Biomarkers of antimony resistance: need for expression analysis of multiple genes to distinguish resistance phenotype in clinical isolates of Leishmania donovani.

Resistance to antimony is a major cause of failure to therapy in a large proportion of visceral leishmaniasis cases. Methods to distinguish resistant and sensitive parasite are urgently needed as the standard in vitro intracellular drug susceptibility assays are cumbersome and time consuming. Differential expression profiling studies have led to the identification of several antimony resistance-associated genes; however, their efficacy as a potential biomarker for monitoring antimony resistance remains imprecise. We analysed the expression of eight genes [antimony metabolism-associated genes - multidrug resistance protein A (MRPA), γ-glutamylcysteine synthetase (γ-GCS) and aquaporin-1 (AQP1) - and genes identified by proteome/transcriptome profiling—heat shock protein 83, mitogen-activated protein kinase 1 and histones H1, H2A and H4) in antimony-resistant (n=10) and antimony-sensitive (n=4) clinical isolates of Leishmania donovani by quantitative real-time PCR, in comparison with a lab-generated resistant and a standard sensitive isolate. We observed a significant differential expression of MRPA, histone H1 (p<0.01), γ-GCS, HSP83 (p<0.005) and histone H2A and H4 (p<0.0001) in a group of sodium antimony gluconate-resistant isolates compared to sensitive isolates. Preferential AQP1 expression was observed in all the sensitive isolates (p<0.0001). Overall, expression profile in field isolates for all the genes studied showed altered expression in majority of isolates, while in some, the expression was static. All the isolates showed a mosaic of expression pattern of the genes analysed indicating constellation of genes contributes towards the drug susceptibility of parasite. As none of the genes exhibit an absolute correlation with phenotype, targeted expression analysis of a set of genes should be considered as biomarker for distinguishing the antimony-resistant and antimony-sensitive parasite.