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Background  Echinostomiasis is one of the major food-borne trematodiases and the species  Echinostoma caproni  serves as a useful model for trematocidal drug discovery. The current  in vitro  drug sensitivity assay uses adult  E. caproni  worms that are incubated with candidate drugs and scored microscopically for viability at 72 hrs. The aim of this study was to investigate the use of newly excysted larvae (NEL) of  E. caproni  for  in vitro  drug testing, which would be faster, more cost effective and more ethical compared to adult worm assays.    Methods  Larvae were obtained by collecting metacercariae from snails and triggering their excystation using the trypsin-bile salt excystation method. Studies concerning various parameters of this chemical transformation process as well as appropriate NEL culturing conditions were carried out and findings evaluated. NEL and adult worms were incubated with praziquantel, tribendimidine, albendazole and quinine and evaluated microscopically 72 hrs post-incubation. In addition, the colorimetric markers resazurin, CellTiter-Glo® and Vybrant® were tested as an alternative assay read-out method.    Results  The chemical excystation method successfully induced  E. caproni  metacercariae to excyst at a rate of about 20-60%. NEL remained viable in culture medium for 5–7 days. The results of an  in vitro  drug assay using NEL mirrored the results of an assay using adult worms incubated with the same drugs. None of the markers could reliably produce signals proportional to NEL viability or cytotoxicity without significant complications.    Conclusion  NEL are adequate for  in vitro  drug testing. Challenges remain in further improving the excystation yield and the practicability of the assay setup. Resolving these issues could also improve read-outs using colorimetric markers. Using NEL is in alignment with the 3 R rules of the ethical use of laboratory animals and can greatly increase the rate and affordability with which drugs are screened  in vitro  against this intestinal trematode.

Development of an in vitro drug sensitivity assay based on newly excysted larvae of Echinostoma caproni