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.