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

Whooping cough (pertussis) has re-emerged despite high vaccine coverage in Australia and many other countries worldwide, partly attributable to genetic adaptation of the causative organism, to vaccines. Therefore, genomic surveillance has become essential to monitor circulating strains for these genetic changes. However, increasing uptake of PCR for the diagnosis of pertussis has affected the availability of cultured isolates for typing. In this study, we evaluated the use of targeted multiplex PCR (mPCR) amplicon sequencing and shotgun metagenomic sequencing for culture-independent typing of directly from respiratory swabs. We developed a nine-target mPCR amplicon assay that could accurately type major lineages [ non- , , non- and epidemic lineages (ELs) 1-5] circulating in Australia. Validation using DNA from isolates and 178 residual specimens collected in 2010-2012 ( = 87) and 2019 ( = 91) showed that mPCR amplicon sequencing was highly sensitive with a limit of detection of 4.6 copies [IS cycle threshold (Ct) 27.3]. Shotgun metagenomic sequencing was successful in genotyping in 84% of clinical specimens with PCR Ct < 24 and was concordant with mPCR typing results. The results revealed an expansion of EL4 strains from 2010 to 2012 to 2019 in Australia and identified unrecognized co-circulating cases of . This study provides valuable insight into the circulating lineages in Australia prior to the COVID-19 pandemic during which border closure and other interventions reduced pertussis cases to an all-time low, and paves the way for the genomic surveillance of in the era of culture-independent PCR-based diagnosis. In this paper, we evaluated the use of targeted multiplex PCR (mPCR) amplicon sequencing and shotgun metagenomic sequencing for culture-independent typing of 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 species called . These fi