High-throughput screening of amastigotes of Leishmania donovani clinical isolates against drugs using a colorimetric beta-lactamase assay.

Abstract

A simple colorimetric beta-lactamase assay for quantifying Leishmania amastigotes in macrophages grown in microtiter plates has been reported. The beta-lactamase gene was integrated into the rRNA region of the genome, thereby allowing for high-level stable expression of the enzyme. Both visceral leishmaniasis (VL) and post-kala azar dermal leishmaniasis (PKDL) isolates were transfected with beta-Lactamase gene. These beta-lactamase-expressing promastigotes were used for infecting intracellular J774A.1 macrophages in vitro. Quantification was done by a colorimetric readout with CENTA beta-lactamase as substrate and with an optical density plate reader. The assay was carried out in 96-well plates. Results obtained demonstrate that this methodology could be a valuable high-throughput screening assay for checking efficacy of anti-leishmanial drugs in the clinical isolates.