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Increased miltefosine tolerance in clinical isolates of Leishmania donovani is associated with reduced drug accumulation, increased infectivity and resistance to oxidative stress
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 invest...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5470736/ https://www.ncbi.nlm.nih.gov/pubmed/28575060 http://dx.doi.org/10.1371/journal.pntd.0005641 |
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author | Deep, Deepak Kumar Singh, Ruchi Bhandari, Vasundhra Verma, Aditya Sharma, Vanila Wajid, Saima Sundar, Shyam Ramesh, V. Dujardin, Jean Claude Salotra, Poonam |
author_facet | Deep, Deepak Kumar Singh, Ruchi Bhandari, Vasundhra Verma, Aditya Sharma, Vanila Wajid, Saima Sundar, Shyam Ramesh, V. Dujardin, Jean Claude Salotra, Poonam |
author_sort | Deep, Deepak Kumar |
collection | PubMed |
description | 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 IC(50) 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. |
format | Online Article Text |
id | pubmed-5470736 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-54707362017-06-26 Increased miltefosine tolerance in clinical isolates of Leishmania donovani is associated with reduced drug accumulation, increased infectivity and resistance to oxidative stress Deep, Deepak Kumar Singh, Ruchi Bhandari, Vasundhra Verma, Aditya Sharma, Vanila Wajid, Saima Sundar, Shyam Ramesh, V. Dujardin, Jean Claude Salotra, Poonam PLoS Negl Trop Dis Research Article 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 IC(50) 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. Public Library of Science 2017-06-02 /pmc/articles/PMC5470736/ /pubmed/28575060 http://dx.doi.org/10.1371/journal.pntd.0005641 Text en © 2017 Deep et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Deep, Deepak Kumar Singh, Ruchi Bhandari, Vasundhra Verma, Aditya Sharma, Vanila Wajid, Saima Sundar, Shyam Ramesh, V. Dujardin, Jean Claude Salotra, Poonam Increased miltefosine tolerance in clinical isolates of Leishmania donovani is associated with reduced drug accumulation, increased infectivity and resistance to oxidative stress |
title | Increased miltefosine tolerance in clinical isolates of Leishmania donovani is associated with reduced drug accumulation, increased infectivity and resistance to oxidative stress |
title_full | Increased miltefosine tolerance in clinical isolates of Leishmania donovani is associated with reduced drug accumulation, increased infectivity and resistance to oxidative stress |
title_fullStr | Increased miltefosine tolerance in clinical isolates of Leishmania donovani is associated with reduced drug accumulation, increased infectivity and resistance to oxidative stress |
title_full_unstemmed | Increased miltefosine tolerance in clinical isolates of Leishmania donovani is associated with reduced drug accumulation, increased infectivity and resistance to oxidative stress |
title_short | Increased miltefosine tolerance in clinical isolates of Leishmania donovani is associated with reduced drug accumulation, increased infectivity and resistance to oxidative stress |
title_sort | increased miltefosine tolerance in clinical isolates of leishmania donovani is associated with reduced drug accumulation, increased infectivity and resistance to oxidative stress |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5470736/ https://www.ncbi.nlm.nih.gov/pubmed/28575060 http://dx.doi.org/10.1371/journal.pntd.0005641 |
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