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Coordinated implementation of a conventional PCR assay to detect all Ebola and Marburg virus species in a European laboratory network
KC. Heimsch, T. Bleicker, TD. Best, LD. Presser, R. Molenkamp, AJ. Jääskeläinen, A. Milewska, J. Šmahelová, C. Baronti, S. Pappa, I. Tabain, R. Cordeiro, G. Marsili, K. Huik, V. Pinho Dos Reis, L. Barzon, P. Maes, C. Drosten, VM. Corman
Jazyk angličtina Země Nizozemsko
Typ dokumentu časopisecké články, multicentrická studie, validační studie
- MeSH
- diagnostické techniky molekulární metody MeSH
- hemoragická horečka Ebola * diagnóza virologie MeSH
- lidé MeSH
- limita detekce MeSH
- marburgská horečka * diagnóza virologie MeSH
- polymerázová řetězová reakce * metody MeSH
- RNA virová genetika MeSH
- senzitivita a specificita MeSH
- virus Ebola * izolace a purifikace genetika MeSH
- virus Marburg * izolace a purifikace genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- multicentrická studie MeSH
- validační studie MeSH
- Geografické názvy
- Evropa MeSH
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 laboratories revealed variability challenges due to reagents, human performance, and sequencing capacity, emphasizing the need for more training and standardization.
Croatian Institute of Public Health Zagreb Croatia
Department of Microbiology Immunology and Transplantation Rega Institute KU Leuven Leuven Belgium
Department of Molecular Medicine University of Padova Padova Italy
Department of Viroscience Erasmus MC Rotterdam the Netherlands
German Center for Infection Research associated partner Charité Berlin Germany
Helsinki University and Helsinki University Hospital HUS Diagnostic Center Helsinki Finland
Institute of Diagnostic Virology Friedrich Loeffler Institute Greifswald Insel Riems Germany
Labor Berlin Charité Vivantes GmbH Berlin Germany
Malopolska Centre of Biotechnology Jagiellonian University Gronostajowa 7A 30 387 Krakow Poland
Microbiology and Virology Unit Padova University Hospital Padova Italy
Citace poskytuje Crossref.org
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- $a Heimsch, K C $u Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt, Universität zu Berlin, Institute of Virology, Berlin, Germany. Electronic address: kim.heimsch@charite.de
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- $a Coordinated implementation of a conventional PCR assay to detect all Ebola and Marburg virus species in a European laboratory network / $c KC. Heimsch, T. Bleicker, TD. Best, LD. Presser, R. Molenkamp, AJ. Jääskeläinen, A. Milewska, J. Šmahelová, C. Baronti, S. Pappa, I. Tabain, R. Cordeiro, G. Marsili, K. Huik, V. Pinho Dos Reis, L. Barzon, P. Maes, C. Drosten, VM. Corman
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- $a 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 laboratories revealed variability challenges due to reagents, human performance, and sequencing capacity, emphasizing the need for more training and standardization.
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- $a Best, T D $u Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt, Universität zu Berlin, Institute of Virology, Berlin, Germany. Electronic address: till-dominik.best@charite.de
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- $a Milewska, A $u Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7A, 30-387 Krakow, Poland. Electronic address: aleksandra.milewska@uj.edu.pl
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- $a Baronti, C $u Unite des Virus Emergents (UVE: Aix-Marseille University, Universita di Corsica, IRD 190, Inserm 1207, IRBA), France. Electronic address: cecile.baronti@univ-amu.fr
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- $a Pappa, S $u Laboratory of Microbiology, School of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece
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- $a Tabain, I $u Croatian Institute of Public Health, Zagreb, Croatia. Electronic address: irena.tabain@hzjz.hr
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- $a Cordeiro, R $u Emergency Response and Biopreparedness Unit, Department of Infectious Diseases, National Institute of Health Doutor Ricardo Jorge (INSA), Lisbon, Portugal. Electronic address: rita.cordeiro@insa.min-saude.pt
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- $a Marsili, G $u National Reference Laboratory for Arbovirus, Unit of Arbo, Hanta and Emerging viruses, Department of Infectious diseases, National Health Institute of Italy (ISS), Rome, Italy. Electronic address: giulia.marsili@iss.it
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- $a Huik, K $u Department of Microbiology, Institute of Biomedicine and Translational Medicine, Faculty of Medicine, University of Tartu, Tartu, Estonia. Electronic address: kristi.huik@ut.ee
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- $a Pinho Dos Reis, V $u Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Greifswald, Insel Riems, Germany. Electronic address: Vinicius.PinhodosReis@fli.de
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- $a Barzon, L $u Department of Molecular Medicine, University of Padova, Padova, Italy; Microbiology and Virology Unit, Padova University Hospital, Padova, Italy. Electronic address: luisa.barzon@unipd.it
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- $a Maes, P $u Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven, Leuven, Belgium. Electronic address: piet.maes@kuleuven.be
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