Establishment and application of unbiased in vitro drug screening assays for the identification of compounds against Echinococcus granulosus sensu stricto

Author summaryTapeworms of the genus Echinococcus cause severe zoonotic diseases in humans and livestock, which goes along with substantial burden on human and veterinary health and big economic losses. Most relevant to human health are the species E. multilocularis and E. granulosus s.l., which are two closely related parasites that cause alveolar and cystic echinococcosis, respectively. Both diseases have limited treatment options, which is why there is a strong need for new drugs against these neglected tropical diseases.Efficacy of new drugs against E. multilocularis can be assessed via various standardized in vitro assays. For E. granulosus, activity of drugs is currently mainly assessed via optical readout methods, which are time consuming and are not easily carried out in a blinded manner.We here describe the application of several standardized in vitro drug screening assays targeting different parasite stages and isolated germinal layer cells of E. granulosus s.s. We have performed these assays in a comparative manner including E. multilocularis and tested various standard drugs with known activity in these assays.All assays could be applied to E. granulosus s.s. and this will allow for standardized, higher throughput testing against this parasite in the future. Echinococcus multilocularis and E. granulosus s.l. are the causative agents of alveolar and cystic echinococcosis, respectively. Drug treatment options for these severe and neglected diseases are limited to benzimidazoles, which are not always efficacious, and adverse side effects are reported. Thus, novel and improved treatments are needed. In this study, the previously established platform for E. multilocularis in vitro drug assessment was adapted to E. granulosus s.s. In a first step, in vitro culture protocols for E. granulosus s.s. were established. This resulted in the generation of large amounts of E. granulosus s.s. metacestode vesicles as well as germinal layer (GL) cells. In vitro culture of these cells formed metacestode vesicles displaying structural characteristics of metacestode cysts generated in vivo. Next, drug susceptibilities of E. multilocularis and E. granulosus s.s. protoscoleces, metacestode vesicles and GL cells were comparatively assessed employing established assays including (i) metacestode vesicle damage marker release assay, (ii) metacestode vesicle viability assay, (iii) GL cell viability assay, and (iv) protoscolex motility assay. The standard drugs albendazole, buparvaquone, mefloquine, MMV665807, monepantel, niclosamide and nitazoxanide were included. MMV665807, niclosamide and nitazoxanide were active against the parasite in all four assays against both species. MMV665807 and monepantel were significantly more active against E. multilocularis metacestode vesicles, while albendazole and nitazoxanide were significantly more active against E. multilocularis GL cells. Albendazole displayed activity against E. multilocularis GL cells, but no effects were seen in albendazole-treated E. granulosus s.s. GL cells within five days. Treatment of protoscoleces with albendazole and monepantel had no impact on motility. Similar results were observed for both species with praziquantel and its enantiomers against protoscoleces. In conclusion, in vitro culture techniques and drug screening methods previously established for E. multilocularis were successfully implemented for E. granulosus s.s., allowing comparisons of drug efficacy between the two species. This study provides in vitro culture techniques for the reliable generation of E. granulosus s.s. metacestode vesicles and GL cell cultures and describes the validation of standardized in vitro drug screening methods for E. granulosus s.s.