Prevalence and factors associated with intestinal schistosomiasis and human fascioliasis among school children in Amhara Regional State, Ethiopia

Background Schistosomiasis and fascioliasis are digenean parasitic infections and are among the neglected tropical diseases that have both medical and veterinary importance. They are found mainly in areas having limited access to safe water supply and improved sanitation. Methods A cross-sectional study was conducted to determine the prevalence of Schistosoma mansoni and Fasciola species infections and to identify associated risk factors among school children in Amhara Regional State, Ethiopia. Stool specimens were collected from 798 children (419 males, 379 females) and processed using Kato-Katz and formol-ether concentration techniques. A semi-structured questionnaire was used to collect socio-demographic and other exposure information to explore potential risk factors for the infections. Results The overall prevalence of S. mansoni and Fasciola species infections was 25.6% (95% confidence interval (CI): 22.5-28.6) and 5.5% (95% CI: 3.9-7.1), respectively. S. mansoni was present in all surveyed schools with the prevalence ranging from 12.8% (16/125; 95% CI = 5.6-20.0) to 39.7% (64/161; 95% CI = 32.2-47.2) while Fasciola species was identified in five schools with the prevalence ranging from 2.5% (4/160; 95% CI = 0.001–4.9) to 9.8% (13/133; 95% CI = 4.7–14.8). The prevalence of S. mansoni infection was significantly associated with swimming in rivers (Adjusted odds ratio (AOR): 1.79, 95% CI, 1.22–2.62; P=0.003), bathing in open freshwater bodies (AOR, 2.02; 95% CI, 1.39–2.94; P<0.001) and engaging in irrigation activities (AOR, 1.69; 95% CI, 1.19-2.39; P=0.004), and was higher in children attending Addis Mender (AOR, 2.56; 95% CI, 1.20–5.46; P=0.015 ) and Harbu schools (AOR, 3.53; 95% CI, 1.64–7.59; P=0.001). Fasciola species infection was significantly associated with consumption of raw vegetables (AOR, 2.47; 95% CI, 1.23-4.97; P=0.011) and drinking water from unimproved sources (AOR, 2.28; 95% CI, 1.11–4.70; P=0.026). Conclusion Both intestinal schistosomiasis and human fascioliasis are prevalent in the study area, affecting school children. Behaviors and access to unimproved water and sanitation are among significant risk factors. The findings are instrumental for targeted interventions.


Background
Neglected tropical diseases (NTDs) pose a substantial threat to global health, affecting billions of population and causing more than 25 million disability-adjusted life years (DALYs) [1,2]. Diseases caused by digenean parasitic trematodes, including the blood flukes Schistosoma mansoni, S. haematobium, S. japonicum, S. mekongi, S. guineensis, and S. intercalatum, and liver flukes Fasciola hepatica and F. gigantica, are among the neglected tropical diseases in the tropics and subtropics regions [3]. Worldwide, schistosomiasis and fascioliasis infect more than 200 million and 2.4 million people, respectively, with 850 million people being at the risk of both infections [3]. These infections typically occur in areas of low-and middle-income countries (LMICs) with lack of improved water supply and adequate sanitation [1] and their transmission is linked to environmental factors, such as temperature, rainfall, and water bodies, which can affect the distribution of intermediate snail hosts and alter the development rate of the parasites [1,2].
Schistosomiasis is a water-based parasitic disease caused by trematode worms of the genus Schistosoma and transmitted via contact with freshwater bodies contaminated by cercariae [3]. In Ethiopia, there are two schistosome species known to infect humans: S. mansoni and S. haematobium. The former, causing intestinal schistosomiasis, is widely distributed across many areas of the country, whereas the latter, causing urogenital schistosomiasis is restricted to lower altitude areas of the country [4]. It is estimated that more than 5 million people are infected with schistosomes and about 37.5 million, particularly those living in irrigated agricultural areas, are at risk of infection in Ethiopia [5]. Previous studies conducted in several regions of the country revealed a correlation between schistosome transmission and recent environmental changes resulting from extensive water resource developments [4,5]. In addition, anthropogenic changes such as increased population growth and movements, altered agricultural practices, lack of adequate drinking water, and poor living conditions have played an important role in spreading schistosomiasis throughout the country [4,6].
Fascioliasis is a food-or water-borne trematodiasis that can be transmitted via consumption of uncooked or unwashed aquatic vegetables or through drinking water contaminated with infective metacercariae cysts [7]. In the past, public health impact associated with Fasciola species infection remains less well understood, in part due to lack of data on the parasites' distribution and their infection status in humans [8]. However, recent studies conducted in many countries show that the infection has a vast geographic distribution throughout Europe, South America, and Oceania, where only F. hepatica infection commonly occurs, and in Africa and Asia, where the infection is caused by either F. hepatica or F. gigantica [7,8]. Owing to its widespread distribution, the World Health Organization(WHO) recognized human fascioliasis as one of the most important foodborne parasitic diseases and included in the list of neglected tropical diseases in 2010 [9].
Although schistosomiasis is a widely reported parasitic disease in many areas of the country [4,5], information on prevalence, intensity, and determinants of S. mansoni infection in the present study area are lacking. In addition, although bovine fascioliasis is highly prevalent and widely distributed, causing considerable economic loss to the livestock production in Ethiopia [10], only limited studies reported on human fascioliasis [10][11][12]. Data on human infections by these parasites in understudied but suspected regions are needed to inform targeted surveillance and/or intervention. Therefore, this study was initiated to assess the prevalence and factors associated with S. mansoni and Fasciola species infections among school children in selected areas of Amhara Regional State, Ethiopia.

Study setting
A cross-sectional survey was conducted between December 2017 and February 2018 in six primary schools in three districts-Kalu (South Wello), Fogera (South Gondar administrative zone), and Dembia (Central Gondar administrative zone)-Amhara Regional States, Ethiopia (Fig. 1). The area has tropical climatic conditions, characterized by a short rainy season from March to May, a long rainy season from June to September, and a dry season from November to February. The area has an elevation of 1484-2100 meters above sea level with a mean annual rainfall ranging from 750 to 1500 mm and a mean temperature ranging from 10°C to 30°C. People living in the area earn their livelihood through producing crops (e.g., rice, bean, and maize) and ranching livestock, mainly cattle, sheep, and goats. The main vegetables grown and consumed in this area are cabbage cauliflower, green chili, cherry tomatoes, and capsicum annum, which are often eaten raw or washed using water from unimproved water sources. Inhabitants have limited access to improved sanitation facilities [13]. The area has many permanent water collections, swampy areas, and rivers which contain the intermediate host snails such as Biomphalaria pfeifferi, Lymnaea truncatula, and L.natalenesis [14,15].

Study design and sample size
A school-based cross-sectional survey was conducted to assess the prevalence and factors associated with S. mansoni and Fasciola species infections among school children in six primary schools (Addis Mender, Harbu, Avia Kokit, Robite, Zengaj Robit, and Wawa) of Amhara Regional State. Sample size was computed using statistical formula n ¼ [16] where, n is sample size, Z is standard score for level of confidence, P is expected prevalence, and d is margin of error. By taking prevalence of 49% for S. mansoni infection from the previous study [17], 95% confidence level and 5% margin of error, the calculated sample size became 384. Considering a design effect of 2 and non-response rate of 10%, the final sample size was estimated to be 845.

Sampling technique
The study area was selected based on presence of favorable environment for the breeding of snail hosts, practice of small-scale irrigations, suspected high burden of schistosomiasis (based on the 2013/2014 national mapping of NTDs) [18], and endemicity for animal fascioliasis. Class roster containing names, age, and sex of the children attending grades 1-4 were obtained from the head teachers of all schools in order to get the number of children aged 6 to 15 years. Thereafter, a total of 845 school children (the desired sample size) were proportionally allocated to the six schools based on the number of eligible children in each school. Within each school, a systematic sampling technique was used to select the study participants using class registers as the sampling frame. Of the eligible children, 36 did not provide any stool specimens and 9 provided insufficient stool specimens-were excluded from the study. This resulted in 798 children who were included in final analysis.

Questionnaire survey
A pre-tested questionnaire assessing risks for S. mansoni and Fasciola species infections was administered to the childrens' parents and/or guardians in the household setting, while the participating children were interviewed at their schools. The parents (mothers or head of the household) were interviewed to collect information on socio-demographic characteristics, source of drinking water, children's habit of consuming vegetables, irrigation practices, and ownership of domestic animals. In addition, they were asked if their children had consumed raw liver 2 weeks prior to conducting this study to rule out the occurrences of false positive fascioliasis. Children were asked about their water contact habits, such as washing clothes, fetching water, water body contact while crossing, swimming, bathing in open water bodies, and whether they engaged in irrigation farming while assisting their parents. Trained study team members and school teachers conducted the interview using the local langue (Amharic).

Specimen collection and processing
Children aged 6 to 15 years who obtained written informed consent from their parents/guardians were given clean plastic sheet with wooden applicator stick and asked to provide their own feces (approximately 5 g). Double Kato-Katz thick smears (using 41.7 mg template) were prepared from each specimen within 3 h of specimen collection in the study area. The slides were examined after 24 h of preparation by experienced laboratory technicians. The remaining portion of specimen (approximately 3 g) was preserved in 10% formalin solution before being processed using the formol-ether concentration technique [19]. The processed specimens were then examined within 2 h of sample preparation at the Medical Parasitology Laboratory of the Aklilu Lemma Institute of Pathobiology, Addis Ababa University. The formol-ether concentration technique was used for detection of S. mansoni and Fasciola species eggs, and the Kato-Katz technique was used for detection and quantification of eggs of both parasites. To estimate infection intensity, the number of eggs counted in both Kato-Katz slides was converted into eggs per gram of stool (epg) by multiplying the mean of eggs counted in both slides by a factor of 24 [19,20]. Infection intensity of S. mansoni was classified as light (1-99 epg), moderate (100-399 epg), and heavy (> 400 epg), based on the WHO guideline [20]. The detection of S. mansoni and Fasciola species eggs by either Kato-Katz or formol-ether concentration technique was considered positive and the absence of at least 1 egg for each parasite was recorded as negative. To ensure consistency in egg counting, 10% of the Kato-Katz smears were re-examined by a senior laboratory technician.

Data analysis
Data were computerized in Microsoft Excel version 2007 (MS Corporation, Washington, USA) and analyzed using the Statistical Package for Social Scientists (SPSS) version 20. (SPSS Inc., Chicago, IL, USA). Prevalences of S. mansoni and Fasciola species infections were determined by using descriptive statistical analysis. Student's t test was used to test any statistical difference in infection intensity for S. mansoni and Fasciola species infections between sex and age groups. Univariate and multivariate logistic regression analysis were used to identify factors associated with the infections. Any association with a P values less than 5% was considered statistically significant.

Discussion
Schistosomiasis continues to be a major public health problem in Ethiopia, especially in rural areas where agro-industrial activities are rapidly intensifying and expanding [21,22]. The prevalence of intestinal schistosomiasis observed in the study area was 25.6 %, in alignment with prevalence of 25.8% in Southern Ethiopia [23], 29.9% in Zegie (Northwest Ethiopia) [24], 24% in Manna District (Southwest Ethiopia) [25], and 29% in Methara (Eastern Ethiopia) [26]. In contrast, the prevalence of S. mansoni infection observed in this study was lower than a study conducted in Sanja area, northern Ethiopia (82.8%) [27], Bushulo village, southern Ethiopia (73.7%) [28], and Yachi areas, Southwest Ethiopia (42.9%) [29]. This could be attributed to difference in contamination of water bodies with cercarial stage, the climate of the areas, water contact patterns of the study participants, and socio-economic characteristics.
Our results showed that prevalence and intensity of S. mansoni infection were higher among male children than the females. This finding corroborated with other previous studies conducted in South Wello [30], Wondo Genet Zuria [31], Western Abaya [32], Yachi areas [29], and Wolaita Zone [33]. This could be partially explained by the difference in gender-specific water contact activities. In the study area, the male students are likely to engage more in outdoor activities such as playing/swimming in rivers and watering animals while the females are involved more in indoor activities (e.g., cooking and food preparation) [34], which may explain the differences in their exposures to cercariae-infected water.
The prevalence of S. mansoni infection was higher in children attending Addis Mender and Harbu Primary Schools. The high prevalence noted in these schools could be related to proximity to nearby water bodies, rendering the children greater opportunities to contaminated water exposure. This is in agreement with findings of earlier studies conducted elsewhere in Ethiopia [29] and Kenya [35], which reported proximity of schools to natural freshwater bodies as an important risk factor for intestinal schistosomiasis. The variation in prevalence between schools could also be related to the difference in other factors, such as fecal contamination of the water bodies and frequency of exposure to open water bodies. Open defecation in field and near water sources is still most commonly practiced in the present study area [13,36].
The relationship between water contact activities and the occurrence of schistosomiasis is well documented [3,37]. In our study, the prevalence of S. mansoni infection was significantly higher among children swimming in rivers and bathing in open freshwater bodies. These water contact activities are a potential risk factor for the children's exposure to free-swimming cercariae and subsequent infection. This is consistent with the findings of earlier studies that reported a significant correlation between S. mansoni infection rate and contact with freshwater bodies [27,33].
The result of the present study also showed that irrigation practice was significantly associated with S. mansoni infection. This observation corroborates previous study which showed that prevalence of S. mansoni infection was higher among children who engaged in irrigation activities [11]. Communities in the study area practice small-scale irrigation activities for production of cash crops and cultivation of vegetables. Notably, large water bodies and irrigation canals used for farming and domestic purposes are found in the study area. Such ideal environment will create suitable habitat for the snail intermediate hosts to breed and cercariae to infect the children who often come in contact with contaminated water.
The prevalence of Fasciola species infection observed in the present study was 5.5%. This agrees with findings of studies conducted in Lake Tana basin (Northwest Ethiopia) (3.3%) [11] and Kemissie areas (Northeast Ethiopia) (2%) [10]. However, the present study observed a lower prevalence of infection compared to studies conducted in Egypt (12.8%) [38], Vietnam (7.7%) [39], and Peru (9.6%) [40]. These variations could be attributed to differences in ecological variations, dietary habits of the study participants, hygiene, level of environmental contamination, and diagnostic test used. According to the epidemiological classification set for human fascioliasis, areas are considered of hypo-endemic, meso-endemic, and hyper-endemic when they present a prevalence of Fasciola species infection below 1%, between 1 and 10%, and greater than 10%, respectively [41]. Hence, the present study area is regarded as meso-endemic region for the disease and this might suggest that the pattern of human fascioliasis distribution is changing from a situation of sporadic human cases to a growing public health problem in the region, highlighting the need for improved surveillance in areas where animal fascioliasis is endemic and irrigation farming are extensively practiced.
The result of the present study shows that there was a significant association between consumption of raw vegetables and Fasciola species infection. This result agrees with the study conducted in the Northern Bolivian Altiplano which showed that ingestion of raw aquatic vegetables was a plausible risk factor for the occurrences of human fascioliasis [42]. This could be due to contamination of raw vegetables with metacercariae of Fasciola species, whereby the children who frequently eat this kind of food could be infected by these parasites and are able to contaminate the environment through excreting infected feces. Accessing water from unimproved source in the present study was also associated with human Fasciola infection. This result is consistent with findings from Egypt [42] and Bolivia [43], which showed that drinking water from unimproved water supplies was linked with human fascioliasis. This could be probably related to the use of contaminated water for drinking and growing vegetables in the area. Lack of improved water supply is the major poverty-related problem reported in the current study area, where majority of the inhabitants use unprotected open water bodies (river, spring, or well) for dinking and recreational purposes, as well as for domestic chores like washing utensils, bathing, and drinking animals. Interventions targeting on the provision of improved water supply and sanitation in the area are needed to reduce the risk of transmission of human fascioliasis and other parasitic infections.
Although the study did not find significant correlation between S. mansoni and Fasciola infections among the school children across the schools, the spatial distribution of the two diseases shows that five out of six schools were identified as transmission foci of both fascioliasis and schistosomiasis, suggesting a significant overlap of the two parasites which might be of public health implications. This observation is in agreement with earlier studies from Egypt which showed that Fasciola species and S. mansoni share similar geographic distribution [38,43]. The overlap in distribution of fascioliasis and schistosomiasis is correlated with the availability of conditions that favor co-existence of both diseases, such as lack of access to safe water supplies, improved hygiene, good sanitation, and existence of favorable environmental conditions for survival of parasites, among others. Given that S. mansoni and Fasciola species share key similarities in disease transmission and biology, there is a need for further researches that assess the comorbidities associated with fascioliasis and schistosomiasis co-infection among affected populations. It is also important to highlight the need for integrating surveillance and control of both diseases, especially in areas where they are co-endemic and public health concerns.
Our study has limitations that warrant further studies. First, the data obtained in this study might not be representative of other areas in the country because the distribution of intestinal schistosomiasis and human fascioliasis vary across localities based on ecology and population dynamics of snail hosts. Second, study participants were recruited only from few primary schools. However, human fascioliasis has been shown to cluster within familial groups that share contaminated vegetables or drink from the same water sources [7]. This might have resulted in underestimation of the prevalence of Fasciola species infection in the study area. Thus, further population-based surveys involving all age groups are suggested to provide a clearer picture of the diseases in the study area.

Conclusion
Our study showed that intestinal schistosomiasis and human fascioliasis are prevalent among school children in the study area. Swimming in rivers, bathing in open freshwater bodies, and engaging in irrigation activities were identified as possible risk factors for acquiring intestinal schistosomiasis while consumption of raw vegetables and drinking from unimproved water sources were associated with the prevalence of Fasciola species infection. Hence, setting targeted interventions, including administration of anthelmintic drugs, health education, snail control, provision of improved water, and adequate sanitation are needed to control these parasitic infections.