Bordetella pertussis, the main etiological agent of whooping cough, is highly infectious and despite widespread vaccination, is re-merging as a significant pathogen globally. In the pre-vaccination era, B. pertussis the leading case of infantile death due to an infectious disease globally. In the decades following the introduction of vaccination in Australia there were on average 5000 notifications per year, in 2011 there were over 38,000 and questions remain as to why widespread vaccination cannot control this highly infectious disease. Thus, molecular surveillance of currently circulating strains becomes paramount to the control of B. pertussis by providing information on strains and for future vaccine development.
However, surveillance of B. pertussis relies on laboratory isolates, yet since the early 1990s, B. pertussis is primarily detected in clinical samples by PCR. Thus, live culture isolates are rarely collected and there is now little material for molecular surveillance.
The objective was to develop a method, independent of culture, to detect and type B. pertussis in respiratory specimens to provide molecular surveillance data.
The study compared commercial depletions kits and specimen-processing methods using selective lysis detergents and determine which method produced optimised data yield by assessing DNA concentration and purity. Commercial depletion kits included the MolYsis Kit, Qiagen Microbiome Kit, NEBNext Kit on B. pertussis spiked nasopharygeal aspirates, following manufacturer protocols. Saponin was used as published previously by Hasan et al (2016). Samples were then tested by rtPCR for ERV3 and IS481, then sequenced and analysed.
Results showed that while commercial kits significantly reduced the human DNA within the sample, it also reduced the concentration of B. pertussis as well. However, selective lysis with Saponin resulted in an almost undetectable level of human DNA, with minimal loss of bacterial DNA. Read depth improved with a 500% increase in sequencing reads that belonged to B. pertussis. This investigation drew upon the need for new methods of surveillance of B. pertussis and delivered a potential protocol.