Ventilator-associated pneumonia (VAP) is a healthcare-acquired infection arising from the invasion of the lower respiratory tract by opportunistic pathogens in ventilated patients. The current method of diagnosis requires the culture of an airway sample such as bronchoalveolar lavage, which is invasive to obtain and may take up to seven days to identify a causal pathogen, or indeed rule out infection. While awaiting results, patients are administered empirical antibiotics; risks of this approach include lack of effect on the causal pathogen, contribution to the development of antibiotic resistance and downstream effects such as increased length of intensive care stay, cost, morbidity and mortality. Specific biomarkers which could identify causal pathogens in a timely manner are needed as they would allow judicious use of the most appropriate antimicrobial therapy. Volatile organic compound (VOC) analysis in exhaled breath is proposed as an alternative due to its non-invasive nature and its potential to provide rapid diagnosis at the patient’s bedside. VOCs in exhaled breath originate from exogenous, endogenous, as well as microbial sources. To identify potential markers, VAP-associated pathogens Escherichia coli , Klebsiella pneumoniae , Pseudomonas aeruginosa , and Staphylococcus aureus were cultured in both artificial sputum medium and nutrient broth, and their headspaces were sampled and analysed for VOCs. Previously reported volatile markers were identified in this study, including indole and 1-undecene, alongside compounds that are novel to this investigation, cyclopentanone and 1-hexanol. We further investigated media components (substrates) to identify those that are essential for indole and cyclopentanone production, with potential implications for understanding microbial metabolism in the lung.

Early and accurate recognition of respiratory pathogens is crucial to prevent increased risk of mortality in critically ill patients. Microbial-derived volatile organic compounds (mVOCs) in exhaled breath could be used as noninvasive biomarkers of infection to support clinical diagnosis. In this study, we investigated the diagnostic potential of in vitro–confirmed mVOCs in the exhaled breath of patients under mechanical ventilation from the BreathDx study. Samples were analyzed by thermal desorption–gas chromatography–mass spectrometry. Pathogens from bronchoalveolar lavage (BAL) cultures were identified in 45 of 89 patients and Staphylococcus aureus was the most commonly identified pathogen (n = 15). Of 19 mVOCs detected in the in vitro culture headspace of 4 common respiratory pathogens (S. aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli), 14 were found in exhaled breath samples. Higher concentrations of 2 mVOCs were found in the exhaled breath of patients infected with S. aureus compared to those without (3-methylbutanal: P < .01, area under the receiver operating characteristic curve [AUROC] = 0.81–0.87; and 3-methylbutanoic acid: P = .01, AUROC = 0.79–0.80). In addition, bacteria identified from BAL cultures that are known to metabolize tryptophan (E. coli, Klebsiella oxytoca, and Haemophilus influenzae) were grouped and found to produce higher concentrations of indole compared to breath samples with culture-negative (P = .034) and other pathogen-positive (P = .049) samples. This study demonstrates the capability of using mVOCs to detect the presence of specific pathogen groups with potential to support clinical diagnosis. Although not all mVOCs were found in patient samples within this small pilot study, further targeted and qualitative investigation is warranted using multicenter clinical studies.

The exhaled breath represents an ideal matrix for noninvasive biomarker discovery, and exhaled metabolomics have the potential to be clinically useful in the era of precision medicine. In this concise translational review, we specifically address volatile organic compounds in the breath, with a view toward fulfilling the promise of these as actionable biomarkers, in particular, for lung diseases. We review the literature paying attention to seminal work linked to key milestones in breath research; discuss potential applications for breath biomarkers across disease areas and healthcare systems, including the perspectives of industry; and outline critical aspects of study design that will need to be considered for any pivotal research going forward if breath analysis is to provide robust validated biomarkers that meet the requirements for future clinical implementation.

Methods for breath sampling and analysis require robust quality assessment to minimise the risk of false discoveries. Planning large-scale multi-site breath metabolite profiling studies also requires careful consideration of systematic and random variation as a result of sampling and analysis techniques. In this study we use breath sample data from the recent U-BIOPRED cohort to evaluate and discuss some important methodological considerations such as batch variation and correction, variation between sites, storage and transportation, as well as inter-instrument analytical differences. Based on this we provide a summary of recommended best practices for new large scale multi-site studies.

Pulmonary aspergillosis can cause serious complications in people with a suppressed immune system. Volatile metabolites emitted by Aspergillus spp. have shown promise for early detection of pathogenicity. However, volatile profiles require further research, as effective headspace analysis methods are required for extended chemical coverage of the volatome; in terms of both very volatile and semi-volatile compounds. In this study, we describe a novel adaptable sampling method in which fungal headspace samples can be sampled continuously throughout a defined time period using both active (pumped) and passive (diffusive) methods, with the capability for samples to be stored for later off-line analysis. For this method we utilise thermal desorption-gas chromatography-mass spectrometry to generate volatile metabolic profiles using Aspergillus fumigatus as the model organism. Several known fungal-specific volatiles associated with secondary metabolite biosynthesis (including α-pinene, camphene, limonene, and several sesquiterpenes) were identified. A comparison between the wild-type A. fumigatus with a phosphopantetheinyl transferase null mutant strain (ΔpptA) that is compromised in secondary metabolite synthesis, revealed reduced production of sesquiterpenes. We also showed the lack of terpene compounds production during the early growth phase, whilst pyrazines were identified in both early and late growth phases. We have demonstrated that the fungal volatome is dynamic and it is therefore critically necessary to sample the headspace across several time periods using a combination of active and passive sampling techniques to analyse and understand this dynamism.