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

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)26-31
Number of pages6
JournalJournal of Clinical Virology
Volume114
DOIs
Publication statusPublished - May 2019

Bibliographical note

Funding Information:
This project is part of the EbolaMoDRAD (Ebola virus: modern approaches for developing bedside rapid diagnostics) consortium, which has received funding from the Innovative Medicine Initiative 2 Joint Undertaking under grant agreement N°115843. This Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA. This work was also funded by Helsinki University Hospital (HUSLAB; research permit TYH2017257).

Publisher Copyright:
© 2019 Elsevier B.V.

Keywords

  • Bundibugyo
  • Ebola
  • Marburg
  • Pan-Filo
  • Sudan

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