Laboratory confirmed miltefosine resistant cases of visceral leishmaniasis from India

Abstract

Background: Miltefosine unresponsive and relapse cases of visceral leishmaniasis (VL) are increasingly being reported. However, there has been no laboratory confirmed reports of miltefosine resistance in VL. Here, we report two laboratory confirmed cases of VL from India.

Methods: Two patients with VL were referred to us with suspected VL. The first patient was a native of the VL endemic state of Bihar, but residing in Delhi, a VL non-endemic area. He was treated with broad-spectrum antibiotics and antipyretics but was unresponsive to treatment. The second patient was from Jharkhand state in eastern India (adjoining Bihar), another endemic state for VL. He was refractory to anti-leishmanial treatment, which included administration of miltefosine. Following investigation, both patients were serologically positive for VL, and blood buffy coat from both patients grew Leishmania donovani. The isolates derived from both cases were characterized for their drug susceptibility, genetically characterised, and SNPs typed for LdMT and LdROS gene expression. Both patients were successfully treated with amphotericin B.

Results: The in vitro drug susceptibility assays carried out on both isolates showed good IC₅₀ values to amphotericin B (0.1 ± 0.0004 μg/ml and 0.07 ± 0.0019 μg/ml). One isolate was refractory to Sb^III with an IC₅₀ of > 200 μM while the second isolate was sensitive to Sb^III with an IC₅₀ of 36.70 ± 3.2 μM. However, in both the isolates, IC₅₀ against miltefosine was more than 10-fold higher (> 100 μM) than the standard strain DD8 (6.8 ± 0.1181 μM). Furthermore, genetic analyses demonstrated single nucleotide polymorphisms (SNPs) (₃₅₄Tyr↔Phe and ₁₀₇₈Phe↔Tyr) in the LdMT gene of the parasites.

Conclusions: Here, we document two laboratory confirmed cases of miltefosine resistant VL from India. Our finding highlights the urgent need to establish control measures to prevent the spread of these strains. We also propose that LdMT gene mutation analysis could be used as a molecular marker of miltefosine resistance in L. donovani.

Keywords: Bihar; Drug resistance; Jharkhand; Miltefosine; Visceral leishmaniasis.

Fig. 1

Map of India with highlighted VL endemic states of Bihar and Jharkhand (red). The map also shows the places from where two cases of miltefosine resistant VL are reported in this study

Fig. 2

PCR analysis of buffy coat sample from serologically positive VL patients. Specific PCR product for ITS region of Leishmania spp. (~1.1 kb product; arrow). Lanes 1, 2: PCR amplified products of DNA extracted from the buffy coat of LD843 and LD860 patients respectively; Lane 3: positive control (DNA extracted from DD8 strain of L. donovani promastigotes); Lane 4: positive control (DNA extracted from the buffy coat of known positive patient); Lane 5: negative control (DNA from the buffy coat of healthy individual); Lane 6: 1 kb molecular weight DNA ladder

Fig. 3

Representative plots of susceptibility profile (IC₅₀) of parasite isolates from VL cases and standard strain DD8 to (a) Miltefosine, b SAG (Sb^III) and (c) Amphotericin B. Each individual graph represents the mean from three separate assays

Fig. 4

The in vitro miltefosine resistance demonstrated in clinical isolates using J774-A1 cell line. a Uninfected J774-A1 macrophages. b Macrophages cells infected with DD8 strain but untreated with miltefosine showing amastigotes within macrophages. c Miltefosine-treated DD8 infected macrophages showing clearance of amastigotes. d Macrophage cells infected with the clinical isolate but untreated with miltefosine and showing amastigotes within macrophages. e Macrophages infected with clinical isolate and treated with miltefosine showing no effect on the clearance of amastigotes. Giemsa stained macrophages cells were photographed at 1000× magnification using a light microscope

Fig. 5

Representative plot of anti-amastigote assay of parasites isolated from kala-azar cases and standard strain DD8. The graph represents the % inhibition and mean IC₅₀ of the results from three separate assays

Fig. 6

SNPs analysis of LdMT gene after genome sequencing of clinical isolates are indicated above the gene by star: red colour indicates non-synonymous mutations and green colour indicates synonymous mutations