Background Schistosomiasis is a neglected tropical parasitic disease associated with severe pathology, mortality and economic loss worldwide. Programs for disease control may benefit from specific and sensitive diagnostic methods to detect Schistosoma trematodes in aquatic environments. Here we report the development of novel environmental DNA (eDNA) qPCR assays for the presence of the human-infecting species Schistosoma mansoni, S. haematobium and S. japonicum. Methodology/Principal findings We first tested the specificity of the assays across the three species using genomic DNA preparations which showed successful amplification of target sequences with no cross amplification between the three focal species. In addition, we evaluated the specificity of the assays using synthetic DNA of multiple Schistosoma species, and demonstrated a high overall specificity; however, S. japonicum and S. haematobium assays showed cross-species amplification with very closely-related species. We next tested the effectiveness of the S. mansoni assay using eDNA samples from aquaria containing infected host gastropods, with the target species revealed as present in all infected aquaria. Finally, we evaluated the effectiveness of the S. mansoni and S. haematobium assays using eDNA samples from eight discrete natural freshwater sites in Tanzania, and demonstrated strong correspondence between infection status established using eDNA and conventional assays of parasite prevalence in host snails. Conclusions/Significance Collectively, our results suggest that eDNA monitoring is able to detect schistosomes in freshwater bodies, but refinement of the field sampling, storage and assay methods are PLOS NEGLECTED TROPICAL DISEASES

Background: Schistosomiasis is a neglected tropical disease that infects over 200 million people worldwide. Control measures can benefit from improved surveillance methods in freshwaters, with environmental DNA (eDNA) surveys having the potential to offer effective and rapid detection of schistosomes. However, sampling eDNA directly from natural water bodies can lead to inaccurate estimation of infection risk if schistosome eDNA is rare in the environment. Here we report a xenomonitoring method that allows schistosome infections of host snail species to be determined from eDNA in water used to house those snails. Methods: Host snail species were collected and placed in containers of water and allowed to shed cercariae, and then water samples were filtered and tested using qPCR assays specific to the African species Schistosoma mansoni and Schistosoma haematobium. We evaluated this "eDNA-based xenomonitoring" approach by experimentally comparing the results to those obtained from direct qPCR screening of tissue sourced from the snails in the experiment. Results: We found that our method accurately diagnosed the presence of S. mansoni-infected snails in all tests, and S. haematobium-infected snails in 92% of tests. Moreover, we found that the abundance of Schistosoma eDNA in experiments was directly dependent on the number and biomass of infected snails. Conclusions: These results provide a strong indication that this surveillance method combining the utility of eDNAbased monitoring with the reliability of traditional xenomonitoring approaches could be used to accurately assay the presence of Schistosoma species in natural habitats. This approach may be well-suited for epidemiological studies and monitoring in endemic areas, where it can assist schistosomiasis control by indicating infection risk from freshwaters and guiding necessary interventions to eliminate the disease.

The species described in the first paragraph of the results subsection 'Environmental DNA detected the presence of schistosomes from the natural environment' has been reported as B. globosus, but this should read B. pfeifferi instead. The first paragraph of the 'Environmental DNA detected the presence of schistosomes from the natural environment' subsection has now been updated to: "Four out of the eight screened sites were positive for S. mansoni eDNA (sites we named A, B, C and D; Tables 5 & 6, Fig 2). At two of these eDNA positive locations, infected B. pfeifferi gastropods were present (sites A and B), while at two of the locations B. pfeifferi was not found (sites C and D; Table 5). Of the four locations that were eDNA negative, only one had B. pfeifferi present, and these snails were determined to be infected through PCR analysis of tissue DNA (Table 5, Fig 2). Thus, the eDNA assay was congruent with the PCR tests for infected snails in 5/8 (62.5%) of locations. Notably, the estimated numbers of S. mansoni copies resolved in most samples were below the defined LOQ of 100 copies/μl, the LODI of 41.68 copies/μl and the LODIII of 10.84 copies/μl.