qPCR-High resolution melt analysis for drug susceptibility testing of Mycobacterium leprae directly from clinical specimens of leprosy patients.
BACKGROUND:Real-Time PCR-High Resolution Melting (qPCR-HRM) analysis has been recently described for rapid drug susceptibility testing (DST) of Mycobacterium leprae. The purpose of the current study was to further evaluate the validity, reliability, and accuracy of this assay for M. leprae DST in cl...
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ftdoajarticles:oai:doaj.org/article:3e0ee1058b354bf29ea7faa8506a93ac 2023-05-15T15:15:26+02:00 qPCR-High resolution melt analysis for drug susceptibility testing of Mycobacterium leprae directly from clinical specimens of leprosy patients. Sergio Araujo Luiz Ricardo Goulart Richard W Truman Isabela Maria B Goulart Varalakshmi Vissa Wei Li Masanori Matsuoka Philip Suffys Amanda B Fontes Patricia S Rosa David M Scollard Diana L Williams 2017-06-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0005506 https://doaj.org/article/3e0ee1058b354bf29ea7faa8506a93ac EN eng Public Library of Science (PLoS) http://europepmc.org/articles/PMC5453413?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0005506 https://doaj.org/article/3e0ee1058b354bf29ea7faa8506a93ac PLoS Neglected Tropical Diseases, Vol 11, Iss 6, p e0005506 (2017) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2017 ftdoajarticles https://doi.org/10.1371/journal.pntd.0005506 2022-12-31T11:43:00Z BACKGROUND:Real-Time PCR-High Resolution Melting (qPCR-HRM) analysis has been recently described for rapid drug susceptibility testing (DST) of Mycobacterium leprae. The purpose of the current study was to further evaluate the validity, reliability, and accuracy of this assay for M. leprae DST in clinical specimens. METHODOLOGY/PRINCIPAL FINDINGS:The specificity and sensitivity for determining the presence and susceptibility of M. leprae to dapsone based on the folP1 drug resistance determining region (DRDR), rifampin (rpoB DRDR) and ofloxacin (gyrA DRDR) was evaluated using 211 clinical specimens from leprosy patients, including 156 multibacillary (MB) and 55 paucibacillary (PB) cases. When comparing the results of qPCR-HRM DST and PCR/direct DNA sequencing, 100% concordance was obtained. The effects of in-house phenol/chloroform extraction versus column-based DNA purification protocols, and that of storage and fixation protocols of specimens for qPCR-HRM DST, were also evaluated. qPCR-HRM results for all DRDR gene assays (folP1, rpoB, and gyrA) were obtained from both MB (154/156; 98.7%) and PB (35/55; 63.3%) patients. All PCR negative specimens were from patients with low numbers of bacilli enumerated by an M. leprae-specific qPCR. We observed that frozen and formalin-fixed paraffin embedded (FFPE) tissues or archival Fite's stained slides were suitable for HRM analysis. Among 20 mycobacterial and other skin bacterial species tested, only M. lepromatosis, highly related to M. leprae, generated amplicons in the qPCR-HRM DST assay for folP1 and rpoB DRDR targets. Both DNA purification protocols tested were efficient in recovering DNA suitable for HRM analysis. However, 3% of clinical specimens purified using the phenol/chloroform DNA purification protocol gave false drug resistant data. DNA obtained from freshly frozen (n = 172), formalin-fixed paraffin embedded (FFPE) tissues (n = 36) or archival Fite's stained slides (n = 3) were suitable for qPCR-HRM DST analysis. The HRM-based assay was also able to ... Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLOS Neglected Tropical Diseases 11 6 e0005506 |
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Open Polar |
collection |
Directory of Open Access Journals: DOAJ Articles |
op_collection_id |
ftdoajarticles |
language |
English |
topic |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
spellingShingle |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Sergio Araujo Luiz Ricardo Goulart Richard W Truman Isabela Maria B Goulart Varalakshmi Vissa Wei Li Masanori Matsuoka Philip Suffys Amanda B Fontes Patricia S Rosa David M Scollard Diana L Williams qPCR-High resolution melt analysis for drug susceptibility testing of Mycobacterium leprae directly from clinical specimens of leprosy patients. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
BACKGROUND:Real-Time PCR-High Resolution Melting (qPCR-HRM) analysis has been recently described for rapid drug susceptibility testing (DST) of Mycobacterium leprae. The purpose of the current study was to further evaluate the validity, reliability, and accuracy of this assay for M. leprae DST in clinical specimens. METHODOLOGY/PRINCIPAL FINDINGS:The specificity and sensitivity for determining the presence and susceptibility of M. leprae to dapsone based on the folP1 drug resistance determining region (DRDR), rifampin (rpoB DRDR) and ofloxacin (gyrA DRDR) was evaluated using 211 clinical specimens from leprosy patients, including 156 multibacillary (MB) and 55 paucibacillary (PB) cases. When comparing the results of qPCR-HRM DST and PCR/direct DNA sequencing, 100% concordance was obtained. The effects of in-house phenol/chloroform extraction versus column-based DNA purification protocols, and that of storage and fixation protocols of specimens for qPCR-HRM DST, were also evaluated. qPCR-HRM results for all DRDR gene assays (folP1, rpoB, and gyrA) were obtained from both MB (154/156; 98.7%) and PB (35/55; 63.3%) patients. All PCR negative specimens were from patients with low numbers of bacilli enumerated by an M. leprae-specific qPCR. We observed that frozen and formalin-fixed paraffin embedded (FFPE) tissues or archival Fite's stained slides were suitable for HRM analysis. Among 20 mycobacterial and other skin bacterial species tested, only M. lepromatosis, highly related to M. leprae, generated amplicons in the qPCR-HRM DST assay for folP1 and rpoB DRDR targets. Both DNA purification protocols tested were efficient in recovering DNA suitable for HRM analysis. However, 3% of clinical specimens purified using the phenol/chloroform DNA purification protocol gave false drug resistant data. DNA obtained from freshly frozen (n = 172), formalin-fixed paraffin embedded (FFPE) tissues (n = 36) or archival Fite's stained slides (n = 3) were suitable for qPCR-HRM DST analysis. The HRM-based assay was also able to ... |
format |
Article in Journal/Newspaper |
author |
Sergio Araujo Luiz Ricardo Goulart Richard W Truman Isabela Maria B Goulart Varalakshmi Vissa Wei Li Masanori Matsuoka Philip Suffys Amanda B Fontes Patricia S Rosa David M Scollard Diana L Williams |
author_facet |
Sergio Araujo Luiz Ricardo Goulart Richard W Truman Isabela Maria B Goulart Varalakshmi Vissa Wei Li Masanori Matsuoka Philip Suffys Amanda B Fontes Patricia S Rosa David M Scollard Diana L Williams |
author_sort |
Sergio Araujo |
title |
qPCR-High resolution melt analysis for drug susceptibility testing of Mycobacterium leprae directly from clinical specimens of leprosy patients. |
title_short |
qPCR-High resolution melt analysis for drug susceptibility testing of Mycobacterium leprae directly from clinical specimens of leprosy patients. |
title_full |
qPCR-High resolution melt analysis for drug susceptibility testing of Mycobacterium leprae directly from clinical specimens of leprosy patients. |
title_fullStr |
qPCR-High resolution melt analysis for drug susceptibility testing of Mycobacterium leprae directly from clinical specimens of leprosy patients. |
title_full_unstemmed |
qPCR-High resolution melt analysis for drug susceptibility testing of Mycobacterium leprae directly from clinical specimens of leprosy patients. |
title_sort |
qpcr-high resolution melt analysis for drug susceptibility testing of mycobacterium leprae directly from clinical specimens of leprosy patients. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2017 |
url |
https://doi.org/10.1371/journal.pntd.0005506 https://doaj.org/article/3e0ee1058b354bf29ea7faa8506a93ac |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 11, Iss 6, p e0005506 (2017) |
op_relation |
http://europepmc.org/articles/PMC5453413?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0005506 https://doaj.org/article/3e0ee1058b354bf29ea7faa8506a93ac |
op_doi |
https://doi.org/10.1371/journal.pntd.0005506 |
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PLOS Neglected Tropical Diseases |
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11 |
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6 |
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e0005506 |
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1766345802899259392 |