title: Estimating trematode prevalence in snail hosts using a single-step duplex PCR: how badly does cercarial shedding underestimate infection rates?
abstract: BACKGROUND
  Trematode 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.
  
  
  METHODS
  Snails 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.
  
  
  RESULTS
  Overall 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.
  
  
  CONCLUSIONS
  We 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.
authors.affiliation: Cavanilles Institute for Biodiversity and Evolutionary Biology, Science Park, University of Valencia, PO Box 22 085, 46071 Valencia, Spain. a.isabel.born@uv.es.
authors.forename: Ana
authors.initials: A
authors.lastname: Born-Torrijos
authors.forename: Robert
authors.initials: R
authors.lastname: Poulin
authors.forename: Juan Antonio
authors.initials: JA
authors.lastname: Raga
authors.forename: Astrid Sibylle
authors.initials: AS
authors.lastname: Holzer
mesh_terms: D000818:Animals; D060085:Coinfection; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D006790:Host-Parasite Interactions; D016133:Polymerase Chain Reaction / Q000379:methods*; D012908:Snails / Q000469:parasitology*; D013045:Species Specificity; D014200:Trematoda / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*
chemical_list: D021903:DNA, Ribosomal Spacer