Co-infection of Schistosoma mansoni/hepatitis C virus and their associated factors among adult individuals living in fishing villages, north-western Tanzania

Background Schistosoma mansoni and Hepatitis C virus (HCV) are co-existence in sub-Saharan Africa and co-infection is common among humans population. The immunological responses characterized with Th2-immune responses for S. mansoni and Th1-immune responses for HCV are responsible for development hepatic morbidities in infected individuals. However, the co-occurrences of S. mansoni and HCV infection, their related ultrasound detectable morbidities and associated risk factors at community levels have not been examined in fishing communities, north-western Tanzania. In this context, the present study covers that gap. Methods A cross-sectional study was conducted among 1924 asymptomatic individuals aged 15–55 years in four fishing villages (Igombe, Igalagala, Sangabuye and Kayenze) of Northwestern Tanzania. A single stool sample was collected from each study participants and examined for S. mansoni eggs using Kato Katz technique. Hepatitis C surface antigen (HCVsAg) was determined from a finger prick blood sample using a rapid test. Results Overall, 51.8% (997/1924; 95%CI: 49.6–54.1) of the study participants were infected with S. mansoni and had a mean intensity of 223.7epg (95%; 202.4–247.1). Of the study participants, 90 (4.7%) were infected with hepatitis C virus (HCV). Overall, 2. 4% (47/1924) of the study participants were co-infected with S. mansoni and hepatitis C virus. Among the co-infected individuals, 42.6%, 70.2% and 19.1% had splenomegaly, hepatomegaly and periportal fibrosis (PPF). Factors associated with S. mansoni/HCV co-infection were being aged 26–35 years (aRR = 2.67, 95%CI: 1.03–6.93, P < 0.04), 46–55 years (aRR = 2.89, 95%CI: 1.10–7.57, P < 0.03) and having marked hepatomegaly (aRR = 2.32, 95%CI: 1.09–4.9, P < 0.03). Conclusion In this setting, S. mansoni and Hepatitis C are co-endemic and a proportion of individuals were co-infected. Hepatosplenic morbidities characterized with hepatomegaly, splenomegaly, hepatosplenomegaly and PPF were observed in co-infected individuals. These results highlight the need for integrated interventions measures against parasitic and viral diseases.


Background
Intestinal schistosomiasis caused by Schistosoma mansoni infection remains one of the public health concerns in Tanzania [1]. This infection is highly endemic in fishing villages bordering the southern shore and islands within the Lake Victoria [1][2][3]. One third of the estimated 44 million Tanzanian populations are estimated to be infected with S. mansoni [1,4]. Chronic S. mansoni infection is mainly associated with deposition of eggs of the parasites in human body tissues especially the liver [5,6]. Immunological responses against trapped eggs are responsible for the development of granulomas, chronic exposure to which may end with development of hepatosplenic schistosomiasis [5,6]. Hepatosplenic morbidities are characterized by hepatic and splenic enlargement, progressive periportal fibrosis (PPF) which can lead to portal hypertension and its associated sequelae such as liver surface irregularities and portalsystemic venous, with the risk of esophageal varices and haemetemesis [5][6][7].
Conversely, hepatitis C virus (HCV) infection is endemic in sub-Saharan Africa and its prevalence varies from one epidemiological setting to another [8,9]. Worldwide, 185 million individuals are estimated to be infected with HCV based on antibodies testing, with the highest prevalence observed in Asia and Middle East [10]. In sub-Saharan Africa, the prevalence of HCV infection ranges from 1% in Ethiopia to 50% in Egypt [10,11]. It is estimated that, 70% of the individuals infected with HCV go on to develop chronic hepatitis and an approximate of 15-20% of them develop cirrhosis [12,13].
Because of co-endemicity of S. mansoni and HCV infection in sub-Saharan Africa, co-infection does occur, especially in areas where intestinal schistosomiasis is highly endemic [9,14,15]. The pathogenesis of S. mansoni and HCV in co-infected individuals on liver fibrosis is well established [5,[11][12][13]. However, the pathomechanism of the two infections differs by the fact that S. mansoni immunological responses are mainly characterized by Th 2 -CD4 + type immune responses [5]; whereas for HCV infection, a type 1 Th 1 -CD8 + like responses is thought to play role in hepatic inflammatory responses and viral clearance [16]. Because of the immunological differences, the characteristics of hepatic damage resulting from the two infections also differs [5,12,13], with HCV infection causing advanced liver disease, increased incidence of liver cirrhosis and hepatocellular carcinoma [12,13,16].
In Tanzania, S. mansoni [1] and HCV infections [17][18][19] are co-endemic. It is estimated that 52% of the Tanzanian population are infected with schistosomiasis (both S. mansoni and S. haematobium) [4]. In Tanzania, S. haematobium is widely distributed, whereas S. mansoni is mainly restricted to large water bodies and paddy farming areas [20]. Schistosoma mansoni is common in areas surrounding the Lake Victoria shores and islands within the lake [2,21,22]. To date, there are no data on the national prevalence of HCV infection and information from the global burden of diseases indicate that, the annual mortality rate per 100,000 people from Hepatitis C in Tanzania has increased by 17.4% since 1990, an average of 0.8% a year [23]. Despite the fact that the two infections are co-endemic in the country, no community study has studied their co-occurrences and their related morbidities, especially in fishing communities. To justify the public health aspects of integrating interventions measures against parasitic and viral diseases at community level, the aim of the present study was to determine the prevalence of S. mansoni and hepatitis C co-infections, related hepatosplenic morbidities and associated risk factors among study participants.

Study area
The current data are from the secondary analysis of the study that was conducted in northwestern Tanzania [7,24]. Briefly, the study was conducted in fishing villages of Ilemela district, including the villages of Igombe, Igalagala, Sangabuye and Kayenze bordering the Lake Victoria shore on the southern part at Ilemela district, Mwanza region (32-34°E and 2-4 0 S, north-western Tanzania. The area experiences a temperature range from 18°C to 28°C and the mean annual rainfall of 1068 mm. The majority of the inhabitants of these villages are involved in farming and fishing activities. Because of high water contact levels, inhabitants remain at high risk of being infected with S. mansoni [2]. The control of S. mansoni infection in the study area involves mass chemotherapy using praziquantel drug, which is mainly focused in school children to reduce the long-term morbidities associated with the infection. There is no any program focusing on HCV control, screening and treatment at community.

Study design, inclusion and exclusion criteria
This was an analytical cross-sectional study conducted between September 2012 -December 2012 and the data presented in current work are secondary analysis of the work published elsewhere [7,24]. The study included participants (i) aged 15-55 years old and (ii) lived in the study villages for more than 2 years (iii) with no history of HCV infection. Study participants with history of treatment against S. mansoni infection (praziquantel drug) in the past 6 month, on anti-HCV treatment and clinical diagnosed with HCV infection (advised to seek medical care at the district hospital) were excluded from the study.

Sample size, sampling technique and recruitment
Sample size calculation and sampling procedures have been previously described in details in Mazigo et al.,. [24]. Briefly, a total of 2142 study participants were enrolled into the study of which 1924 were eligible for the final analysis after fulfilling inclusion criteria. A two-step sampling procedures was used to select households and household members to participate in the study. A list of households and household members was obtained and from this, a random sampling procedure was used to select eligible individuals from randomly selected households.

Data collection
Parasitological examination of stool for Schistosoma mansoni and S. haematobium eggs A single stool and urine samples was collected from all consented study participants using labeled clean containers. From the stool sample, four Kato Katz thick smears were prepared from different sites of each stool sample using a template of 41.7 mg (Vestergaard Frandsen, Lausanne, Switzerland), following a standard protocol [25]. In brief, four Kato-Katz thick smears were prepared on microscope slides and labeled with a participant identification number. After 24 h, the smears were examined for S. mansoni eggs independently by two experienced laboratory technician at the laboratory of the National Institute for Medical Research, Mwanza. The number of eggs for S. mansoni were counted and recorded separately in the prepared parasitological forms. For quality assurance, a random sample of 10% of the negative and positive Kato Katz thick smears were re-examined by a third technician. For diagnosis of S. haematobium infection, collected urine samples were examined grossly for presence of macrohaematuria and using Hemastrix dipstick for presence of microhaematuria. Then, all urine samples were further examined using urine filtration technique with Nuclepore® membrane according to WHO [26].

Examination of hepatitis-C virus infection
For diagnosis of hepatitis-C virus infection, the finger prick blood sample was collected from each study participants and used for screening of hepatitis-C infection using a qualitative rapid test for detection of hepatitis C antigen in whole blood, (ACON Laboratories, Inc., San Diego, CA) according to manufacturer's instructions at the field site laboratory [27]. Briefly, a drop of blood sample was added in the test strip followed by drop of the provided buffer. The results of the test were read after 15-20 min. The test was regarded as positive if the control and test line appeared after the given time.
The test was regarded negative if only a single control line appeared.

Screening for human immunodeficiency Virus-1
The Tanzanian National HIV algorithms which recommended (currently has been revised) the use of a rapid test Determine (Alere Determine, Chiba, Japan) and Uni-GOLD (Trinity Biotech PLV, Bray, Ireland) was used for HIV diagnosis. Participants were counseled before and after HIV testing as per recommendation [28].

Ultrasonographical examination of hepatosplenic morbidities
All study participants were examined clinically for presence of any organomegally (enlargement of the liver and spleen) [29]. Two medically personnel trained on Niamey protocol examined study participants using a portable ultrasound machine (Aloka, Tokyo) [30]. Identified pathology was classified as per modified Niamey protocol [30]. The liver texture patterns, peripheral portal branches (PPBs), periportal fibrosis (PPF), thickness of PPB walls, spleen size, splenic vein (SV) diameter and ascites were assessed. Periportal fibrosis (PPF) was defined according to WHO [15] and the degree of PPF was categorized as A, B, C, D, E and F [30]. Periportal fibrosis grade A and B were classified as normal.

Data analysis
A CSPro system was used for double data entry and data analysis was performed using Stata Version 12 (Stata Corp, college station, Texas, USA). Categorical variables were summarized by numbers and percentages. Comparison of proportions/categorical variables was done using chi-square (χ 2 )/fisher exact where appropriate. For continuous variables descriptive statistics were reported as means with standard deviation for normally distributed variable and medians with interquartile ranges (IQR) for variables that were not normally distributed. The arithmetic mean of S. mansoni egg counts for each participant was calculated from the counts of four Kato Katz thick smears and multiplied by 24 to obtain individual eggs per gram of faeces. Schistosoma mansoni egg counts were logarithmically transformed to allow calculation of the geometric mean egg per gram of feaces (GM-epg), which was calculated as an antilog of the mean of the transformed egg counts. Geometric mean egg counts for S. mansoni between sex and age were compared using Student-t-test (two groups) or ANOVA (more than two groups). Intensity of infection was categorized according to WHO criteria as: 1-99 epg, 100-399 epg, ≥400 epg defined as low, moderate and heavy intensities of infection respectively [31].
The categorization of the ultrasound measurements was based on the Niamey protocol [30]. To identify factors associated with S. mansoni/HCV co-infection, binomial regression model was constructed. At bivariate analysis, factors with P-values of 0.2 were considered for multiple binomial regression analysis. Because PPF grades, splenomegaly and left liver lobe hepatomegaly were related, only left liver lobe hepatomegaly was considered for multiple binomial regressions. A P-value of <0.05 was considered significant.

Ethical consideration
Ethical approval was sought from the Research and Ethics Committees of Bugando University College of Health Sciences and Allied Sciences-Institutional Review Board, (BREC/001/32/2011). Ethical clearance was granted by the National Ethical Review Committee, National Institute for Medical Research, Tanzania. Swahili translated informed assent and consent forms were used to obtain children and adult participants' consent respectively. For illiterate individuals, a thumb print was used to sign the assent and consent forms after a clear description of the study objective was explained to them and accepted to participate.

Demographic characteristics of the study participants
A total of 1924 study participants aged 15-55 years were enrolled into the present study (Fig. 1). Of these participants, 46.7% (n = 899/1924) and 53.3% (n = 1025) were male and female respectively. The mean age of the study participants was 32.75 ± 11.15 years. The main economic activities of the study participants were farming (72.5%, n = 1396), fishing (13.9%, n = 268) and small-scale business (13.5%, n = 260). Overall, 33.3% (n = 638) and 66.7% (n = 1284) of the study participants were illiterate and literate respectively. Table 1 shows demographic characteristics of the study participants.

Prevalence and intensity of Schistosoma mansoni infection
The overall prevalence of S. mansoni was 51.8% (997/1924, 95%CI: 49.6-54.1) with male in individuals having the highest prevalence compared to female individuals (44.1% versus 60.6%, P < 0.001) ( Table 2). Similarly, the youngest age group (15-25 years) having the highest prevalence compared to older age groups (χ 2 = 77.0276, P < 0.001). In relation to village of residence, study participants from Igalagala and Kayenze villages had the highest prevalence of S. mansoni infection than participants from other participating villages (χ 2 = 42.4633, P < 0.001). Similarly, in relation to occupation, participants reported to be involved in fishing activities had the highest prevalence compared to study participants involved in farming and small scale business (χ 2 = 30.8409, P < 0.001) ( Table 2).

Seroprevalence of hepatitis C virus infection
The overall prevalence of Hepatitis C virus (HCV) infection was 4.7% (90/1924, 95%CI: 3.7-5.7). There was no sex difference in prevalence of HCV (χ 2 = 0.0001, P = 0.99) ( Table 3). Age difference in prevalence of HCV infection was observed with the oldest age group having the highest prevalence (χ 2 = 25.0304, P < 0.001). Similarly, study participants from Kayenze village recorded the highest prevalence of HCV infection compared to participants from other participating villages (χ 2 = 11.9520, P < 0.01).

Co-infection of Schistosoma mansoni and hepatitis C virus
The overall prevalence of co-infection of S. mansoni and hepatitis C was 2.4% (47/1924). The overall GMepg of the study participants who were co-infected with S. mansoni/HCV infection was 206.95GMepg (95%CI: 128.4-333.6). Among the co-infected, a sex difference was observed with male individual's having the GMepg of 225.9epg (95%CI; 106.9-477.1) and for female individuals had GMepg of 188.9epg (95%CI: 98.9-360.6).

Discussion
The findings of the present study on S. mansoni infection confirms the report of the previous reports in northwestern Tanzania [2,3,32], which demonstrated high prevalence of S. mansoni and infection intensity among adult population [2,3]. Furthermore, our findings confirm high infection intensity among male individuals and young age groups [2,3]. The variation in exposure to risk areas such as the lake and time spent in water Fig. 1 Flow chart detailing participation and adherence of study participants in the study sources (lake) partly explain the observed differences in prevalence and infection intensity between sex and between age groups [33][34][35]. In addition, a noted difference in prevalence of S. mansoni infection between villages was observed. Partly, this observation is explained by geographical location of the villages from the Lake Victoria shores, with Kayenze and Igalagala villages are located at the shoreline of the lake [2]. The geographical variations of S. mansoni infection even in villages located in the same area have been noted elsewhere in Africa [6,33,35].
Our findings noted a low prevalence of HCV infection among adult population in fishing communities. Population based studies in health adult individuals have reported a prevalence of 0.7% of HCV infection in Dar Es Salaam [36] and 1.2% in Northwestern Tanzania based on antibodies detection using ELISA technique [18]. In special groups such as blood donors, lower prevalence of HCV infection (1.5%) was reported in Dar Es Salaam, Tanzania [19]. Similarly, a lower prevalence of HCV antibodies (1.2%) was reported among health workers [37]. In contrast, a high seroprevalence of HCV infection (57% based on HCV antibody) was reported among a cohort of opioid treatment patients in Dar Es Salaam, Tanzania [38]. Compared to findings from other African countries, a prevalence of 8.1% [39], 12.8% [40] and 13.7% [41] of anti-HCV infection has been reported in Angola Nigeria and Democratic Republic of Congo. The observed variation in prevalence among different groups mainly reflects different levels of exposure to risk factors [38]. Hepatitis C virus infection is transmitted predominantly through exposure to contaminated blood and body fluids, thus, groups of individuals with high   exposure to body fluids such as health workers and drug abusers always present with high prevalence of HCV infection [12,38]. Our analysis on prevalence of HCV infection noted age difference in the prevalence of this viral infection. The older age groups (≥36 years) had the highest prevalence of HCV infection. The report of global burden of diseases indicates that hepatitis C virus infection is more common in the older age groups (75-79 years) in Tanzania [23]. This observation depicts the chronicity of the viral infection [12,13]. Perhaps, the infection starts at young ages and its related morbidities and mortality are observed at older age groups [12].
In the present study a proportion of study participants were co-infected and had different patterns of hepatosplenic morbidities related to S. mansoni and Hepatitis C virus infection. Similar findings have been observed in endemic areas of sub-Saharan Africa, where both S. mansoni and HCV are co-endemic [8][9][10]14]. However, the prevalence of co-infection of S. mansoni and HCV observed in the present study was comparable to findings of similar study in Ethiopia (4%) [15] and lower than what was observed in Egypt (33% and 40.2%) [42,43]. Specifically, in Egypt, the prevalence of HCV is very high compared to many of the sub-Saharan African countries [8,10], thus, co-infection of S. mansoni and HCV is also very high [8,10]. It is worthwhile noting that, the present study used rapid diagnostic test to detected hepatitis C surface antigens (HCVsAg) which may have lower sensitivity than HCV antibodies detection methods used in previous studies [44]. Perhaps in these setting, the prevalence could be very high if HCV antibodies detection method could have been used. This is an open area calling for further studies using the more reliable diagnostic technique with improved sensitivity and preferably with a large sample size to establish the associated effects.
In the present study, study participants had hepatosplenic morbidities characterized by hepatic and splenic enlargements and periportal fibrosis characterized with different grades. Similar observations with different PPF grades and hepatosplenomegaly have been reported by previous studies [2,3]. In co-infected individuals studies have shown that, S. mansoni infections increases HCV morbidities and chronicity of the liver pathology [45], increases HCV RNA titres, incidence of cirrhosis/hepatocellular carcinoma and higher mortality rates [45]. The effects of S. mansoni infection in HCV infected patients have been reviewed in details elsewhere [8]. However, the role of S. mansoni infection in exacerbating hepatic morbidities related to HCV infection remain a topic of debate, with other studies reporting no evidence that S. mansoni infection affect the outcome of HCV in infected individuals [46]. Given the low prevalence of HCV infection observed in the present study population, this HCV infection is likely to account for only a small proportion of chronic liver disease in the studied population. In addition, it is difficult to draw a solid conclusion on the role of S. mansoni infection in HCV co-infected patients in a study which had only 17 study participants who were coinfected and had hepatosplenic morbidities [45]. Large sample sizes will be needed in further studies to add more evidence. The main risk factors associated with S. mansoni/ HCV infection were mainly being of older age group (46-55 years), village of residence and having left liver lobe hepatomegaly. The association of these infections with older ages indicates that, these are chronic infections, which needs time for their obvious related morbidities such as hepatomegaly and fibrosis/ liver cirrhosis to manifest [6,12,13]. The association with village of residence mainly defines the presence of exposure factors in these villages [7,15]. Individually, epidemiological studies have shown that, S. mansoni infection is associated with village of residence, age, occupation, specifically fishing, being male and hepatosplenic morbidities [1,6,7,47]. Older age groups, being male and residing in rural areas have been demonstrated to be associated with HCV infection [11].
The present study was subject to limitation, being a cross-sectional study in nature, partly may contribute to lack of temporal association between the S. mansoni/ HCV as the main outcomes with some of the study variables. Also, the use of only one diagnostic technique to diagnosed HCV infection (HCVsAg), partly, may have underestimated the prevalence of HCV infection in the study population. In addition, the use of a single stool sample to examine for S. mansoni infection owing the day to day variability of parasite eggs output and low sensitivity of the Kato Katz technique in detecting individuals with light infection intensity may have under estimated the prevalence of S. mansoni infection in the studied population.

Conclusion
The present study setting is endemic to S. mansoni and Hepatitis C virus infections and a small proportional of individuals are co-infected. Schistosoma mansoni related PPF grades, hepatomegaly, splenomegaly and hepatosplenomegaly were also present in co-infected individuals. Co-infection of S. mansoni/HCV was mainly associated with older age, village of residence and having hepatomegaly. Further studies are recommended in these areas to understanding the impact of S. mansoni/HCV coinfection before implementation of integrated interventions measures can be thought.