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. 2017 Jun 2;11(6):e0005641.
doi: 10.1371/journal.pntd.0005641. eCollection 2017 Jun.

Increased miltefosine tolerance in clinical isolates of Leishmania donovani is associated with reduced drug accumulation, increased infectivity and resistance to oxidative stress

Deepak Kumar Deep  1   2 , Ruchi Singh  1 , Vasundhra Bhandari  1 , Aditya Verma  1 , Vanila Sharma  1 , Saima Wajid  2 , Shyam Sundar  3 , V Ramesh  4 , Jean Claude Dujardin  5 , Poonam Salotra  1
Affiliations

Increased miltefosine tolerance in clinical isolates of Leishmania donovani is associated with reduced drug accumulation, increased infectivity and resistance to oxidative stress

Deepak Kumar Deep et al. PLoS Negl Trop Dis. .

Abstract

Background: Miltefosine (MIL) is an oral antileishmanial drug used for treatment of visceral leishmaniasis (VL) in the Indian subcontinent. Recent reports indicate a significant decline in its efficacy with a high rate of relapse in VL as well as post kala-azar dermal leishmaniasis (PKDL). We investigated the parasitic factors apparently involved in miltefosine unresponsiveness in clinical isolates of Leishmania donovani.

Methodology: L. donovani isolated from patients of VL and PKDL at pretreatment stage (LdPreTx, n = 9), patients that relapsed after MIL treatment (LdRelapse, n = 7) and parasites made experimentally resistant to MIL (LdM30) were included in this study. MIL uptake was estimated using liquid chromatography coupled mass spectrometry. Reactive oxygen species and intracellular thiol content were measured fluorometrically. Q-PCR was used to assess the differential expression of genes associated with MIL resistance.

Results: LdRelapse parasites exhibited higher IC50 both at promastigote level (7.92 ± 1.30 μM) and at intracellular amastigote level (11.35 ± 6.48 μM) when compared with LdPreTx parasites (3.27 ± 1.52 μM) and (3.85 ± 3.11 μM), respectively. The percent infectivity (72 hrs post infection) of LdRelapse parasites was significantly higher (80.71 ± 5.67%, P<0.001) in comparison to LdPreTx (60.44 ± 2.80%). MIL accumulation was significantly lower in LdRelapse parasites (1.7 fold, P<0.001) and in LdM30 parasites (2.4 fold, P<0.001) when compared with LdPreTx parasites. MIL induced ROS levels were significantly lower (p<0.05) in macrophages infected with LdRelapse while intracellular thiol content were significantly higher in LdRelapse compared to LdPreTx, indicating a better tolerance for oxidative stress in LdRelapse isolates. Genes associated with oxidative stress, metabolic processes and transporters showed modulated expression in LdRelapse and LdM30 parasites in comparison with LdPreTx parasites.

Conclusion: The present study highlights the parasitic factors and pathways responsible for miltefosine unresponsiveness in VL and PKDL.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. In vitro MIL susceptibility, percent infectivity and MIL uptake in L. donovani parasites.
(A) MIL susceptibility at promastigotes stage of LdPreTx, LdRelapse and LdM30 with each individual value represented as mean IC50±SD from three separate assays. (B) MIL susceptibility at intracellular amastigote stage LdPreTx, LdRelapse and LdM30 with each individual value represented as mean IC50 ± SD from three separate assays (C) Mice peritoneal derived macrophages infected with LdPreTx or LdRelapse parasites at a 1:10 (cell/parasite) ratio. The percent infectivity was determined at 24h, 48h, and 72h post infection by counting number of infected cells out of 100 macrophages at 1000X magnification after staining with Diff-Quik. Data represents mean ± SD of three independent experiments each in duplicate. (D) Percent metacyclogenesis of promastigote population estimated based on negative selection of peanut agglutinin (%PNA- promastigote). Values represent mean ± SD of two independent experiments (E) MIL uptake, estimated using LC-MS in 1x108 promastigotes of LdPreTx, LdRelpase and LdM30. Data represents mean ± SD of two independent experiments, each in duplicate. Asterisks indicate significance (**P<0.01; and ***P<0.001).
Fig 2
Fig 2. MIL induced oxidative stress (ROS level) and intracellular thiol content in L. donovani.
(A) Dose dependent accumulation of ROS in LdPreTx, LdRelapse and LdM30 at promastigote stage was assayed fluorometrically at 495 nm excitation and 535 nm emission wavelength using cell permeable probe H2DCF-DA (40nM). Data represents mean ± SD of three independent experiments, each performed in triplicate. Asterisks indicate significance (**P<0.01; and ***P<0.001). (B) Accumulation of ROS in macrophages infected with LdPreTx, LdRelapse, LdM30 parasites before and after MIL exposure (20μM), assayed fluorometrically at 495 nm excitation and 535 nm emission wavelength using cell permeable probe H2DCF-DA (30μM). Data represents mean ± SD of three independent experiments, each in triplicate. Asterisks indicate significance (*P<0.05; **P<0.01; and ***P<0.001). (C) Intracellular thiol content in LdPreTx, LdRelapse and LdM30 promastigotes, measured fluorometrically at 390 nm excitation and 520 nm emission wavelength. Data represents mean ± SD of two independent experiments each performed in triplicate. Asterisks indicate significance (**P<0.01; and ***P<0.001).
Fig 3
Fig 3. Expression analysis of selected genes by real time PCR in clinical isolates of L. donovani at pretreatment (LdPreTx), relapse (LdRelapse), LdM30 parasites.
Fold change represents expression of target genes normalized to internal control (GAPDH and α-Tubulin) genes and relative to LdAG83. Data represents mean ± SD of two separate assays, each performed in triplicate. Asterisks indicate significance (*P<0.05; **P<0.01; and *** P<0.001).
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Grants and funding

This work was supported by EC-FP7-222895, https://ec.europa.eu/research/fp7, Indian Council of Medical Research, www.icmr.nic.in DKD is grateful to Council for Scientific and Industrial Research, India for providing research fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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