Assessment of breath volatile organic compounds in acute cardiorespiratory breathlessness: a protocol describing a prospective real-world observational study

Introduction Patients presenting with acute undifferentiated breathlessness are commonly encountered in admissions units across the UK. Existing blood biomarkers have clinical utility in distinguishing patients with single organ pathologies but have poor discriminatory power in multifactorial presentations. Evaluation of volatile organic compounds (VOCs) in exhaled breath offers the potential to develop biomarkers of disease states that underpin acute cardiorespiratory breathlessness, owing to their proximity to the cardiorespiratory system. To date, there has been no systematic evaluation of VOC in acute cardiorespiratory breathlessness. The proposed study will seek to use both offline and online VOC technologies to evaluate the predictive value of VOC in identifying common conditions that present with acute cardiorespiratory breathlessness. Methods and analysis A prospective real-world observational study carried out across three acute admissions units within Leicestershire. Participants with self-reported acute breathlessness, with a confirmed primary diagnosis of either acute heart failure, community-acquired pneumonia and acute exacerbation of asthma or chronic obstructive pulmonary disease will be recruited within 24 hours of admission. Additionally, school-age children admitted with severe asthma will be evaluated. All participants will undergo breath sampling on admission and on recovery following discharge. A range of online technologies including: proton transfer reaction mass spectrometry, gas chromatography ion mobility spectrometry, atmospheric pressure chemical ionisation-mass spectrometry and offline technologies including gas chromatography mass spectroscopy and comprehensive two-dimensional gas chromatography-mass spectrometry will be used for VOC discovery and replication. For offline technologies, a standardised CE-marked breath sampling device (ReCIVA) will be used. All recruited participants will be characterised using existing blood biomarkers including C reactive protein, brain-derived natriuretic peptide, troponin-I and blood eosinophil levels and further evaluated using a range of standardised questionnaires, lung function testing, sputum cell counts and other diagnostic tests pertinent to acute disease. Ethics and dissemination The National Research Ethics Service Committee East Midlands has approved the study protocol (REC number: 16/LO/1747). Integrated Research Approval System (IRAS) 198921. Findings will be presented at academic conferences and published in peer-reviewed scientific journals. Dissemination will be facilitated via a partnership with the East Midlands Academic Health Sciences Network and via interaction with all UK-funded Medical Research Council and Engineering and Physical Sciences Research Council molecular pathology nodes. Trial registration number NCT0367299

Domiciliary exhaled nitric oxide and eosinophilic airway inflammation in adults with asthma

Exhaled nitric oxide home monitoring is a promising surrogate marker of eosinophilic airway inflammation in asthma

Sputum mediator profiling and relationship to airway wall geometry imaging in severe asthma

Background: Severe asthma is a heterogeneous disease and the relationship between airway inflammation and airway remodelling is poorly understood. We sought to define sputum mediator profiles in severe asthmatics categorised by CT-determined airway geometry and sputum differential cell counts. Methods: In a single centre cross-sectional observational study we recruited 59 subjects with severe asthma that underwent sputum induction and thoracic CT. Quantitative CT analysis of the apical segment of the right upper lobe (RB1) was performed. Forty-one mediators in sputum samples were measured of which 21 mediators that were assessable in >50% of samples were included in the analyses. Results: Independent of airway geometry, sputum MMP9 and IL-1β were elevated in those groups with a high sputum neutrophil count while sputum ICAM was elevated in those subjects with a low sputum neutrophil count. In contrast, sputum CCL11, IL-1α and fibrinogen were different in groups stratified by both sputum neutrophil count and airway geometry. Sputum CCL11 concentration was elevated in subjects with a low sputum neutrophil count and high luminal and total RB1 area, whereas sputum IL1α was increased in subjects with a high sputum neutrophil count and low total RB1 area. Sputum fibrinogen was elevated in those subjects with RB1 luminal narrowing and in those subjects with neutrophilic inflammation without luminal narrowing. Conclusions: We have demonstrated that sputum mediator profiling reveals a number of associations with airway geometry. Whether these findings reflect important biological phenotypes that might inform stratified medicine approaches requires further investigation

Volatile organic compounds in a headspace sampling system and asthmatics sputum samples.

Background:The headspace of a biological sample contains exogenous VOCs present within the sampling environment which represent the background signal.Study aims:This study aimed to characterise the background signal generated from a headspace sampling system in a clinical site, to evaluate intra- and inter-day variation of background VOC and to understand the impact of a sample itself upon commonly reported background VOC using sputum headspace samples from severe asthmatics.Methods:The headspace, in absence of a biological sample, was collected hourly from 11am to 3pm within a day (time of clinical samples acquisition), and from Monday to Friday in a week, and analysed by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). Chemometric analysis identified 1120 features, 37 of which were present in at least the 80% of all the samples. The analyses of intra- and inter-day background variations were performed on thirteen of the most abundant features, ubiquitously present in headspace samples. The concentration ratios relative to background were reported for the selected abundant VOC in 36 asthmatic sputum samples, acquired from 36 stable severe asthma patients recruited at Glenfield Hospital, Leicester, UK.Results:The results identified no significant intra- or inter-day variations in compounds levels and no systematic bias of z-scores, with the exclusion of benzothiazole, whose abundance increased linearly between 11am and 3pm with a maximal intra-day fold change of 2.13. Many of the identified background features are reported in literature as components of headspace of biological samples and are considered potential biomarkers for several diseases. The selected background features were identified in headspace of all severe asthma sputum samples, albeit with varying levels of enrichment relative to background.Conclusion:Our observations support the need to consider the background signal derived from the headspace sampling system when developing and validating headspace biomarker signatures using clinical samples

Sputum Inflammatory Mediators Are Increased in Aspergillus fumigatus Culture-Positive Asthmatics

Aspergillus fumigatus sensitization and culture in asthma are associated with disease severity and lung function impairment, but their relationship with airway inflammation is poorly understood. We investigated the profile of 24 sputum inflammatory mediators in A. fumigatus culture-positive or-negative moderate-to-severe asthmatics. Fifty-two subjects were recruited from a single center. A. fumigatus was cultured from 19 asthmatics. Asthma control, symptom score, lung function, and sputum cell count were not significantly different between the asthmatics with and without a positive A. fumigatus culture. All of the sputum mediators were numerically increased in subjects with a positive versus negative sputum A. fumigatus culture. Sputum TNF-R2 was significantly elevated (P=0.03) and the mediator that best distinguished A. fumigatus culture-positive from culture-negative subjects (receiver-operator characteristic area under the curve 0.66 [95% CI: 0.51 to 0.82, P=0.045]). A. fumigates-positive culture in moderate-to-severe asthma is associated with increased inflammatory sputum mediators

Volatile organic compounds in a headspace sampling system and asthmatics sputum samples

The headspace of a biological sample contains exogenous volatile organic compounds (VOCs) present within the sampling environment which represent the background signal. This study aimed to characterise the background signal generated from a headspace sampling system in a clinical site, to evaluate intra- and inter-day variation of background VOC and to understand the impact of a sample itself upon commonly reported background VOC using sputum headspace samples from severe asthmatics. The headspace, in absence of a biological sample, was collected hourly from 11am to 3pm within a day (time of clinical samples acquisition), and from Monday to Friday in a week, and analysed by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). Chemometric analysis identified 1120 features, 37 of which were present in at least the 80% of all the samples. The analyses of intra- and inter-day background variations were performed on 13 of the most abundant features, ubiquitously present in headspace samples. The concentration ratios relative to background were reported for the selected abundant VOC in 36 asthmatic sputum samples, acquired from 36 stable severe asthma patients recruited at Glenfield Hospital, Leicester, UK. The results identified no significant intra- or inter-day variations in compounds levels and no systematic bias of z-scores, with the exclusion of benzothiazole, whose abundance increased linearly between 11am and 3pm with a maximal intra-day fold change of 2.13. Many of the identified background features are reported in literature as components of headspace of biological samples and are considered potential biomarkers for several diseases. The selected background features were identified in headspace of all severe asthma sputum samples, albeit with varying levels of enrichment relative to background. Our observations support the need to consider the background signal derived from the headspace sampling system when developing and validating headspace biomarker signatures using clinical samples

Lung Computational Models and the Role of the Small Airways in Asthma

Rationale: Asthma is characterized by disease within the small airways. Several studies have suggested that forced oscillation technique–derived resistance at 5 Hz (R5) 2 resistance at 20 Hz (R20) is a measure of small airway disease; however, there has been limited validation of this measurement to date. Objectives: To validate the use of forced oscillation R5 2 R20 as a measure of small airway narrowing in asthma, and to investigate the role that small airway narrowing plays in asthma. Methods: Patient-based complete conducting airway models were generated from computed tomography scans to simulate the impact of different degrees of airway narrowing at different levels of the airway tree on forced oscillation R5 2 R20 (n = 31). The computational models were coupled with regression models in an asthmatic cohort (n = 177) to simulate the impact of small airway narrowing on asthma control and quality of life. The computational models were used to predict the impact on small airway narrowing of type-2 targeting biologics using pooled data from two similarly design randomized, placebo-controlled biologic trials (n = 137). Measurements and Main Results: Simulations demonstrated that narrowing of the small airways had a greater impact on R5 2 R20 than narrowing of the larger airways and was associated (above a threshold of approximately 40% narrowing) with marked deterioration in both asthma control and asthma quality of life, above the minimal clinical important difference. The observed treatment effect on R5 2 R20 in the pooled trials equated to a predicted small airway narrowing reversal of approximately 40%. Conclusions: We have demonstrated, using computational modeling, that forced oscillation R5 2 R20 is a direct measure of anatomical narrowing in the small airways and that small airway narrowing has a marked impact on both asthma control and quality of life and may be modified by biologics

The variability of volatile organic compounds in the indoor air of clinical environments

The development of clinical breath-analysis is confounded by the variability of background volatile organic compounds (VOC). Reliable interpretation of clinical breath-analysis at individual, and cohort levels requires characterisation of clinical-VOC levels and exposures. Active-sampling with thermal-desorption/gas chromatography-mass spectrometry recorded and evaluated VOC concentrations in 245 samples of indoor air from three sites in a large NHS provider trust in the UK over 27 months. Data deconvolution, alignment and clustering isolated 7344 features attributable to VOC and described the variability (composition and concentration) of respirable clinical VOC. 328 VOC were observed in more than 5% of the samples and 68 VOC appeared in more than 30% of samples. Common VOC were associated with exogenous and endogenous sources and 17 VOC were identified as seasonal differentiators. The presence of metabolites from the anaesthetic sevoflurane, and putative-disease biomarkers in room air, indicated that exhaled VOC were a source of background-pollution in clinical breath-testing activity. With the exception of solvents, and PPE waxes, exhaled VOC concentrations above 3 µg m-3 are unlikely to arise from room air contamination, and in the absence of extensive survey-data, this level could be applied as a threshold for inclusion in studies, removing a potential environmental confounding-factor in developing breath-based diagnostics

Use of the ReCIVA device in breath sampling of patients with acute breathlessness: a feasibility study

Introduction: Investigating acute multifactorial undifferentiated breathlessness and understanding the driving inflammatory processes can be technically challenging in both adults and children. Being able to validate non-invasive methods such as breath analysis would be a huge clinical advance. The ReCIVA ® device allows breath samples to be collected directly onto sorbent tubes at the bedside for analysis of exhaled volatile organic compounds (eVOCs). We aimed to assess the feasibility of using this device in acutely breathless patients. Methods: Adults hospitalised with acute breathlessness and children aged 5-16 years with acute asthma or chronic stable asthma as well as healthy adult and child volunteers were recruited. Breath samples were collected onto sorbent tubes using the ReCIVA® device and sent for analysis by means of two dimensional gas chromatography-mass spectrometry (GCxGC-MS). The NASA Task Load Index (NASA-TLX) was used to assess the perceived task workload of undertaking sampling from the patients’ perspective. Results: Data was available for 65 adults and 61 children recruited. In total, 98.4% of adults and 75.4% of children were able to provide the full target breath sample using the ReCIVA ® device. NASA TLX measurements was available in the adult population with mean values of 3.37 for effort, 2.34 for frustration, 3.8 for mental demand, 2.8 for performance, 3.9 for physical demand and 2.8 for temporal demand. Discussion: This feasibility study demonstrates it is possible and acceptable to collect breath samples from both adults and children at the bedside for breathomics analysis using the ReCIVA® device

Visualisation of exhaled breath metabolites reveals distinct diagnostic signatures for acute cardiorespiratory breathlessness

Breath analysis can be a useful noninvasive way to detect disease. Here, Ibrahim et al. studied the volatile organic compound (VOC) signatures associated with acute cardiorespiratory diseases in patients presenting breathlessness. Using two-dimensional gas chromatography and mass spectrometry, the authors found clusters of VOCs associated with acute heart failure, asthma, chronic obstructive pulmonary disease, and pneumonia. These breath biomarkers correlated with blood-based biomarkers. An acute disease VOC score based on a 101-biomarker panel was associated with 2-year all-cause mortality. This study demonstrates how breathomics can help diagnose disease and further our understanding of metabolic subgroups