Anne J Jääskeläinen1, Tarja Sironen2, Cheikh Tidiane Diagne3, Moussa Moïse Diagne3, Martin Faye3, Oumar Faye3, Ousmane Faye3, Roger Hewson4, Markos Mölsä5, Manfred W Weidmann6, Robert Watson4, Amadou Alpha Sall3, Olli Vapalahti7. 1. Helsinki University and Helsinki University Hospital (HUSLAB), Department of Virology, Finland. Electronic address: anne.jaaskelainen@helsinki.fi. 2. University of Helsinki, Department of Virology, Helsinki, Finland; Faculty of Veterinary Medicine, Department of Veterinary Biosciences, University of Helsinki, Finland. 3. Institut Pasteur de Dakar, Pôle de virologie, Dakar, Senegal. 4. National Infection Service, Public Health England, Porton Down, Salisbury, United Kingdom. 5. National Institute for Health and Welfare, Biothreat unit, Centre for Military Medicine, Helsinki, Finland Centres for Biothreat Preparedness and for Military Medicine, Finnish Defence Forces, Finland. 6. University of Stirling, Institute of Aquaculture, Stirling, United Kingdom. 7. Helsinki University and Helsinki University Hospital (HUSLAB), Department of Virology, Finland; University of Helsinki, Department of Virology, Helsinki, Finland; Faculty of Veterinary Medicine, Department of Veterinary Biosciences, University of Helsinki, Finland.
Abstract
BACKGROUND: During the five decades since their discovery, filoviruses of four species have caused human hemorrhagic fever outbreaks: Marburg (MARV) marburgvirus, and Zaire (EBOV), Sudan (SUDV) and Bundybugyo (BDBV) ebolaviruses. The largest, devastating EBOV epidemic in West Africa in 2014-16, has been followed by outbreaks of MARV in Uganda, 2017, and EBOV in Democratic Republic of Congo, 2018, emphasizing the need to develop preparedness to diagnose all filoviruses. OBJECTIVES: The aim of this study was to optimize a new filovirus RT-qPCR to detect all filoviruses, define its limits of detection (LOD) and perform a field evaluation with outbreak samples. STUDY DESIGN: A pan-filovirus RT-qPCR targeting the L gene was developed and evaluated within the EbolaMoDRAD (Ebola virus: modern approaches for developing bedside rapid diagnostics) project. Specificity and sensitivity were determined and the effect of inactivation and PCR reagents (liquid and lyophilized format) were tested. RESULTS: The LODs for the lyophilized pan-filovirus L-RT-qPCR assay were 9.4 copies per PCR reaction for EBOV, 9.9 for MARV, 1151 for SUDV, 65 for BDBV and 289 for Taï Forest virus. The test was set at the Pasteur Institute, Dakar, Senegal, and 83 Ebola patient samples, with viral load ranging from 5 to 5 million copies of EBOV per reaction, were screened. The results for the patient samples were in 100% concordance with the reference EBOV-specific assay. DISCUSSION: Overall, the assay showed good sensitivity and specificity, covered all filoviruses known to be human pathogens, performed well both in lyophilized and liquid-phase formats and with EBOV outbreak clinical samples.
BACKGROUND: During the five decades since their discovery, filoviruses of four species have caused humanhemorrhagic fever outbreaks: Marburg (MARV) marburgvirus, and Zaire (EBOV), Sudan (SUDV) and Bundybugyo (BDBV) ebolaviruses. The largest, devastating EBOV epidemic in West Africa in 2014-16, has been followed by outbreaks of MARV in Uganda, 2017, and EBOV in Democratic Republic of Congo, 2018, emphasizing the need to develop preparedness to diagnose all filoviruses. OBJECTIVES: The aim of this study was to optimize a new filovirus RT-qPCR to detect all filoviruses, define its limits of detection (LOD) and perform a field evaluation with outbreak samples. STUDY DESIGN: A pan-filovirus RT-qPCR targeting the L gene was developed and evaluated within the EbolaMoDRAD (Ebola virus: modern approaches for developing bedside rapid diagnostics) project. Specificity and sensitivity were determined and the effect of inactivation and PCR reagents (liquid and lyophilized format) were tested. RESULTS: The LODs for the lyophilized pan-filovirus L-RT-qPCR assay were 9.4 copies per PCR reaction for EBOV, 9.9 for MARV, 1151 for SUDV, 65 for BDBV and 289 for Taï Forest virus. The test was set at the Pasteur Institute, Dakar, Senegal, and 83 Ebolapatient samples, with viral load ranging from 5 to 5 million copies of EBOV per reaction, were screened. The results for the patient samples were in 100% concordance with the reference EBOV-specific assay. DISCUSSION: Overall, the assay showed good sensitivity and specificity, covered all filoviruses known to be human pathogens, performed well both in lyophilized and liquid-phase formats and with EBOV outbreak clinical samples.
Authors: Laura C Bonney; Robert J Watson; Gillian S Slack; Andrew Bosworth; Nadina I Vasileva Wand; Roger Hewson Journal: PLoS Negl Trop Dis Date: 2020-07-31