Human breath offers several benefits for diagnostic applications, including simple, noninvasive collection. Breath is a rich source of clinically-relevant biological information; this includes a volatile fraction, where greater than 1,000 volatile organic compounds (VOCs) have been described so far, and breath aerosols that carry nucleic acids, proteins, signaling molecules, and pathogens. Many of these factors, especially VOCs, are delivered to the lung by the systemic circulation, and diffusion of candidate biomarkers from blood into breath allows systematic profiling of organismal health. Biomarkers on breath offer the capability to advance early detection and precision medicine in areas of global clinical need. Breath tests are noninvasive and can be performed at home or in a primary care setting, which makes them well-suited for the kind of public screening program that could dramatically improve the early detection of conditions such as lung cancer. Since measurements of VOCs on breath largely report on metabolic changes, this too aids in the early detection of a broader range of illnesses and can be used to detect metabolic shifts that could be targeted through precision medicine. Furthermore, the ability to perform frequent sampling has envisioned applications in monitoring treatment responses. Breath has been investigated in respiratory, liver, gut, and neurological diseases and in contexts as diverse as infectious diseases and cancer. Preclinical research studies using breath have been ongoing for some time, yet only a few breath-based diagnostics tests are currently available and in widespread clinical use. Most recently, tests assessing the gut microbiome using hydrogen and methane on breath, in addition to tests using urea to detect Helicobacter pylori infections have been released, yet there are many more applications of breath tests still to be realized. Here, we discuss the strengths of breath as a clinical sampling matrix and the technical challenges to be addressed in developing it for clinical use. Historically, a lack of standardized methodologies has delayed the discovery and validation of biomarker candidates, resulting in a proliferation of early-stage pilot studies. We will explore how advancements in breath collection and analysis are in the process of driving renewed progress in the field, particularly in the context of gastrointestinal and chronic liver disease. Finally, we will provide a forward-looking outlook for developing the next generation of clinically relevant breath tests and how they may emerge into clinical practice.

Liver diseases are currently diagnosed through liver biopsy. Its invasiveness, costs, and relatively low diagnostic accuracy require new techniques to be sought. Analysis of volatile organic compounds (VOCs) in human bio-matrices has received a lot of attention. It is known that a musty odour characterises liver impairment, resulting in the elucidation of volatile chemicals in the breath and other body fluids such as urine and stool, which may serve as biomarkers of a disease. Aims: This study aims to review all the studies found in the literature regarding VOCs in liver diseases, and to summarise all the identified compounds that could be used as diagnostic or prognostic biomarkers. The literature search was conducted on ScienceDirect and PubMed, and each eligible publication was qualitatively assessed by two independent evaluators using the SANRA critical appraisal tool. Results: In the search, 58 publications were found, and 28 were kept for inclusion: 23 were about VOCs in the breath, one in the bile, three in urine, and one in faeces. Each publication was graded from zero to ten. A graphical summary of the metabolic pathways showcasing the known liver disease-related VOCs and suggestions on how VOC analysis on liver impairment could be applied in clinical practice are given.

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.

The gold standard method for chronic liver diseases diagnosis and staging remains liver biopsy, despite the spread of less invasive surrogate modalities based on imaging and blood biomarkers. Still, more than 50% of chronic liver disease cases are detected at later stages when patients exhibit episodes of liver decompensation. Breath analysis represents an attractive means for the development of non-invasive tests for several pathologies, including chronic liver diseases. In this perspective review, we summarize the main findings of studies that compared the breath of patients with chronic liver diseases against that of control subjects and found candidate biomarkers for a potential breath test. Interestingly, identified compounds with best classification performance are of exogenous origin and used as flavoring agents in food. Therefore, random dietary exposure of the general population to these compounds prevents the establishment of threshold levels for the identification of disease subjects. To overcome this limitation, we propose the exogenous volatile organic compounds (EVOCs) probe approach, where one or multiple of these flavoring agent(s) are administered at a standard dose and liver dysfunction associated with chronic liver diseases is evaluated as a washout of ingested compound(s). We report preliminary results in healthy subjects in support of the potential of the EVOC Probe approach.

Background: Cirrhosis detection in primary care relies on low-performing biomarkers. Consequently, up to 75% of subjects with cirrhosis receive their first diagnosis with decompensation when causal treatments are less effective at preserving liver function. We investigated an unprecedented approach to cirrhosis detection based on dynamic breath testing. Methods: We enrolled 29 subjects with cirrhosis (Child–Pugh A and B), and 29 controls. All subjects fasted overnight. Breath samples were taken using Breath Biopsy^® before and at different time points after the administration of 100 mg limonene. Absolute limonene breath levels were measured using gas chromatography–mass spectrometry. Results: All subjects showed a >100-fold limonene spike in breath after administration compared to baseline. Limonene breath kinetics showed first-order decay in >90% of the participants, with higher bioavailability in the cirrhosis group. At the Youden index, baseline limonene levels showed classification performance with an area under the roc curve (AUROC) of 0.83 ± 0.012, sensitivity of 0.66 ± 0.09, and specificity of 0.83 ± 0.07. The best performing timepoint post-administration was 60 min, with an AUROC of 0.91, sensitivity of 0.83 ± 0.07, and specificity of 0.9 ± 0.06. In the cirrhosis group, limonene bioavailability showed a correlation with MELD and fibrosis indicators, and was associated with signs of portal hypertension. Conclusions: Dynamic limonene breath testing enhances diagnostic performance for cirrhosis compared to static testing. The correlation with disease severity suggests potential for monitoring therapeutic interventions. Given the non-invasive nature of breath collection, a dynamic limonene breath test could be implemented in primary care.