Marc Kaethner (Bern / CH), Matías Preza (Bern / CH), Tobias Kämpfer (Bern / CH), Pascal Zumstein (Bern / CH), Claudia Tamponi (Bern / CH), Antonio Varcasia (Bern / CH), Prof. Dr. Andrew Hemphill (Bern / CH), Prof. Dr. Klaus Brehm (Würzburg / DE), Britta Lundström-Stadelmann (Bern / CH)
Abstract text
Introduction
Cestodes of the genus Echinococcus cause the zoonotic diseases alveolar echinococcosis (AE) and cystic echinococcosis (CE). An in vitro drug screening pipeline is established for AE, but drug efficacy assessments against E. granulosus are mainly performed via subjective eosin exclusion test on protoscoleces (PSCs), which are not the disease-causing stage. Thus, there is the urgent need for unbiased drug tests against E. granulosus.
Objectives
Our objectives were to validate whether the current in vitro drug screening assays for E. multilocularis metacestodes, protoscoleces and primary cells can be applied to E. granulosus. Furthermore, we aimed to establish E. granulosus metacestodes cultures that can be maintained in vitro for prolonged periods of time and to investigate whether proliferative primary cell cultures of E. granulosus could form new metacestode vesicles.
Material and methods
We applied an in vitro screening cascade established for E. multilocularis to E. granulosus to compare the efficacy of several standard drugs (niclosamide, nitazoxanide, albendazole, monepantel, mefloquine, buparvaquone and MMV665807). Efficacy against metacestodes was measured by damage marker release and vesicle viability assays. Efficacy against PSCs was assessed by motility assay, and viability assay was employed to investigate the impact on stem cells. Proliferation of primary cells was assessed by EdU incorporation and metacestode vesicle formation assays.
Results
In vitro cultured E. granulosus metacestodes exhibited an intact laminated layer. Comparing drug efficacy against E. multilocularis and E. granulosus in vitro, the damage marker release assay and the metacestode viability assay showed similar drug responses for most tested drugs, while buparvaquone and MMV665807 showed significantly higher activity to E. multilocularis metacestodes. Albendazole had a higher impact on E. granulosus PSCs. Stem cell assays showed similar activities for most tested drugs, but nitazoxanide, albendazole and monepantel showed higher activity to E. multilocularis stem cells. E. granulosus primary cells were successfully cultured for four weeks, underwent proliferation and formed novel metacestodes.
Conclusion
Our results show that established drug screening assays can be applied to E. granulosus and that its stem cells can be cultured to form novel metacestodes in vitro. This allows in vitro screening of drugs against E. granulosus in an unbiased and objective manner.