Analysis of volatile biomarkers from in vitro cell cultures and exhaled breath for ventilator-associated pneumonia diagnosis

  • Oluwasola Lawal

Student thesis: Phd


Ventilator-associated pneumonia (VAP) is one of the most prevalent nosocomial infections in intensive care units. The current diagnostic methodology consists of culture enrichment, and selective and differential plating methods which are limited in terms of accuracy and speed. Furthermore, the specimen used for these processes is invasively obtained. Consequently, this contributes towards increased morbidity and mortality, antibiotic resistance, and financial burden. Exhaled breath volatile organic compounds (VOCs) are postulated as a suitable diagnostic alternative which would eliminate the aforementioned disadvantages. These VOCs originate from exogenous, endogenous, and microbial sources. Of clinical interest are endogenous and microbial VOCs. This is because the former can potentially provide information on the metabolic state of individuals, whilst the latter may help with pathogen distinction. Thus several investigations were conducted to elucidate these metabolites. To elucidate VOCs of microbial origin, a range of in vitro studies were conducted utilising a combination of headspace, thermal desorption-gas chromatography-mass spectrometry, and statistical analysis. Firstly, commonly implicated VAP pathogens (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus) were cultured separately for discriminating VOCs. Unique VOCs for each bacterium incorporating both previously reported (indole for E. coli, 1-undecene for P. aeruginosa) and novel VOCs (1-hexanol, cyclopentanone) were observed to distinguish these bacterial species. Secondly, a study to investigate the potential to differentiate between mono- and co-cultures was conducted and yielded unique VOCs (2-methylbutyl acetate and methyl 2-methylbutyrate) in the latter and may potentially translate to distinction between mono- and poly-microbial infections. Thirdly, a simple model using A549 epithelial cells and P. aeruginosa to mimic the potential interaction of bacteria and the epithelial lining of the airways was also explored to elucidate discriminatory VOCs (tert-butyl ethyl ether and methyl tert-butyl ether). Furthermore, an aspect of the host immune response was mimicked by culturing A549 epithelial cells with hydrogen peroxide, an oxidant produced during neutrophilic response. An increase in alkane-based VOCs was observed. A clinical study (BreathDx) exploring the potential of VAP diagnosis through exhaled breath VOC measurement was on-going in parallel to these experiments. Before participant recruitment began, method development was performed: different sorbents were tested using a VOC standard mixture and ‘pooled’ breath from healthy volunteers. Breakthrough volume experiments were also conducted to ascertain the sampling volume. Tenax GR was the sorbent choice and a sampling volume of 1200 mL to be used. Early findings from the BreathDx study are also reported. Jointly, the research reported in this thesis demonstrates the potential for VOCs to be utilised in the diagnosis of VAP and beyond.
Date of Award1 Aug 2019
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorRoyston Goodacre (Supervisor) & Stephen Fowler (Supervisor)

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