Population genetic structure and hybridization of schistosomes in schoolchildren in Côte d’Ivoire

Background: Schistosomiasis is a water-based chronic parasitic disease caused by trematode worms of the genus Schistosoma. Considered as a neglected tropical disease by the World Health Organization (WHO), it affects more than 250 million people, primarily in sub-Saharan Africa. In Côte d’Ivoire, both Schistosoma haematobium (causing urogenital schistosomiasis) and Schistosoma mansoni (causing intestinal schistosomiasis) co-exist. The chronic disease is characterized by anaemia, abdominal pain, bloody stool or urine, portal hypertension, swelling of the liver, enlarged spleen, and degradation of intestinal or urogenital organs. The main strategy for control endorsed by WHO is preventive chemotherapy using praziquantel. Despite mass drug administration campaigns, the prevalence of schistosomiasis remains high in many parts of Côte d’Ivoire. Investigating the population genetics of schistosomes, using molecular markers, may help elucidate its dynamic epidemiology and provide better insight into parasite gene flow.
Objectives: This Ph.D. thesis had two major aims. The first was to expand our understanding of the parasite’s transmission dynamics to update epidemiological data on schistosomiasis by determining the prevalence and associated risk factors of the disease. The second aim was to better understand the genetic structure and diversity of S. haematobium in schoolchildren.
Methods: Urine samples were examined from schoolchildren in four sampling sites (Adzopé, Agboville, Duekoué and Sikensi). A filtration method was used to identify S. haematobium eggs, while stool samples were subjected to duplicate Kato-Katz thick smears to identify and quantify eggs of S. mansoni and soil-transmitted helminths. Data on sociodemographic, socioeconomic and environmental factors were obtained using a pretested questionnaire. Eggs were hatched and miracidia were individually collected and stored on Whatman® FTA cards for molecular analysis. Individual miracidia were molecularly characterized by analysis of mitochondrial cox1 gene and nuclear internal transcribed spacer 2 (ITS 2) DNA region. The cox1 and ITS 2 amplicons were Sanger sequenced from 40 randomly selected miracidia to confirm species and hybrids status. To achieve our second aim on the genetic distribution of S. haematobium, we systematically first reviewed the scientific literature of studies focused on methods used for population genetic structuring and diversity of S. haematobium and we genotyped and analysed miracidia using a set of 18 microsatellite markers.
Results: Of 1,187 schoolchildren, the overall prevalence was 14.0% for S. haematobium and 6.1% for S. mansoni. In the southern part of Côte d’Ivoire, the prevalence of S. haematobium was 16.1% with a particularly high prevalence in Sikensi (35.6%), while S. mansoni was most prevalent in Agboville (11.2%). We found that fishing and washing clothes in open freshwater bodies and swimming were positively associated with schistosomiasis. Among the 33 cox1 sequences analysed, we identified 15 S. haematobium sequences (45.5%) belonging to seven haplotypes and 18 S. bovis sequences (54.5%) belonging to 12 haplotypes. Of 40 ITS2 sequences analysed, 77.5% were assigned to pure S. haematobium, 10.0% to pure S. bovis and 12.5% to S. haematobium x S. bovis hybrids. The literature review elucidated that few methods are available for the population genetics of S. haematobium. Variable genetic diversity and population structure were observed within and across countries. We found that microsatellite markers are commonly used for Schistosoma population genetics research and they remain the best tool for population genetic analysis. Of 2,164 miracidia successfully amplified in both cox1 and ITS2, 1,966 (90.9%) yielded reliable peak calls for at least ten microsatellite loci and were retained for population genetic analysis. Significant differences were found between sampling sites in all genetic diversity indices and a genetic structure was observed. The analysis of cox1-ITS profile showed that no significant genetic clustering was observed between S. haematobium and hybrids S. haematobium x S. bovis. We also observed a clear separation between "pure" S. bovis (Sb_SbSb) and the other cox1-ITS profiles.
Conclusion: Our study confirms that schistosomiasis remains prevalent in the southern and western parts of Côte d’Ivoire, and we reported that S. haematobium x S. bovis hybrids are common in Côte d’Ivoire. The current results provide novel insights about the population genetics of S. haematobium in Côte d’Ivoire and reiterate the utility of microsatellite markers designed for this parasite. A genetic diversity and population structure were observed and "pure" S. bovis, previously thought to infect bovines, was identified in children samples. Hence, intense investigation in domestic and wild animals is warranted to determine whether zoonotic transmission occurs. These findings could serve as a benchmark to enhance schistosomiasis control and surveillance to measure the effect of treatment on parasite population genetics in Côte d’Ivoire. New research is needed on population genetics in human and animal schistosomes to evaluate the parasite’s gene flow.