Development, validation and clinical evaluation of a broad-range pan-filovirus RT-qPCR

Anne J Jääskeläinen; Tarja Sironen; Cheikh Tidiane Diagne; Moussa Moïse Diagne; Martin Faye; Oumar Faye; Ousmane Faye; Roger Hewson; Markos Mölsä; Manfred W Weidmann; Robert Watson; Amadou Alpha Sall; Olli Vapalahti

doi:10.1016/j.jcv.2019.03.010

Development, validation and clinical evaluation of a broad-range pan-filovirus RT-qPCR

J Clin Virol. 2019 May:114:26-31. doi: 10.1016/j.jcv.2019.03.010. Epub 2019 Mar 19.

Affiliations

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.

PMID: 30904708
DOI: 10.1016/j.jcv.2019.03.010

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.

Keywords: Bundibugyo; Ebola; Marburg; Pan-Filo; Sudan.

Publication types

Evaluation Study
Research Support, Non-U.S. Gov't
Validation Study

MeSH terms

Animals
Ebolavirus
Filoviridae
Filoviridae Infections / diagnosis*
Filoviridae Infections / virology
Freeze Drying
Hemorrhagic Fever, Ebola / diagnosis
Hemorrhagic Fever, Ebola / virology
Humans
Limit of Detection
Marburg Virus Disease / diagnosis
Marburg Virus Disease / virology
Marburgvirus
Real-Time Polymerase Chain Reaction / methods*
Sensitivity and Specificity
Viral Proteins / genetics*

Substances

Viral Proteins