Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis.
Leprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae, an obligate intracellular bacterium. Timely detection is a challenge in leprosy diagnosis, relying on clinical examination and trained health professionals. Furthermore, adequate care and transmission control depend on...
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ftdoajarticles:oai:doaj.org/article:9eb1604e210244ddab1884dd60d1b4c4 2023-05-15T15:09:17+02:00 Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis. Fernanda Saloum de Neves Manta Thiago Jacomasso Rita de Cássia Pontello Rampazzo Suelen Justo Maria Moreira Najua M Zahra Stewart T Cole Charlotte Avanzi Thyago Leal-Calvo Sidra Ezidio Gonçalves Vasconcellos Phillip Suffys Marcelo Ribeiro-Alves Marco Aurelio Krieger Alexandre Dias Tavares Costa Milton Ozório Moraes 2022-02-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0009850 https://doaj.org/article/9eb1604e210244ddab1884dd60d1b4c4 EN eng Public Library of Science (PLoS) https://doi.org/10.1371/journal.pntd.0009850 https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0009850 https://doaj.org/article/9eb1604e210244ddab1884dd60d1b4c4 PLoS Neglected Tropical Diseases, Vol 16, Iss 2, p e0009850 (2022) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2022 ftdoajarticles https://doi.org/10.1371/journal.pntd.0009850 2022-12-31T12:59:47Z Leprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae, an obligate intracellular bacterium. Timely detection is a challenge in leprosy diagnosis, relying on clinical examination and trained health professionals. Furthermore, adequate care and transmission control depend on early and reliable pathogen detection. Here, we describe a qPCR test for routine diagnosis of leprosy-suspected patients. The reaction simultaneously amplifies two specific Mycobacterium leprae targets (16S rRNA and RLEP), and the human 18S rRNA gene as internal control. The limit of detection was estimated to be 2.29 copies of the M. leprae genome. Analytical specificity was evaluated using a panel of 20 other skin pathogenic microorganisms and Mycobacteria, showing no cross-reactivity. Intra- and inter-operator Cp variation was evaluated using dilution curves of M. leprae DNA or a synthetic gene, and no significant difference was observed between three operators in two different laboratories. The multiplex assay was evaluated using 97 patient samples with clinical and histopathological leprosy confirmation, displaying high diagnostic sensitivity (91%) and specificity (100%). Validation tests in an independent panel of 50 samples confirmed sensitivity and specificity of 97% and 98%, respectively. Importantly, assay performance remained stable for at least five months. Our results show that the newly developed multiplex qPCR effectively and specifically detects M. leprae DNA in skin samples, contributing to an efficient diagnosis that expedites the appropriate treatment. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLOS Neglected Tropical Diseases 16 2 e0009850 |
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Open Polar |
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Directory of Open Access Journals: DOAJ Articles |
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ftdoajarticles |
language |
English |
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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 Fernanda Saloum de Neves Manta Thiago Jacomasso Rita de Cássia Pontello Rampazzo Suelen Justo Maria Moreira Najua M Zahra Stewart T Cole Charlotte Avanzi Thyago Leal-Calvo Sidra Ezidio Gonçalves Vasconcellos Phillip Suffys Marcelo Ribeiro-Alves Marco Aurelio Krieger Alexandre Dias Tavares Costa Milton Ozório Moraes Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
Leprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae, an obligate intracellular bacterium. Timely detection is a challenge in leprosy diagnosis, relying on clinical examination and trained health professionals. Furthermore, adequate care and transmission control depend on early and reliable pathogen detection. Here, we describe a qPCR test for routine diagnosis of leprosy-suspected patients. The reaction simultaneously amplifies two specific Mycobacterium leprae targets (16S rRNA and RLEP), and the human 18S rRNA gene as internal control. The limit of detection was estimated to be 2.29 copies of the M. leprae genome. Analytical specificity was evaluated using a panel of 20 other skin pathogenic microorganisms and Mycobacteria, showing no cross-reactivity. Intra- and inter-operator Cp variation was evaluated using dilution curves of M. leprae DNA or a synthetic gene, and no significant difference was observed between three operators in two different laboratories. The multiplex assay was evaluated using 97 patient samples with clinical and histopathological leprosy confirmation, displaying high diagnostic sensitivity (91%) and specificity (100%). Validation tests in an independent panel of 50 samples confirmed sensitivity and specificity of 97% and 98%, respectively. Importantly, assay performance remained stable for at least five months. Our results show that the newly developed multiplex qPCR effectively and specifically detects M. leprae DNA in skin samples, contributing to an efficient diagnosis that expedites the appropriate treatment. |
format |
Article in Journal/Newspaper |
author |
Fernanda Saloum de Neves Manta Thiago Jacomasso Rita de Cássia Pontello Rampazzo Suelen Justo Maria Moreira Najua M Zahra Stewart T Cole Charlotte Avanzi Thyago Leal-Calvo Sidra Ezidio Gonçalves Vasconcellos Phillip Suffys Marcelo Ribeiro-Alves Marco Aurelio Krieger Alexandre Dias Tavares Costa Milton Ozório Moraes |
author_facet |
Fernanda Saloum de Neves Manta Thiago Jacomasso Rita de Cássia Pontello Rampazzo Suelen Justo Maria Moreira Najua M Zahra Stewart T Cole Charlotte Avanzi Thyago Leal-Calvo Sidra Ezidio Gonçalves Vasconcellos Phillip Suffys Marcelo Ribeiro-Alves Marco Aurelio Krieger Alexandre Dias Tavares Costa Milton Ozório Moraes |
author_sort |
Fernanda Saloum de Neves Manta |
title |
Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis. |
title_short |
Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis. |
title_full |
Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis. |
title_fullStr |
Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis. |
title_full_unstemmed |
Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis. |
title_sort |
development and validation of a multiplex real-time qpcr assay using gmp-grade reagents for leprosy diagnosis. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2022 |
url |
https://doi.org/10.1371/journal.pntd.0009850 https://doaj.org/article/9eb1604e210244ddab1884dd60d1b4c4 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 16, Iss 2, p e0009850 (2022) |
op_relation |
https://doi.org/10.1371/journal.pntd.0009850 https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0009850 https://doaj.org/article/9eb1604e210244ddab1884dd60d1b4c4 |
op_doi |
https://doi.org/10.1371/journal.pntd.0009850 |
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PLOS Neglected Tropical Diseases |
container_volume |
16 |
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2 |
container_start_page |
e0009850 |
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