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...
| Main Authors: | , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2022-02-01
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| Series: | PLoS Neglected Tropical Diseases |
| Online Access: | https://doi.org/10.1371/journal.pntd.0009850 |
| _version_ | 1819123806923390976 |
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-22T07:14:13Z |
| format | Article |
| id | doaj.art-9eb1604e210244ddab1884dd60d1b4c4 |
| institution | Directory Open Access Journal |
| issn | 1935-2727 1935-2735 |
| language | English |
| last_indexed | 2024-12-22T07:14:13Z |
| publishDate | 2022-02-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS Neglected Tropical Diseases |
| spelling | doaj.art-9eb1604e210244ddab1884dd60d1b4c42022-12-21T18:34:25ZengPublic Library of Science (PLoS)PLoS Neglected Tropical Diseases1935-27271935-27352022-02-01162e000985010.1371/journal.pntd.0009850Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis.Fernanda Saloum de Neves MantaThiago JacomassoRita de Cássia Pontello RampazzoSuelen Justo Maria MoreiraNajua M ZahraStewart T ColeCharlotte AvanziThyago Leal-CalvoSidra Ezidio Gonçalves VasconcellosPhillip SuffysMarcelo Ribeiro-AlvesMarco Aurelio KriegerAlexandre Dias Tavares CostaMilton Ozório MoraesLeprosy 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.https://doi.org/10.1371/journal.pntd.0009850 |
| spellingShingle | 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. PLoS Neglected Tropical Diseases |
| title | 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_short | 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 |
| url | https://doi.org/10.1371/journal.pntd.0009850 |
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