BackgroundIn vitromodels for studying interactions betweenAcanthamoebaand host cells are crucial for understanding the pathomechanism ofAcanthamoebaand assessing differences between strains and cell types. The virulence ofAcanthamoebastrains is usually assessed and monitored by using cell cytotoxicity assays. The aim of the present study was to evaluate and compare the most widely used cytotoxicity assays for their suitability to assessAcanthamoebacytopathogenicity.MethodsThe viability of human corneal epithelial cells (HCECs) after co-culture withAcanthamoebawas evaluated in phase contrast microscopy.ResultsIt was shown thatAcanthamoebais unable to considerably reduce the tetrazolium salt and the NanoLuc®Luciferase prosubstrate to formazan and the luciferase substrate, respectively. This incapacity helped to generate a cell density-dependent signal allowing to accurately quantifyAcanthamoebacytotoxicity. The lactate dehydrogenase (LDH) assay led to an underestimation of the cytotoxic effect ofAcanthamoebaon HCECs since their co-incubation negatively affected the lactate dehydrogenase activity.DiscussionOur findings demonstrate that cell-based assays using the aqueous soluble tetrazolium-formazan, and the NanoLuc®Luciferase prosubstrate products, in contrast to LDH, are excellent markers to monitor the interaction ofAcanthamoebawith human cell lines and to determine and quantify effectively the cytotoxic effect induced by the amoebae. Furthermore, our data indicate that protease activity may have an impact on the outcome and thus the reliability of these tests.