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Coordinated implementation of a conventional PCR assay to detect all Ebola and Marburg virus species in a European laboratory network
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Resumo(s)
Background: Filoviruses, including Ebola and Marburg viruses, cause severe hemorrhagic fever in humans and primates. These viruses pose significant threats to public health, making rapid and sensitive detection critical for controlling outbreaks. We developed and validated a hemi-nested generic PanFilo assay to detect all Ebola virus species, Marburg viruses, and recently discovered bat filoviruses. This assay was deployed to 15 European laboratories and evaluated through testing of eight non-infectious samples.
Objectives: Laboratories were asked to determine the detection limit of positive controls and test all samples using the assay provided. The deployed assay enables direct Nanopore sequencing of PCR products, by using tagged primers during the second round of PCR. Sequencing of the samples was carried out on a voluntary basis.
Results: Multicenter validation revealed a 95 % limit of detection of 5309 RNA copies/μL for Ebola, 10,273 copies/μL for Marburg, and 2145 copies/μL for Mengla virus. In an implementation quality assessment, 93.3 % (84/90) of samples containing filovirus RNA were correctly identified and 100 % (30/30) of filovirus-negative samples were correctly identified. Thirteen laboratories sequenced PCR products, with nine identifying all positive samples correctly.
Conclusion: The assay enables rapid and reliable detection of filoviruses, with sequencing capabilities for identifying both known and novel variants. This assay might be used for detection during the initial phase of an emerging filovirus outbreak, before a specific assay has been developed. However, our distribution across 15.
Highlights: A generic PanFilo PCR was deployed to 15 laboratories across 12 European countries within weeks. -The assay enables direct Nanopore sequencing of PCR products. -Successful deployment of the assay was evaluated through testing of blinded samples. -Eleven laboratories correctly identified all blinded samples. -Variability in laboratory performance highlights the need for better training and standardization.
Highlights: A generic PanFilo PCR was deployed to 15 laboratories across 12 European countries within weeks. -The assay enables direct Nanopore sequencing of PCR products. -Successful deployment of the assay was evaluated through testing of blinded samples. -Eleven laboratories correctly identified all blinded samples. -Variability in laboratory performance highlights the need for better training and standardization.
Descrição
Palavras-chave
Diagnostic Testing Filovirus Nested PCR Quality Improvement Infecções Sistémicas e Zoonoses
Contexto Educativo
Citação
Multicenter Study J Clin Virol. 2025 Jun:178:105808. doi: 10.1016/j.jcv.2025.105808. Epub 2025 May 28
Editora
Elservier