Molecular assays for determining Mycobacterium leprae viability in tissues of experimentally infected mice.
BACKGROUND: The inability of Mycobacterium leprae to grow on axenic media has necessitated specialized techniques in order to determine viability of this organism. The purpose of this study was to develop a simple and sensitive molecular assay for determining M. leprae viability directly from infect...
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ftdoajarticles:oai:doaj.org/article:dec6ab604c7140589703845b08d5f51c 2023-05-15T15:16:36+02:00 Molecular assays for determining Mycobacterium leprae viability in tissues of experimentally infected mice. Grace L Davis Nashone A Ray Ramanuj Lahiri Thomas P Gillis James L Krahenbuhl Diana L Williams Linda B Adams 2013-01-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0002404 https://doaj.org/article/dec6ab604c7140589703845b08d5f51c EN eng Public Library of Science (PLoS) http://europepmc.org/articles/PMC3750008?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0002404 https://doaj.org/article/dec6ab604c7140589703845b08d5f51c PLoS Neglected Tropical Diseases, Vol 7, Iss 8, p e2404 (2013) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2013 ftdoajarticles https://doi.org/10.1371/journal.pntd.0002404 2022-12-31T15:55:26Z BACKGROUND: The inability of Mycobacterium leprae to grow on axenic media has necessitated specialized techniques in order to determine viability of this organism. The purpose of this study was to develop a simple and sensitive molecular assay for determining M. leprae viability directly from infected tissues. METHODOLOGY/PRINCIPLE FINDINGS: Two M. leprae-specific quantitative reverse transcription PCR (qRT-PCR) assays based on the expression levels of esxA, encoding the ESAT-6 protein, and hsp18, encoding the heat shock 18 kDa protein, were developed and tested using infected footpad (FP) tissues of both immunocompetent and immunocompromised (athymic nu/nu) mice. In addition, the ability of these assays to detect the effects of anti-leprosy drug treatment on M. leprae viability was determined using rifampin and rifapentine, each at 10 mg/kg for 1, 5, or 20 daily doses, in the athymic nu/nu FP model. Molecular enumeration (RLEP PCR) and viability determinations (qRT-PCR) were performed via Taqman methodology on DNA and RNA, respectively, purified from ethanol-fixed FP tissue and compared with conventional enumeration (microscopic counting of acid fast bacilli) and viability assays (radiorespirometry, viability staining) which utilized bacilli freshly harvested from the contralateral FP. Both molecular and conventional assays demonstrated growth and high viability of M. leprae in nu/nu FPs over a 4 month infection period. In contrast, viability was markedly decreased by 8 weeks in immunocompetent mice. Rifapentine significantly reduced bacterial viability after 5 treatments, whereas rifampin required up to 20 treatments for the same efficacy. Neither drug was effective after a single treatment. In addition, host gene expression was monitored with the same RNA preparations. CONCLUSIONS: hsp18 and esxA qRT-PCR are sensitive molecular indicators, reliably detecting viability of M. leprae in tissues without the need for bacterial isolation or immediate processing, making these assays applicable for in vivo drug ... Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLoS Neglected Tropical Diseases 7 8 e2404 |
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Directory of Open Access Journals: DOAJ Articles |
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English |
topic |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Grace L Davis Nashone A Ray Ramanuj Lahiri Thomas P Gillis James L Krahenbuhl Diana L Williams Linda B Adams Molecular assays for determining Mycobacterium leprae viability in tissues of experimentally infected mice. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
BACKGROUND: The inability of Mycobacterium leprae to grow on axenic media has necessitated specialized techniques in order to determine viability of this organism. The purpose of this study was to develop a simple and sensitive molecular assay for determining M. leprae viability directly from infected tissues. METHODOLOGY/PRINCIPLE FINDINGS: Two M. leprae-specific quantitative reverse transcription PCR (qRT-PCR) assays based on the expression levels of esxA, encoding the ESAT-6 protein, and hsp18, encoding the heat shock 18 kDa protein, were developed and tested using infected footpad (FP) tissues of both immunocompetent and immunocompromised (athymic nu/nu) mice. In addition, the ability of these assays to detect the effects of anti-leprosy drug treatment on M. leprae viability was determined using rifampin and rifapentine, each at 10 mg/kg for 1, 5, or 20 daily doses, in the athymic nu/nu FP model. Molecular enumeration (RLEP PCR) and viability determinations (qRT-PCR) were performed via Taqman methodology on DNA and RNA, respectively, purified from ethanol-fixed FP tissue and compared with conventional enumeration (microscopic counting of acid fast bacilli) and viability assays (radiorespirometry, viability staining) which utilized bacilli freshly harvested from the contralateral FP. Both molecular and conventional assays demonstrated growth and high viability of M. leprae in nu/nu FPs over a 4 month infection period. In contrast, viability was markedly decreased by 8 weeks in immunocompetent mice. Rifapentine significantly reduced bacterial viability after 5 treatments, whereas rifampin required up to 20 treatments for the same efficacy. Neither drug was effective after a single treatment. In addition, host gene expression was monitored with the same RNA preparations. CONCLUSIONS: hsp18 and esxA qRT-PCR are sensitive molecular indicators, reliably detecting viability of M. leprae in tissues without the need for bacterial isolation or immediate processing, making these assays applicable for in vivo drug ... |
format |
Article in Journal/Newspaper |
author |
Grace L Davis Nashone A Ray Ramanuj Lahiri Thomas P Gillis James L Krahenbuhl Diana L Williams Linda B Adams |
author_facet |
Grace L Davis Nashone A Ray Ramanuj Lahiri Thomas P Gillis James L Krahenbuhl Diana L Williams Linda B Adams |
author_sort |
Grace L Davis |
title |
Molecular assays for determining Mycobacterium leprae viability in tissues of experimentally infected mice. |
title_short |
Molecular assays for determining Mycobacterium leprae viability in tissues of experimentally infected mice. |
title_full |
Molecular assays for determining Mycobacterium leprae viability in tissues of experimentally infected mice. |
title_fullStr |
Molecular assays for determining Mycobacterium leprae viability in tissues of experimentally infected mice. |
title_full_unstemmed |
Molecular assays for determining Mycobacterium leprae viability in tissues of experimentally infected mice. |
title_sort |
molecular assays for determining mycobacterium leprae viability in tissues of experimentally infected mice. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2013 |
url |
https://doi.org/10.1371/journal.pntd.0002404 https://doaj.org/article/dec6ab604c7140589703845b08d5f51c |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 7, Iss 8, p e2404 (2013) |
op_relation |
http://europepmc.org/articles/PMC3750008?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0002404 https://doaj.org/article/dec6ab604c7140589703845b08d5f51c |
op_doi |
https://doi.org/10.1371/journal.pntd.0002404 |
container_title |
PLoS Neglected Tropical Diseases |
container_volume |
7 |
container_issue |
8 |
container_start_page |
e2404 |
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1766346898887671808 |