Comparison and clinical validation of qPCR assays targeting Leishmania 18S rDNA and HSP70 genes in patients with American Tegumentary Leishmaniasis.
Leishmaniasis is a worldwide neglected disease, encompassing asymptomatic infections and different clinical forms, such as American Tegumentary Leishmaniasis (ATL) which is part of the complex of diseases caused by protozoan parasites from Leishmania genus, transmitted by sand fly vectors. As a negl...
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ftdoajarticles:oai:doaj.org/article:abc5dc86a40e43fcb1ff2db92b00f213 2023-05-15T15:15:29+02:00 Comparison and clinical validation of qPCR assays targeting Leishmania 18S rDNA and HSP70 genes in patients with American Tegumentary Leishmaniasis. Camila Patricio Braga Filgueira Otacilio Cruz Moreira Lilian Motta Cantanhêde Heloísa Martins Teixeira de Farias Renato Porrozzi Constança Britto Mariana Côrtes Boité Elisa Cupolillo 2020-10-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0008750 https://doaj.org/article/abc5dc86a40e43fcb1ff2db92b00f213 EN eng Public Library of Science (PLoS) https://doi.org/10.1371/journal.pntd.0008750 https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0008750 https://doaj.org/article/abc5dc86a40e43fcb1ff2db92b00f213 PLoS Neglected Tropical Diseases, Vol 14, Iss 10, p e0008750 (2020) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2020 ftdoajarticles https://doi.org/10.1371/journal.pntd.0008750 2022-12-31T07:46:37Z Leishmaniasis is a worldwide neglected disease, encompassing asymptomatic infections and different clinical forms, such as American Tegumentary Leishmaniasis (ATL) which is part of the complex of diseases caused by protozoan parasites from Leishmania genus, transmitted by sand fly vectors. As a neglected disease, much effort is still needed in treatment and diagnosis. Currently, ATL diagnosis is mainly made by parasite detection by microscopy. The sensitivity of the method varies, and factors such as collection procedures interfere. Molecular approaches, specially based on Real Time PCR (qPCR) technique, has been widely used to detect Leishmania infection and to quantify parasite load, once it is a simple, rapid and sensitive methodology, capable to detect low parasite concentrations and less prone to variability. Although many studies have been already published addressing the use of this technique, an improvement on these methodologies, including an analytical validation, standardization and data association is demanded. Moreover, a proper validation by the assay by the use of clinical samples is still required. In this sense, the purpose of the present work is to compare the performance of qPCR using two commonly used targets (18S rDNA and HSP70) with an internal control (RNAse P) in multiplex reactions. Additionally, we validated reactions by assaying 88 samples from patients presenting different clinical forms of leishmaniasis (cutaneous, mucosal, recent and old lesions), representing the diversity found in Brazil's Amazon Region. Following the methodology proposed herein, the results indicate the use of both qPCR assays, 18S rDNA and HSP70, to achieve a very good net sensitivity (98.5%) and specificity (100%), performing simultaneous or sequential testing, respectively. With this approach, our main goal is to conclude the first step of a further multicenter study to propose the standardization of detection and quantification of Leishmania. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLOS Neglected Tropical Diseases 14 10 e0008750 |
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Open Polar |
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Directory of Open Access Journals: DOAJ Articles |
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language |
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 Camila Patricio Braga Filgueira Otacilio Cruz Moreira Lilian Motta Cantanhêde Heloísa Martins Teixeira de Farias Renato Porrozzi Constança Britto Mariana Côrtes Boité Elisa Cupolillo Comparison and clinical validation of qPCR assays targeting Leishmania 18S rDNA and HSP70 genes in patients with American Tegumentary Leishmaniasis. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
Leishmaniasis is a worldwide neglected disease, encompassing asymptomatic infections and different clinical forms, such as American Tegumentary Leishmaniasis (ATL) which is part of the complex of diseases caused by protozoan parasites from Leishmania genus, transmitted by sand fly vectors. As a neglected disease, much effort is still needed in treatment and diagnosis. Currently, ATL diagnosis is mainly made by parasite detection by microscopy. The sensitivity of the method varies, and factors such as collection procedures interfere. Molecular approaches, specially based on Real Time PCR (qPCR) technique, has been widely used to detect Leishmania infection and to quantify parasite load, once it is a simple, rapid and sensitive methodology, capable to detect low parasite concentrations and less prone to variability. Although many studies have been already published addressing the use of this technique, an improvement on these methodologies, including an analytical validation, standardization and data association is demanded. Moreover, a proper validation by the assay by the use of clinical samples is still required. In this sense, the purpose of the present work is to compare the performance of qPCR using two commonly used targets (18S rDNA and HSP70) with an internal control (RNAse P) in multiplex reactions. Additionally, we validated reactions by assaying 88 samples from patients presenting different clinical forms of leishmaniasis (cutaneous, mucosal, recent and old lesions), representing the diversity found in Brazil's Amazon Region. Following the methodology proposed herein, the results indicate the use of both qPCR assays, 18S rDNA and HSP70, to achieve a very good net sensitivity (98.5%) and specificity (100%), performing simultaneous or sequential testing, respectively. With this approach, our main goal is to conclude the first step of a further multicenter study to propose the standardization of detection and quantification of Leishmania. |
format |
Article in Journal/Newspaper |
author |
Camila Patricio Braga Filgueira Otacilio Cruz Moreira Lilian Motta Cantanhêde Heloísa Martins Teixeira de Farias Renato Porrozzi Constança Britto Mariana Côrtes Boité Elisa Cupolillo |
author_facet |
Camila Patricio Braga Filgueira Otacilio Cruz Moreira Lilian Motta Cantanhêde Heloísa Martins Teixeira de Farias Renato Porrozzi Constança Britto Mariana Côrtes Boité Elisa Cupolillo |
author_sort |
Camila Patricio Braga Filgueira |
title |
Comparison and clinical validation of qPCR assays targeting Leishmania 18S rDNA and HSP70 genes in patients with American Tegumentary Leishmaniasis. |
title_short |
Comparison and clinical validation of qPCR assays targeting Leishmania 18S rDNA and HSP70 genes in patients with American Tegumentary Leishmaniasis. |
title_full |
Comparison and clinical validation of qPCR assays targeting Leishmania 18S rDNA and HSP70 genes in patients with American Tegumentary Leishmaniasis. |
title_fullStr |
Comparison and clinical validation of qPCR assays targeting Leishmania 18S rDNA and HSP70 genes in patients with American Tegumentary Leishmaniasis. |
title_full_unstemmed |
Comparison and clinical validation of qPCR assays targeting Leishmania 18S rDNA and HSP70 genes in patients with American Tegumentary Leishmaniasis. |
title_sort |
comparison and clinical validation of qpcr assays targeting leishmania 18s rdna and hsp70 genes in patients with american tegumentary leishmaniasis. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2020 |
url |
https://doi.org/10.1371/journal.pntd.0008750 https://doaj.org/article/abc5dc86a40e43fcb1ff2db92b00f213 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 14, Iss 10, p e0008750 (2020) |
op_relation |
https://doi.org/10.1371/journal.pntd.0008750 https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0008750 https://doaj.org/article/abc5dc86a40e43fcb1ff2db92b00f213 |
op_doi |
https://doi.org/10.1371/journal.pntd.0008750 |
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PLOS Neglected Tropical Diseases |
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e0008750 |
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