Functionality of drug efflux pumps in antimonial resistant Leishmania donovani field isolates.

The recent upsurge of antimony (Sb) resistance is a major impediment to successful chemotherapy of visceral leishmaniasis (VL). Mechanisms involved in antimony resistance have demonstrated an upregulation of drug efflux pumps; however, the biological role drug efflux pumps in clinical isolates remains to be substantiated. Thus, in this study, the functionality of drug efflux pumps was measured in promastigotes and axenic amastigotes isolated from VL patients, who were either Sb-sensitive (AG83, 2001 and MC9) or resistant (NS2, 41 and GE1) using rhodamine123 as a substrate for multidrug resistant (MDR) pumps and calcein as a substrate for multidrug resistance-associated proteins (MRP) respectively; their specificity was confirmed using established blockers. Sb-resistant (Sb-R) isolates accumulated higher amounts of R123, as compared to Sb-sensitive (Sb-S) isolates. Verapamil, a MDR inhibitor failed to alter R123 accumulation, suggesting absence of classical MDR activity. In Sb-R isolates, both promastigotes and axenic amastigotes accumulated significantly lower amounts of calcein than Sb-S isolates and probenecid, an established pan MRP blocker, marginally increased calcein accumulation. Depletion of ATP dramatically increased calcein accumulation primarily in Sb-R isolates, indicating existence of a MRP-like pump, which was more active in Sb-R isolates. In conclusion, our data suggested that overfunctioning of a MRP-like pump contributed towards generation of Sb-R phenotype in L. donovani field isolates.

Drug resistance mechanisms in clinical isolates of Leishmania donovani.

Leishmania are protozoan parasites distributed worldwide. About 1.5-2.0 million cases are reported in the world annually from this disease and the death toll is estimated to be 57,000. Along with Brazil, Sudan and Bangladesh, India contributes to 90 per cent of the global burden of visceral leishmaniasis (VL). The absence of effective vaccines and vector control programmes, makes chemotherapy the most widely used tool against leishmaniasis. Chemotherapy based on pentavalent antimonials has been used for more than 50 years and remains the mainstay for treatment of leishmaniasis. Clinical resistance to pentavalent antimonials, in the form of sodium antimony gluconate (SAG), has become a major problem in the treatment of kala-azar (visceral leishmaniasis) in India. The mechanism of resistance is unclear in these clinical isolates although a lot of work has been carried out with Leishmania mutants selected in vitro by step-wise increasing drug concentration using the antimony related metal arsenic and more recently sodium antimony gluconate. We for the first time, investigated the molecular aspect of drug resistance in clinically confirmed sodium antimony gluconate resistant field isolates and found that the parasite evaded cytotoxic effects of therapy by enhanced efflux of drugs through overexpressed membrane proteins belonging to the superfamily of ABC (ATP-binding cassette) transporters. Additionally, our study also points towards cell surface changes in resistant isolates.