Assessment of Leishmania promastigote growth in vitro by means of nucleoside hydrolase activity determination

Abstract

Nucleoside hydrolases (NH) are involved in the purine salvage pathway of protozoan cells for the biosynthesis of nucleic acids. We developed a simple and reliable microassay based on N-ribohydrolase dosage using 4-nitrophenyl-β-d-ribofuranoside (NPR) substrate for the quantification of Leishmania infantum. The free promastigote stage of L. infantum contains high amounts of NH capable of cleaving NPR, but the parasitic amastigote does not. The method allows reliable quantification of viable parasites over a wide range of concentrations (5 × 10⁴–2 × 10⁸ parasites ml⁻¹) in a single assay. No difference in NH activity was observed between various strains at equivalent concentrations and growth curves determined with NH dosage were similar to optical counts. Samples can be stored at −20 °C for weeks before use in this unique assay without significant loss of NH activity. The method is particularly simple and versatile and proves well adapted for the determination of growth characteristics and drug screening studies of L. infantum promastigotes in vitro.