A novel field-based molecular assay to detect validated artemisinin-resistant k13 mutants
Abstract Background Given the risk of artemisinin resistance spreading from the Greater Mekong sub-region, prospective monitoring in sub-Saharan Africa should be expedited. Molecular biology techniques used for monitoring rely on the detection of k13 validated mutants by using PCR and Sanger sequenc...
| Main Authors: | , , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
BMC
2018-04-01
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| Series: | Malaria Journal |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12936-018-2329-y |
| _version_ | 1819126861056180224 |
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| author | Laurence Vachot-Ganée Nimol Khim Alexandra Iannello Eric Legrand Saorin Kim Rotha Eam Chanra Khean Malen Ken Elizabeth Ashley Kyaw Myo Tun Mehul Dhorda François Nosten Issa Mahamat Souleymane Sophie Blein Alexandre Pachot Frédéric Ariey Karine Kaiser Didier Ménard |
| author_facet | Laurence Vachot-Ganée Nimol Khim Alexandra Iannello Eric Legrand Saorin Kim Rotha Eam Chanra Khean Malen Ken Elizabeth Ashley Kyaw Myo Tun Mehul Dhorda François Nosten Issa Mahamat Souleymane Sophie Blein Alexandre Pachot Frédéric Ariey Karine Kaiser Didier Ménard |
| author_sort | Laurence Vachot-Ganée |
| collection | DOAJ |
| description | Abstract Background Given the risk of artemisinin resistance spreading from the Greater Mekong sub-region, prospective monitoring in sub-Saharan Africa should be expedited. Molecular biology techniques used for monitoring rely on the detection of k13 validated mutants by using PCR and Sanger sequencing approach, usually not available in malaria endemic areas. Methods A semi-automated workflow based on the easyMAG® platform and the Argene Solution® (bioMérieux, Marcy l’Etoile, France) as a field-based surveillance tool operable at national level was developed in four steps. Clinical and analytical performances of this tool detecting five of the most frequent and validated k13 mutants (Y493H, I543T, R539T, F446I and C580Y) from dried blood spots (DBS) were compared to the gold standard approach (PCR and Sanger sequencing). Results By using the ARMS (amplification-refractory mutation system) strategy, the best multiplexing options were found in 3 separate real-time PCR duplexes (IC as internal control/I543T, C580Y/Y493H and F446I/R539T) with limits of detection ranging from 50 (C580Y) to 6.25 parasites/µL (Y493H). In field conditions, using 642 clinical DBS (from symptomatic patients and asymptomatic individuals) collected from Cambodia, Myanmar and Africa (Chad), the overall sensitivity and specificity of the K13 bMx prototype assay developed by bioMérieux were ≥ 90%. Areas under the ROC curves were estimated to be > 0.90 for all k13 mutants in samples from symptomatic patients. Conclusion The K13 ready-to-use bMx prototype assay, considered by the end-users as a user-friendly assay to perform (in shorter time than the K13 reference assay) and easy to interpret, was found to require less budget planning and had fewer logistical constraints. Its excellent performance qualifies the prototype as a reliable screening tool usable in malaria endemic countries recognized to be at risk of emergence or spread of validated k13 mutants. Additional multi-site studies are needed to evaluate the performances of the K13 bMx prototype assay in different epidemiological contexts such as Africa, India, or South America. |
| first_indexed | 2024-12-22T08:02:45Z |
| format | Article |
| id | doaj.art-b7dcfd73d4424c03b659c2e74929c978 |
| institution | Directory Open Access Journal |
| issn | 1475-2875 |
| language | English |
| last_indexed | 2024-12-22T08:02:45Z |
| publishDate | 2018-04-01 |
| publisher | BMC |
| record_format | Article |
| series | Malaria Journal |
| spelling | doaj.art-b7dcfd73d4424c03b659c2e74929c9782022-12-21T18:33:13ZengBMCMalaria Journal1475-28752018-04-0117111210.1186/s12936-018-2329-yA novel field-based molecular assay to detect validated artemisinin-resistant k13 mutantsLaurence Vachot-Ganée0Nimol Khim1Alexandra Iannello2Eric Legrand3Saorin Kim4Rotha Eam5Chanra Khean6Malen Ken7Elizabeth Ashley8Kyaw Myo Tun9Mehul Dhorda10François Nosten11Issa Mahamat Souleymane12Sophie Blein13Alexandre Pachot14Frédéric Ariey15Karine Kaiser16Didier Ménard17Medical Diagnostic Discovery Department (MD3), bioMérieuxMalaria Molecular Epidemiology Unit, Institute Pasteur in CambodiaMedical Diagnostic Discovery Department (MD3), bioMérieuxGenetics and Genomics of Insect Vectors Unit, Institute PasteurMalaria Molecular Epidemiology Unit, Institute Pasteur in CambodiaMalaria Molecular Epidemiology Unit, Institute Pasteur in CambodiaMalaria Molecular Epidemiology Unit, Institute Pasteur in CambodiaMalaria Molecular Epidemiology Unit, Institute Pasteur in CambodiaMahidol–Oxford Tropical Medicine Research UnitDepartment of Preventive & Social Medicine, Defence Services Medical AcademyMahidol–Oxford Tropical Medicine Research UnitCentre for Tropical Medicine and Global Health, Nuffield Department of Medicine Research Building, University of OxfordProgramme National de Lutte Contre le Paludisme au TchadMedical Diagnostic Discovery Department (MD3), bioMérieuxMedical Diagnostic Discovery Department (MD3), bioMérieuxInstitute Cochin Inserm U1016, Université Paris-Descartes, Sorbonne Paris CitéMedical Diagnostic Discovery Department (MD3), bioMérieuxMalaria Molecular Epidemiology Unit, Institute Pasteur in CambodiaAbstract Background Given the risk of artemisinin resistance spreading from the Greater Mekong sub-region, prospective monitoring in sub-Saharan Africa should be expedited. Molecular biology techniques used for monitoring rely on the detection of k13 validated mutants by using PCR and Sanger sequencing approach, usually not available in malaria endemic areas. Methods A semi-automated workflow based on the easyMAG® platform and the Argene Solution® (bioMérieux, Marcy l’Etoile, France) as a field-based surveillance tool operable at national level was developed in four steps. Clinical and analytical performances of this tool detecting five of the most frequent and validated k13 mutants (Y493H, I543T, R539T, F446I and C580Y) from dried blood spots (DBS) were compared to the gold standard approach (PCR and Sanger sequencing). Results By using the ARMS (amplification-refractory mutation system) strategy, the best multiplexing options were found in 3 separate real-time PCR duplexes (IC as internal control/I543T, C580Y/Y493H and F446I/R539T) with limits of detection ranging from 50 (C580Y) to 6.25 parasites/µL (Y493H). In field conditions, using 642 clinical DBS (from symptomatic patients and asymptomatic individuals) collected from Cambodia, Myanmar and Africa (Chad), the overall sensitivity and specificity of the K13 bMx prototype assay developed by bioMérieux were ≥ 90%. Areas under the ROC curves were estimated to be > 0.90 for all k13 mutants in samples from symptomatic patients. Conclusion The K13 ready-to-use bMx prototype assay, considered by the end-users as a user-friendly assay to perform (in shorter time than the K13 reference assay) and easy to interpret, was found to require less budget planning and had fewer logistical constraints. Its excellent performance qualifies the prototype as a reliable screening tool usable in malaria endemic countries recognized to be at risk of emergence or spread of validated k13 mutants. Additional multi-site studies are needed to evaluate the performances of the K13 bMx prototype assay in different epidemiological contexts such as Africa, India, or South America.http://link.springer.com/article/10.1186/s12936-018-2329-yMalariaPlasmodium falciparumArtemisinin resistancek13 mutation detectionSurveillance |
| spellingShingle | Laurence Vachot-Ganée Nimol Khim Alexandra Iannello Eric Legrand Saorin Kim Rotha Eam Chanra Khean Malen Ken Elizabeth Ashley Kyaw Myo Tun Mehul Dhorda François Nosten Issa Mahamat Souleymane Sophie Blein Alexandre Pachot Frédéric Ariey Karine Kaiser Didier Ménard A novel field-based molecular assay to detect validated artemisinin-resistant k13 mutants Malaria Journal Malaria Plasmodium falciparum Artemisinin resistance k13 mutation detection Surveillance |
| title | A novel field-based molecular assay to detect validated artemisinin-resistant k13 mutants |
| title_full | A novel field-based molecular assay to detect validated artemisinin-resistant k13 mutants |
| title_fullStr | A novel field-based molecular assay to detect validated artemisinin-resistant k13 mutants |
| title_full_unstemmed | A novel field-based molecular assay to detect validated artemisinin-resistant k13 mutants |
| title_short | A novel field-based molecular assay to detect validated artemisinin-resistant k13 mutants |
| title_sort | novel field based molecular assay to detect validated artemisinin resistant k13 mutants |
| topic | Malaria Plasmodium falciparum Artemisinin resistance k13 mutation detection Surveillance |
| url | http://link.springer.com/article/10.1186/s12936-018-2329-y |
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