Tetrazolium salt based methods for high-throughput evaluation of anti-parasite chemotherapy

Abstract

This study demonstrates the first standardization of the MTS/PMS tetrazolium-based method in T. cruzi and G. duodenalis, and its application for chemotherapy studies and drug screening. We improved the detection limit and compared the MTT, XTT/PMS and MTS/PMS methods, in T. cruzi epimastigotes, demonstrating that MTS/PMS is more reproducible, sensitive, faster and easier to manipulate than XTT/PMS and MTT. Oligomycin (10 μg ml⁻¹) reduced T. cruzi epimastigote metabolic activity by 60% measured by the MTS/PMS method, which is in accordance with flow cytometry measurements. To validate the MTS/PMS method, an IC₅₀ of 10.5 μM was estimated for the drug amiodarone in T. cruzi and an IC₅₀ of 6.2 μM for metronidazole in G. duodenalis, corroborating the IC₅₀ values found in the literature. In G. duodenalis, a protozoan that has remnant mitochondria, the MTS/PMS method displayed higher sensitivity than T. cruzi epimastigotes. The detection limit was 10⁴ for G. duodenalis trophozoites and 2.5 × 10⁵ for T. cruzi epimastigotes. The intensity of the colored MTS/PMS formazan derivative is proportional to the number of metabolically active protozoa, regardless of whether used in a mitochondrial or an “amitochondrial” organism.