The Role of Exhaled Breath Condensate in Chronic Inflammatory and Neoplastic Diseases of the Respiratory Tract.

Asthma and chronic obstructive pulmonary disease (COPD) are among the most common chronic respiratory diseases. Chronic inflammation of the airways leads to an increased production of inflammatory markers by the effector cells of the respiratory tract and lung tissue. These biomarkers allow the assessment of physiological and pathological processes and responses to therapeutic interventions. Lung cancer, which is characterized by high mortality, is one of the most frequently diagnosed cancers worldwide. Current screening methods and tissue biopsies have limitations that highlight the need for rapid diagnosis, patient differentiation, and effective management and monitoring. One promising non-invasive diagnostic method for respiratory diseases is the assessment of exhaled breath condensate (EBC). EBC contains a mixture of volatile and non-volatile biomarkers such as cytokines, leukotrienes, oxidative stress markers, and molecular biomarkers, providing significant information about inflammatory and neoplastic states in the lungs. This article summarizes the research on the application and development of EBC assessment in diagnosing and monitoring respiratory diseases, focusing on asthma, COPD, and lung cancer. The process of collecting condensate, potential issues, and selected groups of markers for detailed disease assessment in the future are discussed. Further research may contribute to the development of more precise and personalized diagnostic and treatment methods.

Clinical Application of Exhaled Breath Condensate in Diagnosis of Diseases.

Respiratory diseases are highly prevalent in the general population, and the morbidity, mortality, and healthcare burden on society at large have been on the rise worldwide. For example, lung cancer is a major contributor to cancer-related mortality around the globe, and identifying clinically relevant biomarkers for lung cancer detection at both early and metastatic stages has been a pressing need. Human metabolism is complicated and may vary with different individuals. Despite advances in the treatment and the early screening of respiratory diseases, most diagnoses are established at a late stage, i.e., when genetic and epigenetic changes have developed. A promising source of biomarkers indicative of the pathogenesis of respiratory diseases is exhaled breath condensate (EBC), a biological fluid and a natural matrix of the respiratory tract. Molecules, such as DNAs, RNAs, proteins, metabolites, and others, are found in EBC, and their presence/absence or changes in concentrations can serve as biomarkers. This review discusses the exhaled breath composition, candidate EBC biomarkers, and the potential to use EBC for diagnosing diseases, therapeutic monitoring, and screening high-risk individuals.

Oxylipin concentration shift in exhaled breath condensate (EBC) of SARS-CoV-2 infected patients.

Infection of airway epithelial cells with severe acute respiratory coronavirus 2 (SARS-CoV-2) can lead to severe respiratory tract damage and lung injury with hypoxia. It is challenging to sample the lower airways non-invasively and the capability to identify a highly representative specimen that can be collected in a non-invasive way would provide opportunities to investigate metabolomic consequences of COVID-19 disease. In the present study, we performed a targeted metabolomic approach using liquid chromatography coupled with high resolution chromatography (LC-MS) on exhaled breath condensate (EBC) collected from hospitalized COVID-19 patients (COVID+) and negative controls, both non-hospitalized and hospitalized for other reasons (COVID-). We were able to noninvasively identify and quantify inflammatory oxylipin shifts and dysregulation that may ultimately be used to monitor COVID-19 disease progression or severity and response to therapy. We also expected EBC-based biochemical oxylipin changes associated with COVID-19 host response to infection. The results indicated ten targeted oxylipins showing significative differences between SAR-CoV-2 infected EBC samples and negative control subjects. These compounds were prostaglandins A2 and D2, LXA4, 5-HETE, 12-HETE, 15-HETE, 5-HEPE, 9-HODE, 13-oxoODE and 19(20)-EpDPA, which are associated with specific pathways (i.e. P450, COX, 15-LOX) related to inflammatory and oxidative stress processes. Moreover, all these compounds were up-regulated by COVID+, meaning their concentrations were higher in subjects with SAR-CoV-2 infection. Given that many COVID-19 symptoms are inflammatory in nature, this is interesting insight into the pathophysiology of the disease. Breath monitoring of these and other EBC metabolites presents an interesting opportunity to monitor key indicators of disease progression and severity.

A non-invasive method for the detection of glucose in human exhaled breath by condensation collection coupled with ion chromatography.

An analytical method for the detection of glucose in human exhaled breath by non-invasive condensation collection coupled with ion chromatography was developed. A self-designed exhaled breath condensation device was constructed, through which human exhaled breath was condensed and collected. And the glucose in collected human exhaled breath condensate (EBC) was analyzed by ion chromatography with a pulsed amperometric instrument. The standard EBC collection method was established, and the key factors such as cooling temperature and sampling flow rate during condensation collection were investigated deeply, and a good linear correlation between blood glucose levels and exhaled breath glucose levels was obtained. The intra-day precision was 3.37%, and the inter-day precision was 3.83%. Furthermore, EBC from healthy people and diabetic patients was collected in fasting state and after meal. We found the breath glucose level in healthy volunteers was 0.11-2.34 ng/L and that in diabetic patients was 0.23-135.92 ng/L. In after meal samples, the breath glucose level is 10-100 times higher than that of healthy subjects, which offering the prospect of a non-invasive approach to the monitoring of diabetes.

Exhaled Breath Condensate (EBC) analysis of circulating tumour DNA (ctDNA) using a lung cancer specific UltraSEEK oncogene panel.

INTRODUCTION: Small diagnostic tissue samples can be inadequate in testing an expanding list of validated oncogenic driver alterations and fail to reflect intratumour heterogeneity (ITGH) in lung cancer. Liquid biopsies are non-invasive and may better reflect ITGH. Most liquid biopsies are performed in the context of circulating tumour DNA (ctDNA) in plasma but Exhaled Breath Condensate (EBC) shows promise as a lung-specific liquid biopsy. METHODS: In this prospective, proof-of-concept study we carried out targeted Next Generation Sequencing (NGS) on diagnostic tissue samples from 125 patients with lung cancer and compared results to plasma and EBC for 5 oncogenic driver mutations (EGFR, KRAS, PIK3CA, ERBB2, BRAF) using an ultrasensitive PCR technique (UltraSEEK™ Lung Panel on the MassARRAY® System, Agena Bioscience, San Diego, CA, USA). RESULTS: There was a significantly higher failure rate due to unamplifiable DNA in tissue NGS (57/125, 45.6%) compared to plasma (27/125, 21.6%, p < 0.001 and EBC (26/125,20.8%, p ≤ 0.001. Consequently, both plasma and EBC identified higher number of mutations compared to tissue NGS. Specifically, there were significantly higher numbers of mutations detected in EGFR, KRAS and PIK3CA in plasma (p = 9.82 × 10-3, p = 3.14 × 10-5, p = 1.95 × 10-3) and EBC (p = 2.18 × 10-3, p = 2.28 × 10-4,p = 0.016) compared to tissue NGS. There was considerable divergence in mutation profiles between plasma and EBC with 34/76 (44%) mutations detected in plasma and 37/74 (41.89%) in EBC unique to their respective liquid biopsy. CONCLUSIONS: The results suggest that EBC is effective in identifying clinically relevant alterations in patients with lung cancer using UltraSEEK™ and has a potential role as an adjunct to plasma testing.

Insights into the Profile of the Human Expiratory Microbiota and Its Associations with Indoor Microbiotas.

Microorganisms residing in the human respiratory tract can be exhaled, and they constitute a part of environmental microbiotas. However, the expiratory microbiota community and its associations with environmental microbiotas remain poorly understood. Here, expiratory bacteria and fungi and the corresponding microbiotas from the living environments were characterized by DNA amplicon sequencing of residents' exhaled breath condensate (EBC) and environmental samples collected from 14 residences in Nanjing, China. The microbiotas of EBC samples, with a substantial heterogeneity, were found to be as diverse as those of skin, floor dust, and airborne microbiotas. Model fitting results demonstrated the role of stochastic processes in the assembly of the expiratory microbiota. Using a fast expectation-maximization algorithm, microbial community analysis revealed that expiratory microbiotas were differentially associated with other types of microbiotas in a type-dependent and residence-specific manner. Importantly, the expiratory bacteria showed a composition similarity with airborne bacteria in the bathroom and kitchen environments with an average of 12.60%, while the expiratory fungi showed a 53.99% composition similarity with the floor dust fungi. These differential patterns indicate different relationships between expiratory microbiotas and the airborne microbiotas and floor dust microbiotas. The results here illustrated for the first time the associations between expiratory microbiotas and indoor microbiotas, showing a potential microbial exchange between the respiratory tract and indoor environment. Thus, improved hygiene and ventilation practices can be implemented to optimize the indoor microbial exposome, especially in indoor bathrooms and kitchens.

Review of non-invasive detection of SARS-CoV-2 and other respiratory pathogens in exhaled breath condensate.

In 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged to cause high viral infectivity and severe respiratory illness in humans (COVID-19). Worldwide, limited pandemic mitigation strategies, including lack of diagnostic test availability, resulted in COVID-19 overrunning health systems and spreading throughout the global population. Currently, proximal respiratory tract (PRT) specimens such as nasopharyngeal swabs are used to diagnose COVID-19 because of their relative ease of collection and applicability in large scale screening. However, localization of SARS-CoV-2 in the distal respiratory tract (DRT) is associated with more severe infection and symptoms. Exhaled breath condensate (EBC) is a sample matrix comprising aerosolized droplets originating from alveolar lining fluid that are further diluted in the DRT and then PRT and collected via condensation during tidal breathing. The COVID-19 pandemic has resulted in recent resurgence of interest in EBC collection as an alternative, non-invasive sampling method for the staging and accurate detection of SARS-CoV-2 infections. Herein, we review the potential utility of EBC collection for detection of SARS-CoV-2 and other respiratory infections. While much remains to be discovered in fundamental EBC physiology, pathogen-airway interactions, and optimal sampling protocols, EBC, combined with emerging detection methods, presents a promising non-invasive sample matrix for detection of SARS-CoV-2.

Inactivation of SARS-CoV-2 in clinical exhaled breath condensate samples for metabolomic analysis.

Exhaled breath condensate (EBC) is routinely collected and analyzed in breath research. Because it contains aerosol droplets, EBC samples from SARS-CoV-2 infected individuals harbor the virus and pose the threat of infectious exposure. We report for the first time a safe and consistent method to fully inactivate SARS-CoV-2 in EBC samples and make EBC samples safe for processing and analysis. EBC samples containing infectious SARS-CoV-2 were treated with several concentrations of acetonitrile. The most commonly used 10% acetonitrile treatment for EBC processing failed to completely inactivate the virus in samples and viable virus was detected by the assay of SARS-CoV-2 infection of Vero E6 cells in a biosafety level 3 laboratory. Treatment with either 50% or 90% acetonitrile was effective to completely inactivate the virus, resulting in safe, non-infectious EBC samples that can be used for metabolomic analysis. Our study provides SARS-CoV-2 inactivation protocol for the collection and processing of EBC samples in the clinical setting and for advancing to metabolic assessments in health and disease.

Exhaled breath biomarkers of influenza infection and influenza vaccination.

Respiratory viral infections are considered a major public health threat, and breath metabolomics can provide new ways to detect and understand how specific viruses affect the human pulmonary system. In this pilot study, we characterized the metabolic composition of human breath for an early diagnosis and differentiation of influenza viral infection, as well as other types of upper respiratory viral infections. We first studied the non-specific effects of planned seasonal influenza vaccines on breath metabolites in healthy subjects after receiving the immunization. We then investigated changes in breath content from hospitalized patients with flu-like symptoms and confirmed upper respiratory viral infection. The exhaled breath was sampled using a custom-made breath condenser, and exhaled breath condensate (EBC) samples were analysed using liquid chromatography coupled to quadruplole-time-of-flight mass spectrometer (LC-qTOF). All metabolomic data was analysed using both targeted and untargeted approaches to detect specific known biomarkers from inflammatory and oxidative stress biomarkers, as well as new molecules associated with specific infections. We were able to find clear differences between breath samples collected before and after flu vaccine administration, together with potential biomarkers that are related to inflammatory processes and oxidative stress. Moreover, we were also able to discriminate samples from patients with flu-related symptoms that were diagnosed with confirmatory respiratory viral panels (RVPs). RVP positive and negative differences were identified, as well as differences between specific viruses defined. These results provide very promising information for the further study of the effect of influenza A and other viruses in human systems by using a simple and non-invasive specimen like breath.

A mask-based diagnostic platform for point-of-care screening of Covid-19.

Diagnostics of SARS-CoV-2 infection using real-time reverse-transcription polymerase chain reaction (RT-PCR) on nasopharyngeal swabs is now well-established, with saliva-based testing being lately more widely implemented for being more adapted for self-testing approaches. In this study, we introduce a different concept based on exhaled breath condensate (EBC), readily collected by a mask-based sampling device, and detection with an electrochemical biosensor with a modular architecture that enables fast and specific detection and quantification of COVID-19. The face mask forms an exhaled breath vapor containment volume to hold the exhaled breath vapor in proximity to the EBC collector to enable a condensate-forming surface, cooled by a thermal mass, to coalesce the exhaled breath into a 200-500 µL fluid sample in 2 min. EBC RT-PCR for SARS-CoV-2 genes (E, ORF1ab) on samples collected from 7 SARS-CoV-2 positive and 7 SARS-CoV-2 negative patients were performed. The presence of SARS-CoV-2 could be detected in 5 out of 7 SARS-CoV-2 positive patients. Furthermore, the EBC samples were screened on an electrochemical aptamer biosensor, which detects SARS-CoV-2 viral particles down to 10 pfu mL-1 in cultured SARS-CoV-2 suspensions. Using a "turn off" assay via ferrocenemethanol redox mediator, results about the infectivity state of the patient are obtained in 10 min.

Novel LC-MS-TOF method to detect and quantify ascorbic and uric acid simultaneously in different biological matrices.

Ascorbic acid (AA) and uric acid (UA) are known as two of the major antioxidants in biological fluids. We report a novel liquid chromatography-mass spectrometry with time-of-flight (LC-MS-TOF) method for the simultaneous quantification of ascorbic and uric acids using MPA, antioxidant solution and acetonitrile as a protein precipitating agent. Both compounds were separated from interferences using a reverse phase C18 column with water and acetonitrile gradient elution (both with formic acid) and identified and quantified by MS in the negative ESI mode. Isotope labeled internal standards were also added to ensure the accuracy of the measures. The method was validated for exhaled breath condensate (EBC), nasal lavage (NL) and plasma samples by assessing selectivity, linearity, accuracy and precision, recovery and matrix effect and stability. Sample volumes below 250 µL were used and linear ranges were determined between 1 - 25 and 1 - 40 µg/mL for ascorbic and uric acid, respectively, for plasma samples, and between 0.05 - 5 (AA) and 0.05 - 7.5 (UA) µg/mL for EBC and NL samples. The new method was successfully applied to real samples from subjects that provided each of the studied matrices. Results showed higher amounts determined in plasma samples, with similar profiles for AA and UA in EBC and NL but at much lower concentrations.

Effects of continuous positive airway pressure on resolvin and matrix metalloproteinase-9 in patients with obstructive sleep apnea.

PURPOSE: Resolvin is a checkpoint controller in inflammation. Matrix metalloproteinase-9 (MMP-9) is an airway remodeling regulator. We evaluated the levels of resolvin and MMP-9 protein in the serum and exhaled breath condensate (EBC) before and after continuous positive airway pressure (CPAP) treatment. METHOD: We enrolled 20 non-OSA snorers and 40 patients with moderate to severe OSA scheduled for CPAP treatment. ELISA was used to assess resolvin and MMP-9 levels in the serum and EBC. All patients underwent sleep assessment at baseline and 3 months after CPAP. RESULTS: There was no between-group difference; moreover, there were no differences in the pre- and post-treatment serum levels of resolvin and MMP-9 in patients with OSA. Compared with non-OSA snorers, patients with OSA had lower resolvin and higher MMP-9 levels in the EBC. After CPAP treatment, the EBC levels of resolvin and MMP-9 in patients with OSA returned to normal. CONCLUSIONS: Successful OSA treatment by CPAP can normalize EBC levels of resolvin and MMP-9.

Occupational exposure to graphene and silica nanoparticles. Part II: pilot study to identify a panel of sensitive biomarkers of genotoxic, oxidative and inflammatory effects on suitable biological matrices.

The available biomonitoring studies on workers producing/handling nanomaterials (NMs) focused on potential effects on respiratory, immune and cardio-vascular system. Aim of this study was to identify a panel of sensitive biomarkers and suitable biological matrices to evaluate particularly genotoxic and oxidative effects induced on workers unintentionally exposed to graphene or silica nanoparticles during the production process. These nanomaterials have been chosen for 'NanoKey' project, integrating the workplace exposure assessment (reported in part I) with the biomonitoring of exposed workers reported in the present work. Simultaneously to workplace exposure characterization, we monitored the workers using: Buccal Micronucleus Cytome (BMCyt) assay, fpg-comet test (lymphocytes), oxidized DNA bases 8-oxoGua, 8-oxoGuo and 8-oxodGuo measurements (urine), analysis of oxidative stress biomarkers in exhaled breath condensate (EBC), FENO measurement and cytokines release detection (serum). Since buccal cells are among the main targets of NM occupational exposure, particular attention was posed to the BMCyt assay that represents a noninvasive assay. This pilot study, performed on 12 workers vs.11 controls, demonstrates that BMCyt and fpg-comet assays are the most sensitive biomarkers of early, still reparable, genotoxic and oxidative effects. The findings suggest that these biomarkers could represent useful tools for the biomonitoring of workers exposed to nanoparticles, but they need to be confirmed on a high number of subjects. However, such biomarkers don't discriminate the effects of NM from those due to other chemicals used in the NM production process. Therefore, they could be suitable for the biomonitoring of workers exposed to complex scenario, including nanoparticles exposure.

Effects of continuous positive airway pressure on exhaled transforming growth factor-ß and vascular endothelial growth factor in patients with obstructive sleep apnea.

BACKGROUND: Both transforming growth factor ß (TGF-ß) and vascular endothelial growth factor (VEGF) are master regulators of airway remodeling; however, their pathological roles in obstructive sleep apnea (OSA) remain unclear. The aim of the present study was to evaluate the expression of TGF-ß and VEGF protein in the serum and exhaled breath condensate (EBC) before and after continuous positive airway pressure (CPAP) treatment in OSA patients. METHODS: Forty patients with moderate to severe OSA requiring CPAP and 20 healthy subjects were prospectively recruited. The concentrations of TGF-ß and VEGF protein in the serum and EBC were evaluated by enzyme-linked immunosorbent assay. All OSA patients underwent a sleep study that was repeated 3 months after receiving CPAP therapy. RESULTS: Protein concentrations of TGF-ß and VEGF in the serum did not differ between healthy controls and OSA patients before CPAP treatment. There was also no difference in the serum protein concentrations of TGF-ß and VEGF of the OSA patients before and after CPAP treatment. However, both the TGF-ß and VEGF protein concentrations in the EBC were higher in the OSA patients than those in control subjects, and recovered to normal levels after CPAP. CONCLUSIONS: Successful treatment of OSA by CPAP can restore the TGF-ß and VEGF protein concentrations in the EBC.

NanoTiO2 Sunscreen Does Not Prevent Systemic Oxidative Stress Caused by UV Radiation and a Minor Amount of NanoTiO2 is Absorbed in Humans.

The present pilot study tested the efficiency of nanoTiO2 sunscreen to prevent the oxidative stress/inflammation caused by ultraviolet (UV) radiation using biomarkers in subjects' blood, urine, and exhaled breath condensate (EBC). In addition, the skin absorption of nanoTiO2 was studied. Six identical subjects participated in three tests: (A) nanoTiO2 sunscreen, (B) UV radiation, and (C) sunscreen + UV. The first samples were collected before the test and the second after sunscreen application and/or UV exposure. On day 4, the third samples were collected, and the sunscreen was washed off, and the fourth samples were collected on day 11. The following biomarkers were measured: malondialdehyde, 4-hydroxy-trans-hexenal, 4-hydroxy-trans-nonenal, aldehydes C6-C12, 8-iso-Prostaglandin F2α, o-tyrosine, 3-chlorotyrosine, 3-nitrotyrosine, 8-hydroxy-2-deoxyguanosine, 8-hydroxyguanosine, 5-hydroxymethyl uracil, and leukotrienes, using liquid chromatography-electrospray ionisation-tandem mass spectrometry. Titania was measured using inductively coupled plasma mass spectrometry and TiO2 nanoparticles by transmission and scanning electron microscopy. Sunscreen alone did not elevate the markers, but UV increased the biomarkers in the plasma, urine, and EBC. The sunscreen prevented skin redness, however it did not inhibit the elevation of oxidative stress/inflammatory markers. Titania and nanoTiO2 particles were found in the plasma and urine (but not in the EBC) in all sunscreen users, suggesting their skin absorption.

Deep Airway Inflammation and Respiratory Disorders in Nanocomposite Workers.

Thousands of researchers and workers worldwide are employed in nanocomposites manufacturing, yet little is known about their respiratory health. Aerosol exposures were characterized using real time and integrated instruments. Aerosol mass concentration ranged from 0.120 mg/m³ to 1.840 mg/m³ during nanocomposite machining processes; median particle number concentration ranged from 4.8 × 104 to 5.4 × 105 particles/cm³. The proportion of nanoparticles varied by process from 40 to 95%. Twenty employees, working in nanocomposite materials research were examined pre-shift and post-shift using spirometry and fractional exhaled nitric oxide (FeNO) in parallel with 21 controls. Pro-inflammatory leukotrienes (LT) type B4, C4, D4, and E4; tumor necrosis factor (TNF); interleukins; and anti-inflammatory lipoxins (LXA4 and LXB4) were analyzed in their exhaled breath condensate (EBC). Chronic bronchitis was present in 20% of researchers, but not in controls. A significant decrease in forced expiratory volume in 1 s (FEV1) and FEV1/forced vital capacity (FVC) was found in researchers post-shift (p ˂ 0.05). Post-shift EBC samples were higher for TNF (p ˂ 0.001), LTB4 (p ˂ 0.001), and LTE4 (p ˂ 0.01) compared with controls. Nanocomposites production was associated with LTB4 (p ˂ 0.001), LTE4 (p ˂ 0.05), and TNF (p ˂ 0.001), in addition to pre-shift LTD4 and LXB4 (both p ˂ 0.05). Spirometry documented minor, but significant, post-shift lung impairment. TNF and LTB4 were the most robust markers of biological effects. Proper ventilation and respiratory protection are required during nanocomposites processing.

Effect of temperature control on the metabolite content in exhaled breath condensate.

The non-invasive, quick, and safe collection of exhaled breath condensate makes it a candidate as a diagnostic matrix in personalized health monitoring devices. The lack of standardization in collection methods and sample analysis is a persistent limitation preventing its practical use. The collection method and hardware design are recognized to significantly affect the metabolomic content of EBC samples, but this has not been systematically studied. Here, we completed a series of experiments to determine the sole effect of collection temperature on the metabolomic content of EBC. Temperature is a likely parameter that can be controlled to standardize among different devices. The study considered six temperature levels covering two physical phases of the sample; liquid and solid. The use of a single device in our study allowed keeping saliva filtering and collector surface effects as constant parameters and the temperature as a controlled variable; the physiological differences were minimized by averaging samples from a group of volunteers and a period of time. After EBC collection, we used an organic solvent rinse to collect the non-water-soluble compounds from the condenser surface. This additional matrix enhanced metabolites recovery, was less dependent on temperature changes, and may possibly serve as an additional pointer to standardize EBC sampling methodologies. The collected EBC samples were analyzed with a set of mass spectrometry methods to provide an overview of the compounds and their concentrations present at each temperature level. The total number of volatile and polar non-volatile compounds slightly increased in each physical phase as the collection temperature was lowered to minimum, 0 °C for liquid and -30, -56 °C for solid. The low-polarity non-volatile compounds showed a weak dependence on the collection temperature. The metabolomic content of EBC samples may not be solely dependent on temperature but may be influenced by other phenomena such as greater sample dilution due to condensation from the ambient air at colder temperatures, or due to adhesion properties of the collector surface and occurring chemical reactions. The relative importance of other design parameters such as condenser coating versus temperature requires further investigation.

Dataset of breath research manuscripts curated using PubMed search strings from 1995-2016.

The data contained in this article are PubMed search strings and search string builders used to curate breath research manuscripts published from 1995-2016 and the respective number of articles found that satisfied the search requirements for selected categories. Breath sampling represents a non-invasive technique that has gained usefulness for public health, clinical, diagnostic, and environmental exposure assessment applications over the years. This data article includes search strings that were utilized to retrieve publications through the PubMed database for different breath research-related topics that were related to the analysis of exhaled breath, exhaled breath condensate (EBC), and exhaled breath aerosol (EBA) as well as the analysis of cellular headspace. Manuscripts were curated for topics including EBC, EBA, Direct MS, GC-MS, LC-MS, alcohol, and sensors. A summary of the number of papers published per year for the data retrieved using each of the search strings is also included. These data can be utilized to discern trends in the number of breath research publications in each of the different topics over time. A supplementary Appendix A containing the titles, author lists, journal names, publication dates, PMID numbers, and EntrezUID numbers for each of the journal articles curated using the finalized search strings for the seven breath research-related topics can also be found within this article. The selected manuscripts can be used to explore the impact that breath research has had on expanding the scientific knowledge in each of the investigated topics.

Evolution of clinical and environmental health applications of exhaled breath research: Review of methods and instrumentation for gas-phase, condensate, and aerosols.

Human breath, along with urine and blood, has long been one of the three major biological media for assessing human health and environmental exposure. In fact, the detection of odor on human breath, as described by Hippocrates in 400 BC, is considered the first analytical health assessment tool. Although less common in comparison to contemporary bio-fluids analyses, breath has become an attractive diagnostic medium as sampling is non-invasive, unlimited in timing and volume, and does not require clinical personnel. Exhaled breath, exhaled breath condensate (EBC), and exhaled breath aerosol (EBA) are different types of breath matrices used to assess human health and disease state. Over the past 20 years, breath research has made many advances in assessing health state, overcoming many of its initial challenges related to sampling and analysis. The wide variety of sampling techniques and collection devices that have been developed for these media are discussed herein. The different types of sensors and mass spectrometry instruments currently available for breath analysis are evaluated as well as emerging breath research topics, such as cytokines, security and airport surveillance, cellular respiration, and canine olfaction.