Laboratory diagnosis and molecular surveillance of Bordetella pertussis

Pertussis or whooping cough is a highly contagious respiratory disease mainly caused by the bacterium Bordetella pertussis. Despite childhood immunisations having been widely implemented, pertussis remains one of the world’s leading causes of vaccine-preventable deaths, with epidemics occurring every three to five years in many countries. In 2024, around 210 000 cases of pertussis were reported by 29 EU/EEA countries. This clearly highlights the importance of standardised rapid diagnosis of the disease and continuous surveillance of circulating bacteria in Europe for data reporting/comparison and evaluation of the effectiveness of current vaccination policies. 

This laboratory handbook, which is an update of a previous version, provides an overview of recommended laboratory diagnostic and molecular surveillance methods for the detection, identification and typing of B. pertussis, including key aspects of laboratory safety. It highlights the appropriate use of different approaches throughout the course of the disease, which is essential for accurate diagnosis. The handbook has been developed by members of the European Reference Laboratory for Public Health on Diphtheria and Pertussis (EURL-PH-DIPE) consortium in collaboration with counterparts at ECDC. 

For the purposes of diagnosis, the handbook describes the core methodology for nasopharyngeal sampling, culture, PCR, and antibody detection, including clearly defined targets, cut-offs, and guidance for interpretation. In addition, novel diagnostic techniques, such as point-of-care assays, are introduced. 

For the purposes of bacterial surveillance, the handbook outlines fundamental approaches, with particular focus on genes encoding vaccine antigens. It also includes an easy-to-use protocol for measuring vaccine antigen expressions of the bacteria. Given the global increase in macrolide-resistant B. pertussis , special emphasis is placed on the rapid detection of such resistance in clinical samples and retrospective analysis of existing isolates. To support this, both PCR- and sequencing-based methods, and culture-based susceptibility testing are presented, along with possible cut-off values for macrolide resistance, although no such recommendations have been established in Europe to date. 

Whole genome sequencing (WGS) is currently widely used for bacterial typing. Therefore, this handbook includes a dedicated chapter covering the methodology, from DNA extraction and instrumentation to data analysis and comparison of the results using the BIGSdb-Pasteur Bordetella database. 

The handbook also provides scientific context for the various approaches described, guiding laboratories in their decision-making processes. The correct application and interpretation of different diagnostic methodologies, and WGS will be crucial for standardised diagnosis and bacterial surveillance, and to enable meaningful comparisons of pertussis data within and between European countries.
It should be noted that it is not possible to cover all existing methods and protocols within the scope of this handbook. In addition, although the protocols included in the appendices are detailed, they may need to be
adapted for implementation in different laboratory settings.

What’s new in the 2026 edition

This updated edition of the laboratory handbook introduces several important revisions reflecting the evolving epidemiology of pertussis and advances in diagnostic and molecular surveillance technologies. Following the substantial resurgence of pertussis cases, the appearance of macrolide resistant Bordetella pertussis after the COVID-19 pandemic and the emergence of new laboratory needs across Europe, the handbook has been revised to incorporate more timely, sensitive and scalable diagnostic approaches. 

Readers will find a clear transition away from several legacy methods—such as routine MLVA and PFGE typing— towards modern genomic platforms, including expanded use of WGS, updated antigen-genotyping schemes, and improved PCR-based and point-of-care diagnostic solutions. The new version also introduces strengthened guidance on sampling quality, updates to PCR targets, expanded protocols for macrolide-resistance detection, and enhanced biosafety and biosecurity considerations. Together, these updates provide a more robust, harmonised framework to support accurate diagnosis, high-resolution molecular surveillance, and rapid detection of evolving B. pertussis lineages across EU Member States.

Description of EURL-PH-DIPE

The aim of the European Reference Laboratory for Public Health on Diphtheria and Pertussis is to strengthen the guidance and support to ECDC and the NRL network. The EURL activities are organised in seven work packages (WP) with clearly defined objectives over the course of seven years. This includes three network meetings; two updates of the laboratory handbook for pertussis; 10 rounds of External Quality Assessment (EQA) (seven for pertussis and three for diphtheria); a European Union/European Economic Area (EU/EEA) seroprevalence study of anti-PT, anti-DT and anti-TT antibodies; two Gap analyses for diphtheria; four wet-lab trainings; 14 twining training visits and seven on-line scientific seminars (one per year). 

The EURL-PH-DIPE consortium is led by the University of Turku (UTU), Finland, which manages network coordination, the organisation of EQA studies, training activities, and seroprevalence studies. Other members include Institut Pasteur (IP), which leads the activities related to B. pertussis and C. diphtheriae whole genome sequencing (WGS) analysis; Vrije Universiteit Brussel (VUB) and Sciensano (SCI) which jointly contribute to both pertussis and diphtheria activities, particularly in diagnostic methodologies and seroprevalence studies, and Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit (LGL) – a WHO Collaborating Centre for Diphtheria which contributes to most diphtheria-related activities.