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O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis

BACKGROUND: Onchocerciasis is a parasitic disease caused by the filarial nematode Onchocerca volvulus. In endemic areas, the diagnosis is commonly confirmed by microscopic examination of skin snip samples, though this technique is considered to have low sensitivity. The available melting-curve based...

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Autores principales: Mekonnen, Solomon A., Beissner, Marcus, Saar, Malkin, Ali, Solomon, Zeynudin, Ahmed, Tesfaye, Kassahun, Adbaru, Mulatu G., Battke, Florian, Poppert, Sven, Hoelscher, Michael, Löscher, Thomas, Bretzel, Gisela, Herbinger, Karl-Heinz
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5625774/
https://www.ncbi.nlm.nih.gov/pubmed/28969662
http://dx.doi.org/10.1186/s13071-017-2382-3
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author Mekonnen, Solomon A.
Beissner, Marcus
Saar, Malkin
Ali, Solomon
Zeynudin, Ahmed
Tesfaye, Kassahun
Adbaru, Mulatu G.
Battke, Florian
Poppert, Sven
Hoelscher, Michael
Löscher, Thomas
Bretzel, Gisela
Herbinger, Karl-Heinz
author_facet Mekonnen, Solomon A.
Beissner, Marcus
Saar, Malkin
Ali, Solomon
Zeynudin, Ahmed
Tesfaye, Kassahun
Adbaru, Mulatu G.
Battke, Florian
Poppert, Sven
Hoelscher, Michael
Löscher, Thomas
Bretzel, Gisela
Herbinger, Karl-Heinz
author_sort Mekonnen, Solomon A.
collection PubMed
description BACKGROUND: Onchocerciasis is a parasitic disease caused by the filarial nematode Onchocerca volvulus. In endemic areas, the diagnosis is commonly confirmed by microscopic examination of skin snip samples, though this technique is considered to have low sensitivity. The available melting-curve based quantitative real-time PCR (qPCR) using degenerated primers targeting the O-150 repeat of O. volvulus was considered insufficient for confirming the individual diagnosis, especially in elimination studies. This study aimed to improve detection of O. volvulus DNA in clinical samples through the development of a highly sensitive qPCR assay. METHODS: A novel hydrolysis probe based qPCR assay was designed targeting the specific sequence of the O. volvulus O-5S rRNA gene. A total of 200 clinically suspected onchocerciasis cases were included from Goma district in South-west Ethiopia, from October 2012 through May 2013. Skin snip samples were collected and subjected to microscopy, O-150 qPCR, and the novel O-5S qPCR. RESULTS: Among the 200 individuals, 133 patients tested positive (positivity rate of 66.5%) and 67 negative by O-5S qPCR, 74 tested positive by microscopy (37.0%) and 78 tested positive by O-150 qPCR (39.0%). Among the 133 O-5S qPCR positive individuals, microscopy and O-150 qPCR detected 55.6 and 59.4% patients, respectively, implying a higher sensitivity of O-5S qPCR than microscopy and O-150 qPCR. None of the 67 individuals who tested negative by O-5S qPCR tested positive by microscopy or O-150 qPCR, implying 100% specificity of the newly designed O-5S qPCR assay. CONCLUSIONS: The novel O-5S qPCR assay is more sensitive than both microscopic examination and the existing O-150 qPCR for the detection of O. volvulus from skin snip samples. The newly designed assay is an important step towards appropriate individual diagnosis and control of onchocerciasis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13071-017-2382-3) contains supplementary material, which is available to authorized users.
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spelling pubmed-56257742017-10-12 O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis Mekonnen, Solomon A. Beissner, Marcus Saar, Malkin Ali, Solomon Zeynudin, Ahmed Tesfaye, Kassahun Adbaru, Mulatu G. Battke, Florian Poppert, Sven Hoelscher, Michael Löscher, Thomas Bretzel, Gisela Herbinger, Karl-Heinz Parasit Vectors Research BACKGROUND: Onchocerciasis is a parasitic disease caused by the filarial nematode Onchocerca volvulus. In endemic areas, the diagnosis is commonly confirmed by microscopic examination of skin snip samples, though this technique is considered to have low sensitivity. The available melting-curve based quantitative real-time PCR (qPCR) using degenerated primers targeting the O-150 repeat of O. volvulus was considered insufficient for confirming the individual diagnosis, especially in elimination studies. This study aimed to improve detection of O. volvulus DNA in clinical samples through the development of a highly sensitive qPCR assay. METHODS: A novel hydrolysis probe based qPCR assay was designed targeting the specific sequence of the O. volvulus O-5S rRNA gene. A total of 200 clinically suspected onchocerciasis cases were included from Goma district in South-west Ethiopia, from October 2012 through May 2013. Skin snip samples were collected and subjected to microscopy, O-150 qPCR, and the novel O-5S qPCR. RESULTS: Among the 200 individuals, 133 patients tested positive (positivity rate of 66.5%) and 67 negative by O-5S qPCR, 74 tested positive by microscopy (37.0%) and 78 tested positive by O-150 qPCR (39.0%). Among the 133 O-5S qPCR positive individuals, microscopy and O-150 qPCR detected 55.6 and 59.4% patients, respectively, implying a higher sensitivity of O-5S qPCR than microscopy and O-150 qPCR. None of the 67 individuals who tested negative by O-5S qPCR tested positive by microscopy or O-150 qPCR, implying 100% specificity of the newly designed O-5S qPCR assay. CONCLUSIONS: The novel O-5S qPCR assay is more sensitive than both microscopic examination and the existing O-150 qPCR for the detection of O. volvulus from skin snip samples. The newly designed assay is an important step towards appropriate individual diagnosis and control of onchocerciasis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13071-017-2382-3) contains supplementary material, which is available to authorized users. BioMed Central 2017-10-02 /pmc/articles/PMC5625774/ /pubmed/28969662 http://dx.doi.org/10.1186/s13071-017-2382-3 Text en © The Author(s). 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Mekonnen, Solomon A.
Beissner, Marcus
Saar, Malkin
Ali, Solomon
Zeynudin, Ahmed
Tesfaye, Kassahun
Adbaru, Mulatu G.
Battke, Florian
Poppert, Sven
Hoelscher, Michael
Löscher, Thomas
Bretzel, Gisela
Herbinger, Karl-Heinz
O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis
title O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis
title_full O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis
title_fullStr O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis
title_full_unstemmed O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis
title_short O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis
title_sort o-5s quantitative real-time pcr: a new diagnostic tool for laboratory confirmation of human onchocerciasis
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5625774/
https://www.ncbi.nlm.nih.gov/pubmed/28969662
http://dx.doi.org/10.1186/s13071-017-2382-3
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