New Viability Assays for <i>Acanthamoeba castellanii</i> Trophozoites and Cysts Allow Rapid Assessment of Drug Activity

Pathogenic free-living amoebae 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, coupled with optimized techniques for measuring trophozoite viability and pseudocyst formation, were used to screen the activity of 16 clinically relevant drugs and disinfectants. Four agents, including caspofungin, were active against both trophozoites and cysts.