The oral microbiome and its effect on exhaled breath volatile analysis-the elephant in the room.

The rapid transfer of volatiles from alveolar blood into the lungs and then out of the body in exhaled breath leads to the common and natural conclusion that these volatiles provide information on health and metabolic processes, with considerable potential as biomarkers for use in the screening, diagnosis and monitoring of diseases. Whilst these exhaled volatiles could well serve as biomarkers for human metabolic processes, thereby providing insights into the clinical and nutritional status of individuals, there exist various confounding factors that limit their easy application. A major confounding factor is the introduction of microbially produced oral volatiles into the exhaled breath, yet these volatiles are often ignored in discovery volatile research studies. Here, we provide a comparative cross-sectional study of selected volatiles commonly found in exhaled breath. Using gas chromatography-ion mobility spectrometry, we monitored these selected volatiles in nasal and oral end-tidal exhaled breath samples from twenty-one volunteers. The signal intensities from untargeted volatile detection were analysed for variances using principal component analysis (PCA), revealing a clear separation correlated with the sampling method. Four compounds representing sampling method-independent (acetone, isoprene, methanol, and 2-pentanone) and four corresponding to sampling method-dependent (1-propanol, 2-propanol, ethanol, and acetoin) were identified and selected based on their high PCA loadings. These compounds are further analysed and discussed to illustrate the extent to which the oral microbiome can influence volatile concentrations in exhaled breath. An additional noteworthy finding of this study is that the nasally sampled selected exhaled volatiles are little influenced by the inhalation route (oral or nasal). The outcome from this study is clear, namely that in order to reduce the influence of the oral microbiome on untargeted discovery breath research studies, end-tidal exhaled nasal breath samples should be taken for endogenous volatile analysis, otherwise oral microbial volatiles could be falsely identified as biomarkers. This is particularly important given the continuous rise in the use of machine learning algorithms and artificial intelligence to identify variations in volatilomes. The development and commercialisation of simple, user-friendly and comfortable end-tidal exhaled nasal sample collection devices are required for nasal sampling to become widely adopted.