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  • TECHNICAL REPORT

    External quality assessment for the detection of Bordetella pertussis

    by PCR, 2018 On behalf of EUPert-LabNet network

    www.ecdc.europa.eu

  • ECDC TECHNICAL REPORT

    External quality assessment for the detection of Bordetella pertussis by PCR, 2018 On behalf of EUPert-LabNet network

  • ii

    This report was commissioned by the European Centre for Disease Prevention and Control (ECDC), referring to Specific Contract No. 4 ECD.7289 implementing Framework Contract no. ECDC/2015/009 coordinated by Dr Sabrina Bacci, Dr Csaba Ködmön and Dr Lucia Pastore Celentano and produced by members of the EUPert-LabNet consortium as part of the coordination of activities for laboratory surveillance of whooping cough in Member States/EEA countries.

    Authors Dr Norman K. Fry, Dr David Litt, Public Health England – National Infection Service, London, UK; Dr Kevin Markey, National Institute for Biological Standards and Control, NIBSC, Potters Bar, UK; Alex-Mikael Barkhoff, Prof Qiushui He, University of Turku, Turku, Finland.

    Acknowledgements We acknowledge the expert technical assistance of John Duncan, Public Health England - National Infection Service, London, UK, in the preparation, testing and dispatch of this EQA panel. We also gratefully acknowledge the participation from all the laboratories in the EU/EEA Member States that took part in this study.

    Suggested citation: European Centre for Disease Prevention and Control. External quality assessment for the detection of Bordetella pertussis by PCR, 2018 – On behalf of EUPert-LabNet network. Stockholm: ECDC; 2019.

    Stockholm, January 2019

    ISBN 978-92-9498-301-5 DOI 10.2900/172389 Catalogue number TQ-02-19-016-EN-N

    © European Centre for Disease Prevention and Control, 2019

    Cover picture: © National Institute for Biological Standards and Control/Science Photo Library

    Reproduction is authorised, provided the source is acknowledged.

    For any use or reproduction of photos or other material that is not under the EU copyright, permission must be sought directly from the copyright holders.

  • TECHNICAL REPORT EQA for the detection of Bordetella pertussis by PCR, 2018 – On behalf of EUPert-LabNet network

    iii

    Contents Abbreviations ............................................................................................................................................... iv Executive summary ........................................................................................................................................ 1 1 Introduction ............................................................................................................................................... 2 2 Study design and methods ........................................................................................................................... 4

    2.1 Organisation ....................................................................................................................................... 4 2.2 Panel characteristics ............................................................................................................................ 4 2.3 Data analysis ...................................................................................................................................... 5

    3 Results ....................................................................................................................................................... 6 Bordetella pertussis dilution series .............................................................................................................. 6 Bordetella holmesii .................................................................................................................................... 6 Bordetella parapertussis and Bordetella bronchiseptica ................................................................................. 6 Negative/no template controls ................................................................................................................... 6 Haemophilus influenzae ............................................................................................................................. 6 Reproducibility .......................................................................................................................................... 7

    4 Discussion ................................................................................................................................................ 10 Sensitivity, specificity and reproducibility ................................................................................................... 10 Evidence of good laboratory practice of PCR technique by checking for evidence of contamination ................. 11 Strengths ............................................................................................................................................... 11 Limitations ............................................................................................................................................. 11

    5 Conclusions .............................................................................................................................................. 12 References .................................................................................................................................................. 14 Annex 1. Invitation letter to participants for Bordetella pertussis PCR EQAssurance program, reply form and short questionnaire (2018) .................................................................................................................................... 16 Annex 2. List of participants .......................................................................................................................... 18 Annex 3. EUPert-LabNet Bordetella pertussis PCR EQA reply and result submission forms ................................... 19

    Tables Table 1. Characteristics of strains used to prepare genomic DNA for inclusion in second EUPert-LabNet Bordetella pertussis PCR EQA (February 2018) ................................................................................................. 4 Table 2. Intended results for the second EUPert-LabNet Bordetella pertussis PCR EQA (February 2018) ................ 7 Table 3. Number of correct qualitative results achieved per panel member and analytical technology type ............. 7 Table 4. Summary of responses to questionnaire .............................................................................................. 8

  • EQA for the detection of Bordetella pertussis by PCR, 2018 – On behalf of EUPert-LabNet network TECHNICAL REPORT

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    Abbreviations BP485 Reported as specific target sequence for Bordetella pertussis Cq Crossing threshold (for quantitative PCR) DSM (DSMZ) Deutsche Sammlung von Mikroorganismen und Zellkulturen, German Collection of

    Microorganisms and Cell Cultures, Braunschweig, Germany EUPert-LabNet European Pertussis Laboratory Surveillance Network EUVAC.NET European surveillance network for selected vaccine-preventable diseases fla Flagellin gene IS Insertion sequence element hIS1001 Bordetella holmesii-specific IS1001 target NTC No template control PCR Polymerase chain reaction pilT Bordetella bronchiseptica twitching mobility gene ptxA Pertussis toxin subunit A ptxP Pertussis toxin promoter region qPCR Real-time (quantitative) PCR recA Housekeeping gene

  • TECHNICAL REPORT EQA for the detection of Bordetella pertussis by PCR, 2018 – On behalf of EUPert-LabNet network

    1

    Executive summary Laboratory confirmation of pertussis is integral to surveillance, especially to monitor the effectiveness of vaccination strategies and inform any changes to national policies. Although there are now at least two commercially available molecular (DNA-based) external quality assessment (EQA) schemes for Bordetella pertussis, the European Centre for Disease Prevention and Control (ECDC) has organised previous and current studies in order to ensure maximum participation of microbiology reference laboratories across EU/EEA countries and highlight any issues of concern and make recommendations.

    This report presents the results of the second European Pertussis Laboratory Surveillance Network (EUPert-LabNet) EQA scheme for Bordetella pertussis PCR funded by ECDC on behalf of EUPert-LabNet. The EQA study was conducted between February and March 2018.

    The panel included dilutions of purified genomic DNA from Bordetella pertussis at three concentrations designated ‘high’, ‘medium’ and ‘low’. The ‘low’ dilution was designed to be challenging. Duplicate samples of the B. pertussis ‘high’ dilution were included to test the reproducibility of polymerase chain reaction (PCR) assays. Genomic DNA from other Bordetella species — B. parapertussis, B. holmesii, and B. bronchiseptica, which can cause pertussis-like symptoms in humans — were also included in the panel, together with DNA from Haemophilus influenzae. Two ‘blank’ samples, i.e. with no added DNA, were included to check for potential contamination.

    Twenty-eight laboratories from 27 EU/EEA countries participated in the current EQA round. One laboratory submitted two datasets – one using an in-house assay and one using a commercial assay – with both using real- time PCR (qPCR). As a result, 29 datasets were submitted.

    Of the 29 datasets, all reported the successful detection of a