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

Nucleoside hydrolases (NH) are involved in the purine salvage pathway of pr otozoan cells for the biosynthesis of nucleic acids. We developed a simple and reliable microassay based on N-ribohydrolase dosage using 4-nitrophenyl -beta -D-ribofuranoside (NPR) substrate for the quantification of Leishmani a infantum. The free promastigote stage of L. infantum contains high amount s of NH capable of cleaving NPR, but the parasitic amastigote does not. The method allows reliable quantification of viable parasites over a wide rang e of concentrations (5 x 10(4-)2 x 10(8) parasites ml(-1)) in a single assa y. No difference in NH activity was observed between various strains at equ ivalent concentrations and growth curves determined with NH dosage were sim ilar to optical counts. Samples can be stored at -20 degreesC for weeks bef ore use in this unique assay without significant loss of NH activity. The m ethod is particularly simple and versatile and proves well adapted for the determination of growth characteristics and drug screening studies of L. in fantum promastigotes in vitro.