Optimization of an in vitro drug screening assay for anti-Trypanosoma cruzi activity using epimastigotes

Chagas disease, caused by Trypanosoma cruzi, poses a significant global health burden. Current treatment relies solely on benznidazole and nifurtimox, both with limited efficacy and significant adverse effects. In vitro phenotype-based screening enables identification of alternative treatments. While epimastigote-based screening is not suitable for primary drug discovery, it provides valuable complementary data for compound evaluation. However, the lack of standardized experimental conditions across studies compromises reproducibility and inter-laboratory comparability. Here, we optimized experimental conditions for T. cruzi epimastigote drug susceptibility assays. Trypanosoma cruzi epimastigotes (strain Y) were cultured under systematically varied conditions to optimize medium composition, serum supplementation, medium replacement frequency, inoculum density, and treatment exposure time. Epimastigote growth peaked on day 8, declining thereafter. Low initial parasite inoculum showed reduced growth and high variability, while high parasite densities formed aggregates. Growth rates were similar across media, but Roswell Park Memorial Institute medium could promote trypomastigote-like forms. Fetal bovine serum (FBS) enhanced growth rates, with a positive correlation between serum concentration and proliferation. Three-day medium replacement maintained >98% epimastigotes. Benznidazole exhibited an IC₅₀ of 16.61 ± 0.38 µM under optimized conditions. Optimal conditions include: Liver Infusion Tryptose or Brain Heart Infusion medium with 10% FBS, medium replacement every 3 days, 1 × 10⁶ parasites/ml inoculum, and 72-h treatment at 28°C. The assay demonstrated robustness across microplate designs, producing consistent results with both U-bottom and flat-bottom 96-well plates. Under these optimized conditions, benznidazole serves as an effective positive control.