The epimastigote form of the parasite is cultivated in liver infusion tryptose medium,12 supplemented with 10% new born calf serum at 28°C with strong (120 rpm) agitation. Cultures are initiated at a cell density of 2×10⁶ epimastigotes mL-1, and drugs are added at a cell density of 0.5?1.0 ×10⁷ epimastigotes mL-1. Cell densities are measured by using an electronic particle counter as well as by direct counting with a hemocytometer. Cell viability is followed by Trypan blue exclusion, using light microscopy. Amastigotes are cultured in Vero cells maintained in minimal essential medium supplemented with 1% fetal calf serum in a humidified atmosphere (95% air?5% CO₂) at 37°C, as described previously. Cells are infected with 10 tissue culture-derived trypomastigotes per cell for 2 h and then washed three times with phosphate-buffered saline (PBS) to remove nonadherent parasites. Fresh medium with and without drugs is added, and the cells are incubated for 96 h with a medium change at 48 h. The percent of infected cells and the numbers of parasites per cell are determined directly using light microscopy, and a statistical analysis of the results is carried out as described previously. IC₅₀ values are calculated by nonlinear regression, using the program GraFit. Cytoplasmic free Ca²⁺ concentrations in control and drug-treated extracellular epimastigotes are determined by fluorimetric methods, using Fura-2, again as described previously. Subcellular Ca²⁺ levels and mitochondrial membrane potentials are monitored on individual Vero cells infected with T. cruzi amastigotes by using time-scan confocal microscopy, as described in detail elsewhere. Briefly, Vero cells heavily infected (72 h) with T. cruzi amastigotes are plated onto 22×40 mm glass coverslips (0.15 mm thickness) and incubated simultaneously with 10 μM cell-permeant Rhod-2 and 10 μg/mL Rhodamine-123 for 50 min at 37°C in culture medium and then washed and incubated with Ringer's solution, with or without amiodarone. Under the conditions used, fluorescence of Rhod-2 comes mainly from intracellular Ca²⁺-rich compartments, like mitochondria, since its low affinity for Ca²⁺ limits its fluorescence in the Ca²⁺-poor cytoplasm of the Vero cells or amastigotes. Rhodamine-123 is a mitochondrion-specific cationic dye, which distributes across the inner mitochondrial membranes strictly according to their membrane potential.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

In vivo studies are carried out by using the murine model of acute Chagas' disease in which female NMRI?IVIC mice (20?25 g) are infected with 10⁵ or 10³ bloodstream trypomastigotes and drug treatment is started 24 h later. Treatments are given for 30 consecutive days at 20 mg/kg/d for posaconazole (30 doses) and/or at 50 mg/kg every other day for amiodarone (15 doses). Negative controls (i.e. untreated animals) receive only the vehicle, while positive controls are treated with the anti-T. cruzi compound, nifurtimox, at 50 mg/kg/d for 30 days. Survival is followed daily and parasitemia weekly, the latter by direct microscopic examination. Animals are observed for 60 days postinfection, after which time parasitological cures are evaluated by using a combination of hemoculture, xenodiagnosis, and blood PCR tests.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

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