External quality assurance scheme for Bordetella pertussis antimicrobial susceptibility testing, 2022

Pertussis (whooping cough) is an acute bacterial infection usually caused by Bordetella pertussis, which can affect people of all ages [1-2]. Macrolides [erythromycin (ERY) and azithromycin (AZT)] are the recommended drugs to treat B. pertussis infection in many countries [2, 3]. To date, macrolide resistant B. pertussis isolates have mainly been found in China, but such isolates have also been found sporadically in Europe, the Middle East and in North and South America [4-10]. In a previous ECDC-funded (ECDC/2015/009) European Pertussis Laboratory Surveillance Network (EUPert-LabNet) project, training for antimicrobial susceptibility testing was provided to reference laboratories from a number of European countries.

This report presents the results of the first external quality assurance (EQA) scheme for B. pertussis antimicrobial susceptibility testing by ECDC as part of the European Reference Laboratory Network for Pertussis (ERLNPert-Net) consortium. Sixteen laboratories in 16 countries (out of 28 invited, 57.1%) participated in the EQA scheme, however results were only obtained from 13 of the 16 (81.3%) laboratories as three (18.9%) were unable to perform testing. Participating laboratories were from or working on behalf of 16 EU/EEA countries.

The specific aims of this B. pertussis AST EQA scheme were to: evaluate the ability of participating laboratories to perform genotypic antimicrobial susceptibility testing of B. pertussis based on nucleic acid analysis; to assess differences in methodology, interpretation and reporting of results; to identify future training needs; and to assist with the establishment of ‘best practice’ in current assays, interpretation and reporting.

In this EQA, the test panel included 11 samples. These samples contained DNA extracted from macrolide sensitive and resistant B. pertussis (in different concentrations), DNA from clinical samples which tested negative for B. pertussis and PBS (as no DNA sample). The EQA scheme was designed for genotypic identification of sensitive/resistant B. pertussis (PCR/Sanger sequencing/WGS based). This strategy was feasible as the only known mechanism for B. pertussis macrolide resistance is associated with a single point mutation (A2047G) within its 23S rRNA gene.

For identification of macrolide resistant B. pertussis, different protocols were used. Of the 13 laboratories that provided the results, 11 (84.6%) used block-based PCR, one (7.7%) used qPCR and 2 (15.4%) used Sanger sequencing and two (15.4%) used whole genome sequencing (WGS). Block-based PCR was used solely by nine (69.2%) laboratories and Sanger sequencing by two (15.4%) laboratories. The other two (15.4%) laboratories used multiple techniques.

Results for the first EQA on antimicrobial susceptibility testing of macrolide sensitive/resistant B. pertussis were encouraging as nine (69.2%) laboratories had only one or no samples incorrectly reported: five out of 13 (38.5%) laboratories scored 11/11 (100%, intended results) correctly and four (30.8%) laboratories scored 10/11 correctly. The remaining four laboratories had five to nine panel samples correctly identified. The results indicate that low DNA concentration was a major obstacle for application of the WGS approach for identification of resistant/sensitive B. pertussis in EQA samples, and that PCR based methods provided better results.

Pertussis national reference laboratories (NRLs) should be able to correctly perform antimicrobial susceptibility testing (AST) based on genotyping and phenotyping. This EQA round highlighted training needs in the identification of macrolide resistant B. pertussis by DNA based approaches. Furthermore, guidelines, such as proper test controls, importance of DNA concentration suitable for detection with different methods and how to interpret the results would provide added value.