Hugo Knobel

A European Respiratory Society technical standard: exhaled biomarkers in lung disease

Breath tests cover the fraction of nitric oxide in expired gas (FENO), volatile organic compounds (VOCs), variables in exhaled breath condensate (EBC) and other measurements. For EBC and for FENO, official recommendations for standardised procedures are more than 10 years old and there is none for exhaled VOCs and particles. The aim of this document is to provide technical standards and recommendations for sample collection and analytic approaches and to highlight future research priorities in the field. For EBC and FENO, new developments and advances in technology have been evaluated in the current document. This report is not intended to provide clinical guidance on disease diagnosis and management. Clinicians and researchers with expertise in exhaled biomarkers were invited to participate. Published studies regarding methodology of breath tests were selected, discussed and evaluated in a consensus-based manner by the Task Force members. Recommendations for standardisation of sampling, analysing and reporting of data and suggestions for research to cover gaps in the evidence have been created and summarised. Application of breath biomarker measurement in a standardised manner will provide comparable results, thereby facilitating the potential use of these biomarkers in clinical practice. ERS technical standard: exhaled biomarkers in lung disease http://ow.ly/mAjr309DBOP

Exhaled breath metabolomics as a noninvasive diagnostic tool for acute respiratory distress syndrome

There is a need for biological markers of the acute respiratory distress syndrome (ARDS). Exhaled breath contains hundreds of metabolites in the gas phase, some of which reflect (patho)physiological processes. We aimed to determine the diagnostic accuracy of metabolites in exhaled breath as biomarkers of ARDS. Breath from ventilated intensive care unit patients (n=101) was analysed using gas chromatography and mass spectrometry during the first day of admission. ARDS was defined by the Berlin definition. Training and temporal validation cohorts were used. 23 patients in the training cohort (n=53) had ARDS. Three breath metabolites, octane, acetaldehyde and 3-methylheptane, could discriminate between ARDS and controls with an area under the receiver operating characteristic curve (AUC) of 0.80. Temporal external validation (19 ARDS cases in a cohort of 48) resulted in an AUC of 0.78. Discrimination was insensitive to adjustment for severity of disease, a direct or indirect cause of ARDS, comorbidities, or ventilator settings. Combination with the lung injury prediction score increased the AUC to 0.91 and improved net reclassification by 1.17. Exhaled breath analysis showed good diagnostic accuracy for ARDS, which was externally validated. These data suggest that exhaled breath analysis could be used for the diagnostic assessment of ARDS. Metabolites in the breath of ventilated patients may be used to diagnose the acute respiratory distress syndrome http://ow.ly/uWHF1

BreathDx – molecular analysis of exhaled breath as a diagnostic test for ventilator–associated pneumonia: protocol for a European multicentre observational study

BackgroundThe diagnosis of ventilator-associated pneumonia (VAP) remains time-consuming and costly, the clinical tools lack specificity and a bedside test to exclude infection in suspected patients is unavailable. Breath contains hundreds to thousands of volatile organic compounds (VOCs) that result from host and microbial metabolism as well as the environment. The present study aims to use breath VOC analysis to develop a model that can discriminate between patients who have positive cultures and who have negative cultures with a high sensitivity.Methods/designThe Molecular Analysis of Exhaled Breath as Diagnostic Test for Ventilator-Associated Pneumonia (BreathDx) study is a multicentre observational study. Breath and bronchial lavage samples will be collected from 100 and 53 intubated and ventilated patients suspected of VAP. Breath will be analysed using Thermal Desorption – Gas Chromatography – Mass Spectrometry (TD-GC-MS). The primary endpoint is the accuracy of cross-validated prediction for positive respiratory cultures in patients that are suspected of VAP, with a sensitivity of at least 99% (high negative predictive value).DiscussionTo our knowledge, BreathDx is the first study powered to investigate whether molecular analysis of breath can be used to classify suspected VAP patients with and without positive microbiological cultures with 99% sensitivity.Trial registrationUKCRN ID number 19086, registered May 2015; as well as registration at www.trialregister.nl under the acronym ‘BreathDx’ with trial ID number NTR 6114 (retrospectively registered on 28 October 2016).

Exhaled breath profiles in the monitoring of loss of control and clinical recovery in asthma

Asthma is a chronic inflammatory airway disease, associated with episodes of exacerbations. Therapy with inhaled corticosteroids (ICS) targets airway inflammation, which aims to maintain and restore asthma control. Clinical features are only modestly associated with airways inflammation. Therefore, we hypothesized that exhaled volatile metabolites identify longitudinal changes between clinically stable episodes and loss of asthma control.

Levels of cytokines in broncho-alveolar lavage fluid, but not in plasma, are associated with levels of markers of lipid peroxidation in breath of ventilated ICU patients.

Alkanes and alkenes in the breath are produced through fatty acid peroxidation, which is initialized by reactive oxygen species. Inflammation is an important cause and effect of reactive oxygen species. We aimed to evaluate the association between fatty acid peroxidation products and inflammation of the alveolar and systemic compartment in ventilated intensive care unit (ICU) patients.Volatile organic compounds were measured by gas chromatography and mass spectrometry in the breath of newly ventilated ICU patients within 24 h after ICU admission. Cytokines were measured in non-directed bronchial lavage fluid (NBL) and plasma by cytometric bead array. Correlation coefficients were calculated and presented in heatmaps.93 patients were included. Peroxidation products in exhaled breath were not associated with markers of inflammation in plasma, but were correlated with those in NBL. IL-6, IL-8, IL-1β and TNF-α concentration in NBL showed inverse correlation coefficients with the peroxidation products of fatty acids. Furthermore, NBL IL-10, IL-13, GM-CSF and IFNγ demonstrated positive associations with breath alkanes and alkenes. Correlation coefficients for NBL cytokines were high regarding peroxidation products of n-6, n-7 and particularly in n-9 fatty acids.Levels of lipid peroxidation products in the breath of ventilated ICU patients are associated with levels of inflammatory markers in NBL, but not in plasma. Alkanes and alkenes in breath seems to be associated with an anti-inflammatory, rather than a pro-inflammatory state in the alveoli.

Exhaled Breath Metabolomics for the Diagnosis of Pneumonia in Intubated and Mechanically-Ventilated Intensive Care Unit (ICU)-Patients

The diagnosis of hospital-acquired pneumonia remains challenging. We hypothesized that analysis of volatile organic compounds (VOCs) in exhaled breath could be used to diagnose pneumonia or the presence of pathogens in the respiratory tract in intubated and mechanically-ventilated intensive care unit patients. In this prospective, single-centre, cross-sectional cohort study breath from mechanically ventilated patients was analysed using gas chromatography-mass spectrometry. Potentially relevant VOCs were selected with a p-value < 0.05 and an area under the receiver operating characteristics curve (AUROC) above 0.7. These VOCs were used for principal component analysis and partial least square discriminant analysis (PLS-DA). AUROC was used as a measure of accuracy. Ninety-three patients were included in the study. Twelve of 145 identified VOCs were significantly altered in patients with pneumonia compared to controls. In colonized patients, 52 VOCs were significantly different. Partial least square discriminant analysis classified patients with modest accuracy (AUROC: 0.73 (95% confidence interval (CI): 0.57–0.88) after leave-one-out cross-validation). For determining the colonization status of patients, the model had an AUROC of 0.69 (95% CI: 0.57–0.82) after leave-one-out cross-validation. To conclude, exhaled breath analysis can be used to discriminate pneumonia from controls with a modest to good accuracy. Furthermore breath profiling could be used to predict the presence and absence of pathogens in the respiratory tract. These findings need to be validated externally.

Volatile organic compound signature from co-culture of lung epithelial cell line with Pseudomonas aeruginosa

Bacteria are found ubiquitously within and on nearly every site within humans, including the airways. Microbes interact with airway epithelial cells in lung infections such as ventilator-associated pneumonia (VAP). Development of infection results in the production of oxidants such as hydrogen peroxide that may further damage the epithelium. VAP is difficult to diagnose and associated with significant mortality. Current methods are invasive and time consuming impacting on appropriate therapy, antimicrobial resistance and financial costs. Volatile organic compound (VOC) analysis in exhaled breath is proposed as a tool for early detection due to its non-invasive property and potential to facilitate timely diagnosis. To investigate potential early VOC markers, A549 epithelial cells that were originally isolated from human alveoli were cultured with and without Pseudomonas aeruginosa, and the headspace of the culture vessel analysed using sorbent-based capture of VOCs followed by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS) in order to identify potential discriminatory VOCs. A549 cells were also cultured with hydrogen peroxide to induce oxidative stress in order to investigate potential biomarkers of epithelial cell damage. Previously reported VOCs including acetone and ethanol were observed from the infection experiment along with novel bacterial markers, which we identified as mostly ether based compounds. Alkanes such as decane and octane were also found to be elevated after hydrogen peroxide treatment of A549 cells, likely as a result of peroxidation of oleic acids.

Methodological considerations for large-scale breath analysis studies: lessons from the U-BIOPRED severe asthma project

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.

Analysing exhaled breath during endovenous laser ablation of varicose veins using an electronic nose and gas chromatography-mass spectrometry.

During the last two decades, several minimally invasive techniques have been developed to treat the incompetent great saphenous vein (GSV). In the late 1990s, various endovenous laser treatments were generally used. During this procedure the temperature of the venous wall tissue and blood remaining in the vein can reach temperatures of over 8008C. This causes carbonization and a release of steam bubbles and gases. During and after the endovenous laser ablation (EVLA) procedure patients often report a barbeque-like taste. Even the treating surgeon can sometimes smell an indistinct odour. The sensation of flavour is due to three main chemoreceptor systems: olfaction (sense of smell by nose), gustation (sense of taste by tongue) and trigeminal (sense of irritation of trigeminal receptors). The sense of smell is used to detect patterns of volatile compounds, while the sense of taste is used to detect non-volatile chemicals. Electronic noses (e-noses) have been developed as a simplified ‘electronic’ model of the human olfactory system and were used in the present study to analyse the breath produced during EVLA. In our centre EVLA 980 nm bare tip is being used for the treatment of the GSV and small saphenous vein, using tumescent anaesthesia only. In this study we hypothesized that e-nose assessment can detect changes in exhaled breath molecular profiles during the EVLA procedure. We also hypothesized that non-physiological and elevated volatile organic compounds (VOCs) can be identified and quantified by gas chromatography-mass spectrometry (GC-MS) during EVLA. We aimed to associate these observations with the subjective tastes and odours as reported by the subjects. This is an experimental study in patients eligible for EVLA, and agreed to have local tumescent anaesthesia. Inclusion and exclusion criteria for surgery and method of treatment have been fully described in a previous publication. The study was approved by the medical ethical committee Noord-Holland, The Netherlands, and a written informed consent was signed by all patients. Exhaled breath of 12 patients was analysed using the Cyranose 320 e-nose (Smiths Detection, Pasadena, CA, USA), a hand-held portable chemical vapour analyser, containing a 32-polymer nanocomposite sensor array. Patients needed to be sober and non-smoking for at least three hours, in order to avoid confounding factors for breath analysis. Fifteen minutes before the EVLA, they breathed for five minutes through a nonrebreathing valve (Hans Rudolph, Kansas City, MO, USA) using an inspiratory VOC filter (A2, North Safety, Middelburg, The Netherlands) to avoid contamination by environmental VOCs. An expiratory silica reservoir was used to dry the expired air and a nose clip was used to prevent nasal air leakage. One single vital capacity manoeuvre was done while the expiratory port was connected to a 10-L Tedlar bag. The subject exhaled the full expiratory vital capacity into the bag with an expiratory resistance of 20 cm H2O. 12 The same procedure was repeated during the actual EVLA procedure 3–5 minutes after the start of the heating of the laser wire. Then a second Tedlar bag was filled with a single vital capacity volume. Within ten minutes the Tedlar bag was connected to the e-nose for analysis. Patients recorded whether they did have (yes 1⁄4 1) or did not have (no 1⁄4 0) a ‘barbeque-like’ taste and/or smell during the procedure. The breathprints were analysed using SPSS software (version 18). A paired t-test was assessed to calculate the difference in breathprint measured at both timetables. Then correlation coefficients of these results and changes in perceived taste and smell were calculated. As an explorative procedure, in two out of 12 patients, GC-MS was introduced to perform specific compound analysis.