Molecular detection and epidemiological studies of atypical bacteria causing respiratory tract infections

Abstract: Respiratory infections are common causes of morbidity and mortality. Chlamydia pneumoniae, Mycoplasma pneumoniae and Bordetella pertussis cause respiratory infection, often with similar symptoms. Molecular diagnostic methods are preferred since these bacteria are difficult to culture. The aim of this thesis was to evaluate and improve the diagnostics and knowledge of the epidemiology of these bacteria.A real-time polymerase chain reaction (PCR) method targeting the IS481 element present in the genome of B. pertussis was compared to culture and serology results, and a duplex real-time PCR method was constructed for detecting C. pneumoniae and M. pneumoniae, which was compared to two endpoint PCR methods. Both real-time PCR methods showed high sensitivity and specificity.Typing of 624 M. pneumoniae samples, collected from 1996 to 2017 from four counties, was performed by P1 typing and multiple-locus variable number tandem repeat analysis (MLVA). A polyclonal distribution of strains was seen over all epidemic periods, but strains of P1 type 2/variant 2 and MLVA types 3-5-6-2 and 4-5-7-2 predominated in 2010−2013. A shift from type 2 strains to different variant 2 strains was seen and a new variant, 2e, was detected in 2016−2017. An A2063G mutation associated with macrolide resistance was detected by a fluorescence resonance energy transfer (FRET) PCR method in one (0.16%) of 608 M. pneumoniae strains.Molecular characterisation using whole-genome sequencing of 93 B. pertussis isolates, collected between 1986 and 2016 from three counties showed that there were polyclonal strains in the county of Dalarna, Gävleborg and Uppsala in the years 2014−2016. Changes in virulence-related genes were detected: a shift from isolates harbouring the ptxP3 allele in favour of ptxP1 was seen, and almost all isolates had a disrupted prn gene. No detection of macrolide resistance in B. pertussis was detected.In conclusion, the validated real-time PCR methods for detection of B. pertussis, C. pneumoniae and M. pneumoniae have led to improved diagnostic methods for use in clinical laboratories. The molecular characterisation of M. pneumoniae and B. pertussis strains has contributed to the wider understanding of the genetic changes that has occurred over the epidemic periods, but further studies is needed.