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. 2012;6(5):e1657.
doi: 10.1371/journal.pntd.0001657. Epub 2012 May 22.

Drug susceptibility in Leishmania isolates following miltefosine treatment in cases of visceral leishmaniasis and post kala-azar dermal leishmaniasis

Vasundhra Bhandari  1 , Arpita Kulshrestha, Deepak Kumar Deep, Olivia Stark, Vijay Kumar Prajapati, V Ramesh, Shyam Sundar, Gabriele Schonian, Jean Claude Dujardin, Poonam Salotra
Affiliations

Drug susceptibility in Leishmania isolates following miltefosine treatment in cases of visceral leishmaniasis and post kala-azar dermal leishmaniasis

Vasundhra Bhandari et al. PLoS Negl Trop Dis. 2012.

Abstract

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 IC(50)±SD = 2.43±1.44 µM), was comparable (p>0.05) whereas that from relapses (n = 3, mean IC(50) = 4.72±1.99 µM) was significantly higher (p = 0.04) to that of the pre-treatment group (n = 6, mean IC(50) = 1.86±0.75 µM). In PKDL, post-treatment isolates (n = 3, mean IC(50) = 16.13±2.64 µM) exhibited significantly lower susceptibility (p = 0.03) than pre-treatment isolates (n = 5, mean IC(50) = 8.63±0.94 µM). Overall, PKDL isolates (n = 8, mean IC(50) = 11.45±4.19 µM) exhibited significantly higher tolerance (p<0.0001) to MIL than VL isolates (n = 22, mean IC(50) = 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 IC(50) = 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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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. In vitro miltefosine susceptibility of parasite isolates from VL and PKDL cases before and after Mil treatment.
Sensitivity of VL and PKDL isolates at intracellular amastigote stage were determined by infection in murine macrophage cell line J774A.1. Each individual value represents mean IC50±SD of the results from two separate assays.
Figure 2
Figure 2. PMM susceptibility profile of VL and PKDL isolates exposed or non-exposed to MIL treatment.
Susceptibility of VL and PKDL isolates at intracellular amastigote stage was determined by infection in murine macrophage cell line J774A.1. Each individual value represents mean IC50±SD of the results from two separate assays.
Figure 3
Figure 3. Expression of LdMT and LdRos3 in clinical isolates of VL and PKDL.
Real-time reverse-transcription PCR expression analysis of L. donovani MIL transporter genes (LdMT and LdRos3) was performed using GAPDH as internal control. Graph shows the expression index, defined as ratio of gene expression relative to that of strain LdAG83. Data represent the mean±SD of the results of three independent experiments.
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