Development of environmental loop-mediated isothermal amplification (eLAMP) diagnostic tool for Bulinus truncatus field detection.

BACKGROUND: Global changes are reshaping the distribution of vector-borne diseases by spreading vectors to previously non-endemic areas. Since 2013, urogenital schistosomiasis has emerged in Corsica and threatens European countries. Gastropod vectors release schistosome larvae that can infect humans who come into contact with freshwater bodies. Monitoring schistosomiasis host vectors is a prerequisite to understand and subsequently to control this pathogen transmission. Because malacological surveys are time consuming and require special expertise, the use of a simple molecular method is desirable. METHODS: The aim of this study is to develop a ready-to-use protocol using the LAMP (loop-mediated isothermal amplification) method to detect environmental DNA of Bulinus truncatus, vector of Schistosoma haematobium. Interestingly, LAMP method possesses all the characteristics required for adaptability to field conditions particularly in low-income countries: speed, simplicity, lyophilized reagents, low cost and robustness against DNA amplification inhibitors. We have tested this new method on Corsican water samples previously analysed by qPCR and ddPCR. RESULTS: We demonstrate that our diagnostic tool B. truncatus eLAMP (Bt-eLAMP) can detect the eDNA of Bulinus truncatus as effectively as the two other methods. Bt-eLAMP can even detect 1/4 of positive samples not detectable by qPCR. Moreover, the complete Bt-eLAMP protocol (sampling, sample pre-process, amplification and revelation) does not require sophisticated equipment and can be done in 1 ½ h. CONCLUSIONS: LAMP detection of environmental DNA provides large-scale sensitive surveillance of urogenital schistosomiasis possible by identifying potentially threatened areas. More generally, eLAMP method has great potential in vector-borne diseases and ecology.

A duplex tetra-primer ARMS-PCR assay to discriminate three species of the Schistosoma haematobium group: Schistosoma curassoni, S. bovis, S. haematobium and their hybrids.

BACKGROUND: The use of applications involving single nucleotide polymorphisms (SNPs) has greatly increased since the beginning of the 2000s, with the number of associated techniques expanding rapidly in the field of molecular research. Tetra-primer amplification refractory mutation system-PCR (T-ARMS-PCR) is one such technique involving SNP genotyping. It has the advantage of amplifying multiple alleles in a single reaction with the inclusion of an internal molecular control. We report here the development of a rapid, reliable and cost-effective duplex T-ARMS-PCR assay to distinguish between three Schistosoma species, namely Schistosoma haematobium (human parasite), Schistosoma bovis and Schistosoma curassoni (animal parasites), and their hybrids. This technique will facilitate studies of population genetics and the evolution of introgression events. METHODS: During the development of the technique we focused on one of the five inter-species internal transcribed spacer (ITS) SNPs and one of the inter-species 18S SNPs which, when combined, discriminate between all three Schistosoma species and their hybrid forms. We designed T-ARMS-PCR primers to amplify amplicons of specific lengths for each species, which in turn can then be visualized on an electrophoresis gel. This was further tested using laboratory and field-collected adult worms and field-collected larval stages (miracidia) from Spain, Egypt, Mali, Senegal and Ivory Coast. The combined duplex T-ARMS-PCR and ITS + 18S primer set was then used to differentiate the three species in a single reaction. RESULTS: The T-ARMS-PCR assay was able to detect DNA from both species being analysed at the maximum and minimum levels in the DNA ratios (95/5) tested. The duplex T-ARMS-PCR assay was also able to detect all hybrids tested and was validated by sequencing the ITS and the 18S amplicons of 148 of the field samples included in the study. CONCLUSIONS: The duplex tetra-primer ARMS-PCR assay described here can be applied to differentiate between Schistosoma species and their hybrid forms that infect humans and animals, thereby providing a method to investigate the epidemiology of these species in endemic areas. The addition of several markers in a single reaction saves considerable time and is of long-standing interest for investigating genetic populations.

Genetic profiles of Schistosoma haematobium parasites from Malian transmission hotspot areas.

BACKGROUND: Although schistosomiasis is a public health issue in Mali, little is known about the parasite genetic profile. The purpose of this study was to analyze the genetic profile of the schistosomes of Schistosoma haematobium group in school-aged children in various sites in Mali. METHODS: Urine samples were collected from 7 to 21 November 2021 and subjected to a filtration method for the presence S. haematobium eggs. The study took place in two schistosomiasis endemic villages (Fangouné Bamanan and Diakalèl), qualified as hotspots according to the World Health Organization (WHO) definition. Molecular genotyping on both Cox1 and ITS2/18S was used for eggs' taxonomic assignation. RESULTS: A total of 970 miracidia were individually collected from 63 school-aged children and stored on Whatman FTA cards for molecular analysis. After genotyping 42.0% (353/840) and 58.0% (487/840) of miracidia revealed Schistosoma bovis and S. haematobium Cox1 profiles, respectively; 95.7 (885/925) and 4.3% (40/925) revealed S. haematobium and S. haematobium/S. curassoni profiles for ITS/18S genes, respectively. There was a significant difference in the Cox1 and ITS2/18S profile distribution according to the village (P < 0.0001). Overall, 45.6% (360/789) were hybrids, of which 72.0% (322/447) were from Diakalèl. Three hybrids' profiles (Sb/Sc_ShxSc with 2.3%; Sb/Sc_ShxSh with 40.5%; Sh_ShxSc with 2.8%) and one pure profile (Sh_ShxSh with 54.4%) were identified. CONCLUSION: Our findings show, for the first time to our knowledge, high prevalence of hybrid schistosomes in Mali. More studies are needed on population genetics of schistosomes at the human and animal interface to evaluate the parasite's gene flow and its consequences on epidemiology of the disease as well as the transmission to humans.

Hybridization increases genetic diversity in Schistosoma haematobium populations infecting humans in Cameroon.

BACKGROUND: Hybrids between Schistosoma haematobium (Sh) and S. bovis (Sb) have been found in several African countries as well as in Europe. Since the consequences of this hybridization are still unknown, this study aims to verify the presence of such hybrids in Cameroonian humans, to describe the structure of S. haematobium populations on a large geographic scale, and to examine the impact of these hybrids on genetic diversity and structure of these populations. METHODS: From January to April 2019, urine from infected children was collected in ten geographically distinct populations. Miracidia were collected from eggs in this urine. To detect the presence of hybrids among these miracidia we genotyped both Cox1 (RD-PCR) and ITS2 gene (PCR-RFLP). Population genetic diversity and structure was assessed by genotyping each miracidium with a panel of 14 microsatellite markers. Gene diversity was measured using both heterozygosity and allelic richness indexes, and genetic structure was analyzed using paired Fst, PCA and Bayesian approaches. RESULTS: Of the 1327 miracidia studied, 88.7% were identified as pure genotypes of S. haematobium (Sh_Sh/Sh) while the remaining 11.3% were hybrids (7.0% with Sh_Sh/Sb, 3.7% with Sb_Sb/Sh and 0.4% with Sb_Sh/Sb). No miracidium has been identified as a pure genotype of S. bovis. Allelic richness ranged from 5.55 (Loum population) to 7.73 (Matta-Barrage) and differed significantly between populations. Mean heterozygosity ranged from 53.7% (Loum) to 59% (Matta Barrage) with no significant difference. The overall genetic differentiation inferred either by a principal component analysis or by the Bayesian approach shows a partial structure. Southern populations (Loum and Matta Barrage) were clearly separated from other localities but genetic differentiation between northern localities was limited, certainly due to the geographic proximity between these sites. CONCLUSIONS: Hybrids between S. haematobium and S. bovis were identified in 11.3% of miracidia that hatched from eggs present in the urine of Cameroonian schoolchildren. The percentages of these hybrids are correlated with the genetic diversity of the parasite, indicating that hybridization increases genetic diversity in our sampling sites. Hybridization is therefore a major biological process that shapes the genetic diversity of S. haematobium.