Indian Leishmania donovani isolates (n ‫؍‬ 19) from regional zones representing various levels of antimony resistance displayed significantly (P < 0.01) correlated results with respect to in vitro susceptibility to the antileishmanial drugs sodium antimony gluconate, amphotericin B, and Miltefosine, raising the possibility of cross-resistance mechanisms operating in the field isolates. The results of gene expression analysis of LdMT and LdRos3 were suggestive of alternate mechanisms of Miltefosine susceptibility in the isolates.A high (Ͼ60%) proportion of non-antimony-responsive cases of Kala azar in India and the anthroponotic mode of transmission of the parasite causing the disease increase the chances of the generation and spreading of drug-resistant parasites (15, 17). The second-line antileishmanials amphotericin B (AmB) and Miltefosine (MIL) are highly effective for treatment of antimony-resistant patients but are of limited utility because of adverse reactions and high cost. A recent report of unresponsiveness to Ambisome in Sudanese patients of VL is worrisome and indicates the emergence of AmB-resistant parasites (9). Preliminary data from a phase IV trial with MIL suggested a doubling of the relapse rate, indicating lower drug efficacy than in phase II and III trials and providing a warning about the emergence of resistance (3,18,19).Earlier studies using isolates from responsive and nonresponsive patients indicated that resistance to antimonials is an intrinsic property of the parasite (4,8,15,16). Antimony resistance varies among zones representing differing levels of endemicity, emphasizing the acquired nature of resistance in the region (15). Sodium antimony gluconate (SAG)-resistant isolates exhibited cross-resistance to AmB and MIL, with HSP83 and a calpain-related protein being implicated in resistance by modulating drug-induced programmed cell death (21). Since the use of MIL for VL treatment has been introduced only recently, resistance has not yet been reported in the field; however, a wide range of 50% effective doses (ED 50 ) of MIL has been observed for parasite isolates from Nepal and Peru (23). The results of earlier studies revealed a role in MIL uptake and susceptibility for the LdMT-LdRos3-dependent flippase machinery at the plasma membrane (10-12). The present study was aimed at (i) evaluating the in vitro natural susceptibility of field isolates of Leishmania donovani to SAG, AmB, and MIL and (ii) correlating MIL susceptibility with the mRNA expression of LdMT and LdRos3 to explore their role in MIL resistance and potential as markers of MIL resistance in field isolates.The present study considered 19 L. donovani isolates from VL patients representing regional zones with various degrees of disease endemicity. In vitro susceptibility of parasites from SAG-treated patients (responsive and nonresponsive) and AmB-treated patients (all responded to treatment, and no clinical resistance was observed) was studied. Informed consent based on the guidelines of the Ethical Committee, Safdarjun...

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.