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PLoS Negl Trop Dis. 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.

Author information

1
National Institute of Pathology (ICMR), Safdarjung Hospital Campus, New Delhi, India.
2
Department of Biotechnology, Faculty of Science, Jamia Hamdard, New Delhi, India.
3
Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.
4
Dermatology Department, Safdarjung Hospital and Vardhman Mahavir Medical College (VMMC), New Delhi, India.
5
Unit of Molecular Parasitology, Department of Parasitology, Institute of Tropical Medicine, Antwerp, Belgium.

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.

PMID:
28575060
PMCID:
PMC5470736
DOI:
10.1371/journal.pntd.0005641
[Indexed for MEDLINE]
Free PMC Article

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