Determination of ravuconazole and benznidazole interactions against the amastigote forms of the Y and Colombian T. cruzi strains.

The in vitro assays were carried out using the H9c2 rat cardiomyoblast line infected with highly invasive trypomastigote forms of the Y and Colombian T. cruzi strains, which have been shown to infect at least 50% of exposed H9c2 cells. The drug interactions were assessed using a fixed-ratio method (15). Host cells were dispensed into 24-well tissue culture plates, containing coverslips, at 1 × 10⁴/well. After 24 h, the cells were infected with trypomastigotes obtained from tissue culture at a ratio of 10 parasites per cell (incubation for 24 h). Predetermined 50% effective concentration (EC₅₀) values were used to determine the maximum concentrations of the individual drugs to ensure that the EC₅₀ fell near the midpoint of a six-point 2-fold dilution series. The maximum concentrations used were 20 μM for benznidazole and 24 nM for ravuconazole. These concentrations were used to prepare fixed-ratio solutions at ratios of 5:0, 4:1, 3:2, 2:3, 1:4, and 0:5 of benznidazole to ravuconazole. All tissue culture slides were maintained at 37°C in a 5% CO₂-air mixture. After 72 h, the cultures were fixed with methanol, stained with Giemsa, and microscopically examined to determine the percentage of cells infected in treated and untreated controls.