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

BACKGROUND: 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. OBJECTIVES: To determine whether exhaled volatile organic compounds (VOCs) as measured by gas-chromatography/mass-spectrometry (GC/MS) and electronic nose (eNose) technology discriminate between clinically stable and unstable episodes of asthma. METHODS: Twenty-three patients with (partly) controlled mild to moderate persistent asthma using ICS were included in this prospective steroid withdrawal study. Exhaled metabolites were measured at baseline, during loss of control and after recovery. Standardized sampling of exhaled air was performed, after which samples were analysed by GC/MS and eNose. Univariate analysis of covariance (ANCOVA), followed by multivariate principal component analysis (PCA) was used to reduce data dimensionality. Next paired t tests were utilized to analyse within-subject breath profile differences at the different time-points. Finally, associations between exhaled metabolites and sputum inflammation markers were examined. RESULTS: Breath profiles by eNose showed 95% (21/22) correct classification for baseline vs loss of control and 86% (19/22) for loss of control vs recovery. Breath profiles using GC/MS showed accuracies of 68% (14/22) and 77% (17/22) for baseline vs loss of control and loss of control vs recovery, respectively. Significant associations between exhaled metabolites captured by GC/MS and sputum eosinophils were found (Pearson r≥.46, P<.01). CONCLUSIONS & CLINICAL RELEVANCE: Loss of asthma control can be discriminated from clinically stable episodes by longitudinal monitoring of exhaled metabolites measured by GC/MS and particularly eNose. Part of the uncovered biomarkers was associated with sputum eosinophils. These findings provide proof of principle for monitoring and identification of loss of asthma control by breathomics.

Detection of Airway Colonization by Aspergillus fumigatus by Use of Electronic Nose Technology in Patients with Cystic Fibrosis.

Currently, there is no noninvasive test that can reliably diagnose early invasive pulmonary aspergillosis (IA). An electronic nose (eNose) can discriminate various lung diseases through an analysis of exhaled volatile organic compounds. We recently published a proof-of-principle study showing that patients with prolonged chemotherapy-induced neutropenia and IA have a distinct exhaled breath profile (or breathprint) that can be discriminated with an eNose. An eNose is cheap and noninvasive, and it yields results within minutes. We determined whether Aspergillus fumigatus colonization may also be detected with an eNose in cystic fibrosis (CF) patients. Exhaled breath samples of 27 CF patients were analyzed with a Cyranose 320. Culture of sputum samples defined the A. fumigatus colonization status. eNose data were classified using canonical discriminant analysis after principal component reduction. Our primary outcome was cross-validated accuracy, defined as the percentage of correctly classified subjects using the leave-one-out method. The P value was calculated by the generation of 100,000 random alternative classifications. Nine of the 27 subjects were colonized by A. fumigatus. In total, 3 subjects were misclassified, resulting in a cross-validated accuracy of the Cyranose detecting IA of 89% (P = 0.004; sensitivity, 78%; specificity, 94%). Receiver operating characteristic (ROC) curve analysis showed an area under the curve (AUC) of 0.89. The results indicate that A. fumigatus colonization leads to a distinctive breathprint in CF patients. The present proof-of-concept data merit external validation and monitoring studies.

Loss of asthma control and activation of coagulation and fibrinolysis.

BACKGROUND: Epidemiologic studies have shown that patients with severe asthma have increased risk of pulmonary embolism, in particular patients with frequent asthma exacerbations. Therefore, we hypothesized that asthma exacerbations are associated with increased haemostatic activity. OBJECTIVE: To investigate whether induced loss of asthma control is associated with changes in coagulation and fibrinolytic parameters in peripheral blood. METHODS: We performed a prospective, inhaled steroid withdrawal study in 23 patients with moderate to moderately severe asthma, consisting of a baseline visit and a visit after loss of asthma control. During the visits, we measured asthma control questionnaire (ACQ), atopy, lung function, inflammatory markers (eosinophils and neutrophils), and haemostatic parameters in plasma. RESULTS: Complete cessation of inhaled corticosteroids led to a loss of asthma control in 22 of 23 patients. We found increased asthma symptoms (ACQ 0.9 vs. 2.9, P < 0.01), significantly reduced lung function (forced expiratory volume in 1 s (FEV1) 3.51L vs. 3.13L, P < 0.01) and increased levels of eosinophils in plasma (0.26 × 10(E9)/L vs. 0.16 × 10(E9)/L, P = 0.03) in patients after loss of asthma control. However, we observed no significant changes in the coagulation and fibrinolysis parameters. CONCLUSION: Loss of asthma control after cessation of inhaled corticosteroids does not lead to increased haemostatic activation in patients with moderate to moderately severe asthma. This suggests that more severe inflammation or additional risk factors are required for activation of coagulation or reduction of fibrinolysis in asthma.

Integration of electronic nose technology with spirometry: validation of a new approach for exhaled breath analysis.

New 'omics'-technologies have the potential to better define airway disease in terms of pathophysiological and clinical phenotyping. The integration of electronic nose (eNose) technology with existing diagnostic tests, such as routine spirometry, can bring this technology to 'point-of-care'. We aimed to determine and optimize the technical performance and diagnostic accuracy of exhaled breath analysis linked to routine spirometry. Exhaled breath was collected in triplicate in healthy subjects by an eNose (SpiroNose) based on five identical metal oxide semiconductor sensor arrays (three arrays monitoring exhaled breath and two reference arrays monitoring ambient air) at the rear end of a pneumotachograph. First, the influence of flow, volume, humidity, temperature, environment, etc, was assessed. Secondly, a two-centre case-control study was performed using diagnostic and monitoring visits in day-to-day clinical care in patients with a (differential) diagnosis of asthma, chronic obstructive pulmonary disease (COPD) or lung cancer. Breathprint analysis involved signal processing, environment correction based on alveolar gradients and statistics based on principal component (PC) analysis, followed by discriminant analysis (Matlab2014/SPSS20). Expiratory flow showed a significant linear correlation with raw sensor deflections (R(2) = 0.84) in 60 healthy subjects (age 43 ± 11 years). No correlation was found between sensor readings and exhaled volume, humidity and temperature. Exhaled data after environment correction were highly reproducible for each sensor array (Cohen's Kappa 0.81-0.94). Thirty-seven asthmatics (41 ± 14.2 years), 31 COPD patients (66 ± 8.4 years), 31 lung cancer patients (63 ± 10.8 years) and 45 healthy controls (41 ± 12.5 years) entered the cross-sectional study. SpiroNose could adequately distinguish between controls, asthma, COPD and lung cancer patients with cross-validation values ranging between 78-88%. We have developed a standardized way to integrate eNose technology with spirometry. Signal processing techniques and environmental background correction ensured that the multiple sensor arrays within the SpiroNose provided repeatable and interchangeable results. SpiroNose discriminated controls and patients with asthma, COPD and lung cancer with promising accuracy, paving the route towards point-of-care exhaled breath diagnostics.

Exhaled breath analysis by electronic nose in airways disease. Established issues and key questions.

Exhaled air contains many volatile organic compounds (VOCs) that are the result of normal and disease-associated metabolic processes anywhere in the body. Different omics techniques can assess the pattern of these VOCs. One such omics technique suitable for breath analysis is represented by electronic noses (eNoses), providing fingerprints of the exhaled VOCs, called breathprints. Breathprints have been shown to be altered in different disease states, including in asthma and COPD. This review describes the current status on clinical validation and application of breath analysis by electronic noses in the diagnosis and monitoring of chronic airways diseases. Furthermore, important methodological issues including breath sampling, modulating factors and incompatibility between eNoses are raised and discussed. Next steps towards clinical application of electronic noses are provided, including further validation in suspected disease, assessment of the influence of different comorbidities, the value in longitudinal monitoring of patients with asthma and COPD and the possibility to predict treatment responses. Eventually, a Breath Cloud may be constructed, a large database containing disease-specific breathprints. When collaborative efforts are put into optimization of this technique, it can provide a rapid and non-invasive first line diagnostic test.

Effect of transportation and storage using sorbent tubes of exhaled breath samples on diagnostic accuracy of electronic nose analysis.

Many (multi-centre) breath-analysis studies require transport and storage of samples. We aimed to test the effect of transportation and storage using sorbent tubes of exhaled breath samples for diagnostic accuracy of eNose and GC-MS analysis. As a reference standard for diagnostic accuracy, breath samples of asthmatic patients and healthy controls were analysed by three eNose devices. Samples were analysed by GC-MS and eNose after 1, 7 and 14 days of transportation and storage using sorbent tubes. The diagnostic accuracy for eNose and GC-MS after storage was compared to the reference standard. As a validation, the stability was assessed of 15 compounds known to be related to asthma, abundant in breath or related to sampling and analysis. The reference test discriminated asthma and healthy controls with a median AUC (range) of 0.77 (0.72-0.76). Similar accuracies were achieved at t1 (AUC eNose 0.78; GC-MS 0.84), t7 (AUC eNose 0.76; GC-MS 0.79) and t14 (AUC eNose 0.83; GC-MS 0.84). The GC-MS analysis of compounds showed an adequate stability for all 15 compounds during the 14 day period. Short-term transportation and storage using sorbent tubes of breath samples does not influence the diagnostic accuracy for discrimination between asthma and health by eNose and GC-MS.

External validation of exhaled breath profiling using an electronic nose in the discrimination of asthma with fixed airways obstruction and chronic obstructive pulmonary disease.

BACKGROUND: Fixed airflow limitation can be found both in asthma and chronic obstructive pulmonary disease (COPD), posing a day-to-day diagnostic challenge. OBJECTIVE: We aimed to determine the external validity of metabolomic analysis of exhaled air by electronic nose for distinguishing asthma and COPD in patients with fixed airways obstruction. METHODS: One hundred patients were included in a cross-sectional design: 60 asthma patients: 21 with fixed airways obstruction (fixed asthma), 39 with reversible airways obstruction (classic asthma) and 40 COPD patients (GOLD stages II-III). Standardized sampling of exhaled breath was performed and volatile organic compounds were captured using an electronic nose resulting in breathprints. External validity in newly recruited patients (validation sets) was tested using a previous and independent training set. Breathprints were analysed by principal component and canonical discriminant analysis and area under the curve (AUC) of receiver operating characteristic curves. RESULTS: External validity of breathprints showed 88% accuracy for distinguishing fixed asthma from COPD (AUC 0.95, 95% CI 0.84-1.00, sensitivity 85%, specificity 90%) and 83% for classic asthma (AUC 0.93, 95% CI 0.87-1.00, sensitivity 91%, specificity 90%) (both P<0.001). Discriminative accuracy was not confounded by current smoking. CONCLUSIONS AND CLINICAL RELEVANCE: External validation of exhaled breath molecular profiling shows high accuracy in distinguishing asthma and COPD in newly recruited patients with fixed airways obstruction. Exhaled air analysis may therefore reduce misdiagnosis in obstructive airways diseases, potentially leading to more appropriate management.

Exhaled air molecular profiling in relation to inflammatory subtype and activity in COPD.

Eosinophilic inflammation in chronic obstructive pulmonary disease (COPD) is predictive for responses to inhaled steroids. We hypothesised that the inflammatory subtype in mild and moderate COPD can be assessed by exhaled breath metabolomics. Exhaled compounds were analysed using gas chromatography and mass spectrometry (GC-MS) and electronic nose (eNose) in 28 COPD patients (12/16 Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage I/II, respectively). Differential cell counts, eosinophil cationic protein (ECP) and myeloperoxidase (MPO) were measured in induced sputum. Relationships between exhaled compounds, eNose breathprints and sputum inflammatory markers were analysed and receiver operating characteristic (ROC) curves were constructed. Exhaled compounds were highly associated with sputum cell counts (eight compounds with eosinophils, 17 with neutrophils; p < 0.01). Only one compound (alkylated benzene) overlapped between eosinophilic and neutrophilic profiles. GC-MS and eNose breathprints were associated with markers of inflammatory activity in GOLD stage I (ECP: 19 compounds, p < 0.01; eNose breathprint r = 0.84, p = 0.002) (MPO: four compounds, p < 0.01; eNose r = 0.72, p = 0.008). ROC analysis for eNose showed high sensitivity and specificity for inflammatory activity in mild COPD (ECP: area under the curve (AUC) 1.00; MPO: AUC 0.96) but not for moderate COPD. Exhaled molecular profiles are closely associated with the type of inflammatory cell and their activation status in mild and moderate COPD. This suggests that breath analysis may be used for assessment and monitoring of airway inflammation in COPD.