Flynn, C. A.; Harmez, A.; Colon-Rios, R.; Flynn, W. F.; Kazmierczak, B. I.

Pathogenic free-living amoebae (FLA) such as Acanthamoeba castellanii are present in soil and water worldwide. A. castellanii causes systemic infections with very high mortality rates, yet drugs specifically targeting this pathogen are not available. Methods to reliably generate and assay cysts, which drive infection recurrence and drug resistance, are unavailable in a high-throughput format suitable for drug screening and testing. In this study, we developed a robust and reproducible protocol for encysting A. castellanii as well as a high-throughput, quantitative cyst viability assay using fluorescent live/dead staining coupled with microscopy and automated image analysis. These methods were used to screen the cysticidal activity of 17 clinically relevant drugs and disinfectants and identified five agents, including caspofungin, as active against cysts.