O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis
Abstract 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-cur...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2017-10-01
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| Series: | Parasites & Vectors |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13071-017-2382-3 |
| _version_ | 1811210161077878784 |
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| author | Solomon A. Mekonnen Marcus Beissner Malkin Saar Solomon Ali Ahmed Zeynudin Kassahun Tesfaye Mulatu G. Adbaru Florian Battke Sven Poppert Michael Hoelscher Thomas Löscher Gisela Bretzel Karl-Heinz Herbinger |
| author_facet | Solomon A. Mekonnen Marcus Beissner Malkin Saar Solomon Ali Ahmed Zeynudin Kassahun Tesfaye Mulatu G. Adbaru Florian Battke Sven Poppert Michael Hoelscher Thomas Löscher Gisela Bretzel Karl-Heinz Herbinger |
| author_sort | Solomon A. Mekonnen |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-04-12T04:51:29Z |
| format | Article |
| id | doaj.art-9ad3834ca8334d30899dda8153c4a617 |
| institution | Directory Open Access Journal |
| issn | 1756-3305 |
| language | English |
| last_indexed | 2024-04-12T04:51:29Z |
| publishDate | 2017-10-01 |
| publisher | BMC |
| record_format | Article |
| series | Parasites & Vectors |
| spelling | doaj.art-9ad3834ca8334d30899dda8153c4a6172022-12-22T03:47:18ZengBMCParasites & Vectors1756-33052017-10-011011910.1186/s13071-017-2382-3O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasisSolomon A. Mekonnen0Marcus Beissner1Malkin Saar2Solomon Ali3Ahmed Zeynudin4Kassahun Tesfaye5Mulatu G. Adbaru6Florian Battke7Sven Poppert8Michael Hoelscher9Thomas Löscher10Gisela Bretzel11Karl-Heinz Herbinger12Department of Medical Laboratory Sciences and Pathology, College of Health Sciences, Jimma UniversityDepartment of Infectious Diseases and Tropical Medicine, Medical Center of the University of MunichDepartment of Infectious Diseases and Tropical Medicine, Medical Center of the University of MunichDepartment of Medical Laboratory Sciences and Pathology, College of Health Sciences, Jimma UniversityDepartment of Medical Laboratory Sciences and Pathology, College of Health Sciences, Jimma UniversityDepartment Microbial, Cellular and Molecular Biology, Addis Ababa UniversityDepartment of Medical Laboratory Sciences and Pathology, College of Health Sciences, Jimma UniversityDr. Battke SCIENTIA GmbHUniversity Medical Center Hamburg-Eppendorf, University of HamburgDepartment of Infectious Diseases and Tropical Medicine, Medical Center of the University of MunichDepartment of Infectious Diseases and Tropical Medicine, Medical Center of the University of MunichDepartment of Infectious Diseases and Tropical Medicine, Medical Center of the University of MunichDepartment of Infectious Diseases and Tropical Medicine, Medical Center of the University of MunichAbstract 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.http://link.springer.com/article/10.1186/s13071-017-2382-3ConfirmationMicroscopyMolecular diagnosisOnchocerciasisO-5S qPCRO-150 qPCR |
| spellingShingle | Solomon A. Mekonnen Marcus Beissner Malkin Saar Solomon Ali Ahmed Zeynudin Kassahun Tesfaye Mulatu G. Adbaru Florian Battke Sven Poppert Michael Hoelscher Thomas Löscher Gisela Bretzel Karl-Heinz Herbinger O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis Parasites & Vectors Confirmation Microscopy Molecular diagnosis Onchocerciasis O-5S qPCR O-150 qPCR |
| 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 | Confirmation Microscopy Molecular diagnosis Onchocerciasis O-5S qPCR O-150 qPCR |
| url | http://link.springer.com/article/10.1186/s13071-017-2382-3 |
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