Pathogen identification in lower respiratory tract infection

Thesis

Treatment of lower respiratory tract infection (pneumonia and pleural infection) relies on the use of empirical broad spectrum antibiotics, primarily because reliable pathogen identification occurs infrequently. Another consequence of poor rates of pathogen identification is that our understanding of the microbiology of these infections is incomplete.

This thesis addresses some of these issues by combining the acquisition of high quality lower respiratory tract samples, free from nasooropharyngeal contamination, with novel molecular microbiological techniques in an attempt to increase rates of pathogen identification.

Four main areas are examined;

1. The role of so-called ‘atypical pneumonia’ bacteria in causing pleural infection. These pathogens have been previously identified in the pleural space infrequently and routine culture usually fails to isolate such bacteria. High sensitivity nested polymerase chain reaction (PCR) is a culture-independent technique which is used to undertake a systematic evaluation for these pathogens in pleural infection samples.
2. The role of Pneumocystis jirovecii in pleural infection, either as a co-infecting pathogen or in monomicrobial infection. This fungus causes severe pneumonia, particularly in the immunosuppressed, but is increasingly recognised as a co-pathogen in community-acquired pneumonia, and is frequently isolated in the upper and lower respiratory tract in health. A high sensitivity real-time PCR assay is used to examine for this fungus.
3. Ultra-deep sequencing of the 16S rRNA gene is used to perform a comprehensive microbial survey in samples taken from the multi-centre MIST2 study of pleural infection. The techniques employed allow analysis of polymicrobial samples and give very high taxonomic resolution, whilst incorporating methods to control for potential contamination. Further, these techniques provide confirmation of the results from the ‘atypical’ bacteria nested PCR study.
4. Bedside ultrasound-guided percutaneous transthoracic needle aspiration (TNA) of consolidated lung is undertaken in patients with pneumonia, as part of the PIPAP study. An evaluation is undertaken of the efficacy and acceptability of TNA. Aspirate samples acquired are also processed using ultra-deep sequencing of the 16S rRNA gene. Other samples obtained as part of the PIPAP study, such as ‘control’ lung aspirates and ‘control’ pleural fluid samples, are similarly processed to enable calculation of sensitivity and specificity of the sequencing methodology.

Authors: Wrightson, JM

• Publication date: 2014
• DOI: 10.5287/ora-o88o2xrq0
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: 16S rRNA; diagnostics; pneumonia; pleural infection; microbiology; empyema
• Subjects: Clinical laboratory sciences; Respiratory medicine; Medical Sciences; Clinical microbiology; Infectious diseases