Comparative Assessment of colorimetric assays in evaluating intracellular drug susceptibility of Leishmania tropica against conventional antileishmanial drugs.

Abstract

This study aims to identify the most sensitive colorimetric test for assessing intracellular drug susceptibility of Leishmania tropica to conventional antileishmanial drugs. To this end, the efficacy of four colorimetric methods-MTT, XTT, MTS, and WST-8-was compared using reference L. tropica promastigotes. The intracellular drug susceptibility was further evaluated using the test with the widest absorbance range on isolates from Türkiye CL patients: two responsive to a single course of meglumine antimoniate (MA) and two that showed no clinical improvement after two treatments. CL isolates were identified via real-time PCR targeting the ITS1 region. Promastigote suspensions at standardized densities (0.08 × 10⁶ to 10 × 10⁶ promastigotes/well) were prepared in both RPMI (phenol red-containing) and RPMIØRP (phenol red-free) media, then analyzed with ELISA-based MTT, XTT, MTS, and WST-8 to identify the method with the broadest specific absorbance range. Intracellular drug susceptibility of CL isolates was subsequently assessed in a macrophage/amastigote model by infecting THP-1 macrophages with promastigotes from both reference and patient isolates, followed by treatment with MA, sodium stibogluconate (SSG), miltefosine (MTF), pentamidine (PMD), and amphotericin B (AmB). Promastigotes obtained from parasite rescue and transformation assays were analyzed using the most sensitive colorimetric method to determine IC₅₀ values. Species identification confirmed all four CL isolates as L. tropica, and the XTT assay provided the widest absorbance range in RPMIØRP media. IC₅₀ values for both treatment-responsive and unresponsive isolates were similar to those of the reference isolate, showing susceptibility to all tested drugs without statistically significant differences. Expanding the isolate set is necessary to further evaluate the predictive value of Sb^V (pentavalent antimonials) susceptibility for treatment outcomes. The identification of XTT as the most sensitive method for intracellular antileishmanial susceptibility testing is expected to aid in standardizing laboratory models and provide valuable insights for researchers and clinicians managing treatment-unresponsive CL cases.