Evaluation of Parameters Impacting Drug Susceptibility in Intracellular Trypanosoma cruzi Assay Protocols

In order to understand the key parameters influencing drug susceptibility, different Trypanosoma cruzi assay protocols were evaluated using a comparative assay design. The assays compared in this study were an image-based intracellular T. cruzi assay quantified through an image-mining algorithm and an intracellular assay utilizing a β-galactosidase-expressing T. cruzi strain. Thirty-one reference compounds known to exhibit activities against intracellular T. cruzi were used as benchmarks. Initial comparison using EC50 values from two assays showed a very poor correlation, with an R2 value of 0.005. Nitroheterocyclics and CYP51 inhibitors were inactive in an image-based assay, but were highly active in a colorimetric assay. In order to identify the differentiating factor, we synchronized the compound–parasite incubation times or the sequential cell and compound seeding schemes between assays, but the correlation remained low. A high correlation (R2 = 0.86) was observed only after both compound incubation t...