There are ample data to suggest that early-life dysbiosis of both the gut and/or airway microbiome can predispose a child to develop along a trajectory toward asthma. Although individual studies show clear associations between dysbiosis and asthma development, it is less clear what (collection of) bacterial species is mechanistically responsible for the observed effects. This is partly due to issues related to the asthma diagnosis and the broad spectrum of anatomical sites, sample techniques, and analysis protocols that are used in different studies. Moreover, there is limited attention for potential differences in the genetics of individuals that would affect the outcome of the interaction between the environment and that individual. Despite these challenges, the first bacterial components were identified that are able to affect the transcriptional state of human cells, ergo the immune system. Such molecules could in the future be the basis for intervention studies that are now (necessarily) restricted to a limited number of bacterial species. For this transition, it might be prudent to develop an ex vivo human model of a local mucosal immune system to better and safer explore the impact of such molecules. With this approach, we might move beyond association toward understanding of causality.

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.

Background and Aims:The prevalence of chronic liver disease in adults exceeds 30% in some countries and there is significant interest in developing tests and treatments to help control disease progression and reduce healthcare burden. Breath is a rich sampling matrix that offers non-invasive solutions suitable for early-stage detection and disease monitoring. Having previously investigated targeted analysis of a single biomarker, here we investigated a multiparametric approach to breath testing that would provide more robust and reliable results for clinical use. Methods:To identify candidate biomarkers we compared 46 breath samples from cirrhosis patients and 42 from controls. Collection and analysis used Breath Biopsy OMNI™, maximizing signal and contrast to background to provide high confidence biomarker detection based upon gas chromatography mass spectrometry (GC-MS). Blank samples were also analyzed to provide detailed information on background volatile organic compounds (VOCs) levels. Results:A set of 29 breath VOCs differed significantly between cirrhosis and controls. A classification model based on these VOCs had an area under the curve (AUC) of 0.95±0.04 in cross-validated test sets. The seven best performing VOCs were sufficient to maximize classification performance. A subset of 11 VOCs was correlated with blood metrics of liver function (bilirubin, albumin, prothrombin time) and separated patients by cirrhosis severity using principal component analysis. Conclusions:A set of seven VOCs consisting of previously reported and novel candidates show promise as a panel for liver disease detection and monitoring, showing correlation to disease severity and serum biomarkers at late stage.

Background Breath analysis is a burgeoning field, with interest in volatile organic compounds (VOCs) as a noninvasive diagnostic tool or an outcome measure, but no randomised controlled trials (RCTs) have yet evaluated this technology in a clinical trial longitudinally. In a pilot RCT, our exploratory objectives were feasibility of measuring VOCs via multiple techniques, assessing relationships between VOCs and Haemophilus colonisation and whether CXCR2 antagonism with danirixin altered lung microbiome composition in individuals with COPD. Method 43 participants had VOCs and sputum biomarkers evaluated. VOCs and induced sputum were collected after 6 h of fasting at screening and at days 1, 7 and 14. VOCs were analysed via gas chromatography mass spectrometry (GC-MS), field asymmetric ion mobility spectrometry (FAIMS) and eNose. The primary outcome for these analyses was the relationship between VOCs and Haemophilus abundance determined by 16S rRNA sequencing. Results A joint-effects model demonstrated a modest relationship between four exhaled VOCs and Haemophilus relative abundance (R2=0.55) measured only by GC-MS, but not as measured using gas chromtaography FAIMS or eNose. There was considerable variability in absolute quantities of individual VOCs longitudinally. Conclusions VOC measurement in clinical trials to identify subsets of COPD is feasible, but assessment of new VOC technologies must include concurrent GC-MS validation. Further work to standardise collection of VOCs and measuring a background or “housekeeper” VOC is required to understand and normalise individual VOC quantities.

Many preschool children present with complaints of wheezing during their first years of life. One third of these children will develop asthma later in childhood[1]. Accurate techniques for the early diagnosis of asthma would provide important prognostic information, and by identifying those at risk for developing asthma, allow for targeted studies on interventions for preventing and early treatment of asthma.

Face masks and personal respirators are used to curb the transmission of SARS-CoV-2 in respiratory droplets; filters embedded in some personal protective equipment could be used as a non-invasive sample source for applications, including at-home testing, but information is needed about whether filters are suited to capture viral particles for SARS-CoV-2 detection. In this study, we generated inactivated virus-laden aerosols of 0.3–2 microns in diameter (0.9 µm mean diameter by mass) and dispersed the aerosolized viral particles onto electrostatic face mask filters. The limit of detection for inactivated coronaviruses SARS-CoV-2 and HCoV-NL63 extracted from filters was between 10 to 100 copies/filter for both viruses. Testing for SARS-CoV-2, using face mask filters and nasopharyngeal swabs collected from hospitalized COVID-19-patients, showed that filter samples offered reduced sensitivity (8.5% compared to nasopharyngeal swabs). The low concordance of SARS-CoV-2 detection between filters and nasopharyngeal swabs indicated that number of viral particles collected on the face mask filter was below the limit of detection for all patients but those with the highest viral loads. This indicated face masks are unsuitable to replace diagnostic nasopharyngeal swabs in COVID-19 diagnosis. The ability to detect nucleic acids on face mask filters may, however, find other uses worth future investigation.

Introduction Asthma is one of the most common chronic diseases in childhood and is generally characterized by exercise induced bronchoconstriction (EIB). Assessing EIB is time consuming and expensive as it requires a fully equipped pulmonary function laboratory. Analysis of volatile organic compounds (VOCs) in breath is a novel technique for examining biomarkers which may associate with asthma features. The aim of this pilot study was to identify potential markers in the relationship between EIB and VOCs. Methods Children between four and 14 years old were asked to provide a breath sample prior to undergoing an exercise challenge test to assess for EIB. Results Breath samples were collected and analyzed in 46 asthmatic children, 21 with EIB and 25 without EIB (NO-EIB). Molecular features (MFs) were not significantly different between EIB and NO-EIB controls. 29 of the 46 children were corticosteroid naïve, 10 with EIB and 13 without. In the corticosteroid naïve group EIB was associated with increased MF23 and MF14 in the lower breath sample (p-value < 0.05). Conclusion This pilot study shows that EIB was related to an increased MF14 and MF23 in corticosteroid naïve children. The tentative identities of these compounds are octanal and dodecane/tetradecane respectively. These candidate biomarkers have a potential to enable non-invasive diagnosis of EIB in steroid-naïve children. Trial registration This study is registered in the Netherlands trial register (trial no. NL6087) at 14 February 2017.

3TR, the largest IMI consortium ever in immune diseases, brings clinical researchers and scientists from several disease areas together, in an endeavour to increase the clinical impact of targeted immune-mediated therapies, including asthma and COPDhttps://bit.ly/3kPq0xI

INTRODUCTION: Liver cirrhosis and its complication — hepatocellular carcinoma (HCC) — have been associated with increased exhaled limonene. It is currently unclear whether this increase is more strongly associated with the presence of HCC or with the severity of liver dysfunction. METHODS: We compared the exhaled breath of 40 controls, 32 cirrhotic patients, and 12 cirrhotic patients with HCC using the Breath Biopsy platform. Breath samples were analyzed by thermal desorption–gas chromatography–mass spectrometry. Limonene levels were compared between the groups and correlated to bilirubin, albumin, prothrombin time international normalized ratio, and alanine aminotransferase. RESULTS: Breath limonene concentration was significantly elevated in subjects with cirrhosis-induced HCC (M: 82.1 ng/L, interquartile range [IQR]: 16.33–199.32 ng/L) and cirrhosis (M: 32.6 ng/L, IQR: 6.55–123.07 ng/L) compared with controls (M: 6.2 ng/L, IQR: 2.62–9.57 ng/L) (P value = 0.0005 and 0.0001, respectively) with no significant difference between 2 diseased groups (P value = 0.37). Levels of exhaled limonene correlated with serum bilirubin (R2 = 0.25, P value = 0.0016, r = 0.51), albumin (R2 = 0.58, P value = 5.3e-8, r = −0.76), and international normalized ratio (R2 = 0.29, P value = 0.0003, r = 0.51), but not with alanine aminotransferase (R2 = 0.01, P value = 0.36, r = 0.19). DISCUSSION: Exhaled limonene levels are primarily affected by the presence of cirrhosis through reduced liver functional capacity, as indicated by limonene correlation with blood metrics of impaired hepatic clearance and protein synthesis capacity, without further alterations observed in subjects with HCC. This suggests that exhaled limonene is a potential non-invasive marker of liver metabolic capacity (see Visual abstract, Supplementary Digital Content 1, http://links.lww.com/CTG/A388).

A systematic review of pharmacogenomic studies capturing adverse drug reactions (ADRs) related to asthma medications was undertaken, and a survey of Pharmacogenomics in Childhood Asthma (PiCA) consortia members was conducted. Studies were eligible if genetic polymorphisms were compared with suspected ADR(s) in a patient with asthma, as either a primary or secondary outcome. Five studies met the inclusion criteria. The ADRs and polymorphisms identified were change in lung function tests (rs1042713), adrenal suppression (rs591118), and decreased bone mineral density (rs6461639) and accretion (rs9896933, rs2074439). Two of these polymorphisms were replicated within the paper, but none had external replication. Priorities from PiCA consortia members (representing 15 institution in eight countries) for future studies were tachycardia (SABA/LABA), adrenal suppression/crisis and growth suppression (corticosteroids), sleep/behaviour disturbances (leukotriene receptor antagonists), and nausea and vomiting (theophylline). Future pharmacogenomic studies in asthma should collect relevant ADR data as well as markers of efficacy.

The clinical trial is a tool that most breath researchers will use at some point to address a scientific question. Each clinical study requires a bespoke design that is tailor-made to address the specific study objective(s). As such, this chapter does not list suggested designs, but rather provides a three-step approach toward designing a bespoke trial. Firstly, a framework is provided to help set realistically achievable and useful objectives and subsequently define minimum requirements to address these. The next step is to list constraints within which to operate, as well as specific strengths that can be leveraged. This provides a clear framework for the actual step of designing the study and maximizing its chances of success. This includes considerations on the relevance of understanding clinical utility, applying appropriate quality controls, and performing independent multicenter validation. Research is currently filled with examples of bad trial design that limits the progress of this important field of study toward clinical practice. Adhering to the framework outlined in this chapter will aid in preventing some of the common pitfalls.

Introduction: Accurate and timely diagnosis of exercise induced bronchoconstriction (EIB) is important for disease management, especially in children. Exhaled Volatile Organic Compounds (VOCs) can reflect underlying disease activity in the airways potentially enabling non-invasive diagnosis. Objectives: In this cross-sectional study we investigated whether exhaled VOCs can discriminate between children with and without EIB. Methods: Exhaled breath was collected in 46 children with a clinical suspicion of EIB using the Breath Biopsy platform (Owlstone Medical). EIB was assessed at OCON sports-medical clinic by a standardised exercise challenge with pulmonary function testing prior to and after exercise. EIB was defined as a post-exercise drop in FEV1>13% (on ICS) or a >20% FEV1 reduction (ICS naïve). VOCs were extracted from the chromatogram and matched to library-IDs. Significantly discriminating VOCs were input into a Canonical Discriminant Analysis to subsequently generate a ROC-Curve with AUC value. Results: VOCs did not differentiate between EIB (n= 21, age 8.7±2.2yrs) and controls (n=25, age 8.4±3.1 years), AUC 0.42±0.31. Similar results were found in post-hoc analysis when limiting the analysis to subjects taking ICS (n=23, AUC 0.65±0.12). However, in ICS naïve subjects a significant difference was found (n=23, AUC 0.86±0.08), this was driven by 5 VOCs. Conclusions: Analysis of exhaled VOCs only discriminates patients with and without EIB if they are steroid naïve. Speculatively, this could be related to ICS related suppression of inflammation although results require confirmation in a larger study.

Lung cancer remains the most common cause of cancer related death in both the UK and USA. Development of diagnostic approaches that have the ability to detect lung cancer early are a research priority with potential to improve survival. Analysis of exhaled breath metabolites, or volatile organic compounds (VOCs) is an area of considerable interest as it could fulfil such requirements. Numerous studies have shown that VOC profiles are different in the breath of patients with lung cancer compared to healthy individuals or those with non-malignant lung diseases. This review provides a scientific and clinical assessment of the potential value of a breath test in lung cancer. It discusses the current understanding of metabolic pathways that contribute to exhaled VOC production in lung cancer and reviews the research conducted to date. Finally, we highlight important areas for future research and discuss how a breath test could be incorporated into various clinical pathways.

Breath research has almost invariably focussed on the identification of endogenous volatile organic compounds (VOCs) as disease biomarkers. After five decades, a very limited number of breath tests measuring endogenous VOCs is applied to the clinic. In this perspective article, we explore some of the factors that may have contributed to the current lack of clinical applications of breath endogenous VOCs. We discuss potential pitfalls of experimental design, analytical challenges, as well as considerations regarding the biochemical pathways that may impinge on the application of endogenous VOCs as specific disease biomarkers. We point towards several lines of evidence showing that breath analysis based on administration of exogenous compounds has been a more successful strategy, with several tests currently applied to the clinic, compared to measurement of endogenous VOCs. Finally, we propose a novel approach, based on the use of exogenous VOC (EVOC) probes as potential strategy to measure the activity of metabolic enzymes in vivo, as well as the function of organs, through breath analysis. We present longitudinal data showing the potential of EVOC probe strategies in breath analysis. We also gathered important data showing that administration of EVOC probes induces significant changes compared to previous exposures to the same compounds. EVOC strategies could herald a new wave of substrate-based breath tests, potentially bridging the gap between research tools and clinical applications.

Breath biopsy enables the non-invasive collection and analysis of volatile organic compounds (VOCs) in exhaled breath, providing valuable information about disease processes occurring in the body. Metabolic changes occur in cancer cells at the earliest stages of disease. We discuss progress in the use of breath biopsy for discovery of breath-based biomarkers for early detection of cancer, and potential applications for breath biopsy in enabling precision medicine in cancer.

The Breath Biopsy platform enables the collection and analysis of breath samples, in order to look for volatile organic compound (VOC) biomarkers of disease. The LuCID (lung cancer indicator detection) project is one of the first major deployments of the platform in a research setting, and is currently the largest breath-based trial in the world, recruiting up to 4000 patients. The aim of the LuCID project is to discover VOC biomarkers in breath for early detection of lung cancer, which could improve patient outcomes and save lives. In this talk, we will give an introduction to the LuCID program, then go on to describe the biology underlying the VOC biomarkers, before discussing the inherent challenges associated with breath sampling and analysis. We will conclude by giving an update on the progress of the LuCID trial to date. The LuCID Study: LuCID is an international multi-centre prospective case-control cohort study (ClinicalTrials.gov ID NCT02612532) currently in progress, evaluating breath VOCs in patients with a clinical suspicion of lung cancer. A clinical suspicion is based on symptoms and/or suspicious finding on incidental imaging. Using tidal breathing, patients breathe into the ReCIVA Breath Sampler to collect breath samples on stable sorbent tubes for later analysis by Gas Chromatography-Mass Spectrometry and Field Asymmetric Ion Mobility Spectrometry (FAIMS, Owlstone Medical Ltd). One arm of the study is focused on early detection of lung cancer, with the aim of increasing the number of cases diagnosed at Stages 1 and 2, while an additional arm is currently being initiated looking at differences in breath profiles pre- and post-surgery which has the advantage of allowing the patient to act as their own control. The Biology of VOCs: So why would we believe that VOC biomarkers for lung cancer could be discovered during LuCID? Cancer cells undergo profound changes of their metabolism in order to support high energetic demands of uncontrolled proliferation. Several oncogenic mutations have been shown to affect metabolism of cancer cells by converging to common metabolic pathways linked to cell cycle and anabolic growth. The Warburg effect is among well-established cancer metabolic hallmarks and entails the activation of aerobic glycolysis as main pathway for biosynthetic purposes, as opposed to normal cells that exploit mitochondrial metabolism for their energetic needs. These changes in cellular metabolism favor survival in an oxygen deprived environment and result in altered metabolic intermediates that function as the building blocks for new cells, both enabling the growth of rapidly dividing cancer cells, and also altering the profile of VOCs in breath. As these processes are fundamental to cancer cell survival, such altered metabolism occurs as one of the earliest stages of tumorigenesis, hence VOCs are excellent candidate biomarkers for early detection of cancer. Breath Sampling: Challenges and Solutions: The potential of using breath sampling to identify markers of disease has long been recognised, but has to date seen almost no adoption into clinical practice, with only FeNO and H. pylori breath tests in widespread use. This has largely been due to practical considerations that have made large-scale clinical trials impractical to carry out. Most previous tests have involved collecting breath in bags, which - suffer from chemical losses over time - are vulnerable to contamination from ambient air if they are reused incorrectly - are difficult to transport and store. - only allow the collection of smaller volumes, limiting the sensitivity of the analysis. In this section, we will discuss how the ReCIVA breath sampler, a key part of the Breath Biopsy platform, allows these problems to be overcome, and we will present data, including VOC washout curves monitoring changes in VOC levels over time following ingestion of a peppermint capsule, that demonstrate how the ReCIVA performs in practice. Current Status of the LuCID trial: In the concluding section, we will provide an update on the LuCID trial. We have identified some VOCs for which we have observed associations with certain disease processes in our biomarker discovery phase, and we are currently investigating what the mechanisms associating these VOCs with lung cancer initiation and progression might be. Biomarkers, Early Detection, breath analysis

Rationale: Exhaled breath contains >1,000 volatile organic compounds (VOCs) which originate from metabolic processes, the environment or drug metabolites. Analyzing the breath VOC profile over time may provide insight into the kinetics of uptake and breakdown of exogenous compounds such as drugs. Aim: This proof-of-concept study explores longitudinal analysis of VOCs in exhaled breath as a means to investigate the washout period of an ingested exogenous compound. Methods: Breath samples were collected in quadruplicate before ingestion and at 30 minute intervals for up to 8 hours after ingestion of a peppermint capsule. Breath was collected using the ReCIVA Breath Sampler and analyzed using Gas Chromatography Mass Spectrometry. The peak areas of five peppermint-related compounds (ɑ-pinene, β-pinene, limonene, eucalyptol and (±)-menthol) were quantified. The mean average and range of % relative standard deviation (%RSD) of peak area was calculated for each compound to assess intra-sample reproducibility. Results: 30 minutes after ingestion of the peppermint capsule the levels of peppermint-related VOCs in breath increased compared to baseline, for example ɑ-pinene increased 28-fold (peak area 1.11E+08 +/- 3.73E+06 SD) compared to baseline (3.88E+06 +/- 2.54E+05 SD). All five VOCs followed a washout curve, returning to baseline levels after 6.5 hours. Replicates demonstrated high intra-sample reproducibility, for example the mean average %RSD of peak area for ɑ-pinene was 8.05. Conclusions: We demonstrate the feasibility of breath biopsy to monitor volatile metabolites related to exogenous compounds in a longitudinal study, for example to non-invasively study the kinetics of drugs inside the body.

Research in children should strike the right balance between protecting underage study subjects and advancing the medical field. This study gives insight into the emotional burden that common invasive research procedures in asthma research have on young children, both from the child and parent perspective. Puppetry was used to stimulate children (age 5–6 years) to explain their emotional burden prior to and after the research procedures. We operationalised emotional burden as willingness to participate in future research and reluctance towards participation. Parents filled out a questionnaire on this topic. Symptomatic patients as well as healthy controls were analysed. Forty-one children were included. Children’s anticipatory fear for future research showed a clear decrease of 0.7 ± 1.6 on a 5-point Likert scale as a consequence of participation (p = 0.02). Sixty percent of all participating children explicitly indicated willingness to undergo identical research procedures again. Children uninformed by their parents about the venipuncture were significantly more reluctant to the venipuncture after the procedure (p < 0.01), compared to children who had been informed (4.0 ± 0.9 resp. 2.8 ± 1.2).

There is an urgent need for methods to detect lung cancer earlier. If detected early, over half of lung cancer patients could be cured with existing treatments. Therefore, our greatest opportunity lies in increasing rates of early diagnosis through improved cancer screening. Exhaled breath contains over 1,000 Volatile Organic Compounds (VOCs), which are the products of metabolic activity, hence they directly reflect the current state of cells and represent a valuable source of information about the health of an individual. As the earliest stages of tumor development are characterized by profound changes in cellular metabolic activity, VOCs are potential non-invasive biomarkers for early detection of lung cancer. The LuCID study aims to collect breath samples and evaluate VOCs in exhaled breath as non-invasive biomarkers for early detection of lung cancer.