase in the number of cases after the protest (Adu unpublished correspondence). Figure 8 Spread of the 2013-2014 West African EVD outbreak (Torpiano and Pace, 2014). Figure 9 An ebola dead victim lying in the street of Liberia during the 2014 epidemic (Source: Associated Press). Inappropriate or even damaging evolution of effective defenses (“Cytokine storm”) against the virus by the human and non human primate hosts: The balance and timing of early immune responses to infection play a critical role in determining the outcome of infections (Bray and Mahanty, 2003). In the case of septic shock in EVD, fatal infection of humans appears to be associated with an elevation or overproduction of anti-ebola pro inflammatory cytokines. Survival appears to correlate with the development of an antigen-specific immune response, usually signified by the appearance of ZEBOV-specific IgG (Baize et al., 1999; Sanchez et al., 2004). If EBOV elicits damaging host responses, then therapeutic interventions that modify those responses may help the primate immune system to control viral replication and achieve survival.

ntigen-specific immune response, usually signified by the appearance of ZEBOV-specific IgG (Baize et al., 1999; Sanchez et al., 2004). If EBOV elicits damaging host responses, then therapeutic interventions that modify those responses may help the primate immune system to control viral replication and achieve survival. Ease of travel and aviation as potentials for global spread: Frequent air travel is a major risk factor to the global spread of infectious diseases. It is estimated that over 100 million passenger- journeys by air are made annually. It is feasible to visit as many as three countries in a few hours. Road transport offers a more favorable route for transmission. Approximately 50% of all travel in Africa is by public road transport, compared to air travel which represents less than 0.2% of all passenger kilometers traveled. In contrast to airlines, public trains, buses, etc, are rarely fitted with high efficiency particulate air filters (HEPA). It is not only humans that travel: the International Air transport Association estimate that around 80,000 wild caught animals are air freighted each year, many being placed in holding facilities close to populated areas whilst in transit (Askar et al., 2012). The potential routes of transmission of infectious agents on board are mainly by inhalation of airborne droplets nuclei, direct contact with organic residues, indirect contact with respiratory secretions and other biological fluids contaminated surfaces. Spread of ebola cases over longer distances is often associated with ventures for treatment that draws people from rural villages that typify the index case locations to big urban centres with central medical facilities. While this mostly involves domestic land travel (Francesconi et al., 2003). some instances of international importation by air travel have been documented. The first travel-associated case of Ebola was reported on 30th September 2014 in the United States (USA), when an asymptomatic individual travelling from Liberia to Dallas, Texas developed clinical findings consistent with EVD and died five days after arrival in the USA and also infected two Nurses providing him healthcare (CDC, 2014). Following travel of an infectious individual, either secondary clusters of Ebola cases will occur, or transmission will be interrupted by control methods such as patient contacts tracing and quarantine (WHO, 2014c), as witnessed in the first index case in Nigeria (Fig.8).

wo Nurses providing him healthcare (CDC, 2014). Following travel of an infectious individual, either secondary clusters of Ebola cases will occur, or transmission will be interrupted by control methods such as patient contacts tracing and quarantine (WHO, 2014c), as witnessed in the first index case in Nigeria (Fig.8). However, due to the highly incapacitating symptoms of Ebola virus infection, the probability that an affected individual undertake an air travel during the symptomatic phase is very low. Moreover the symptomatic passenger boarding should be prevented by control measures in place by the countries involved in the outbreak at the point ofdeparture. Therefore, in response to public health emergency events of international concern, the airports located in areas of outbreaks must practice a screening of passengers from affected areas by visual inspection, questionnaires, and temperature measurement (thermal scanners and other available methods) rather than the screening made on arrival, to effectively limit the spread of the disease. Possible mechanical transmission by arthropod vectors: The potentials of arthropods to act as mechanical vector is highly probable, particularly by houseflies that visited the dead (a scenario in the abandon of ebola dead on the streets of Liberia) during the overwhelming outbreak (Fig 9).

Therefore, in response to public health emergency events of international concern, the airports located in areas of outbreaks must practice a screening of passengers from affected areas by visual inspection, questionnaires, and temperature measurement (thermal scanners and other available methods) rather than the screening made on arrival, to effectively limit the spread of the disease. Possible mechanical transmission by arthropod vectors: The potentials of arthropods to act as mechanical vector is highly probable, particularly by houseflies that visited the dead (a scenario in the abandon of ebola dead on the streets of Liberia) during the overwhelming outbreak (Fig 9). No vaccines or therapeutics are yet approved for human treatment: The magnitude of the recent outbreaks of EVD and the likelihood of future human exposure and epidemic, underscores the necessity for pre- and postexposure therapeutics. However, none of the developed investigational drugs have been certified for human treatment. Therefore, absence of proven vaccines or drugs constitute opportunities for the virus to deride the host. It is hoped that as soon as an approved drug or vaccine is found the rage of the virus would be curtailed.

therapeutics. However, none of the developed investigational drugs have been certified for human treatment. Therefore, absence of proven vaccines or drugs constitute opportunities for the virus to deride the host. It is hoped that as soon as an approved drug or vaccine is found the rage of the virus would be curtailed. THREATS TO EBOLA VIRUS: Threats are external elements in the environment that could cause trouble for Ebola i.e. external factors, beyond an organization’s (Ebola) control, which could place the virus’ mission or operation at risk. Essentially, the external conditions that are inimical to Ebola virus establishment, and which may be harnessed for elimination of the virus include the followings: Avoidance of direct contact with infected blood, and other body fluids of infected patient: At the very early stages when EVD is asymptomatic, ebola is not contagious but at the symptomatic stage, person-to-person contact should be minimized to interrupt transmission. In addition, direct contact with infected blood, urine, saliva, sweat, semen, breastmilk, vaginal fluids, mucus, phlegm, vomitus, and tears of infected patient should be avoided.

s asymptomatic, ebola is not contagious but at the symptomatic stage, person-to-person contact should be minimized to interrupt transmission. In addition, direct contact with infected blood, urine, saliva, sweat, semen, breastmilk, vaginal fluids, mucus, phlegm, vomitus, and tears of infected patient should be avoided. Appropriate and correct burial practices: Traditional practices regarding patient care and burial rituals often involve high risk conducts, such as washing and preparation of the body for exposure for several days, during which family and friends pay tribute by stroking or hugging the deceased (Hewlett and Amola, 2003). Decontamination can be presented as ablutions that can be associated with the burial rituals; deceased’s clothes could be buried in the grave rather than burnt to prevent stigmatization etc, (Hewlett et al., 2005). Deceased people as a consequence of EVD must be handled using protective clothing and gloves, put into body bags and bury outside city immediately (Fig. 10). No washing or direct touching of the carcass of the dead should be made. Figure 10 Proper handling and burial of ebola dead victims by health personnel. Figure 11 A typical BSL-4 facility (Jean-Philippe, 2014). Figure 12 Crematorium for Ebola casualties in the DRC

Appropriate and correct burial practices: Traditional practices regarding patient care and burial rituals often involve high risk conducts, such as washing and preparation of the body for exposure for several days, during which family and friends pay tribute by stroking or hugging the deceased (Hewlett and Amola, 2003). Decontamination can be presented as ablutions that can be associated with the burial rituals; deceased’s clothes could be buried in the grave rather than burnt to prevent stigmatization etc, (Hewlett et al., 2005). Deceased people as a consequence of EVD must be handled using protective clothing and gloves, put into body bags and bury outside city immediately (Fig. 10). No washing or direct touching of the carcass of the dead should be made. Figure 10 Proper handling and burial of ebola dead victims by health personnel. Figure 11 A typical BSL-4 facility (Jean-Philippe, 2014). Figure 12 Crematorium for Ebola casualties in the DRC Adoption of barrier Nursing: Nosocomial transmission through contaminated and poorly (or not) sterilized medical instruments is a major multiplication factor in most EVD outbreaks, particularly in remote and poor regions. Barrier precautions are used to prevent virus entry via the portals from blood, other body fluids, secretions (including respiratory droplets), or excretions. Barrier nursing procedures, such as protective clothing, hand gloves, face mask, mouth/nose guard, protective booths, shoe covers, proper waste disposals, use of Tyvek suits and respirators by Scientist and Healthcare Personnel are sufficient to rapidly interrupt transmission in hospital settings. Hand hygiene is strongly emphasized and should be performed thoroughly and often, including before and after donning and before and after doffing PPE. Patients isolation, use of the protective clothing and disinfection procedures are mandatory and sufficient to interrupt further transmission of Ebola virus, and thus to control and end outbreak (Raabea and Borcherta, 2012).

rmed thoroughly and often, including before and after donning and before and after doffing PPE. Patients isolation, use of the protective clothing and disinfection procedures are mandatory and sufficient to interrupt further transmission of Ebola virus, and thus to control and end outbreak (Raabea and Borcherta, 2012). Handling of all EBOV samples in BSL-4 facility, or minimum BSL-3: Ebola virus is classified as Biological Level 4 (BSL-4) agents by Health and Safety Executive and as category A biological warfare agents (BWA) by the Centers for Disease Control and Prevention. Blood testing for definitive diagnosis and confirmation should be executed in National and International reference laboratories with BSL-4 facility. The nature of the virus make it suitable as BWA for the high lethality in human infection and for the potential oral and conjunctival transmissions via aerosolized virus suspensions in non-human primates.

nosis and confirmation should be executed in National and International reference laboratories with BSL-4 facility. The nature of the virus make it suitable as BWA for the high lethality in human infection and for the potential oral and conjunctival transmissions via aerosolized virus suspensions in non-human primates. Future development and availability of pre- and post exposure prophylactics and therapeutics.: Several vaccines are being developed but the two most advanced vaccines were based on recombinant vesicular stomatitis virus111 expressing an ebola virus protein VSV-EBOV (50% ZEBOV in Rhesus at 30 min) and the recombinant chimpanzee adenovirus expressing ebola virus protein (ChAd-EBOV). These were tested in humans for safety and efficacy in the United States of America and trials were started in Africa and Europe (Babin et al., 2014). The recombinant vesicular stomatitis virus111 vaccines have shown remarkable usefulness when given as a postexposure treatment against Ebola haemorrhagic fever in non-human primates. However, its use and efficacy in humans needs further studies. Others are FDA-Approved selective estrogen receptor modulators, SERMs (Clomiphene and toremifene) which inhibit EBOV. Recent studies have shown promise for a combination of monoclonal antibodies and for a small interfering RNA compound (BCX4430) as post exposure prophylaxis in non human primates (Warren et al., 2014). Key components of two of the most efficacious mAbs cocktails, titled MB-003 (MappBio) including antibodies c13C6, h13F6, and c6D8 and ZMAb (Defyrus) including antibodies c1H3, c2G4, and c4G7 have been recently combined and are in development for human use as a cocktail named ZMApp. The mAb components of ZMApp include c13C6, c2G4, and c4G7. Each of these antibodies was raised in vaccinated mice, chimerized into human IgG1 scaffolds and are mass produced in tobacco plants (Murin et al., 2014). Cocktail of Zmapp has already been used compassionately in a few patients with ebola. Use of plasma from patients who have recovered from infections, and drugs that affect coagulation such as recombinant nematode anticoagulant protein or recombinant human protein- C have also been tried and reported to be successful (Fieldmann and Geisbert, 2011).

been used compassionately in a few patients with ebola. Use of plasma from patients who have recovered from infections, and drugs that affect coagulation such as recombinant nematode anticoagulant protein or recombinant human protein- C have also been tried and reported to be successful (Fieldmann and Geisbert, 2011). EBOV virulence also depends on its entry to immune cells via its surface glycoprotein, thus Compound 7, a benzodiazepine compound that binds directly to the EBOV entry glycoprotein, blocking infectivity has been developed, while Compound 8a also inhibited EBOV entry (Yemolina et al., 2011). These compounds displayed potent inhibition at low concentrations, but are yet to be evaluated for in vivo efficacy. Eventually, full development, trial, certification and acceptance of these investigational drugs in synergy with multivalent vaccines (properly engineered to overcome possible mutations of the virus that may render vaccination futile) against the 5 strains of ebola virus will constitute breakthroughs at enhancing the virus elimination.

development, trial, certification and acceptance of these investigational drugs in synergy with multivalent vaccines (properly engineered to overcome possible mutations of the virus that may render vaccination futile) against the 5 strains of ebola virus will constitute breakthroughs at enhancing the virus elimination. Managing patient’ Hemorrhage by replacement of blood, platelets, and clotting factors.: Supportive treatment, maintaining oxygen and blood pressure level, fluid and electrolytes replacement, and treating any complications are some of the measures to mitigate ebola virus disease. Most often, palliative treatments are limited to rehydration with sugar solutions preferably orally to avoid injections, but sometimes not realistic because of throat pain, vomiting and intense fatigue. Analgesic, antipyretic, antiemetic, anti-diarrheal and sedatives or antipsychotic drugs are recommended to ease agitated and anxious patients. Improved surveillance to prevent potential spread of epidemic.: Syndromic surveillance should be instituted to identify groups of signs and symptoms that precede diagnosis by the use of Infra-red thermometers that can be simply operated.

Managing patient’ Hemorrhage by replacement of blood, platelets, and clotting factors.: Supportive treatment, maintaining oxygen and blood pressure level, fluid and electrolytes replacement, and treating any complications are some of the measures to mitigate ebola virus disease. Most often, palliative treatments are limited to rehydration with sugar solutions preferably orally to avoid injections, but sometimes not realistic because of throat pain, vomiting and intense fatigue. Analgesic, antipyretic, antiemetic, anti-diarrheal and sedatives or antipsychotic drugs are recommended to ease agitated and anxious patients. Improved surveillance to prevent potential spread of epidemic.: Syndromic surveillance should be instituted to identify groups of signs and symptoms that precede diagnosis by the use of Infra-red thermometers that can be simply operated. Making Available Rapid laboratory equipment and procedures for prompt detection (ELISA, Western Blot, PCR).: A number of diagnostics and techniques are currently available for laboratory diagnosis of EVD. Acute infection is diagnosed by RT-PCR tests or ELISA to detect viral antigens. These tests can be positive from day 3 to day 15 of infection. Antigen can be detected in acute-phase blood by antigen-capture ELISA. The assay time is approximately 3 to 4 hours and the detection of antigen has been successful as compared to using PCR. Antibodies are tested either by direct IgG and IgM ELISA or IgM capture ELISA, IgM antibodies appear in blood by day 3 and disappear by 30 to 150 days. While IgG antibodies appear by day 6 and can remain in blood for many years. IgM or rising IgG titre constitutes a strong presumptive diagnosis (Sanchez et al., 2006). The virus can be isolated from acute-phase blood, liver, and other organs by inoculation into guinea pigs or cell culture (Strong et al., 2006). These technologies and personnel should be made available for use in the endemic areas.

or rising IgG titre constitutes a strong presumptive diagnosis (Sanchez et al., 2006). The virus can be isolated from acute-phase blood, liver, and other organs by inoculation into guinea pigs or cell culture (Strong et al., 2006). These technologies and personnel should be made available for use in the endemic areas. Miscellaneous measures.: Public awareness campaign, Strengthening health infrastructures especially in Africa where healthcare centres caring for people with the disease do not have tap water, Reporting to health authorities, Travel restrictions and system shut down may be instituted. Sterilization or disinfection of equipment and safe disposal of instrument.: Appropriate concentration of calcium or sodium hypochlorite is effective to disinfect the premises, equipment, and other materials exposed to biological contamination from ebola patients (CDC, 2014b). Sodium hypochlorite is a lipid solvent, an inactivating agent which distrupts the viral envelope, the ligands, and in effect subsequent attachment and infectivity. Handy instruments, and fluids should be autoclaved. Sharps, used articles, beddings and linens should be incinerated. Patient’ excretions should be disposed off properly, and wash hands frequently using soap and water, or 70% ethanol.

the viral envelope, the ligands, and in effect subsequent attachment and infectivity. Handy instruments, and fluids should be autoclaved. Sharps, used articles, beddings and linens should be incinerated. Patient’ excretions should be disposed off properly, and wash hands frequently using soap and water, or 70% ethanol. Prompt hospitalization, isolation and quarantine of infected individual.: Quarantine is regarded as enforced isolation of areas or individuals who may be infected. It may involve closure of schools, markets, worship congregations, or total shutdown of the system. These are milestones in reducing transmission of the virus. In the implementation of these threats, the ethical and social aspects should not be overlooked. Isolation of patients, required to avoid contamination, should not be seen as segregation. The family should be able to see and talk to patients, even if they are prevented from touching them. Authorities and medical staff should comply with, as far as possible, funeral rites by providing body bags and coffins for the families (Boumandouki et al., 2005).

void contamination, should not be seen as segregation. The family should be able to see and talk to patients, even if they are prevented from touching them. Authorities and medical staff should comply with, as far as possible, funeral rites by providing body bags and coffins for the families (Boumandouki et al., 2005). Active contact tracing and monitoring.: Contact tracing involves finding everyone who had close contact with infected individuals and watching / monitoring for signs of illness for 21days. If any of the contacts comes down with the ailment, they should be isolated, tested, and treated. The process is then repeated by tracing the contacts’ contacts (CDC, 2014c) such as in the Nigerian index case experience. In respect of bus or air travel, as direct contact is the main route of transmission for Ebola, only the passengers who were seated in direct proximity to the index passenger should be included in the trace-back, i.e. only passengers who were one seat away from the index case (+/- 1 seat in all directions) should be traced back. If the index case occupied an aisle seat, the three passengers seated directly across the aisle from the index case should also be traced back (ECDC, 2010).

ssenger should be included in the trace-back, i.e. only passengers who were one seat away from the index case (+/- 1 seat in all directions) should be traced back. If the index case occupied an aisle seat, the three passengers seated directly across the aisle from the index case should also be traced back (ECDC, 2010). Further research and treatment alternatives.: Considering the known / admitted challenges with vaccines, it is pertinent to consider addressing further research into exploiting, 1) the critical requirement (disulfide bond reduction / redox status) of viruses in cell entry. 2) A shift from the current paradigm in medicine which are heavily tilted toward the profitable Pharma model. We call for a worldwide integrative /alternative / holistic medical practitioners “in the field” for their experiences in treating otherwise difficult to treat infections, and a fair forum of reviewers (disconnected from the Pharma /profit or Pharma influenced universities and agencies) for their consideration. Such enlightened approaches could potentially successfully address other emerging recalcitrant infections which pharmaceutical based medicine fails to cure 3) Practitioners must evolve holistic support for the diseased body itself. From the clinical perspective, we need to explore how to augment the body in scaling up the activities of mediators such as the Reactive Oxygen Intermediates in combating pathogens, ozone therapy, replacement of Intravenous fluid electrolytes, lost macronutrients, optimizing trace minerals (required for normal immune function). 4) Optimization of other nutritional approaches (which might ultimately include IV Vitamin C, glutathione, etc for the ill, if simple ozone does not improve their status) as the African experience has shown that administration of Vitamin A mitigates complications of measles virus infection in children (Clement Adewunmi Personal Communication).

nutritional approaches (which might ultimately include IV Vitamin C, glutathione, etc for the ill, if simple ozone does not improve their status) as the African experience has shown that administration of Vitamin A mitigates complications of measles virus infection in children (Clement Adewunmi Personal Communication). Use of crematorium to dispose of carcasses.: Cremation is one of the best methods to dispose of the dead victims of EVD. However, this should be carried out with caution to avoid stigmatization of family of the deceased.

nutritional approaches (which might ultimately include IV Vitamin C, glutathione, etc for the ill, if simple ozone does not improve their status) as the African experience has shown that administration of Vitamin A mitigates complications of measles virus infection in children (Clement Adewunmi Personal Communication). Use of crematorium to dispose of carcasses.: Cremation is one of the best methods to dispose of the dead victims of EVD. However, this should be carried out with caution to avoid stigmatization of family of the deceased. Conclusion According to John F. Kennedy, “Change is the law of life and those who look only to the past or present are certain to miss the future.” As a paradigm shift, the SWOTs concept can be adopted to help us as Scientists to formulate and apply strategies that augment our research and enable us to understand and combat emerging and/ or recalcitrant infectious diseases. Cumulative evidence from Africa has shown that the continent is developing and not economically buoyant, We think that a concurrent research into natural approaches alongside the synthetic medicines, for the possibility of discovering good treatment (like the wonderful wormwood, the source of artemisinin in malaria therapy) will be worthy of consideration. Adoption of the SWOTs concept is a promising research strategy to the discovery of leeway to mitigate the impact of Ebola virus. The identified capacities and gaps presented in this study are inexhaustive framework to combat the ebola virus. To undermine and overcome the virus, research focus should be aimed at strategically decreasing the identified Strengths and Opportunities, while Increasing on the Weaknesses of, and Threats to the virus (Table 1). Mankind are dealing with a major threat to human life on earth, and a transboundary disease that could easily be turned into a weapon.

virus, research focus should be aimed at strategically decreasing the identified Strengths and Opportunities, while Increasing on the Weaknesses of, and Threats to the virus (Table 1). Mankind are dealing with a major threat to human life on earth, and a transboundary disease that could easily be turned into a weapon. Table 1 Contingency Table of SWOTs Analysis of Ebola virus.

virus, research focus should be aimed at strategically decreasing the identified Strengths and Opportunities, while Increasing on the Weaknesses of, and Threats to the virus (Table 1). Mankind are dealing with a major threat to human life on earth, and a transboundary disease that could easily be turned into a weapon. Table 1 Contingency Table of SWOTs Analysis of Ebola virus. HELPFUL TO ACHIEVING THE PATHOGENIC OBJECTIVE HARMFUL TO ACHIEVING THE PATHOGENIC OBJECTIVE Internal factors (attributes of the ebola virus) STRENGTHS OF EBOLA VIRUS (i) Ebola virus is an RNA virus with inherent capability to mutate, reassort and recombine to generate mutant or reassortant virulent strains. (ii) Ebola virus has a broad cellular tropism. (iii) Natural reservoir of Ebola virus is unconfirmed but fruit bats, arthropods, and plants re hypothesized. (iv) Ebola virus primarily targets and selectively destroys the immune system. (v) Ebola viruses possess accessory proteins that inhibits the hosts’ immune responses. (vi) Secreted glycoprotein (sGP), a truncated soluble protein that triggers immune activation and increased vascular permeability is uniquely associated with ebola virus only. (vii) Ability to effectively cross the species barrier and establish productive infection in humans, non human primates, and other mammals. (viii) Ebola virus attacks every parts of the human body. (ix) Multiple portals of virion entry. (x) Transmission is enhanced by direct intimate person to person contact. (xi) Ebola virus is highly infectious at very low doses and has a short incubation period. (xii) Ebola induces severe haemorrhage and DIC on infection. (xiii) Ebola elicits complications and induces multisystem collapse. (xiv) Absence of carrier state and high mortality. (xv) Confounding pattern of signs, symptoms and illdefined clinical course (xiv) Absence of carrier state and high mortality. (xv) Confounding pattern of signs, symptoms and ill-defined clinical course WEAKNESSES OF EBOLA VIRUS (i) Ebola virus transmission and persistence is severely limited by its virulence. (ii) Ebola virus essentially requires host encoded protein, Niemann-Pick C1 (NPC1) for host’s cell’ entry. (iii) Ebola virus essentially requires host encoded proteins (TIM-1) for cell’ entry. (iv) Relative abundance of Ebola virus Nucleoprotein than the other virion components. External factors OPPORTUNITIES FOR EBOLA VIRUS (i) Lack of infection control practices in African health-care facilities and paucity of health infrastructures, especially in the endemic zones.

ost vaginal surgical procedure. Treatment of BV may also reduce the risk of acquiring sexually transmitted diseases (STDs), including human immunodeficiency virus (HIV) (Schwebke et al., 2004). For this reason, some experts support the concept of treating all women with BV regardless of presence or absence of symptoms. Risk factors for BV include sexual activity, new or multiple sexual partners and early age of sexual intercourse. Epidemiologic studies are strongly supportive of sexual transmission of BV pathogens and most experts believe that BV does not occur in women who have never had vaginal intercourse (Yen et al., 2003). However, BV can develop in women who have never had sexual intercourse, probably because of other factors that may destabilize the normal vaginal flora such as douching and wearing of tight fitting underwear (Papanikolaou et al., 2002; Fethers et al., 2009). Distortion of the vaginal flora may also result from sexual activity alone without necessary transmission of the BV pathogens to susceptible sexual partners (Holzman et al., 2001). Generally, the use of an intrauterine contraceptive device, douching, wearing of tight fitting underwear and personal hygiene are reported as risk factors for BV (Chiaffarino et al., 2004; Klebanoff, 2010). Infection seems to be most common around the time of menstruation. There is a high occurrence of BV and concordance of flora in women who have sex with women, further suggesting sexual transmission is important. It is not clear, however, whether one type of sexual activity may be more important in the pathogenesis of infection than another. As an example, oral-genital sex may be a more important risk factor than penile intromission into the vagina. Although some degree of genetic susceptibility to BV is likely, no association between a gene polymorphism and BV has been established.

ncoded proteins (TIM-1) for cell’ entry. (iv) Relative abundance of Ebola virus Nucleoprotein than the other virion components. External factors OPPORTUNITIES FOR EBOLA VIRUS (i) Lack of infection control practices in African health-care facilities and paucity of health infrastructures, especially in the endemic zones. (ii) Permissiveness of circulating Monocytes, Macrophages and dendritic cells in virus mobilization and dissemination. (iii) Collection, consumption and trade of wild games (bushmeat). (iv) Perturbation and drastic changes in forest ecosystems present opportunities for Ebola virus. (v) Use of dogs in hunting predisposes man and animals to inter-species contact. (vi) Poverty, malnutrition, crowding, social disorder, mobility and political instability. (vii) Ease of travel and aviation as potentials for global spread. (viii) Possible mechanical transmission by arthropods vectors. (ix) Emotional and behavioral disposition of susceptible host by direct contact with the body or funeral apparels of deceased EVD patient. (x) Demographics: Changes in the age, race, gender, culture of residents in areas of outbreaks. (xi) Inappropriate or damaging evolution of effective defenses (cytokine storm) against the virus by the human and non human primates hosts. THREATS TO EBOLA VIRUS (i) Avoidance of direct contact with infected blood and other bodily fluids of infected persons. (ii) Improved surveillance to prevent potential spread of epidemic. (iii) Appropriate and correct burial practices. (iv) Adoption of barrier Nursing. (v) Making available rapid laboratory equipment and procedures for prompt detection (ELISA, Western Blot, PCR). (vi) Sterilization or disinfection of equipment and safe disposal of instrument. (vii) Active contact tracing and monitoring. (viii) Prompt hospitalization, isolation and quarantine of infected individual. (ix) Handling of all EBOV samples in BSL-4 facility, or minimum BSL-3. (x) Future development and availability of pre- and post exposure prophylactics and therapeutics. (xi) Use of crematorium to dispose of carcasses. It is pertinent to state that Ebola virus explores its biological strengths, conserves its weaknesses and exploits the host opportunities to decimate human population. Human induced environmental changes, inter-species contacts, altered social conditions, demography and medical technology affect microbes’ opportunities.

t is pertinent to state that Ebola virus explores its biological strengths, conserves its weaknesses and exploits the host opportunities to decimate human population. Human induced environmental changes, inter-species contacts, altered social conditions, demography and medical technology affect microbes’ opportunities. The academia and the industry should scale up all promising research findings and build more on the threats to the virus. Although ebola is endemic in some African Nations, the recent epidemic experience shows that it has the capacity to garner pandemic proportion if adequate responses and controls are not effected. Therefore, all nations should proactively constitute Ebola Response and Control Committees as one of the proactive measures to curtail the disease. The development or strengthening of strategies and plans of action with timely and effective response in order to reduce the consequences of public health emergencies is imperative. By holistically harmonising all the identified factors, putting them into perspective and implementing our strategies, we can win the Nobel prize for overcoming the Ebola virus. Acknowledgements We gratefully acknowledge the diverse Authors and sources whose publications and materials were synthesized to generate these analyses.

Introduction Klebsiella pneumoniae is a gram negative, rod shaped bacterium belonging to the family Enterobacteriaceae. It has in recent years become an important pathogen in nosocomial infections worldwide. Increasingly, many strains that are extended-spectrum P-lactamase (ESBL) producing as well as Carbapenem resistant are being reported as causing outbreaks in hospitals and particularly in intensive care units (ICUs) and New Born Units (NBUs) where there is great antibiotic pressure (Centers for Disease Control and Prevention, 2003; George AJ et al., 2005). The spread of these nosocomial infections occur from patient to patient, healthcare workers to patients and vice versa as well as contaminated hospital environment and equipment. Most of the infections occur in neonates, in immunocompromised patients such as critically ill patients in ICU, patients with malignancies, patients on chemotherapy, HIV infected patients and diabetic patients (Adamski J et al., 2008; Richards MJ et al., 1999; Winokur PL et al., 2001). Treatment options for multi-drug resistant K. pneumonia (MDR KP) are limited; more so in resource constrained settings. Most studies on MDR KP are from developed countries with scanty data from resource limited settings. The study objective was to describe the epidemiology and antibiotic resistance pattern and trends for K. pneumoniae at Moi Teaching and Referral Hospital (MTRH) in Eldoret, Kenya.

re so in resource constrained settings. Most studies on MDR KP are from developed countries with scanty data from resource limited settings. The study objective was to describe the epidemiology and antibiotic resistance pattern and trends for K. pneumoniae at Moi Teaching and Referral Hospital (MTRH) in Eldoret, Kenya. Materials and Methods Moi Teaching and Referral Hospital (MTRH) is the second largest public hospital in Kenya. It hosts Moi University School of Medicine and serves a catchment with population of about 16 million people. It has a microbiology laboratory that handles the culture and sensitivity specimen in the hospital. Blood specimens were cultured in BACTEC 9120 and BACTEC 9050 (Becton-Dickinson, New Jersey, USA) automated systems. Antibiotic sensitivity at the facility was performed by disc diffusion method and susceptibility reported based on Clinical and Laboratory Standards Institute (CLSI) susceptibility criteria. The laboratory is International Organization for Standardization (ISO 15189) certified and has internal quality management systems in place. The culture results are manually documented in a microbiology register provided by the Kenya Ministry of Health. Ethical approval was sought from the MTRH/ Moi University Ethics and Review Board and from the Director of MTRH before the study was conducted. The data was anonymized through unique study numbers and no patient identifiable information was collected.

ivity may be more important in the pathogenesis of infection than another. As an example, oral-genital sex may be a more important risk factor than penile intromission into the vagina. Although some degree of genetic susceptibility to BV is likely, no association between a gene polymorphism and BV has been established. Bacterial vaginosis is a risk factor for acquisition of herpes simplex virus type 2 (HSV-2), gonorrhea, and chlamydial infection (Cherpes et al., 2003). The incidence of BV has also been associated with a greater occurrence of other sexually transmitted infections like HIV and cytomegalovirus (Joesoef et al., 1995). Materials and Methods Ethics Statement The study was approved by the Institutional Review Board of Saint Monica University Buea, Cameroon while administrative authorization to collect clinical samples was obtained from the CDC Central Clinic, Tiko, where the study was conducted. Written informed assent was obtained from the patients and data was treated with discrete confidentiality.

Materials and Methods Moi Teaching and Referral Hospital (MTRH) is the second largest public hospital in Kenya. It hosts Moi University School of Medicine and serves a catchment with population of about 16 million people. It has a microbiology laboratory that handles the culture and sensitivity specimen in the hospital. Blood specimens were cultured in BACTEC 9120 and BACTEC 9050 (Becton-Dickinson, New Jersey, USA) automated systems. Antibiotic sensitivity at the facility was performed by disc diffusion method and susceptibility reported based on Clinical and Laboratory Standards Institute (CLSI) susceptibility criteria. The laboratory is International Organization for Standardization (ISO 15189) certified and has internal quality management systems in place. The culture results are manually documented in a microbiology register provided by the Kenya Ministry of Health. Ethical approval was sought from the MTRH/ Moi University Ethics and Review Board and from the Director of MTRH before the study was conducted. The data was anonymized through unique study numbers and no patient identifiable information was collected. This was a retrospective analysis of K. pneumoniae isolates in patients of MTRH for the period 2002 to 2013. Data is analyzed using SAS version 9.3 (SAS Institute Inc., Cary, NC). Normality test is conducted using Shapiro and Wilks normality test. Categorical variables are presented as frequencies and percentages while continuous variables are expressed as mean and standard deviation. Bar charts and line graphs are used to present the pictorial distribution of organisms.

stitute Inc., Cary, NC). Normality test is conducted using Shapiro and Wilks normality test. Categorical variables are presented as frequencies and percentages while continuous variables are expressed as mean and standard deviation. Bar charts and line graphs are used to present the pictorial distribution of organisms. Results Majority of the study samples were from female patients (64.8%). The median age was 5 days with interquartile range of 3-15 days (Table i). K pneumoniae accounted for 23 % of the total isolates during the study period (281/1231). It was the most prevalent pathogenic isolate. It constituted 65.5% of NBU growths (205/313) and 50% of ICU growths (5/10). It constituted 17%, 15% and 14 % of the growths in the pediatric wards (22/130), medical wards (13/88) and obstetrics/gynecology wards (1/7) respectively. There was no K. pneumoniae isolated from the surgical wards (Fig i). The NBU K. pneumoniae isolates contributed 83% of all the hospital KP isolates (205/281). The pediatric and medical wards contributed 8% and 5% of the hospital K. pneumoniae isolates respectively (Fig i). K. pneumoniae was constantly a significant growth in the hospital over the 11 year period (Fig ii). Table i Demographic characteristics of patients with K. pneumoniae isolates at MTRH 2002-2013 Characteristics n (%)N=281 Age, yrs; mean(std) 4.8 (11.5) Gender Male 99 (35.2) Female 182 (64.8) Wards* Adults 13 (5) Pediatric 22 (8) NBU 205 (83) Obstetrics/Gynecology 1 (0) ICU 5 (2) Others 6 (2) NBU-New born unit; ICU- Intensive care unit

Table i Demographic characteristics of patients with K. pneumoniae isolates at MTRH 2002-2013 Characteristics n (%)N=281 Age, yrs; mean(std) 4.8 (11.5) Gender Male 99 (35.2) Female 182 (64.8) Wards* Adults 13 (5) Pediatric 22 (8) NBU 205 (83) Obstetrics/Gynecology 1 (0) ICU 5 (2) Others 6 (2) NBU-New born unit; ICU- Intensive care unit * Percentages reflect distribution of K. pneumoniae among the wards. 83% of the K. pneumoniae isolates originated from the NBU. Figure i K. pneumoniae distribution within wards at MTRH 2002-2013 Figure ii Prevalence of K. pneumoniae by year at MTRH 2002-2013 as a percentage of the total hospital blood culture isolates. Most of the isolates were multidrug resistant with highest resistance of over 80% to Penicillins, Cephalosporins, Macrolides, Tetracyclines, Sulphonamides, Lincosamides and Chloramphenicol (Table ii). Aminoglycoside and quinolone resistance was at 49.2% and 41.3% respectively. The lowest resistance rates were documented for Carbapenems (23.2 %). An analysis of the resistance levels to individual commonly prescribed antibiotics indicated resistance of over 80% to Ceftriaxone, Cefipime, and Gentamycin (Table iii). Amikacin and Meropenem had least resistance (21% and 7 % respectively). Table ii K. pneumoniae resistance to antibiotic groups at MTRH 2002-2013

Most of the isolates were multidrug resistant with highest resistance of over 80% to Penicillins, Cephalosporins, Macrolides, Tetracyclines, Sulphonamides, Lincosamides and Chloramphenicol (Table ii). Aminoglycoside and quinolone resistance was at 49.2% and 41.3% respectively. The lowest resistance rates were documented for Carbapenems (23.2 %). An analysis of the resistance levels to individual commonly prescribed antibiotics indicated resistance of over 80% to Ceftriaxone, Cefipime, and Gentamycin (Table iii). Amikacin and Meropenem had least resistance (21% and 7 % respectively). Table ii K. pneumoniae resistance to antibiotic groups at MTRH 2002-2013 ANTIBIOTIC GROUP RESISTANT n (%) SENSITIVE n (%) INTERMEDIATE n (%) TOTAL NO TESTED Penicillin 226 (84.6) 37 (13.9) 4 (1.5) 267 Cephalosporins 511 (81.8) 90 (14.4) 24 (3.8) 625 Aminoglycosides 204 (49.2) 161 (38.8) 50 (12) 415 Macrolides 20 (87) 3 (13) 0 (0) 23 Carbapenems 44 (23.2) 143 (75.3) 3 (1.6) 190 Tetracyclins 38 (95) 2 (5) 0 (0) 40 Sulphonamides 79 (88.8) 9 (10.1) 1 (11) 89 Quinolones 50 (41.3) 71 (58.7) 0 (0) 121 Lincosamides 4 (80) 1 (20) 0 (0) 5 Vancomycin 29 (87.9) 4 (12.1) 0 (0) 33 Oxazolidinones 18 (75) 6 (25) 0 (0) 24 Minocycline 2 (40) 1 (20) 2 (40) 5 Chloramphenicol 46 (93.9) 3 (6.1) 0 (0) 49 Table iii K. pneumoniae resistance pattern to specific antibiotics at MTRH 2002-2013

89 Quinolones 50 (41.3) 71 (58.7) 0 (0) 121 Lincosamides 4 (80) 1 (20) 0 (0) 5 Vancomycin 29 (87.9) 4 (12.1) 0 (0) 33 Oxazolidinones 18 (75) 6 (25) 0 (0) 24 Minocycline 2 (40) 1 (20) 2 (40) 5 Chloramphenicol 46 (93.9) 3 (6.1) 0 (0) 49 Table iii K. pneumoniae resistance pattern to specific antibiotics at MTRH 2002-2013 ANTIBIOTIC GROUP RESISTANT n(%) SENSITIVE n(%) INTERMEDIATE n(%) TOTAL NO TESTED Ceftriaxone 68 (87.2) 9 (11.5) 1 (1.3) 78 Ciprofloxacin 32 (44.4) 40 (55.6) 0 (0.0) 72 Gentamycin 106 (82.8) 19 (14.8) 3 (2.3) 128 Amikacin 48 (21.0) 135 (59.0) 46 (20.1) 229 Meropenem 8 (7.0) 106 (92.2) 1 (0.9) 115 Cefipime 105 (85.4) 12 (9.8) 6 (4.9) 123 Ceftazidime 108 (69.7) 37 (23.9) 10 (6.5) 155 We assessed for resistance pattern over the 11 year period for possible trends. Antibiotic class resistance pattern analysis showed persistent high resistance (>70%) to Cephalosporins and Penicillins throughout the study period. Quinolone resistance was below 40% for the study period except for 2005-2007 when it peaked to above 80%. Cephalosporin resistance declined from 100% in the year 2002-2004 to below 10% for the next 6 years followed by an increase to 20% in the period 2011-2013 (Fig iii). Figure iii K. pneumoniae resistance pattern to antibiotic groups over time at MTRH 2002-2013. Left pane showing trend for Penicilin, Cephalosporins, and Carbapenems; Right pane showing trend for Aminoglycosides, Tetracyclins and Quinolones and Lincosamides antibiotic groups

ANTIBIOTIC GROUP RESISTANT n(%) SENSITIVE n(%) INTERMEDIATE n(%) TOTAL NO TESTED Ceftriaxone 68 (87.2) 9 (11.5) 1 (1.3) 78 Ciprofloxacin 32 (44.4) 40 (55.6) 0 (0.0) 72 Gentamycin 106 (82.8) 19 (14.8) 3 (2.3) 128 Amikacin 48 (21.0) 135 (59.0) 46 (20.1) 229 Meropenem 8 (7.0) 106 (92.2) 1 (0.9) 115 Cefipime 105 (85.4) 12 (9.8) 6 (4.9) 123 Ceftazidime 108 (69.7) 37 (23.9) 10 (6.5) 155 We assessed for resistance pattern over the 11 year period for possible trends. Antibiotic class resistance pattern analysis showed persistent high resistance (>70%) to Cephalosporins and Penicillins throughout the study period. Quinolone resistance was below 40% for the study period except for 2005-2007 when it peaked to above 80%. Cephalosporin resistance declined from 100% in the year 2002-2004 to below 10% for the next 6 years followed by an increase to 20% in the period 2011-2013 (Fig iii). Figure iii K. pneumoniae resistance pattern to antibiotic groups over time at MTRH 2002-2013. Left pane showing trend for Penicilin, Cephalosporins, and Carbapenems; Right pane showing trend for Aminoglycosides, Tetracyclins and Quinolones and Lincosamides antibiotic groups Figure iv K. pneumoniae resistance pattern to individual antibiotics over time at MTRH 2002-2013. Left pane showing trend for Ceftriaxone, Meropenem, Cefipime and Ceftazidimine; Right pane showing trend for Ciprofloxacin, Gentamycin and Amikacin antibiotics.

Figure iii K. pneumoniae resistance pattern to antibiotic groups over time at MTRH 2002-2013. Left pane showing trend for Penicilin, Cephalosporins, and Carbapenems; Right pane showing trend for Aminoglycosides, Tetracyclins and Quinolones and Lincosamides antibiotic groups Figure iv K. pneumoniae resistance pattern to individual antibiotics over time at MTRH 2002-2013. Left pane showing trend for Ceftriaxone, Meropenem, Cefipime and Ceftazidimine; Right pane showing trend for Ciprofloxacin, Gentamycin and Amikacin antibiotics. Among the Cephalosporins, resistance to Ceftriazone showed a steady rise over the study period from no resistance recorded in 2002-2004 to remain constantly above 70% for the remaining period. Resistance to Cefipime and Ceftazidime was high (above 50% and 60% respectively) throughout the study period. For the Aminoglycosides, Gentamycin resistance was constantly above 70% while that of Amikacin dropped from above 70% at the beginning to below 20% in the last 6 years. Likewise, Meropenem resistance steadily reduced from 100% resistance in the first 3 years to below10% in the last 6 years. Ciprofloxacin resistance was at 40% in the first 3 years, peaked during the period 2005-2007 at 84.6% then rapidly declined thereafter.

above 70% at the beginning to below 20% in the last 6 years. Likewise, Meropenem resistance steadily reduced from 100% resistance in the first 3 years to below10% in the last 6 years. Ciprofloxacin resistance was at 40% in the first 3 years, peaked during the period 2005-2007 at 84.6% then rapidly declined thereafter. Discussion Klebsiella pneumonia is normal human intestinal enterobacteria. It is considered an opportunistic human pathogen and is responsible for severe nosocomial infections in immunocompromised patients, in patients with prolonged hospital stay and in patients with various implants (Adamski J et al., 2008; Mehrgan H et al., 2010; Velasco E et al., 2004). It has been found to be a significant cause of neonatal sepsis in the developing countries (Mathure NB et al., 2002; Stephen EM et al., 2013; Tiwari DK et al., 2013; Tumaini VM et al., 2012). In the developed world, multi drug resistant K. pneumoniae (MDR KP) has been documented to cause disease outbreaks leading to significant morbidity and mortality (Lee Ket al., 2010; Patricia AB et al., 2004). It was the commonest pathogenic organism isolated in our hospital during the study period and was most prevalent in the New Born Unit (NBU), the pediatric wards and Intensive Care Unit (ICU). Most of the KP isolates were multidrug resistant with high resistance to Cephalosporins, Penicillins and Gentamycin, relatively high resistance to the Quinolones and relatively moderate sensitivity to Carbepenems. Our study suggests a high prevalence of MDRKP in the hospital with possible cross contamination between the wards.

he Institutional Review Board of Saint Monica University Buea, Cameroon while administrative authorization to collect clinical samples was obtained from the CDC Central Clinic, Tiko, where the study was conducted. Written informed assent was obtained from the patients and data was treated with discrete confidentiality. Study Population The study population was drawn from the population of women attending the CDC Central Clinic, Tiko, South West of Cameroon from January to June 2014. A total of 100 sexually active women aged from 15 to 45 years, who met the inclusion criteria were recruited in this study. After detailed explanation of the study objectives and protocols by the researcher, the women gave their consent to participate in the study by signing informed consent forms. Sexually active women who refused to give their consent or who were menstruating at the time of recruitment were excluded from the study. Socio-demographic Data A structured questionnaire was used to collect participants’ socio-demographic data. Taken into consideration were the participant’s age, marital status, pregnancy, knowledge of BV, clinical data, and other variables.

the KP isolates were multidrug resistant with high resistance to Cephalosporins, Penicillins and Gentamycin, relatively high resistance to the Quinolones and relatively moderate sensitivity to Carbepenems. Our study suggests a high prevalence of MDRKP in the hospital with possible cross contamination between the wards. MDR KBP has been found to be resistant to third generation Cephalosporins, Aminoglycosides and Quinolones but often sensitive to Carbapenems (Bradford PA, 2001; Hirsch EB et al., 2010). In our study, resistance to Cephalosporins was very high (>80% resistance) possibly due to widespread use of ceftriaxone especially in our NBU where the largest burden of this organism was present. Ceftriaxone resistance increased rapidly during the study period to remain constantly above 70%. Cefipime and Ceftazidime, although not frequently prescribed in our set up, had very high resistance throughout the study period. Cephalosporin resistance is conferred from ^-lactamase production, which is plasmid mediated and transferable to other Cephalosporins and to other gram negative enterobacteriacea such as E. coli (Bradford PA, 2001; George AJ et al., 2005). Similar findings were documented in Tanzania where Cefotaxime was the most prescribed antibiotic in the NBU resulting in high overall third generation Cephalosporin resistance (Stephen EM et al., 2013). Data from Korea for the year 2007 showed lower Cephalosporin resistance rates than in our study (Cefotaxime 25%, Cefepime 22%, Ceftazidime 29%, Cefoxitin 21%)) although during an ESBL KP outbreak Ceftazidime resistance rose to 47% (Lee K et al., 2010; Roh KH et al., 2008).

losporin resistance (Stephen EM et al., 2013). Data from Korea for the year 2007 showed lower Cephalosporin resistance rates than in our study (Cefotaxime 25%, Cefepime 22%, Ceftazidime 29%, Cefoxitin 21%)) although during an ESBL KP outbreak Ceftazidime resistance rose to 47% (Lee K et al., 2010; Roh KH et al., 2008). Development of bacterial resistance to Aminoglycosides has been documented to be slowest amongst the antibiotics (Rennie RP et al., 1977). In Toronto, the first Gentamycin resistance was documented 7 years after first use (Curie K et al., 1978). This resistance was transferrable to other Gram Negatives, particularly E. coli. Gentamycin is part of many first line regimens in both developed and developing countries. In our hospital, it is often used as first line antibiotic in neonatal sepsis and has been used in the unit for over 10 years. It recorded a high level of resistance of 83% compared to Amikacin (21% resistance) which is preserved for second-line treatment. In Korea, both Gentamycin and Amikacin had relatively lower resistance of about 30% (Lee K et al., 2010). Data from Tanzania was similar to ours for Gentamycin (77% resistance) and slightly lower for Amikacin (1.45 % resistance) (Tumaini VM et al., 2012). Gentamycin resistant KP species has been found to have higher carriage in the intestinal and urinary tracts and longer durations of shedding than Gentamycin sensitive KP resulting in nosocomial hospital outbreaks (Hart CA et al., 1982).

nce) and slightly lower for Amikacin (1.45 % resistance) (Tumaini VM et al., 2012). Gentamycin resistant KP species has been found to have higher carriage in the intestinal and urinary tracts and longer durations of shedding than Gentamycin sensitive KP resulting in nosocomial hospital outbreaks (Hart CA et al., 1982). K. pneumoniae resistance to Quinolones was 41.3% with Ciprofloxacin resistance being at 44.4 %. Similar findings were reported in India where K. pneumoniae resistance to Ciprofloxacin amongst children below ten years was 35.71% (Tiwari DK et al., 2013). In our study, Ciprofloxacin resistance reduced from over 80% in 2005-2007 to below 30% in 2011 -2013 possibly due to reduction in prescription in our hospital over that period. Quinolone resistance has been noted to be low in NBU compared to other wards due to contra-indication of their use in newborns (n= 25, 38%, p<0.05) (Stephen EM et al., 2013). However, in our study Quinolone resistance in the medical wards was lower than the over-all resistance (36.6% versus 44.4%). Quinolone resistance is plasmid mediated and transferrable from person to person especially amongst patients with long hospital stay. Aminoglycoside exposure has been associated with Quinolone resistance in K. pneumoniae and Pseudomonas aeruginosa, suggesting the need for awareness of the potential cross resistance and thus failure of Quinolones in settings where there is widespread use of Aminoglycosides such as in our hospital (Lautenbach E et al., 2001; Masuda N et al., 1992; Strausbaugh LJ et al., 1996).

resistance in K. pneumoniae and Pseudomonas aeruginosa, suggesting the need for awareness of the potential cross resistance and thus failure of Quinolones in settings where there is widespread use of Aminoglycosides such as in our hospital (Lautenbach E et al., 2001; Masuda N et al., 1992; Strausbaugh LJ et al., 1996). The resistance to Carbapenems was lowest at 23.2%. Resistance to Meropenem was 7.0%. Carbapenem resistant K. pneumoniae is considered the most resistant strain. Carbapenem resistance has been shown to result from changes in membrane permeability, high β lactamase and Cephalosporinase levels and production of carbapenemases. Evidence of carbapenemase production from a unit needs to be handled efficiently as they are associated with P lactamase production resulting in penicillin and cephalosporin resistance (Walsh TR et al., 2012). A prospective study of clinical enterobacteriacea isolates in Morocco found a Carbapenemase production rate of 2.8% (Wartiti MA et al., 2012). Data from several European countries record rates of less than 1% except during outbreaks where rates as high as 17-43% are recorded. (Chetcuti Z et al., 2014; Marina K et al., 2014). In Kenya only seven Carbapenem resistant K. pneumoniae were detected in a two years study at the Aga Khan University Hospital in Nairobi (Nordmann P et al., 2011). Most of the isolates were from urine of adult patients in contrast to our study where the isolates were from blood cultures and significantly from neonates. Our results indicate higher rates of Carbapenem resistance and this should be monitored with possible genomic and epidemiologic studies to curb any further rise. There was 100% resistance to Meropenem in the period 2002-2004 (n=8) and all the samples were from the NBU.

om blood cultures and significantly from neonates. Our results indicate higher rates of Carbapenem resistance and this should be monitored with possible genomic and epidemiologic studies to curb any further rise. There was 100% resistance to Meropenem in the period 2002-2004 (n=8) and all the samples were from the NBU. Diagnosis and treatment of MDR KP infections and colonization in our hospital and similar hospitals in the developing countries is complicated. A multidisciplinary approach is necessary with teams including clinicians, pharmacists, and microbiologists with support from the hospital management. Prevention of spread of resistance by proper aseptic procedures such as hand washing, disinfection of equipment, reduction in ward congestion and isolation need to be intensified. Awareness on MDR organisms should be raised. Early detection of MDR KP through routine surveillance with profiling of the resistance pattern should be encouraged to avoid ineffective therapy in order curb the spread of the strain responsible for the outbreak. In one incidence in New York, increased use of Ceftazidime in treatment of Acinetobacter infections resulted in an outbreak of Ceftazidime resistant K. pneumoniae, and it was after detection that effective therapy was instituted (Kenneth SM et al., 1993). Attempts to reduce the multi-class resistance should also be made to regain susceptibility to cheaper and more readily available drugs. Antibiotic restriction and rotation are possible options for developing countries where access to newer antibiotics is limited. In the USA, an 80% reduction of hospital consumption of Cephalosporins resulted in a 44.0% reduction in Ceftazidime-resistant K. pneumoniae infection and colonization throughout the medical center (P<.01), a 70.9% reduction of cephalosporin resistance within all intensive care units (P<.001), and an 87.5% reduction within the surgical intensive care unit (P<.001) within 1 year of restriction (Rahal JJ et al., 1998).

n in Ceftazidime-resistant K. pneumoniae infection and colonization throughout the medical center (P<.01), a 70.9% reduction of cephalosporin resistance within all intensive care units (P<.001), and an 87.5% reduction within the surgical intensive care unit (P<.001) within 1 year of restriction (Rahal JJ et al., 1998). Conclusion There was a high prevalence of MDR KBP isolates in our hospital with most of the isolates in the NBU. The isolates were highly resistant to third generation Cephalosporins and Gentamycin. Recommendations Stringent infection prevention control measures need to be instituted especially in the NBU to minimize the nosocomial spread of MDRKP. In case of suspected or confirmed infection with MDRKP; Carbapenems are the drug of choice for treatment. Quinolones and Amikacin may be considered where Carbapenems are unavailable. We also recommend further studies to characterize the molecular genetic composition of MDR and Carbapenem resistant K. pneumoniae in our set up. Acknowledgements Mr. Richard Too (Head of the Microbiology Laboratory MTRH) who availed the records of the data and clarifications in an orderly and timely manner. Dr. Wilson Aruasa the Deputy director MTRH, for encouraging and supporting the study. All the people involved in any manner at all stages of this study.

Introduction Reproductive-tract infections (RTI) including bacterial vaginosis, are major public health concerns among sexually–active women in developing countries with bacterial vaginosis alone being responsible for 40-50% of vaginal infections (Spiegel, 1991). Bacterial vaginosis (BV) is a polymicrobial, superficial vaginal infection involving a reduction in the amount of hydrogen peroxide-producing Lactobacillus and overgrowth of anaerobic bacteria. The reduced numbers of Lactobacillus allow overgrowth of anaerobic bacteria, especially Mycoplasma hominis, Bacteroides species, Mobiluncus species and Gardnerella vaginalis. Although most of these organisms are present in small number in the normal vagina, Mobiluncus species are rarely found and indicates a sensitive marker for the diagnosis of BV. However, recent studies suggests that G. vaginalis is the key player in the pathogenesis of BV initiating the development of a biofilm and then becomes the scaffolding to which other species adhere (Verstraelen et al., 2013). Past studies on the microbiota of the epithelial surfaces of vaginal biopsy from women with BV showed that G. vaginalis comprised 90% of the bacteria in the biofilm (Swidsinski et al., 2005).

itiating the development of a biofilm and then becomes the scaffolding to which other species adhere (Verstraelen et al., 2013). Past studies on the microbiota of the epithelial surfaces of vaginal biopsy from women with BV showed that G. vaginalis comprised 90% of the bacteria in the biofilm (Swidsinski et al., 2005). Bacterial vaginosis is an extremely prevalent condition and the number one cause of vaginitis among sexually active women (Yudin and Money, 2008). Although it is not a reportable disease, available data show the prevalence of BV among non-pregnant women to range from 15% to 30% and 50% for pregnant women (Fleury et al., 1981; Shayo et al., 2012). However, the majority of cases of BV are asymptomatic and remain unreported and untreated (Amsel et al., 1983). Fifty to 75 percent of women with bacterial vaginosis (BV) are asymptomatic. Symptomatic women typically present with vaginal discharge and/or vaginal odor. The discharge is off-white, thin, and homogeneous; the odor is an unpleasant “fishy smell” that may be more noticeable after sexual intercourse and during menses.

Fifty to 75 percent of women with bacterial vaginosis (BV) are asymptomatic. Symptomatic women typically present with vaginal discharge and/or vaginal odor. The discharge is off-white, thin, and homogeneous; the odor is an unpleasant “fishy smell” that may be more noticeable after sexual intercourse and during menses. Previously considered a benign condition, BV has been implicated in many gynecologic conditions and complications of pregnancy including pelvic inflammatory disease, post hysterectomy vaginal cuff cellulites, endometriosis, amniotic fluid infection, preterm delivery, preterm labor, premature rupture of the membranes, and possibly spontaneous abortions (Eschenback et al., 1988). A cross sectional study carried out in Bangladesh reported that reproductive tract infections (RTI), including bacterial vaginosis, are a major public-health problem among sexually active women. Among RTI investigated, bacterial vaginosis was responsible for 40 -50% of vaginal infections in sexually -active women (Spiegel, 1991).

Study Population The study population was drawn from the population of women attending the CDC Central Clinic, Tiko, South West of Cameroon from January to June 2014. A total of 100 sexually active women aged from 15 to 45 years, who met the inclusion criteria were recruited in this study. After detailed explanation of the study objectives and protocols by the researcher, the women gave their consent to participate in the study by signing informed consent forms. Sexually active women who refused to give their consent or who were menstruating at the time of recruitment were excluded from the study. Socio-demographic Data A structured questionnaire was used to collect participants’ socio-demographic data. Taken into consideration were the participant’s age, marital status, pregnancy, knowledge of BV, clinical data, and other variables. Samples The samples analyzed were vaginal swabs and were collected aseptically from the women with the use of sterile swab sticks. The participant was instructed to lie on the collection bed in a supine position. With both legs flexed, the labial majora was held apart by the participant. A labeled sterile swab was carefully inserted into the vagina and discharge was collected by gently rolling the inserted swab. The swab sticks were recapped and refrigerated (at 4°C) for further processing at the end of each day.

pine position. With both legs flexed, the labial majora was held apart by the participant. A labeled sterile swab was carefully inserted into the vagina and discharge was collected by gently rolling the inserted swab. The swab sticks were recapped and refrigerated (at 4°C) for further processing at the end of each day. Diagnosis of Bacterial Vaginosis The Nugent criteria for the diagnosis of BV were employed (Nugent et al., 1991). According to this method, three bacterial morphotypes — Lactobacillus, Mobiluncus and Gardnerella were used as markers of BV. Vaginal swabs were used to prepare smears on clean, grease-free slides. The smears were allowed to air-dry and then fixed with 95% alcohol. The smears were then Gram stained using the Jenson’s modified Gram technique as described by Cheesbrough (2006). The Gram stained smears were observed under the light microscope (Olympus) using the oil immersion objective (x100). Lactobacillus morphotypes were reported as large Gram positive bacilli, Mobiluncus as curved Gram negative or Gram variable rods while Gardneralla were reported as small Gram variable bacilli. Using the Nugent scoring technique, scores ranged from 0 to 4 for Lactobacillus; 0 indicating that ≥ 30 organisms were found and 4 indicated that no organisms were found. In contrast, for Gardnerella, a score of 0 indicated that no organisms were found and 4 indicated > 30 organisms. For Mobiluncus, scores ranged from 0 to 2; 0 indicating no organisms and 2 indicating > 5 organisms. A total numerical score was calculated (ranging from 0 to 10) for each sample by summing the scores for each of the three morphotypes. The scores were classified into one of three ranges to define a case of BV; a score in the range 0-3 indicated normal vaginal flora, 4-6 indicated altered vaginal flora which is not consistent with BV, and 7-10 was consistent with BV.

rom 0 to 10) for each sample by summing the scores for each of the three morphotypes. The scores were classified into one of three ranges to define a case of BV; a score in the range 0-3 indicated normal vaginal flora, 4-6 indicated altered vaginal flora which is not consistent with BV, and 7-10 was consistent with BV. Data Analysis Data obtained from this study was entered into the computer Microsoft Excel and analyzed using SPSS version 17.0. The Chi square test was used to determine the differences in the distribution of bacterial vaginosis. Statistical significance was considered at p<0.05. Results Socio-demographic Data Participants’ socio-demographic data are presented in Table 1. Of the 100 participants, 54 (54%) were equally distributed between the age ranges 25-29 and 30-34 years while only a few fell in the age ranges of 15-19 years (5%) and 40 years or more (6%). More married women participated in the study (62%) as well as pregnant women (59%). With regards to educational level, 55% of the participants had attained only primary qualification and only one had never been to school. Table 1 Participants’ sociodemographic data

Results Socio-demographic Data Participants’ socio-demographic data are presented in Table 1. Of the 100 participants, 54 (54%) were equally distributed between the age ranges 25-29 and 30-34 years while only a few fell in the age ranges of 15-19 years (5%) and 40 years or more (6%). More married women participated in the study (62%) as well as pregnant women (59%). With regards to educational level, 55% of the participants had attained only primary qualification and only one had never been to school. Table 1 Participants’ sociodemographic data Parameter Responses Number/100 Age (years) 15-19 5 20-24 14 25-29 27 30-34 27 35-39 21 ≥40 6 Marital Status Married 62 Single 38 Education Never been to school 1 Primary 55 Secondary 24 High school 14 Post high school 6 Pregnancy status Pregnant 59 Not pregnant 41 Participants’ Knowledge of Bacterial Vaginosis and Practices that Pre-dispose them to the Infection Information concerning participants’ knowledge of BV as well as the practices they indulge in that could predispose them to the infection is summarized in Table 2; only 38% of the participants had adequate knowledge of bacterial vaginosis and up to 97% of them indulged in at least one practice that predisposes the infection. Table 2 Participants’ knowledge of BV and practices that predispose them to the infection Assessed Variable Number of Respondents/100 Total Yes No Knowledge of urogenital tract infection 38 62 100 Knowledge of bacterial vaginosis 38 62 100 Knowledge of BV transmission 44 56 100 Knowledge of vaginal douching 32 68 100 Knowledge of risk associated with vaginal douching 40 60 100

Table 2 Participants’ knowledge of BV and practices that predispose them to the infection Assessed Variable Number of Respondents/100 Total Yes No Knowledge of urogenital tract infection 38 62 100 Knowledge of bacterial vaginosis 38 62 100 Knowledge of BV transmission 44 56 100 Knowledge of vaginal douching 32 68 100 Knowledge of risk associated with vaginal douching 40 60 100 Practicing vaginal douching 97 3 100 Use of antiseptics in vaginal douching 43 57 100 donning air-tight pants 33 67 100 Prevalence of Bacterial Vaginosis Of the 100 participants, 38 were positive for bacterial vaginosis, 34 had alteration in vaginal normal flora that was not consistent with BV while 28 were negative giving a prevalence rate of 38%. The prevalence of BV with respect to the various sociodemographic data and patients’ characteristics is presented in Table 3. Although the prevalence of BV was higher in some groups than others, no statistically significant differences were found (P-values ≥ 0.05). Table 3 Prevalence of BV with respect to various sociodemographic characteristics Patient’s Characteristic Count/10 0 BV status Statistics

donning air-tight pants 33 67 100 Prevalence of Bacterial Vaginosis Of the 100 participants, 38 were positive for bacterial vaginosis, 34 had alteration in vaginal normal flora that was not consistent with BV while 28 were negative giving a prevalence rate of 38%. The prevalence of BV with respect to the various sociodemographic data and patients’ characteristics is presented in Table 3. Although the prevalence of BV was higher in some groups than others, no statistically significant differences were found (P-values ≥ 0.05). Table 3 Prevalence of BV with respect to various sociodemographic characteristics Patient’s Characteristic Count/10 0 BV status Statistics No. Negative No. Negative with altered vaginal flora No. Positive (%) Age (years) 15-19 5 2 2 1 (20) χ=9.735 20-24 14 4 6 4 (28.5) P=0.464 25-29 27 5 9 13 (48.1) 30-34 27 7 8 12 (44.4) > 35 27 10 9 8 (29.6) Level of education Uneducated 1 0 0 1 (100) χ=4.562 Primary 55 13 19 23 (41.8) P=0..803 Secondary 24 9 7 8 (33.3) High school 14 4 5 5 (35.7) Post high school 6 2 3 1 (16.6) Knowledge of BV and its spread Adequate 38 12 14 12 (31.6) χ=3.175 Inadequate 62 16 20 26 (41.9) P=0.529 Marital status Married 62 17 19 26 (41.9) χ=1.225 Single 38 11 15 12 (31.6) P=0.542 Pregnancy Pregnant 59 15 17 27 (45.8) χ=3.762 Non-pregnant 41 13 17 11 (26.8) P=0.152 Practice vaginal douching Yes 97 25 35 37 (38.1) χ=5.615 No 3 2 0 1 (33.3) P=0.06 Donne air-tight pants Yes 33 8 10 15 (45.5) χ=1.167 No 67 20 24 23 (34.3) P=0.558 Clinical data of BV/Complications of BV From the data obtained, 78% participants had current or past history of bacterial vaginosis or its complications ranging from normal vaginal itches (40%), through children born with low weight (8%) to miscarriages (30%).

Yes 33 8 10 15 (45.5) χ=1.167 No 67 20 24 23 (34.3) P=0.558 Clinical data of BV/Complications of BV From the data obtained, 78% participants had current or past history of bacterial vaginosis or its complications ranging from normal vaginal itches (40%), through children born with low weight (8%) to miscarriages (30%). Discussion and Conclusion This study reported a prevalence of 38% for bacterial vaginosis among sexually active women in the study area. This finding is comparable to that of Spiegel (1991) in Bangledesh showing a 40-50% prevalence rate for BV. Although little or no information is available in Cameroon on bacterial vaginosis, this condition remains a major public-health problem among sexually active women in Cameroon based on our findings. This problem is made worst in pregnant women due to its sequallae and adverse effects on pregnancy and pregnancy outcome. The impact of bacterial vaginosis in pregnancy for the causation of premature rupture of membranes, preterm delivery and low birth weight is well established (Koumans et al., 2001). Bacterial vaginosis is often asymptomatic, and its diagnosis is inexpensive yet needs technical skill. Failure to detect BV early constitutes a major risk factor for the acquisition of other sexually transmitted infections as previously reported (Joesoef et al., 1995; Cherpes et al., 2003).

shed (Koumans et al., 2001). Bacterial vaginosis is often asymptomatic, and its diagnosis is inexpensive yet needs technical skill. Failure to detect BV early constitutes a major risk factor for the acquisition of other sexually transmitted infections as previously reported (Joesoef et al., 1995; Cherpes et al., 2003). We reported a higher prevalence of bacterial vaginosis among participants who attained only primary education or no education at all, those who practiced vaginal douching and those who wore tight underwear. These findings raise the need for public awareness and education on vaginal infections in general. Emphasis should be laid on proper hygienic practices as well as the bad sides of early sex, multiple sex partners, change of sex partners, use of unprescribed drugs and antiseptics amongst others. The higher prevalence of BV among pregnant women and married women remains a major phobia. Implementation of diagnosis of BV during antenatal care especially during the second and third trimesters of pregnancy can ameliorate the condition by reducing the number or frequency of BV-associated reproductive tract infections and birth defects. However, given the complicated nature of BV diagnosis and considering the generally higher prevalence of BV among pregnant women, syndromic treatment may be an alternative. We suggest that all married women should be screened at least once a year for BV so that positive cases can be detected and treated early enough to reduce subsequent sequellae.

d nature of BV diagnosis and considering the generally higher prevalence of BV among pregnant women, syndromic treatment may be an alternative. We suggest that all married women should be screened at least once a year for BV so that positive cases can be detected and treated early enough to reduce subsequent sequellae. We, therefore, conclude that the prevalence of bacterial vaginosis in our study population is 38% and highest among women aged between 25 and 34 years, pregnant women, married women, less educated women and women who practiced poor vaginal hygiene. Acknowledgements We thank the administration and staff of CDC Central Clinic Tiko for the collaboration and the participants who made the work possible. Declaration of Competing Interests The author(s) declare that they have no competing interests, no financial relationships with any organizations that might have an interest in the submitted work; no other relationships or activities that could appear to have influenced the submitted work. Author Contributions: AEA Conceived, designed, sponsored and supervised the experiments. FFF Contributed reagents and drafted the manuscript. ACA Contributed reagents and performed the experiments. ARA and AMU analyzed the data, Contributed reagents / materials / analysis tools.

Introduction Toxoplasma gondii is a zoonotic intracellular parasite that causes the disease toxoplasmosis (Kopecna et al., 2006). The infection is very common in humans around the world (Lim and Othman, 2014). In immuno-competent individuals, the symptoms may be mild or remain asymptomatic while it causes high rates of morbidity and mortality in immunocompromised individuals (Shen et al., 2016). Toxoplasmosis is one of the most important opportunistic infections detected in HIV patients. According to the U.S centres for disease control and prevention, Toxoplasmosis is an AIDS- defining illness. In HIV infection, toxoplasmic encephalitis occurs due to reactivation of latent Toxoplasma infection (Daryani et al., 2011). However, the epidemiology of toxoplasmosis is not clearly understood in the Northern region of South Africa.

entres for disease control and prevention, Toxoplasmosis is an AIDS- defining illness. In HIV infection, toxoplasmic encephalitis occurs due to reactivation of latent Toxoplasma infection (Daryani et al., 2011). However, the epidemiology of toxoplasmosis is not clearly understood in the Northern region of South Africa. The prevalence of toxoplasmosis varies between regions and is higher in areas where cats are common (Prestrud et al., 2010; Dubey et al., 2016). In South Africa, Harri et al. (2007) reported a prevalence of 8% infection with T. gondii among HIV patients in Johannesburg, whereas another study in the Limpopo province, north of the country, reported a higher prevalence (18.1%) among chronically ill HIV patients (Bessong and Mathomu, 2010). However, both studies did not test for IgM antibodies. In Brazil, the prevalence of Toxplasma IgG was 16% among pregnant women while that of IgM was 1% in the same population (de Quadros et al., 2015). Moreover, in an Ethiopian study, the seroprevalence was found to be 60% and 64% in females and males respectively (Negash et al., 2008). However, they also did not test for T. gondii IgM antibodies.

of Toxplasma IgG was 16% among pregnant women while that of IgM was 1% in the same population (de Quadros et al., 2015). Moreover, in an Ethiopian study, the seroprevalence was found to be 60% and 64% in females and males respectively (Negash et al., 2008). However, they also did not test for T. gondii IgM antibodies. Exposure to Toxoplasma is shown by the detection of Toxoplasma IgG while IgM indicates current infections (Gashout et al., 2016). These infections might be more relevant as they indicate the level of epidemic in a specific community. Current infections among pregnant women are dangerous and could lead to malformation of the newborn or its death (Dubey et al., 2016). Although previous studies have shown that the prevalence of Toxoplasma IgG in South Africa varies between 8% and 16%, very few studies have reported on the prevalence of current infections particularly in the northern part of South Africa. Therefore, the present study sought to determine T. gondii IgG and IgM seroprevalence among patients visiting the HIV clinics in the Vhembe region and to identify factors that might be associated with T. gondii in the region.

ted on the prevalence of current infections particularly in the northern part of South Africa. Therefore, the present study sought to determine T. gondii IgG and IgM seroprevalence among patients visiting the HIV clinics in the Vhembe region and to identify factors that might be associated with T. gondii in the region. Materials and Methods Study participants and design The study was a cross sectional survey of patients visiting HIV clinics at different hospitals in the region and the University of Venda. It was conducted in the Vhembe district in Limpopo province from April 2012 to January 2013. A well-structured questionnaire was used to collect socio-demographic information and possible risk factor information on toxoplasmosis from participants. A total of 161 blood samples were collected from HIV positive (118) and negative (43) patients from the three main hospitals in the Vhembe district, Limpopo Province (Elim, Donald Fraser and Tshilidzini). Some of the samples were collected from the students attending the University of Venda. The samples were transported in cooler boxes filled with ice to the laboratory of Microbiology at the University of Venda for further analysis. Plasma was obtained following centrifugation of blood samples and stored at -20ºc until further use.

samples were collected from the students attending the University of Venda. The samples were transported in cooler boxes filled with ice to the laboratory of Microbiology at the University of Venda for further analysis. Plasma was obtained following centrifugation of blood samples and stored at -20ºc until further use. Serological testing for toxoplasmosis The presence of T. gondii IgG and IgM antibodies in the serum samples was determined by the ELISA method. The test was done using the bioelisa TOXO IgG and TOXO IgM kits (BIOKIT, S.A, Barcelona- Spain) as per the manufacturer’s instructions. The optical densities (OD) values were read using ELISA reader (Bio-Tek INSTRUMENTS, INC) at 450nm and the results were recorded. Ethical considerations The health and ethics committee of the University of Venda approved the study. Ethical clearance was also obtained from the different hospitals where the samples were collected. The objectives of the study were clearly explained to the sample providers and they were requested to sign the consent forms before data and sample collection. The information obtained from the patients was kept confidential. Statistical analysis The data obtained was analysed using SPSS version 18.1. The chi square test was used for all analysis comparing the prevalence of toxoplamosis among the patients according to different demographic characteristics as well as different potential risk factors. The difference was considered significant if the p value was less than 0.05.

ned was analysed using SPSS version 18.1. The chi square test was used for all analysis comparing the prevalence of toxoplamosis among the patients according to different demographic characteristics as well as different potential risk factors. The difference was considered significant if the p value was less than 0.05. Results Socio- demographic and clinical characteristics of the study population Most of the patients tested for Toxoplasma antibodies in the present study, were HIV positive 118 (73.3%) while 34 (26.7%) were HIV negative. Most of them were from Donald Fraser (41%), and 64% of the patients were females [Table 1]. Table 1 Demographic characteristics of the study population

Results Socio- demographic and clinical characteristics of the study population Most of the patients tested for Toxoplasma antibodies in the present study, were HIV positive 118 (73.3%) while 34 (26.7%) were HIV negative. Most of them were from Donald Fraser (41%), and 64% of the patients were females [Table 1]. Table 1 Demographic characteristics of the study population Characteristics Frequency Percent HIV status HIV negative 43 26.7 HIV positive 118 73.3 Hospital Donald Fraser 66 41.0 Elim 12 7.5 LTT 15 9.3 Tshilidzini 25 15.5 Univen 43 26.7 Age group Less than 25 years 39 26.5 25 years- 45 years 65 44.2 Above 45 years 43 29.3 Gender Male 54 36.0 Female 96 64.0 Marital Status Single 81 54.0 Married 49 32.7 Divorced 8 5.3 The occurrence of T. gondii IgG in patients in relation to HIV status, place of infection, hospitals, age, gender and marital status of the patients. The antibodies (IgG) against T. gondii were more common in HIV infected patients (38%) compared to HIV negative patients (16.7%) and the difference was statistically significant (χ2=6.218, p= 0.013). Patients recruited at Elim hospital had a higher prevalence (54.5%) compared to other hospitals and University of Venda students had the least prevalence among all the sites. The antibodies (IgG) against T. gondii were more detected in patients aged above 45, and the least prevalence was found in patients aged 25 or less. In terms of gender, the infection was more commonly detected in females (34.1%) than in males (29.8%) and the difference was not statistically significant. The infection was more common among patients who indicated that they had obtained the HIV infection in the Vhembe district (42.0%) compared to those infected in other places (27.3%) but the difference was not statistically significant (p>0.05) (Table 2). There was no difference in the prevalence of IgG among patients receiving prophylaxis and those who were not receiving prophylaxis.

ey had obtained the HIV infection in the Vhembe district (42.0%) compared to those infected in other places (27.3%) but the difference was not statistically significant (p>0.05) (Table 2). There was no difference in the prevalence of IgG among patients receiving prophylaxis and those who were not receiving prophylaxis. Table 2 The occurrence of T. gondii IgG in patients in relation to HIV status, place of infection, hospitals, age, gender and marital status of the patients.

ey had obtained the HIV infection in the Vhembe district (42.0%) compared to those infected in other places (27.3%) but the difference was not statistically significant (p>0.05) (Table 2). There was no difference in the prevalence of IgG among patients receiving prophylaxis and those who were not receiving prophylaxis. Table 2 The occurrence of T. gondii IgG in patients in relation to HIV status, place of infection, hospitals, age, gender and marital status of the patients. Variables Characteristics Toxo IgG Positive Total Statistics (χ2, p) HIV status HIV negative 7 (16.7%) 42 χ2=6.218, p=0.013 HIV positive 38 (38%) 100 Hospital Donald Fraser 22(40.0%) 55 χ2= 8.963, p=0.062 Elim 6 (54.5%) 11 LTT 4 (26.7%) 15 Tshilidzini 6 (31.6%) 19 Univen 7 (16.7%) 42 Agegroup3 25 or less 6 (16.2%) 37 χ2=7.425, p=0.024 25- 45 20 (35.1%) 57 Above 45 16 (45.7%) 35 Gender Male 14 (29.8%) 47 χ2=0.258, p=0.611 Female 29 (34.1%) 85 Marital status Single 21 (28.8%) 73 χ2=1.258, p= 0.739 Married 15 (35.7%) 42 Divorced 3 (42.9%) 7 Widowed 4 (40.0%) 10 Infected in Vhembe NO 3 (27.3%) 11 χ2=0.871, p=0.351 YES 34(42.0%) 81 Year of recruitment 2010 36 (43.6%) 104 χ2=1.536, p= 0.215 2011 9 (23.7%) 38 WAZ less than -2 Not underweight 36 (30.5%) 118 χ2=2.154, p=0.142 Underweight 4(57.1%) 7 Prophylaxis NO 23 (38.3%) 60 χ2=0.149, p=0.700 YES 14 (42.4%) 33 The occurrence of T. gondii -IgG in HIV patients in relation to ARV treatment and CD4 count Toxoplasma gondii IgG antibodies were more common in patients who were not taking ARV’s (46.2%)[Table 3]. No significant difference was observed between the CD4 level (less than 50) and T. gondii IgG titer. The seroprevalence of Toxoplasma IgG was much higher (58.3%) among patients with high viral load compared to those with low viral load (14.8%) (P=0.005) The patients using alternative medication were not highly infected (33.3%), compared to those who were not taking alternative medication (40.2%) although the difference was not significant. In terms of ARV’s used by these patients, tenofovir was found to be the best ARV because of the low Toxo sero-prevalence in people who were taking it (29.4%), compared to those using other ARV’s, e. g stavudine, lamivudine, nevirapine and efavirenz.

native medication (40.2%) although the difference was not significant. In terms of ARV’s used by these patients, tenofovir was found to be the best ARV because of the low Toxo sero-prevalence in people who were taking it (29.4%), compared to those using other ARV’s, e. g stavudine, lamivudine, nevirapine and efavirenz. Table 3 The occurrence of T. gondii IgG in HIV positive patients in relation to ARV treatment and the CD4 status of the patients

native medication (40.2%) although the difference was not significant. In terms of ARV’s used by these patients, tenofovir was found to be the best ARV because of the low Toxo sero-prevalence in people who were taking it (29.4%), compared to those using other ARV’s, e. g stavudine, lamivudine, nevirapine and efavirenz. Table 3 The occurrence of T. gondii IgG in HIV positive patients in relation to ARV treatment and the CD4 status of the patients Variables Characteristics Toxo IgG Positive Total Statistics ARV NO 18(46.2%) 39 χ2=1.137, p= 0.286 YES 19 (35.2%) 54 Have you stopped NO 6 (33.3%) 18 YES 2(100.0%) 2 CD4 less than 50 NO 29 (39.7%) 73 χ2=0.007, p=0.932 YES 5 (38.5%) 13 CD4 count Less Than 200 NO 16 (37.2%) 43 χ2=0.195, p=0.659 YES 18 (41.9%) 43 CD4 count more than 300 NO 25 (41.7%) 60 χ2= 0.377, p=0.539 YES 9 (34.6%) 26 Viral load range Low 4(14.8%) 27 χ2=7.770, p=0.005 High 7 (58.3%) 12 Bactrim NO 28 (38.9%) 72 χ2= 0.107, P=0.744 YES 9 (42.9%) 21 Zidovudine NO 17(34.7%) 49 χ2= 0.056, P=0.813 YES 2(40.0%) 5 Efavirenz NO 4 (33.3%) 12 χ2= 0.023, P=0.870 YES 15 (35.7%) 42 Lamivudine NO 0 (0.0 1 χ2= 0.553, P=0.457 YES 19(35.8%) 53 Tenofovir NO 14 (37.8%) 37 χ2=0.363, P=0.547 YES 5 (29.4%) 17 Nevirapine NO 15 (33.3%) 45 χ2= 0.406, P=0.524 YES 4(44.4%) 9 Stavudine NO 7 (31.8%) 22 χ2=0.185, P=0.667 YES 12 (37.5%) 32 The prevalence of T. gondii IgG in patients in relation to animal ownership Patients who admitted keeping animals in their households appeared to be less infected compared to those who did not keep animals in the house. Toxoplasma gondii IgG antibodies were more detected in patients who had dogs in their homes (40%) [Table 4].

alence of T. gondii IgG in patients in relation to animal ownership Patients who admitted keeping animals in their households appeared to be less infected compared to those who did not keep animals in the house. Toxoplasma gondii IgG antibodies were more detected in patients who had dogs in their homes (40%) [Table 4]. Table 4 The prevalence of T. gondii IgG in patients who kept animals in the house Variables Characteristics Toxo IgG Positive Total Statistics (x2, p) Animals in house NO 39 (34.5%) 113 χ2=0.920, p=0.337 YES 5 (23.8%) 21 Chicken NO 41(33.1%) 124 χ2=0.538, p=0.463 YES 3 (23.1%) 13 Cattle NO 43 (32.3%) 133 χ2=0.096, p=0.757 YES 1 (25.0%) 4 Dogs NO 42 (31.8%) 132 χ2=0.148, p=0.701 YES 2 (40.0%) 5 Cats NO 44 (32.4%) 13 χ2=0.477, p=0.490 YES 38(23.4%) 13 Goats NO 43 (32.3%) 133 χ2=0.096, p=0.757 YES 1(25.0%) 4 The occurrence of T. gondii IgG among the patients in relation to water sources The patients were using water from different sources; some patients were treating their water before use, while others were not. No statistically significant difference was noted between those using untreated water as opposed to the group of patients using treated water [Table 5]. Toxoplasma gondii IgG antibodies were higher in patients who were storing water for a long time (more than 5 days) than those who were storing water for less than 5 days. However, the difference was not statistically significant. [Table 5]. Table 5 The occurrence of T. gondii IgG in relation to the different water sources used

Variables Characteristics Toxo IgG Positive Total Statistics (x2, p) Animals in house NO 39 (34.5%) 113 χ2=0.920, p=0.337 YES 5 (23.8%) 21 Chicken NO 41(33.1%) 124 χ2=0.538, p=0.463 YES 3 (23.1%) 13 Cattle NO 43 (32.3%) 133 χ2=0.096, p=0.757 YES 1 (25.0%) 4 Dogs NO 42 (31.8%) 132 χ2=0.148, p=0.701 YES 2 (40.0%) 5 Cats NO 44 (32.4%) 13 χ2=0.477, p=0.490 YES 38(23.4%) 13 Goats NO 43 (32.3%) 133 χ2=0.096, p=0.757 YES 1(25.0%) 4 The occurrence of T. gondii IgG among the patients in relation to water sources The patients were using water from different sources; some patients were treating their water before use, while others were not. No statistically significant difference was noted between those using untreated water as opposed to the group of patients using treated water [Table 5]. Toxoplasma gondii IgG antibodies were higher in patients who were storing water for a long time (more than 5 days) than those who were storing water for less than 5 days. However, the difference was not statistically significant. [Table 5]. Table 5 The occurrence of T. gondii IgG in relation to the different water sources used Variables Characteristics Toxo IgG Positive Total Statistics Water source Communal tap 31 (30.7%) 101 χ2=1.159, p=0.763 Tap in the house 9(39.1%) 23 River/Spring 2 (50.0%) 4 Borehole 2 (33.3%) 6 Water storage Two days or less 27 (31.0%) 87 χ2=0.30, p=0.866 3 to 5 days 7 (36.8%) 19 More than 5 days 9 (34.6%) 26 Treated Source NO 4 (40.0%) 10 χ2=0.251, p=0.616 YES 40 (32.3%) 124 Do you treat water YES 42 (32.1%) 131 χ2=1.593, p=0.207 NO 2 (66.7%) 3 Overall T. gondii IgM seroprevalence in the study population The seroprevalence of T. gondii IgM was much lower compared to that of the IgG at 4.9%. There was no significant difference between the seroprevalence of T. gondii IgM among HIV negative (7.1%) and positive individuals (3.9%) The infection with T. gondii was more detected in patients from UNIVEN (7.1%), LTT (6.7%) and Donald Fraser (5.8%), while those from Elim and Tshilidzini were seronegative (P=0.652) (Table 6). The patients in the age group 25 years or less, had a high prevalence (7.9%) compared to others in the age groups 26 to 45 years (3.6%) [Table 6]. The prevalence was higher in males compared to females and in terms of marital status the prevalence was found to be high in divorced patients (12.5%) and those who indicated that they were married had a prevalence of (4.8%). The patients who were infected in the Vhembe district had a lower prevalence (2.4%), than those who were infected from other places and the difference between these two variables was not statistically significant. There was no difference between the prevalence of T. gondii IgM among patients who were recruited in 2010 and 2011. Those who were underweight had a high prevalence of 12.5% compared to those who were not underweight (4.1%). In terms of prophylaxis, those who were receiving prophylaxis had a low prevalence compared to those who were not receiving.

en the prevalence of T. gondii IgM among patients who were recruited in 2010 and 2011. Those who were underweight had a high prevalence of 12.5% compared to those who were not underweight (4.1%). In terms of prophylaxis, those who were receiving prophylaxis had a low prevalence compared to those who were not receiving. Table 6 The occurrence of T. gondii IgM in HIV patients from different hospitals, place of infection, age groups, gender and marital status of patients.

en the prevalence of T. gondii IgM among patients who were recruited in 2010 and 2011. Those who were underweight had a high prevalence of 12.5% compared to those who were not underweight (4.1%). In terms of prophylaxis, those who were receiving prophylaxis had a low prevalence compared to those who were not receiving. Table 6 The occurrence of T. gondii IgM in HIV patients from different hospitals, place of infection, age groups, gender and marital status of patients. Variables Characteristics Toxo IgM Positive Total Statistics HIV status Negative 3 (7.1%) 42 χ2=0.668, p=0.414 Positive 4 (3.9%) 102 Hospital Donald Fraser 3 (5.8%) 52 χ2=2.460, p=0.652 Elim 0 (0.0%) 10 LTT 1(6.7%) 15 Tshilidzini 0(0.0%) 25 Univen 3 (7.1%) 42 Age group 25 or less 3 (7.9%) 38 χ2=1.398, p=0.497 25-45 2 (3.6%) 55 Above 45 1 (2.6%) 38 Gender Male 4 (8.0%) 50 χ2=2.314, p=0.128 Female 2 (2.4%) 84 Marital status Single 3 (4.0%) 75 χ2= 1.674, p= 0.643 Married 2(4.8%) 42 Divorced 1 (12.5%) 8 Widowed 0 (0.0%) 9 Infected in Vhembe NO 1 (11.1%) 9 χ2= 1.985, p= 0.159 YES 2 (2.4%) 84 Year of recruitment 2010 5 (4.9%) 103 χ2= 0.000, p= 0.995 2011 2 (4.9%) 41 WAZ-2 Not underweight 5(4.1%) 121 χ2= 1.185, p= 0.276 Under weight 1 (12.5%) 8 Prophylaxis NO 3 (4.8%) 62 χ2= 1.599, p=0.206 YES 0 (0.0%) 32 The prevalence of T. gondii IgM in HIV patients taking ARV’S and the CD4 status of the study population Toxoplasma gondii IgM was more prevalent in patients taking ARV’s compared to those who were not on ARV’s. Different types of ARV’s were used by the patients on medications. These included: stavudine, lamivudine, tenofovir, efavirenz, tenofovir and nevirapine. Toxoplasma IgM antibodies were found to be high in patients who were not on ARV’s than those who were adhering to treatment. Toxoplasmagondii IgM antibodies were not detected in patients using alternative medication and bactrim. While statistically insignificant, the Toxo-IgM seroprevalence was higher among patients whose CD4 was less than 50 cell/mm3 (7.1%) compared to those who had a CD4 level more than 50 cells/mm3. No significant difference was observed between the CD4 level (less than 50) and T. gondii IgM titer [Table 7].

on and bactrim. While statistically insignificant, the Toxo-IgM seroprevalence was higher among patients whose CD4 was less than 50 cell/mm3 (7.1%) compared to those who had a CD4 level more than 50 cells/mm3. No significant difference was observed between the CD4 level (less than 50) and T. gondii IgM titer [Table 7]. Table 7 The prevalence of Toxoplasma gondii (IgM) in HIV patients taking different ARV’s and the CD4 status the patients.

on and bactrim. While statistically insignificant, the Toxo-IgM seroprevalence was higher among patients whose CD4 was less than 50 cell/mm3 (7.1%) compared to those who had a CD4 level more than 50 cells/mm3. No significant difference was observed between the CD4 level (less than 50) and T. gondii IgM titer [Table 7]. Table 7 The prevalence of Toxoplasma gondii (IgM) in HIV patients taking different ARV’s and the CD4 status the patients. Variables Characteristics Toxo IgM Positive Total Statistics ARV NO 1 (2.3%) 44 χ2=0.226, p=0.635 YES 2 (4.0%) 50 Zidovudine NO 1 (2.2%) 45 χ2=2.932, p=0.087 YES 1 (16.7%) 6 Efavirenz NO 1 (7.7%) 13 χ2=0.658, p=0.417 YES 1 (2.6%) 38 Lamivudine NO 0(0.0%) 2 χ2=0.085, p=0.771 YES 2 (4.1%) 49 Tenofovir NO 2(6.7%) 30 χ2=1.457, p=0.227 YES 0 (0.0%) 21 Nevirapine NO 1 (2.4%) 42 χ2=1.499, p=0.221 YES 1 (11.1%) 9 Alternative medicine NO 3 (3.3%) 91 χ2= 0.102, p=0.749 YES 0 (0.0%) 3 Bactrim NO 3 (4.0%) 75 χ2=0.785, p=0.376 YES 0 (0.0%) 19 Diarrhea at ARV start NO 2 (4.1%) 49 χ2=0.127, p=0.721 YES 0 (0.0%) 3 CD4 less than 50 NO 1 (1.4%) 72 χ2=1.708, p=0.191 YES 1 (7.1%) 14 CD4 count Less Than 200 NO 1 (2.3%) 43 χ2=0.000, p=1.000 YES 1 (2.3%) 43 CD4 More than 300 NO 1 (1.7%) 58 χ2=0.284, p=0.594 YES 1(3.6%) 28 The prevalence of T. gondii IgM in patients keeping domestic animals in the households Toxoplasma gondii IgM antibodies were higher in patients keeping domestic animals in the households. The patients who had chickens were more infected than those who were not with no chickens. Moreover, patients who had goats and cattles had a higher prevalence of T. gondii IgM [Table 8].

estic animals in the households Toxoplasma gondii IgM antibodies were higher in patients keeping domestic animals in the households. The patients who had chickens were more infected than those who were not with no chickens. Moreover, patients who had goats and cattles had a higher prevalence of T. gondii IgM [Table 8]. Table 8 The prevalence of T. gondii IgM in patients who keep animals in their houses

estic animals in the households Toxoplasma gondii IgM antibodies were higher in patients keeping domestic animals in the households. The patients who had chickens were more infected than those who were not with no chickens. Moreover, patients who had goats and cattles had a higher prevalence of T. gondii IgM [Table 8]. Table 8 The prevalence of T. gondii IgM in patients who keep animals in their houses Variables Characteristics Toxo IgM Positive Total Statistics Animals in house NO 5 (4.4%) 114 χ2= 0.001, p=0.973 YES 1(4.5%) 22 Chicken NO 6 (4.8%) 125 χ2= 0.205, p=0.651 YES 1(7.7%) 13 Cattle NO 6(4.5%) 133 χ2=2.401, p= 0.121 YES 1(20.0%) 5 Dogs NO 6 (4.6%) 131 χ2=1.300, p=0.254 YES 1 (14.3%) 7 Cats NO 7(5.1%) 138 YES 0 0 Goats NO 6 (4.5%) 134 χ2=3.397, p=0.065 YES 1 (25.0%) 4 The prevalence of T. gondii IgM in relation to water sources and the quality of water In terms of water usage, the patients who were using water from rivers and streams were be more infected by T. gondii (33.3%). Furthermore, those who were using water from communal tap had a prevalence of 4.9% [Table 9]. No statistically significant difference was noted between those using tap water as opposed to the group of patients using water from the river/borehole. A high prevalence of T. gondii IgM antibodies was detected in patients using water from untreated source compared to the group of patients using water from treated sources (P=0.311). Very few patients were treating their water before use, about 4.5% of T. gondii IgM was detected in patients who were not treating their water and the difference was not statistically significant (p>0.05). In terms of water storage, the patients who stored water for 2 days or less appeared to be more infected (4.7%) and there was no difference between the prevalence of T. gondii among patients who stored water for 3 to 5 days and more than 5 days, the difference was also not statistically significant.

gnificant (p>0.05). In terms of water storage, the patients who stored water for 2 days or less appeared to be more infected (4.7%) and there was no difference between the prevalence of T. gondii among patients who stored water for 3 to 5 days and more than 5 days, the difference was also not statistically significant. Table 9 The prevalence of T. gondii in relation to water source and the quality of water. Variables Characteristics Toxo IgM Positive Total Statistics Water source Communal tap 5(4.9%) 102 χ2=7.439, p=0.311 Tap in the house 0 (0.0%) 25 River/Spring 1 (33.3%) 3 Borehole 0(0.0%) 6 Treated Source NO 1(11.1%) 9 χ2=1.026, p=0.311 YES 5 (3.9%) 127 Do you treat water NO 6 (4.5%) 134 χ2=0.094, p=0.760 YES 0 (0.0%) 2 Water storage Two days or less 4 (4.7%) 85 χ2=0.029, p=0.986 3 to 5 days 1 (4.2%) 24 More than 5 days 1 (4.0%) 25 Discussion Opportunistic infections, with reference to toxoplasmosis are common cause of serious health problems in immune-compromised patients, particularly those with HIV and AIDS (Schurer et al., 2016). In the present study, out of 142 (100 positive and 42 negative) subjects tested, 31.7% were sero-positive for Toxo- IgG antibodies and of the 144 samples tested, 4.9% were sero-positive for Toxo- IgM antibodies. Our study reported a higher prevalence of T. gondii IgG antibodies than the one reported by Bessong and Mathomu, (2010) and by Kistiah et al in (2011).

ive and 42 negative) subjects tested, 31.7% were sero-positive for Toxo- IgG antibodies and of the 144 samples tested, 4.9% were sero-positive for Toxo- IgM antibodies. Our study reported a higher prevalence of T. gondii IgG antibodies than the one reported by Bessong and Mathomu, (2010) and by Kistiah et al in (2011). This study showed that prevalence of T. gondii IgG was higher in HIV positive patients and low in those who were taking ARVs indicating that the use of ARVs might have a positive effect on T. gondii infections. Similar results were found in Ethiopia where Muluye et al. (2013) also described a high prevalence of toxoplasma IgG among HIV and AIDS patients while the prevalence was lower among HIV negative participants. It was also found that both IgG and IgM antibodies are elevated in patients with high viral load, which cause the body to be more susceptible to opportunistic infections. The IgM antibodies prevalence in the study population decreased with age. This shows a decline in the infection rate. Similar results were found in a study conducted by Bata et al. (2009). Their study showed a high prevalence in patients with age group between 36-45 years. Nijem and Al-Amleh (2009) also found the prevalence of toxoplasmosis to be increasing with age and similar results were obtained by another study by Rosso et al. (2008). It has been suggested that old people are more likely to have been exposed to any of the risk factors than younger people as a results of longer exposure time (Endalew et al., 2012)

found the prevalence of toxoplasmosis to be increasing with age and similar results were obtained by another study by Rosso et al. (2008). It has been suggested that old people are more likely to have been exposed to any of the risk factors than younger people as a results of longer exposure time (Endalew et al., 2012) Ingestion of contaminated meat or contact with infected animals (definitive hosts)are some of the risk factors of toxoplasmosis (Foroutan-Rad et al., 2016). People become infected after ingesting soil, water or plant material contaminated with oocysts. In the present study, many participants agreed that they had animals in their house, but Toxo IgG antibodies were less detected in patients who had animals compared to those who didn’t have animals in the house. This is to show that most of the patients, who had past infection, probably did not get it from the animals, but about 40% of these antibodies were found in patients who had dogs, which might serve as one of the potential risks for toxoplasmosis. Similarly, a study in Sri Lanka indicated that animal ownership did not have a significant impact on the seroprevalence of Toxoplasma (Chandrasena et al., 2016). In a study in Spain, Cano-Terriza et al. (2016) found a high prevalence of Toxoplasma IgG in dogs and the prevalence was associated with the age of the dogs.

Similarly, a study in Sri Lanka indicated that animal ownership did not have a significant impact on the seroprevalence of Toxoplasma (Chandrasena et al., 2016). In a study in Spain, Cano-Terriza et al. (2016) found a high prevalence of Toxoplasma IgG in dogs and the prevalence was associated with the age of the dogs. Toxoplasmosis is an important problem among pregnant women presenting the risk of infecting the new born child. Fortunately, in the present study, IgM positivity was absent among pregnant women. Similar results have been described by Capretti et al. (2014). Over the past five years there has been a significant decrease of toxoplasmosis among pregnant women. Similar results were described by Chandrasena et al. (2016). In a study conducted in Saudi Arabia by Elsafi et al., (2015), the seroprevalence of toxoplasma among pregnant women were 28% and 3% for IgG and IgM respectively while more than 75% of the women were unaware of the disease toxoplasmosis. Education of the community members is therefore very important for the implementation of safety measures to reduce infection levels. Contrary to the IgG, the IgM antibodies were more detected in patients who had animals in home. This may be an indication of the potential differences in the prevalence of exposure to toxoplasmosis and current infection trends. It is possible that people who had the animals were at a much higher high risk of current toxoplasmosis. High IgM antibodies were detected in patients who had goats, cattle and the seroprevalence rate decreased among those who had dogs. A study in Mexico found a high seroprevalence of T. gondii in dogs (Cedillo-Peláez et al., 2012). These results suggest that most of the people who are infected are those who come into contact with dogs, cattles and goats. This study also showed that Toxoplasmosis is associated with low hygiene, considering its potential transmission by water and food which have been identified as important risk factors for the transmission of toxoplasmosis (Pereira et al., 2010). The severity of the infection differs among patients depending on host’s immune status and genotypes. Further studies are needed to determine the circulating genotypes in the study population.

ter and food which have been identified as important risk factors for the transmission of toxoplasmosis (Pereira et al., 2010). The severity of the infection differs among patients depending on host’s immune status and genotypes. Further studies are needed to determine the circulating genotypes in the study population. Conclusion Toxoplasmosis is prevalent in the study population. This study has shown that sero-positive status of the patient to HIV, high viral load, non-adherence to ARV therapy, age (>25 years) and the presence of infected animals such as dogs, cattle and goats are the significant risk factors for toxoplasmosis in this study population. Therefore, people should be educated about toxoplasmosis prevention. Acknowledgements The authors would like to thank the hospital management and the patients for their cooperation throughout the study.

Introduction The Human Immunodeficiency Virus (HIV) causes the chronic disease, Acquired Immunodeficiency Syndrome (AIDS) which has remained a public menace despite the efforts being geared towards curtailing it. Globally, over 1.5 million AIDS-related deaths are recorded annually, more than 2 million new infections were recorded in 2013 and 36 million people are still living with the virus (Fowler, 2014; UNAIDS, 2014). In sub-Saharan Africa, 1.5 million new infections and 1.1 million AIDS-related deaths were recorded in 2013 while 24.7 million people are still living with the virus, making the region home to more than two-third of the world’s HIV cases (UNAIDS, 2014). South Africa is one of the countries worse hit by this pandemic. The National HIV Prevalence and Incidence Survey report released by the Human Sciences Research Council (HSRC) of South Africa in 2014 estimated the number of new HIV infections that occurred in South Africa in 2012 at 400,000; raising the proportion of South Africans infected with HIV from 10.6% in 2008 to 12.2% in 2012 (Malan, 2014). According to UNAIDS, 2012 estimates, about 5.6 million people were living with HIV in South Africa in 2011; a figure which is the highest compared with that of other countries (UNAIDS, 2012). Yet, the National HIV Prevalence and Incidence Survey conducted by HSRC reported a higher figure of 6.4 million in 2014 (Malan, 2014).

ing to UNAIDS, 2012 estimates, about 5.6 million people were living with HIV in South Africa in 2011; a figure which is the highest compared with that of other countries (UNAIDS, 2012). Yet, the National HIV Prevalence and Incidence Survey conducted by HSRC reported a higher figure of 6.4 million in 2014 (Malan, 2014). Researches and censuses have shown that women are more vulnerable to contracting the virus compared with their male counterparts (UNAIDS, 2012, 2014). According to UNAIDS, (2008) report, females comprise about 70% of the world’s poor, more than two-thirds of the world’s illiterate and more than half (about 52%) of all the people living with HIV/AIDS. In Sub-Saharan Africa, 58% of the people living with HIV/AIDS are women (UNAIDS, 2014). Illiteracy and risky sexual behaviours are some of the factors that predispose people to contracting the disease (Kowala-Piaskowska et al., 2010). These three factors are inter-related with the possibility of one resulting in the other and vice-versa. With many cultures against female education while advocating early marriage, the resultant illiteracy and poverty put the female child at a disadvantage and a higher risk of poor health condition including contracting HIV/AIDS (Kowala-Piaskowska et al., 2010).

e possibility of one resulting in the other and vice-versa. With many cultures against female education while advocating early marriage, the resultant illiteracy and poverty put the female child at a disadvantage and a higher risk of poor health condition including contracting HIV/AIDS (Kowala-Piaskowska et al., 2010). The informal economy in South Africa is a growing economic sector and a source of employment for those who are marginalized and excluded from formal work opportunities in South African cities and towns for various reasons. One main factor is the inability of the government and the formal business sector to provide sufficient employment opportunities to people in the economically active age categories. Street trading, one of the largest sectors of informal work has absorbed many of these people, especially women (Lund, 1998; Chen et al., 2001). It was estimated that in the year 2000, there were about half a million street traders (hereafter referred to as street vendors) in South Africa, and more than 70% of these were women (International Labour Organization, 2004).

mal work has absorbed many of these people, especially women (Lund, 1998; Chen et al., 2001). It was estimated that in the year 2000, there were about half a million street traders (hereafter referred to as street vendors) in South Africa, and more than 70% of these were women (International Labour Organization, 2004). In the Limpopo Province of South Africa, HIV prevalence is still rising. The HIV prevalence among antenatal clients in Limpopo Province, which reflects the prevalence among females of reproductive age, increased from 20.6% in 2008 to 22.3% in 2012 (Massyn et al., 2014). Following the same trend is the Vhembe District where HIV prevalence among antenatal clients increased progressively from 13.9% in 2005 to 17.7% in 2012 (Massyn et al., 2014), yet, the South African government is still unable to provide drugs for all infected persons (National Department of Health, 2013). More than 13,000 people were newly tested positive for HIV in Vhembe District in 2014. However, the government was able to initiate Intermittent Prophylactic Treatment (IPT) for only 9,759 out of 12,404 eligible HIV positive clients in the district (Department of Health, 2015).

ns (National Department of Health, 2013). More than 13,000 people were newly tested positive for HIV in Vhembe District in 2014. However, the government was able to initiate Intermittent Prophylactic Treatment (IPT) for only 9,759 out of 12,404 eligible HIV positive clients in the district (Department of Health, 2015). Negative attitudes, the most common of which is stigmatization, have been listed among the factors that hinder the control of HIV (Thanavanh et al., 2013). The United Nations Secretary-General, Ban Ki Moo (2008) observed that many people are afraid to see a doctor for HIV testing or seek treatment if they have the infection for fear of being stigmatized. Sayles et al. (2009) claim that HIV infected individuals are more than four times more likely to report poor access to health care than uninfected ones because of stigmatization from health workers. More than 20% of HIV-infected people in Nigeria reported a denial of health care because of their status (UNAIDS, 2012). A study conducted by International Labour Organization and the Global Network of people living with HIV (2012) recorded a discriminatory attitude towards people living with HIV as high as 54% in Malaysia. Holzemer et al.(2009) also reported a high rate of discrimination in five African countries including South Africa.

A study conducted by International Labour Organization and the Global Network of people living with HIV (2012) recorded a discriminatory attitude towards people living with HIV as high as 54% in Malaysia. Holzemer et al.(2009) also reported a high rate of discrimination in five African countries including South Africa. Araoye (2004) noted in his study among female adolescent street vendors in Nigeria that many of them did not desist from sexual intercourse and some of them even have multiple sexual partners despite the fact that most of them were aware of the sexual transmission mode of the virus. He concluded that their risk perception of the virus was poor. This study was conducted in order to assess the attitudes of female street vendors in Thohoyandou, Vhembe district of South Africa about HIV/AIDS and those infected by it.

e the fact that most of them were aware of the sexual transmission mode of the virus. He concluded that their risk perception of the virus was poor. This study was conducted in order to assess the attitudes of female street vendors in Thohoyandou, Vhembe district of South Africa about HIV/AIDS and those infected by it. Materials and Methods The study design and setting A cross-sectional, descriptive design in a quantitative paradigm was employed in this study because it helps to determine cause and effects at the same time, measuring the attitudes of the respondents to HIV and those infected with the virus at the same time. Thohoyandou town was purposively selected for the study because it is the commercial centre of Vhembe District Municipality, surrounded by many rural settlements and rich in maize, tobacco and banana plantations. Most street vendors in Thohoyandou are found trading in Mvuzuludzo Taxi Rank, while others are located in other taxi ranks and some other streets of the town (Arnold, 2013). Most of these are women selling food stuffs (cooked or raw), fruits, vegetables and household items. The major local language is Tshivenda, but there are Tsonga, Sotho and Afrikaans speaking people in a smaller percentage (Siyabonga Africa, 2009).

d in other taxi ranks and some other streets of the town (Arnold, 2013). Most of these are women selling food stuffs (cooked or raw), fruits, vegetables and household items. The major local language is Tshivenda, but there are Tsonga, Sotho and Afrikaans speaking people in a smaller percentage (Siyabonga Africa, 2009). Population and sampling Street vendors in Thohoyandou are registered under four major locations in Thulamela Local Municipality. Participants for this study were selected from the female street vendors (FSV) trading in those locations which are: the Mvuzuludzo taxi rank and its environment, Thulamela taxi rank and its environment, Game shopping mall environment and Phangami mall environment. As at January 2015, 400 female street vendors were registered with the municipality. The Yamane formula {n=N/(1+Ne2)} was used to arrive at the sample size of 200. A stratified random sampling technique was employed to select prospective participants.

nment, Game shopping mall environment and Phangami mall environment. As at January 2015, 400 female street vendors were registered with the municipality. The Yamane formula {n=N/(1+Ne2)} was used to arrive at the sample size of 200. A stratified random sampling technique was employed to select prospective participants. Data collection and Instrument Data were collected using a structured questionnaire which was prepared in English language and translated to Tshivenda. Retranslation studies were carried out to ascertain the accuracy of the instrument. The retranslation showed an accurate translation of the original document to Tshivenda. Both versions were presented to each volunteered participant to choose which one they preferred. The participants who could read and write were given the questionnaires to complete while the researcher and her assistants waited to collect the completed questionnaires. For those who could not read or write, and some who preferred the questions to be read to them, the researcher read the questions to and completed the questionnaires for them. A pretest was conducted using 20 FSV (10% of sample size) afterwhich some adjustments were made on the instrument. During the main data collection for the study, a total of 220 questionnaires were administered to the respondents and 200 most completed questionnaires were selected for analysis.

questionnaires for them. A pretest was conducted using 20 FSV (10% of sample size) afterwhich some adjustments were made on the instrument. During the main data collection for the study, a total of 220 questionnaires were administered to the respondents and 200 most completed questionnaires were selected for analysis. Statistical analysis Data obtained were analysed with Statistical Package for the Social Sciences (SPSS), version 20.0. Descriptive statistics were performed on the data and the results were summarised using frequency distribution tables and figures. Chi-square tests were performed to identify the relationships between the sociodemographic characteristics and the attitudes of the participants to HIV and those infected. A p-value of less than 0.05 was set to be statistically significant. Ethics and Consent Ethical approval was obtained from the University of Venda Health, Safety and Research Ethics Committee (SHS/15/PH/06/1803). Permission to conduct the study was obtained from the Department of Health, Vhembe District and the Manager of Thulamela Local Municipality. An information letter with detailed explanation of the purpose and method of the study was given to each participant to read (or read and explained to her) before being given the consent form to sign or thumbprint and afterwards the questionnaires were administered to them. The information letter also contains other ethical issues like voluntary participation, informed consent, the right to withdraw, confidentiality, anonymity and minimization of risk of harm to participants.

re being given the consent form to sign or thumbprint and afterwards the questionnaires were administered to them. The information letter also contains other ethical issues like voluntary participation, informed consent, the right to withdraw, confidentiality, anonymity and minimization of risk of harm to participants. Results Sample Characteristics The sociodemographic characteristics of the respondents are presented in Table 1 below. Table 1 Socio-demographic Characteristics of the Participants. CHARACTERISTIC n % AGE (YEARS) < 20 6 3.0 20 - 30 52 26.0 31 - 40 59 29.5 >40 83 41.5 TOTAL 200 100 MARITAL STATUS Single 93 46.7 Married 69 34.7 Separated 14 7.0 Divorced 8 4.0 Widowed 15 7.5 Total 199 100 LEVEL OF EDUCATION None 8 4.1 Primary 22 11.2 Secondary 121 61.4 Tertiary 46 23.3 Total 197 100 AVERAGE INCOME PER WEEK <R500 91 65.0 R500 – R1000 32 22.9 >1000 17 12.1 Total 140 100 NUMBER OF DEPENDANTS None 24 12.8 1 – 3 72 38.3 >3 92 48.9 Total 188 100 Attitide towards voluntary HIV testing Almost all the participants (n=181, 90.5%) indicated that they would accept to do a voluntary HIV test. The reasons for decline given by the other respondents are presented in figure 1. Figure 1 Given reasons for not accepting to do a voluntary HIV test Attitudes towards taking antiretroviral drugs (HAART) in pregnancy A total of 151 participants responded to this question out of which 115 (57.5%) indicated that they would take the drugs if they were tested positive during antenatal visit; 36 (18%) said they would not take the drugs while 49 (24.5%) avoided answering the question.

ards taking antiretroviral drugs (HAART) in pregnancy A total of 151 participants responded to this question out of which 115 (57.5%) indicated that they would take the drugs if they were tested positive during antenatal visit; 36 (18%) said they would not take the drugs while 49 (24.5%) avoided answering the question. Attitudes towards transactional sex Most participants (n=177, 88.5%) responded that they will not have sex in exchange for money (Figure 2). Figure 2 Will you have sex in exchange for money? Attitudes towards those infected with HIV Almost all the respondents (91.0%) reported that they can stay in the same house with a relative or friend who tested HIV positive (Figure 3). Figure 3 If a relative or friend is tested positive, can you stay in the same house with him or her? Discussions Almost all the participants in this study indicated that they would accept to do a voluntary HIV test. The high number of the participants who would accept to do a voluntary HIV test found in this study is highly commendable. It is a great improvement on the HSRC’s report on the National HIV Prevalence, Incidence and Behaviour Survey done in South Africa in 2014 in which only about 76% of the participants agreed to be tested for the virus (Malan, 2014). This shows that many more people have developed a positive attitude towards testing for HIV.

s a great improvement on the HSRC’s report on the National HIV Prevalence, Incidence and Behaviour Survey done in South Africa in 2014 in which only about 76% of the participants agreed to be tested for the virus (Malan, 2014). This shows that many more people have developed a positive attitude towards testing for HIV. The reasons given by the few who declined the test ranged from fear of stigmatization, not having time to go for the test and fear of testing positive. Only one participant claims that she does not believe that HIV exists, while two others who also said they would decline the test did not give any reasons for their response. Stigmatization has remained a big problem in the HIV/AIDS debate and a major reason why many still maintain a negative attitude towards being tested for the virus. A high rate of discrimination against HIV positive people has been reported in five African countries including South Africa (Holzemer et al., 2009). Stigmatization is exhibited at every level, even among the health professionals where it is least expected (UNAIDS, 2012; Ban, 2008; Sayles et al., 2009). An underlying fear of subsequent stigmatization may also be the reason for those who claimed they do not have time to go for the test and those who said they are afraid of testing positive.

ted at every level, even among the health professionals where it is least expected (UNAIDS, 2012; Ban, 2008; Sayles et al., 2009). An underlying fear of subsequent stigmatization may also be the reason for those who claimed they do not have time to go for the test and those who said they are afraid of testing positive. Highly Active Antiretroviral Therapy (HAART) are known to retard the progression of HIV to AIDS. Taking antiretroviral drugs in pregnancy is a way of reducing the menace of mother-to-child transmission (MTCT) of HIV. However, only 115 of the participants in this study responded to the question about taking the drugs if they are tested positive during antenatal visit. This is an indication that the respondents were not positively inclined to the benefits of taking antiretroviral drugs in pregnancy if they are tested positive and the possible consequences of not doing so. Those who left the question unanswered (about 24.5%) may be regarded as avoiding such a question because they do not even want to think of being tested positive or they might not have a good understanding of the benefits of taking the drugs during pregnancy. Furthermore, only 76.2% of those who answered the question said they will take antiretroviral drugs if they are tested positive to HIV during antenatal visit; this response is worrisome because more than 90% of cases of paediatrics’ HIV are from vertical transmission, which can be reduced if an infected mother takes antiretroviral drugs in pregnancy (Muula, 2000; Gayle and Hill, 2001).

will take antiretroviral drugs if they are tested positive to HIV during antenatal visit; this response is worrisome because more than 90% of cases of paediatrics’ HIV are from vertical transmission, which can be reduced if an infected mother takes antiretroviral drugs in pregnancy (Muula, 2000; Gayle and Hill, 2001). Though, a negligible number of the participants answered in the affirmative that they would have sex for money, however, more street traders might be involved in such an act, but refuse to publicly admit to it due to the sensitive nature of the question and the societal stigmatization attached to such a behavior. Some respondents to whom the questions were being read felt offended to be asked such a question and refused to give an answer. Previous researches have however shown that many female street traders are involved in transactional sex to support their meager income as many of them are bread-winners in their homes (Lee, 2004; Roever, 2014). The fact that most of the participants in this study have 3 or more dependants and yet earn less than R500 every week on the average (Table 1) makes it suspiscious that at least some of those who said they can not have sex in exchange for money might actually be involved in the act to make ends meet.

04; Roever, 2014). The fact that most of the participants in this study have 3 or more dependants and yet earn less than R500 every week on the average (Table 1) makes it suspiscious that at least some of those who said they can not have sex in exchange for money might actually be involved in the act to make ends meet. Almost all the respondents (91.0%) reported that they can stay in the same house with a relative or friend who tested HIV positive. This finding is in consonance, with some improvements on the report of the Department of Health, Medical Research Council (2007) of South Africa where 85% of women said they would be willing to care for an HIV- infected family member at home. This shows an increasingly positive attitude towards those who are infected with HIV. The level of education of the respondents was found to be significantly associated with their attitudes towards HIV and those infected with the virus (p=0.000) implying that the more educated a woman is, the more likely she would have a positive attitude towards the virus and those infected by it. Unnikrishnan et al. (2010) also recorded a similar finding in their study in Coastal Karnataka where respondents with less than secondary school education had discriminatory attitudes towards the people infected with HIV. This suggests that women education, which is discouraged in many African cultures should rather be encouraged to reduce the menace of HIV/AIDS to the bearest minimum.

study in Coastal Karnataka where respondents with less than secondary school education had discriminatory attitudes towards the people infected with HIV. This suggests that women education, which is discouraged in many African cultures should rather be encouraged to reduce the menace of HIV/AIDS to the bearest minimum. Conclusions With increasing availability of HIV information from the media, handbills and posters, and in many local languages including Tshivenda, many people including the female street vendors in Thohoyandou have developed a satisfactory level of acceptable attitudes towards HIV and those who are infected with it. However, the prevailing problem of stigmatization is yet to be solved. Many female street vendors in Thohoyandou seem not to know the importance of using antiretroviral drugs if tested positive for HIV in pregnancy, however, their saying “no” to the drugs may also be a way of hiding their HIV statuses from relatives for fear of stigmatization as many of those who will not accept to do the test indicated. This attitude definitely needs to be improved upon. HIV information providers and health workers alike should provide health education to women about the importance of taking antiretroviral drugs if tested positive in pregnancy to reduce the chances of the unborn baby getting infected. They should also provide the necessary support for infected pregnant women to take antiretroviral drugs during pregnancy and delivery to reduce the the incidence of mother-to-child transmission of HIV. HIV infected women should not be stigmatized in any way by health workers. Female education should be encouraged as it has a significant impact on the attitude towards HIV/AIDS.

pregnant women to take antiretroviral drugs during pregnancy and delivery to reduce the the incidence of mother-to-child transmission of HIV. HIV infected women should not be stigmatized in any way by health workers. Female education should be encouraged as it has a significant impact on the attitude towards HIV/AIDS. Acknowledgements The authors wish to thank the Research and Publication Committee (RPC) of the University of Venda for funding this study and the female street vendors who volunteered and participated in the study.

Introduction Salmonella is an enteric bacterial pathogen and a major pathogenic bacterium that causes food poisoning. Its routes of infection include contaminated foods and water. Salmonella species are leading causes of acute gastroenteritis in several countries and salmonellosis remains an important public health problem worldwide, particularly in the developing countries (Rotimi et al., 2008). Developing countries are more concerned by a broad range of these diseases among which appears cholera, campylobacteriosis, infections with Escherichia coli, shigellosis, brucellosis, hepatitis A and salmonellosis. In this numerous of foodborne infections, salmonellosis is the most frequent infection with a great number of serotypes and intoxications caused with lethality in 1% cases (Ao et al., 2015; Assi-Claire, 2000). Among the most foodborne infections with Salmonella, the lettuce takes up a significant place.

salmonellosis. In this numerous of foodborne infections, salmonellosis is the most frequent infection with a great number of serotypes and intoxications caused with lethality in 1% cases (Ao et al., 2015; Assi-Claire, 2000). Among the most foodborne infections with Salmonella, the lettuce takes up a significant place. In Burkina Faso, rains shortage leads to the practice of the farming irrigated by barrage or waste water. It is the case of the truck farmer production. The dirty water in particular those of the stoppings and the gutter ones are used for the vegetables irrigation. Theses vegetables mainly lettuce are generally contaminated by the enteric bacteria in particular Salmonella from this contaminated water (Traoré et al., 2015). According to Petterson et al. (2010), the consumption of the fruit and vegetables constitutes a factor of potential risk of infection by bacteria enteropathogens such as Salmonella and Escherichia coli O157. Cases of food poisoning related to the contaminated vegetable ingestion were identified a little everywhere in the world (Wendel et al., 2009). Among the factors generally implicated in the contamination of vegetables appears the irrigation water (Koffi-Nevry et al., 2011).

such as Salmonella and Escherichia coli O157. Cases of food poisoning related to the contaminated vegetable ingestion were identified a little everywhere in the world (Wendel et al., 2009). Among the factors generally implicated in the contamination of vegetables appears the irrigation water (Koffi-Nevry et al., 2011). Drug resistance among Salmonella strains has emerged worldwide, making antimicrobial susceptibility testing an important role in public health laboratories. Antibacterial agents are often recommended for the treatment of suspected salmonellosis. Patients were not responding to the most available antibiotics of choice. Those practices can enhance the antibiotics resistances genes. It is now generally accepted that the main risk factor for the increase of resistance to pathogenic bacteria is the anarchic use of antibiotics. Previous studies in Burkina Faso showed that Salmonellaenterica isolated from meat and several foods is resistant to commonly used antibiotics like amoxicillin/clavulanic-acid, aztreonam, cefalotin, ceftriaxone, cefepim, gentamicin, chloramphenicol, tetracycline, nalidixic-acid and ciprofloxaxin (Bagré et al., 2014; Bsadjo Tchamba et al., 2015). In this study, we examined lettuce samples from garden and stools samples from diarrheic persons to determine Salmonella. Specifically, the aims of this study were (1) to determine the Salmonella serotypes and antimicrobial resistance of the obtained isolates and (2) to compare the serotypes and resistance profiles to those previously obtained from the lettuce and human.

n and stools samples from diarrheic persons to determine Salmonella. Specifically, the aims of this study were (1) to determine the Salmonella serotypes and antimicrobial resistance of the obtained isolates and (2) to compare the serotypes and resistance profiles to those previously obtained from the lettuce and human. Materials and methods Study design, population and settings A total of 94 Salmonella were isolated from 134 lettuce samples collected in 2014 in the surrounding environments of the dam number 3 of Ouagadougou and the university hospital Yalgagado Ouédraogo, the biggest hospital in Burkina Faso. In addition, 60 Salmonella were isolated from 765 (447 in Ouagadougou, 125 in Boromo and 193 in Gourcy) patients with diarrhea between 2009 and 2015 in three regions in Burkina Faso: -Ouagadougou (Hopital du District de Bogodogo (HDB) and Laboratoire National de Santé Publique (LNSP)), which is the capital city located in the center of Burkina Faso; -Gourcy (District Sanitaire de Gourcy (DSG)) -and Boromo (District Sanitaire de Boromo (DSB)), which are rural areas located in northern and western parts of the country. All samples, human and lettuce were analysed at National Public Health Laboratory (LNSP) in Ouagadougou for pathogens isolation and stored for further analysis at -30°C.

-Gourcy (District Sanitaire de Gourcy (DSG)) -and Boromo (District Sanitaire de Boromo (DSB)), which are rural areas located in northern and western parts of the country. All samples, human and lettuce were analysed at National Public Health Laboratory (LNSP) in Ouagadougou for pathogens isolation and stored for further analysis at -30°C. Microbiological analyses The suspected Salmonella samples were placed on Mueller Hinton Agar (Himedia, India) and incubated at 37°C for 18–24h. The colonies were subjected to biochemical reactions using Enteric API 20E according to manufactures’ instructions (BioMerieux, France) for further confirmation. Serotyping was done by slide agglutination using Salmonella polyvalent A, B, C, T, Vi antisera (Bio-Rad, France) according to the Kauffmann-White classification scheme (Popoff et al., 2004). All isolates were also tested for susceptibility to 14 different antimicrobial agents using the disk diffusion method on Mueller Hinton II agar (Bio-Rad France) following the European Committee on Antimicrobial Susceptibility Instructions (EUCAST) guidelines (EUCAST, 2013). E. coli ATCC 25922 and ATCC 35218 were used as a control. The antimicrobial disks (Himedia, India) used were nalidixic-acid (30μg), ciprofloxacin (5μg), ampicillin (10μg), amoxicillin (25μg) cefotaxime (30μg), imipenem (10μg), tetracycline (30μg), gentamicin (10μg), chloramphenicol (30μg), ceftriaxone (30μg), norfloxacin (10μg), ticarcillin (75μg), amoxicillin/clavulanic-acid (30μg) and trimethoprime/sulfamexazol (25μg). Inhibition diameters of the antibiotics were interpreted according to the EUCAST (EUCAST, 2013). The multiresistant is defined as the resistance to at least three different antibiotics family (Magiorakos et al., 2011). Extended-spectrum β-lactamases (ESBL) activity was carried out by using amoxicillin/clavulanic-acid against cefotaxime, ceftriaxone.

ibiotics were interpreted according to the EUCAST (EUCAST, 2013). The multiresistant is defined as the resistance to at least three different antibiotics family (Magiorakos et al., 2011). Extended-spectrum β-lactamases (ESBL) activity was carried out by using amoxicillin/clavulanic-acid against cefotaxime, ceftriaxone. Results Bacterial isolates from humans and lettuce Out of 154 Salmonella isolated, 39 % (60) were from human and 61 % (94) from lettuce samples. Of 60 Salmonella enterica isolates from human, 27 % (16/60) of strains were from rural and 73 % (44/60) from urban clinical samples. This study showed high prevalence of Salmonella from male 57 % than female 43 %. Our results showed that 40 % of Salmonella were isolated from patients aged 12-23 months and 27 % of patients aged 0-11 months reported Salmonella (Table 1). Table 1 Salmonella distribution by locality, age and sex Age (months) 0 - 11 12 - 23 24 - 35 36 – 47 48 – 59 Males HDB 2 7 2 0 1 LNSP 2 5 1 1 2 DSB 6 1 1 0 0 DSG 1 0 1 0 0 Sous-Total 33 (56,89%) 11 13 5 1 3 Females HDB 2 4 4 2 1 LNSP 1 3 1 0 1 DSB 2 2 0 0 0 DSG 0 2 0 0 0 Sous-Total 25 (43,11%) 5 11 5 2 2 Total 16 (27,58%) 24 (41,4%) 10 (17,2%) 3 (5,17%) 5 (8,62%) Legend: %=percentage, HDB= Hopital de District de Bogodogo, LNSP= Laboratoire National de Santé Publique, DSB= District Sanitaire de Boromo, DSG= District Sanitaire de Gourcy.

4 4 2 1 LNSP 1 3 1 0 1 DSB 2 2 0 0 0 DSG 0 2 0 0 0 Sous-Total 25 (43,11%) 5 11 5 2 2 Total 16 (27,58%) 24 (41,4%) 10 (17,2%) 3 (5,17%) 5 (8,62%) Legend: %=percentage, HDB= Hopital de District de Bogodogo, LNSP= Laboratoire National de Santé Publique, DSB= District Sanitaire de Boromo, DSG= District Sanitaire de Gourcy. Salmonella serotypes Of 60 human Salmonella isolates, 77 % (46/60) were serotyped. Globally highest prevalence was observed to serotype Paratyphi B 34 % followed Typhi 20 %, Paratyphi C 13 % and Paratyphi A 10 %. According to the localities, the highest prevalence was observed with Salmonella serotype Paratyphi B: 3/4, 11/25 and 4/12 from DSG, HDB (Ouagadougou) and DSB respectively and then the serotype Typhi: 6/19, 1/4, 2/12 and 3/25 from LNSP, DSG, DSB and HDB respectively (Table 2). Salmonella spp were identified in 23 % (14/60). These isolates were not reacting to antisera used in our study. Table 2 Salmonella serotypes distribution by locality Sites/Serotypes S. Paratyphi A S. Paratyphi B S. Paratyphi C S. Typhi Salmonella. spp TOTAL HDB 01(04%) 11 (44%) 05 (20 %) 03(12 %) 05 (20 %) 25 (100%) LNSP 03 (18%) 02 (12 %) 01 (06%) 06(35%) 05 (29 %) 17(100 %) DSB 02 (17%) 04 (33%) 02 (17%) 02 (17%) 02 (17 %) 12 (100 %) DSG 00 03 (75%) 00 01(25%) 00 04 (100 %) TOTAL 06 (10%) 20 (34%) 08 (14%) 12(21 %) 12(21 %) 58 (100 %) Legend: S= Salmonella, 00 = no prevalence, %=percentage, HDB= Hopital de District de Bogodogo, LNSP= Laboratoire National de Santé Publique, DSB= District Sanitaire de Boromo, DSG= District Sanitaire de Gourcy.

100 %) DSG 00 03 (75%) 00 01(25%) 00 04 (100 %) TOTAL 06 (10%) 20 (34%) 08 (14%) 12(21 %) 12(21 %) 58 (100 %) Legend: S= Salmonella, 00 = no prevalence, %=percentage, HDB= Hopital de District de Bogodogo, LNSP= Laboratoire National de Santé Publique, DSB= District Sanitaire de Boromo, DSG= District Sanitaire de Gourcy. Out of 94 Salmonella enterica isolates from lettuce, 51 % (48/94) reacted to the antisera used. The highest prevalence was observed to Salmonella serotype Paratyphi A 23 % followed by Paratyphi C 18 %, Paratyphi B 8 % and serotype Typhi 1 % (Table 3). Salmonella spp represented 49 % (46/94) of the strains did not react to the antisera used. Table 3 Salmonella serotype from lettuce Serotypes S.Paratyphi A S.Paratyphi B S.Paratyphi C S.Typhi Salmonella.spp Total Prevalence 22 (23 %) 08 (09 %) 17 (18 %) 01 (01 %) 46 (49 %) 94(100 %) Legend: S= Salmonella, %=percentage.+

Out of 94 Salmonella enterica isolates from lettuce, 51 % (48/94) reacted to the antisera used. The highest prevalence was observed to Salmonella serotype Paratyphi A 23 % followed by Paratyphi C 18 %, Paratyphi B 8 % and serotype Typhi 1 % (Table 3). Salmonella spp represented 49 % (46/94) of the strains did not react to the antisera used. Table 3 Salmonella serotype from lettuce Serotypes S.Paratyphi A S.Paratyphi B S.Paratyphi C S.Typhi Salmonella.spp Total Prevalence 22 (23 %) 08 (09 %) 17 (18 %) 01 (01 %) 46 (49 %) 94(100 %) Legend: S= Salmonella, %=percentage.+ Antimicrobial susceptibility testing of human isolates The Salmonella isolates originating from human were all resistant to fourteen (14) antibiotics. A higher frequency of antimicrobial resistance was observed to tetracycline 55 %, ticarcillin 38 %, amoxicillin/clavulanic-acid, ampicillin, amoxicillin 36 % and trimethoprime/sulfamexazol 33 %. Low frequency of resistance was observed to imipenem 3 %, ceftriaxon, cefotaxime 5 %, ciprofloxacin, norfloxacin, gentamicin 7 % and nalidixic-acid 10 % (Table 4). From different serotypes tested, the high resistance was observed with tetracycline 14/20, ampicillin, amoxicillin, amoxicillin/clavulanic-acid, ticarcillin 12/20, trimethoprime/sulfamexazol 11/20 and chloramphenicol 10/20 to Salmonella ParatyphiB. Resistance was observed to imipenem 02/20, ceftriaxone, cefotaxime, ciprofloxacin, norfloxacin, nalidixic-acid, and gentamicin 01/20 with Salmonella ParatyphiB. Extended-spectrum β-lactamases (ESBL) were not observed.

icarcillin 12/20, trimethoprime/sulfamexazol 11/20 and chloramphenicol 10/20 to Salmonella ParatyphiB. Resistance was observed to imipenem 02/20, ceftriaxone, cefotaxime, ciprofloxacin, norfloxacin, nalidixic-acid, and gentamicin 01/20 with Salmonella ParatyphiB. Extended-spectrum β-lactamases (ESBL) were not observed. Table 4 Frequency of antimicrobial resistance in Salmonella isolates from human Antibiotiques Salmonella serotypes S. Paratyphi A N=06 S. Paratyphi B N=20 S. Paratyphi C N=08 S. Typhi N=12 Salmonella. spp N=12 Total N=58 AMP 02(33%) 12(60%) 00 05(42%) 02(17%) 21(36 %) AMX 02(33%) 12(60%) 00 05(42%) 02(17%) 21(36%) AMC 02(33%) 12(60%) 00 05(42%) 02(17%) 21(36%) CTR 00 01(05%) 00 00 02(17%) 3(5%) IMI 00 02(10%) 00 00 00 2(3%) CTX 00 01(05%) 00 00 02(17%) 3(5%) TI 02(33%) 12(60%) 00 06(50%) 02(17%) 22(38%) GEN 01(17%) 01(05%) 00 00 02(17%) 4(7%) COT 02(33%) 11(55%) 00 04(33%) 02(17%) 19(33%) NA 02(33%) 01(05%) 00 01(08%) 02(17%) 6(10%) CIP 01(17%) 01(05%) 00 00 02(17%) 4(07%) NX 01(17%) 01(05%) 00 00 02(17%) 4(07%) C 01(17%) 10(50%) 00 05(42%) 01(08%) 17(29%) TE 02(33%) 14(70%) 03(38%) 06(50%) 07(58%) 32(55%) Legend: AMP= ampicillin, AMX= amoxicillin, AMC = amoxicillin/cluvulanic-acid, CRO = ceftriaxone, CTX= cefotaxime, NX= norfloxacin, COT= trimithoprime/sulfamexazol, C = chloramphenicol, CIP = ciprofloxacin, GEN = gentamicin, IMI = imipenem, NA = nalidixicacid, TE = tetracycline, TC = ticarcillin, % = percentage, S = Salmonella, N=number.

illin, AMX= amoxicillin, AMC = amoxicillin/cluvulanic-acid, CRO = ceftriaxone, CTX= cefotaxime, NX= norfloxacin, COT= trimithoprime/sulfamexazol, C = chloramphenicol, CIP = ciprofloxacin, GEN = gentamicin, IMI = imipenem, NA = nalidixicacid, TE = tetracycline, TC = ticarcillin, % = percentage, S = Salmonella, N=number. Antimicrobial susceptibility testing of lettuce isolates. All 94 lettuce isolates were susceptible to imipenem, gentamicin, ceftriaxone, cefotaxime and ciprofloxacin. We observed a low antimicrobial resistance to tetracycline 22 %, amoxicillin/clavulanic-acid, amoxicillin, ampicillin, chloramphenicol, nalidixic-acid and trimethoprime/sulfamexazol respectively 7 %, 6 %, 5 %, 4 %, 3 %, and 2 %. The resistance was observed to tetracycline, amoxicillin/clavulanic-acid, amoxicillin, ampicillin, ticarcillin and trimethoprime/sulfamexazol, 01/17 with Salmonella Paratyphi C (Table 5). Table 5 Frequency of antimicrobial resistance in Salmonella isolates from lettuce Antibiotiques Salmonella sérotypes

Antimicrobial susceptibility testing of lettuce isolates. All 94 lettuce isolates were susceptible to imipenem, gentamicin, ceftriaxone, cefotaxime and ciprofloxacin. We observed a low antimicrobial resistance to tetracycline 22 %, amoxicillin/clavulanic-acid, amoxicillin, ampicillin, chloramphenicol, nalidixic-acid and trimethoprime/sulfamexazol respectively 7 %, 6 %, 5 %, 4 %, 3 %, and 2 %. The resistance was observed to tetracycline, amoxicillin/clavulanic-acid, amoxicillin, ampicillin, ticarcillin and trimethoprime/sulfamexazol, 01/17 with Salmonella Paratyphi C (Table 5). Table 5 Frequency of antimicrobial resistance in Salmonella isolates from lettuce Antibiotiques Salmonella sérotypes S.Paratyphi A N=22 S. Paratyphi B N=09 S. Paratyphi C N=17 S. Typhi N=01 S. spp N=46 Total N=94 AMP 00 00 01(06%) 00 04(09 %) 5(05 %) AMX 00 00 01(06%) 00 05(11 %) 6(06 %) AMC 00 00 01(06 %) 00 06(13 %) 7(07 %) CTR 00 00 00 00 00 00 IMI 00 00 00 00 00 00 CTX 00 00 00 00 00 00 TI 00 00 01(06%) 00 02(04%) 3(03%) GEN 00 00 00 00 00 00 COT 00 00 01(06%) 00 01(02 %) 2(02 %) NA 00 00 00 00 03(07 %) 3 (03 %) CIP 00 00 00 00 00 00 NX 00 00 00 00 01(02%) 1(01%) C 00 00 00 00 04(09 %) 4 (04 %) TE 03(14%) 01(13 %) 01(06%) 00 16(35 %) 21(22 %) Legend: AMP= ampicillin, AMX= amoxicillin, AMC = amoxicillin/cluvulanic-acid, CRO = ceftriaxone, CTX= cefotaxime, NX= norfloxacin, COT= trimithoprime/sulfamexazol, C = chloramphenicol, CIP = ciprofloxacin, GEN = gentamicin, IMI = imipenem, NA = nalidixicacid, TE = tetracycline, TC = ticarcillin, % = percentage, S = Salmonella, N=number

cillin, AMX= amoxicillin, AMC = amoxicillin/cluvulanic-acid, CRO = ceftriaxone, CTX= cefotaxime, NX= norfloxacin, COT= trimithoprime/sulfamexazol, C = chloramphenicol, CIP = ciprofloxacin, GEN = gentamicin, IMI = imipenem, NA = nalidixicacid, TE = tetracycline, TC = ticarcillin, % = percentage, S = Salmonella, N=number Multi-drugs resistance This study revealed multidrugs resistance (resistance of three or more antibiotics with differents families) with Salmonella Paratyphi B 07/20, Salmonella Typhi 04/12, Salmonella Paratyphi A 01/06 and Salmonella spp 02/12 from the clinical Salmonella isolates. However, Salmonella isolated from lettuce showed multidrugs resistance with Salmonella spp 02/46 (4 %). In the Hospital, multidrugs resistance observed with Salmonella Paratyphi B from DSB 03/04, DSG02/03, LNSP 01/02 and HDB 04/11 followed Salmonella Typhi from DSB, LNSP03/06 and HDB 01/03. Discussion Salmonellosis is one of the major bacterial diseases transmitted from food. In this study, we investigated the distribution of Salmonella serotypes between human and lettuce isolates and highlight the consumption of lettuce as potential source of transmission of Salmonella causing diarrhea among human in Burkina Faso. In Ouagadougou, people use generally waste water for vegetable irrigation. The high number of Salmonella strains isolated from lettuce was expected. Indeed, Salmonella is reported to be an environmentally persistent pathogen capable of surviving and proliferating in diverse environments (Winfield and Groisman, 2003).

Discussion Salmonellosis is one of the major bacterial diseases transmitted from food. In this study, we investigated the distribution of Salmonella serotypes between human and lettuce isolates and highlight the consumption of lettuce as potential source of transmission of Salmonella causing diarrhea among human in Burkina Faso. In Ouagadougou, people use generally waste water for vegetable irrigation. The high number of Salmonella strains isolated from lettuce was expected. Indeed, Salmonella is reported to be an environmentally persistent pathogen capable of surviving and proliferating in diverse environments (Winfield and Groisman, 2003). In addition, our study site was a high risk of pathogens spread in environment. Vegetable farmers in our study locations depend largely on contaminated waste water from dam to irrigate their produce, while also using untreated animal manure as sources of nutrient supply to vegetables. Due to the vicinity, waste water from university hospital Yalgado Ouedraogo, the biggest hospital in the country is released into a canal which flows nearby the dam.

gely on contaminated waste water from dam to irrigate their produce, while also using untreated animal manure as sources of nutrient supply to vegetables. Due to the vicinity, waste water from university hospital Yalgado Ouedraogo, the biggest hospital in the country is released into a canal which flows nearby the dam. The distribution of serotypes of Salmonella from lettuce samples comprised Salmonella Typhi 1%, Salmonella Paratyphi 50% and Salmonella spp (untypeable) 49%. Salmonella Paratyphi has long been reported as a common cause of foodborne gastroenteritis (Hur et al., 2012). Also, the presence of Salmonella Typhi from lettuce is an indication of feacal contamination. Our finding supports the well-documented role of the presence of Salmonella in waste water and animals feces in environmental contamination in Burkina Faso as in some others developing countries (Kagambèga et al., 2013; Traoré et al., 2015). The distribution of serotypes of Salmonella from human samples comprised Salmonella Typhi 21%, Salmonella Paratyphi 58% and Salmonella spp (untypeable) 21%. Salmonella Typhi was the second most common serotype isolated after Salmonella Paratyphi B. The high prevalence of Salmonella Typhi among isolates reveals that this pathogen still constitutes a significant public health importance in the country, therefore, appropriate control measures, such as vaccination and food safety measurements, need to be put in place. Recent study shown that Salmonella serotype Typhi and Paratyphi B were the most serotypes incriminated in diarrheal infections in Burkina Faso (Timbiné, 2014).