Deciphering Bordetella pertussis epidemiology through culture-independent multiplex amplicon and metagenomic sequencing.

Abstract

In this paper, we evaluated the use of targeted multiplex PCR (mPCR) amplicon sequencing and shotgun metagenomic sequencing for culture-independent typing of Bordetella pertussis directly in respiratory swabs. We first developed a novel targeted mPCR amplicon sequencing assay that can type major circulating lineages and validated its accuracy and sensitivity on 178 DNA extracts from clinical swabs. We also demonstrate the feasibility of using deep metagenomic sequencing for determining strain lineage and markers of virulence, vaccine adaptation, macrolide resistance, and co-infections. Our culture-independent typing methods applied to clinical specimens revealed the expansion of a major global epidemic lineage in Australia (termed EL4) just prior to the COVID-19 pandemic. It also detected cases of previously hidden co-infections from another Bordetella species called Bordetella holmesii. These findings offer valuable insight into the circulating pertussis lineages in Australia prior to the COVID-19 pandemic during which border closure and other interventions reduced pertussis cases to an all-time low. It also provides comparative data for future surveillance as pertussis resurgence after the COVID-19 pandemic has been reported this year in Australia and many other countries. Overall, our paper demonstrates the utility, sensitivity, and specificity of mPCR amplicon and metagenomic sequencing-based culture-independent typing of B. pertussis, which not only paves the way for culture-independent genomic surveillance of B. pertussis but also for other pathogens in the era of PCR-based diagnosis.