Background With widespread resistance to antimonials in Visceral Leishmaniasis (VL) in the Indian subcontinent, Miltefosine (MIL) has been introduced as the first line therapy. Surveillance of MIL susceptibility in natural populations of Leishmania donovani is vital to preserve it and support the VL elimination program. Methodology and Principal Findings We measured in vitro susceptibility towards MIL and paromomycin (PMM) in L. donovani isolated from VL and PKDL, pre- and post-treatment cases, using an amastigote-macrophage model. MIL susceptibility of post-treatment isolates from cured VL cases (n = 13, mean IC50±SD = 2.43±1.44 μM), was comparable (p>0.05) whereas that from relapses (n = 3, mean IC50 = 4.72±1.99 μM) was significantly higher (p = 0.04) to that of the pre-treatment group (n = 6, mean IC50 = 1.86±0.75 μM). In PKDL, post-treatment isolates (n = 3, mean IC50 = 16.13±2.64 μM) exhibited significantly lower susceptibility (p = 0.03) than pre-treatment isolates (n = 5, mean IC50 = 8.63±0.94 μM). Overall, PKDL isolates (n = 8, mean IC50 = 11.45±4.19 μM) exhibited significantly higher tolerance (p<0.0001) to MIL than VL isolates (n = 22, mean IC50 = 2.58±1.58 μM). Point mutations in the miltefosine transporter (LdMT) and its beta subunit (LdRos3) genes previously reported in parasites with experimentally induced MIL resistance were not present in the clinical isolates. Further, the mRNA expression profile of these genes was comparable in the pre- and post-treatment isolates. Parasite isolates from VL and PKDL cases were uniformly susceptible to PMM with respective mean IC50 = 7.05±2.24 μM and 6.18±1.51 μM. Conclusion The in vitro susceptibility of VL isolates remained unchanged at the end of MIL treatment; however, isolates from relapsed VL and PKDL cases had lower susceptibility than the pre-treatment isolates. PKDL isolates were more tolerant towards MIL in comparison with VL isolates. All parasite isolates were uniformly susceptible to PMM. Mutations in the LdMT and LdRos3 genes as well as changes in the expression of these genes previously correlated with experimental resistance to MIL could not be verified for the field isolates.
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