Estimating trematode prevalence in snail hosts using a single-step duplex PCR: how badly does cercarial shedding underestimate infection rates?

Abstract

BackgroundTrematode communities often consist of different species exploiting the same host population, with two or more trematodes sometimes co-occuring in the same host. A commonly used diagnostic method to detect larval trematode infections in snails has been based on cercarial shedding, though it is often criticized as inaccurate. In the present study we compare infection prevalences determined by cercarial emission with those determined, for the first time, by molecular methods, allowing us to quantify the underestimation of single and double infections based on cercarial emission. We thus developed a duplex PCR for two host-parasite systems, to specifically differentiate between single and double infections. The Ebro samples include two morphologically similar opecoelids, whereas the Otago samples include two morphologically different larval trematodes.MethodsSnails were screened for infections by incubating them individually to induce cercarial emission, thus determining infection following the “classical” detection method. Snail tissue was then removed and fixed for the duplex PCR. After obtaining ITS rDNA sequences, four species-specific primers were designed for each snail-trematode system, and duplex PCR prevalence was determined for each sample. Results from both methods were statistically compared using the McNemar’s Chi-squared test and Cohen’s Kappa Statistic for agreement between outcomes.ResultsOverall infection prevalences determined by duplex PCR were consistently and substantially higher than those based on cercarial shedding: among Ebro samples, between 17.9% and 60.1% more snails were found infected using the molecular method, whereas in the Otago samples, the difference was between 9.9% and 20.6%. Kappa values generally indicated a fair to substantial agreement between both detection methods, showing a lower agreement for the Ebro samples.ConclusionsWe demonstrate that molecular detection of single and double infections by duplex PCR strongly outcompetes the classical method. Detection failure is most likely due to immature and covert infections, however, the higher incidence of misidentified double infections in the Ebro samples arises from morphological similarity of closely-related species. The higher accuracy of the duplex PCR method also adds to our understanding of community structure of larval trematodes in snail hosts, by providing a clearer assessment of the importance of interspecific interactions within the host.

Seasonal and Spatial Environmental Influence on Opisthorchis viverrini Intermediate Hosts, Abundance, and Distribution: Insights on Transmission Dynamics and Sustainable Control

“…Recent evidence indicates that the routinely used cercarial shedding detection method is greatly outcompeted by molecular detection methods, which identify trematode infection in snails with up to 60.1% higher efficacy. The authors of the study suggested that detection failure is most likely due to the immature and covert infections, which can result in delayed and therefore undetectable (at the time of observation) cercarial shedding [80]. Therefore, the near absence of detected infection in our study may not necessarily be indicative of low snail infection prevalence.…”

Towards interruption of schistosomiasis transmission in sub-Saharan Africa: developing an appropriate environmental surveillance framework to guide and to support ‘end game’ interventions

“…Several pioneering molecular studies have shown that many other species of freshwater snail carry evidence of schistosome infection than traditional methods have inferred previously [75–78]. Another important technical development has been the use of FTA card sampling methods which has enabled convenient storage of schistosome eggs, miracidia and cercariae for more precise genotyping of schistosome populations and species [62, 79–81].…”

Zoonotic Trematodiasis

“…elsewhere [18], however, in spite of these disadvantages, the sensitivity and specificity of this diagnostic test, once performed properly, have no equal when compared to other tests.…”

“…Food-borne trematode zoonoses (FBTZ) start their life cycle as miracidium, a 100-micrometer ciliated developmental stage result of trematodes embryonated eggs, highly mobile in aquatic conditions, upon finding a compatible snail, the miracidium penetrates the tegument of the intermediate host shedding its cilia [17] turning into germinal masses of cells called sporocysts. These sporocysts multiply into several masses of germinal cell each of them originating a redia [18], and these are intermediate germinal stages that develop into cercariae, a highly mobile developmental stage that finally exits the intermediate host by perforation of the snail'st e g ument and then migrates into an aquatic environment, the cercariae, depending on the parasitic trematode species, can penetrate the skin of a second intermediate host and encyst in the muscles of it [19]. Cercariae may also penetrate the skin of the definitive host [6] or lose its tail and become a cyst, an environment-resistant developmental stage on surrounding vegetation or in drinking water.…”