Improved methods for the detection of cystic fibrosis pathogens

Hatfield, Lauren Rebecca (2022) Improved methods for the detection of cystic fibrosis pathogens. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

Lung infection is the leading cause of morbidity and mortality in people with cystic fibrosis (CF). Routine microbiological surveillance is crucial to guide effective antimicrobial therapies. Culture-based methods are currently used for identification and surveillance of CF pathogens in respiratory samples. However, molecular methods such as quantitative PCR (qPCR) and microbiota sequencing can be more sensitive and informative. In this thesis, qPCR assays were optimised for the detection and enumeration of CF pathogens, and then applied along with microbiota sequencing to two clinical studies. During the coronavirus pandemic in-person clinics were reduced, highlighting the need for remote microbiological surveillance. In a postal study, microbiology of fresh and posted samples from adult patients were assessed. Key pathogens were still detected in postal samples with culture (A. xylosoxidans 85%, B. cepacia complex 100%, P. aeruginosa 85%, S. aureus 87%, S. maltophilia 57%) and qPCR (A. xylosoxidans 70%, B. cepacia complex 95%, P. aeruginosa 87%, S. aureus 82%, S. maltophilia 65%). No overall significant difference was seen in pathogen abundances (P>0.05 for all targeted pathogens) between fresh and posted samples, however, there was variation in pathogen abundances and microbiota compositions in individual samples. This indicated posted samples could be utilised for remote microbiological surveillance, but with caution. CF lung disease is characterised by periods of worsening symptoms, known as pulmonary exacerbations, which contribute to progressive loss of lung function. In an exacerbation study, microbiology was retrospectively assessed in sputum samples at start and end of antibiotic treatment for exacerbation, and at follow up. Pathogen targeted qPCR and microbiota sequencing indicated no significant difference (P>0.05) in the presence or abundances of CF pathogens or the bacterial microbiota between samples irrespective of antibiotic therapy. Prospective application of pathogen targeted qPCR could be a useful approach to inform effective treatment for exacerbation, allowing changes in antibiotic regimen to be implemented during treatment. The work presented in this thesis highlights the utility of pathogen targeted qPCR and microbiota sequencing in providing rapid and accurate representation of the CF lung bacteria. Based on this, it is possible to recommend prospective use of such molecular approaches for pathogen detection and surveillance to enhance management of CF lung infections.