Exhaled Breath Condensate Research Articles

Molecular Detection of SARS-CoV-2 Viral Particles in Exhaled Breath Condensate via Engineered Face Masks

In this study, we present a novel face mask engineered for the collection of exhaled breath condensate (EBC) and its application and performance in a clinical study of COVID-19 infection status assessment versus the gold standard polymerase chain reaction (PCR) nasopharyngeal swab testing. EBC was collected within a clinical trial of COVID-19-infected and non-infected patients and analyzed by reverse transcription quantitative (RT-q) PCR, with the results being compared with nasopharyngeal sampling of the same patient. The cycle threshold (Ct) values of the nasopharyngeal samples were generally lower than those of EBC, with viral loads in EBC ranging from 1.2 × 104 to 5 × 108 viral particles mL−1 with 5 min of breathing. From the 60 clinical patients’ samples collected, 30 showed a confirmed SARS-CoV-2 infection. Of these 30 individuals, 22 (73%) had Ct values < 40 (representing the threshold for SARS-CoV-2 infectivity) using both amplification of ORF1a/b and the E-gene. The 30 EBC samples from non-infected participants were all identified as negative, indicating a 100% specificity. These first results encourage the use of the face mask as a noninvasive sampling method for patients with lung-related diseases, especially with a view to equipping the face mask with miniaturized sensing devices, representing a true point-of-care solution in the future.

A wearable exhaled breath condensate (EBC) collector with controllable condensation microfluidics and a branched hydrophilic film

In previous research, exhaled breath condensate (EBC) analysis has emerged as a promising noninvasive method for assessing human health, potentially increasing patient testing acceptance. However, current EBC collection devices require cooling equipment for temperature reduction, leading to issues such as increased size, increased energy consumption, and a lack of real-time collection capability. In this study, an EBC collector was developed that integrates controllable condensation microfluidics with a biomimetic water-collecting film. Microfluids induce an endothermic reaction by rapidly dissolving water and NH4NO3 within the chamber, reducing the surface temperature to 3.5 °C. When positioned inside a mask, exhaled air undergoes condensation and collection on the branched biomimetic film. Human tests were conducted to analyze caffeine and nicotine in the collected EBC samples using LC–MS, demonstrating the device’s advantages in terms of power-free operation, wearable design, and rapid sample collection for metabolite detection.

The optimization of low volume-SPME method for volatilomics analysis of exhaled breath condensate

The analysis of breath can provide insight into the metabolic state of the body. However, the collection, transport, and storage of breath samples present certain challenges. The collection of exhaled breath condensate (EBC) emerges as a promising method for breath analysis. Despite this, the volume of EBC is relatively low, necessitating a sensitive method capable of extracting analytes from a small sample volumes. Therefore, this study aimed to develop and optimize a robust and green solid-phase extraction method for the analysing volatile organic compounds (VOCs) of various polarities in a low-volume EBC using a statistical design of experiment (DoE).Based on the screening experiments and DoE, the optimal conditions were determined to be 15 min equilibration time, and 20 min of extraction at 44 °C using DVB/CAR/PDMS fibre in direct immersion (DI) mode. The method was validated for linearity, precision and accuracy, yielding satisfactory results. The optimized method was applied to quantify the concentration of selected VOCs in real samples collected from a healthy subject, as well as after consumption of coffee and smoking of an electronic cigarette. Many of the selected compounds are associated with diseases and are challenging to detect in specimens from a healthy subject, but this method can measure them as potential disease markers.The proposed method enables the analysis of VOCs is very low-volume samples (200 µL), which is particularly important for studies involving body fluids. Notably, the applied approach demonstrated that different VOCs, representing various chemical classes and polarities, can be detected and quantified.

Early Diagnosis of Bronchopulmonary Dysplasia with E-Nose: A Pilot Study in Preterm Infants

Bronchopulmonary dysplasia (BPD) is the most common respiratory disease in preterm and is still associated with increased mortality and morbidity. The great interest lies in identifying early biomarkers that can predict the development of BPD. This pilot study explores the potential of e-nose for the early identification of BPD risk in premature infants by analyzing volatile organic compounds (VOCs) in the exhaled breath condensate (EBC). Fourteen mechanically ventilated very preterm infants were included in this study. The clinical parameters and EBC were collected within the first 24 h of life. The discriminative ability of breath prints between preterms who did and did not develop BPD was investigated using pattern recognition, a machine learning algorithm, and standard statistical methods. We found that e-nose probes can significantly predict the outcome of “no-BPD” vs. “BPD”. Specifically, a subset of probes (S18, S24, S14, and S6) were found to be significantly predictive, with an AUC of 0.87, 0.89, 0.82, 0.8, and p = 0.019, 0.009, 0.043, 0.047, respectively. The e-nose is an easy-to-use, handheld, non-invasive electronic device that quickly samples breath. Our preliminary study has shown that it has the potential for early prediction of BPD in preterms.

Exhaled Breath Condensate Surveillance for Aspergillus in Acute Leukemia—a Pilot Trial

Abstract Invasive fungal infections in patients with leukemia carry a high mortality rate, but early diagnosis has the potential to modify this natural history. A novel screening method using Aspergillus droplet-digital polymerase chain reaction in exhaled breath condensate may have a similar performance to serum galactomannan screening. Larger studies, including other molds, are necessary.

Circulating Interleukins as Biomarkers in Non-Small Cell Lung Cancer Patients: A Pilot Study Compared to Normal Individuals.

Identifying biomarkers in non-small cell lung cancer (NSCLC) can improve diagnosis and patient stratification. We evaluated plasmas and sera for interleukins (IL)-11, IL-6, IL-8, IL-17A, and IL-33 as biomarkers in primary NSCLC patients undergoing surgical treatment against normal volunteers. Exhaled-breath condensates (EBCs), a potential source without invasive procedures, were explored in normal individuals. Due to separate recruitment criteria and intrinsic cohort differences, the NSCLC and control cohorts were not well matched for age (median age: 65 vs. 40 years; p < 0.0001) and smoking status (p = 0.0058). Interleukins were first assessed through conventional ELISA. IL-11 was elevated in NSCLC plasma compared to controls (49.71 ± 16.90 vs. 27.67 ± 14.06 pg/mL, respectively, p < 0.0001) but undetectable in sera and EBCs by conventional ELISA. Therefore, high-sensitivity PCR-based IL-11 ELISA was repeated, albeit with concentration discrepancies. IL11 gene and protein upregulation by RT-qPCR and immunohistochemistry, respectively, were validated in NSCLC tumors. The lack of detection sensitivity across IL-6, IL-8, IL-17A, and IL-33 suggests the need for further, precise assays. Surprisingly, biomarker concentrations can be dissimilar across paired plasmas and sera. Our results identified a need to optimize detection limits for biomarker detection and caution against over-reliance on just one form of blood sample for biomarker assessment.

Quantification of morphine in exhaled breath condensate using a double network polymeric hybrid hydrogel functionalized with AuNPs

BackgroundMorphine serves as a foundation for creating other opioid derivatives, such as hydro/oxymorphine and heroin, which possess enhanced pain-relieving properties but are also prone to addiction and abuse. In cases of morphine overdose, it not only affects multiple immune functions but can also cause severe health complications. Given these concerns and the widespread use of morphine, it is crucial to develop efficient, uncomplicated, and precise methods for accurately detecting morphine in various biological and pharmaceutical samples.ResultsIn this investigation, a novel gold nanoparticle (AuNPs)-based double network hydrogel (DNH) nanoprobe has been fabricated for sensitive quantification of morphine in exhaled breath condensate samples. For that, gelatin/agarose DNH was fabricated through a one-step heating-cooling method in the presence of AuNPs, providing not only chemical stability but also prevent the AuNPs aggregation during synthesis process. In this method, the absorbance intensity of the nanoprobe gradually decreased with increasing morphine concentration due to the interaction morphine with AuNPs surface plasmon. The aggregation of AuNPs by addition of morphine was verified by UV-Vis spectrophotometry. The sensor displayed high sensitivity with detection limit of 0.006 µg.mL-1 in the linear range from 0.01 to 1.0 µg.mL-1. A reliable performance was attained for the spectrophotometric method for determination of morphine in the real samples.

Measurement of Human and Bovine Exhaled Breath Condensate pH Using Polyaniline-Modified Flexible Inkjet-Printed Nanocarbon Electrodes.

The collection, processing, and electrochemical analysis of exhaled breath condensate (EBC) from healthy human and animal subjects is reported on. EBC is a biospecimen potentially rich in biomarkers of respiratory disease. The EBC pH was analyzed potentiometrically using a disposable polyaniline (PANI)-modified inkjet-printed (IJP) carbon electrode. Comparison measurements were performed using a commercial screen-printed carbon (SPC) electrode. The PANI-modified electrodes exhibited reproducible and near-Nernstian responses for pH values between 2 and 9 with slopes from -50 to -60 mV/dec. The PANI-modified IJP carbon electrode exhibited a faster response time and superior reproducibility to the modified SPC electrode. In proof-of-concept studies, the healthy human EBC pH was found to be 6.57 ± 0.09 and the healthy bovine EBC pH was 5.9 ± 0.2. All pH determined using the PANI-modified electrodes were in good agreement with the pH determined using a micro glass pH electrode. An RTube device was used to collect EBC from humans while a modified device was used to collect EBC from calves in the field. EBC volumes of 0.5-2 mL for 5-6 min of tidal breathing were collected from healthy animals. The pH of EBC from healthy calves (17 animals) depends on their age from 1 to 9 weeks with values ranging from 5.3 to 7.2. A distinct alkaline shift was observed for many animals around 20 days of age. The bovine EBC pH also depends on the ambient temperature and humidity at the time of collection. The results indicate that the PANI-modified IJP carbon electrodes outperform commercial SPC and provide reproducible and accurate measurement of pH across various biospecimen types.

Development of a nanocomposite hydrogel catalyzed H2O2/TMB system for determination of chlordiazepoxide in exhaled breath condensate.

In this study, an enzyme mimic catalyzed H2O2-tetramethylbenzidine system based on UiO-66/Au NPs-PVA nanocomposite hydrogel was employed as an optical probe for chlordiazepoxide sensing. An excellent detection limit of 0.0032 μg mL-1 with a linear range of 0.005-2.0 μg mL-1 was obtained for chlordiazepoxide in exhaled breath condensate samples under optimal conditions. The validated system showed good repeatability, simplicity, and stability toward chlordiazepoxide sensing in the exhaled breath condensate of patients receiving this drug.

P21-36 Effects of air pollution in asthmatic children: personal exposure to pollutants and biomarkers of effect in exhaled breath condensates

P21-36 Effects of air pollution in asthmatic children: personal exposure to pollutants and biomarkers of effect in exhaled breath condensates

Combination of exhaled breath condensate samples with lipoarabinomannan point of care assay for pulmonary tuberculosis (TB) diagnosis: protocol for a diagnostic accuracy study

IntroductionThe WHO estimates a gap of about 30% between the incident (10.6 million) and notified (7.5 million) cases of tuberculosis (TB). Combined with the growing recognition in prevalence surveys of...

Utilizing nitrogen, sulfur, phosphorus, and chlorine co-doped carbon dots as a fluorescent probe for determination of vancomycin in exhaled breath condensate

Vancomycin is employed to treat infections caused by gram-positive bacteria. Ensuring precise vancomycin dosages is essential to avoid the emergence of bacterial resistance. In the current study, a fluorescent nanoprobe was designed for vancomycin determination in exhaled breath condensate samples. The nanoprobe was based on carbon dots (CDs) doped with nitrogen, sulfur, phosphorus, and chlorine (NSPCl-doped CDs). Vancomycin significantly reduced the fluorescence of NSPCl-doped CDs and presented a quenching process in the analytical response of the probe within a concentration range of 0.01–2.0 μg mL−1 due to forming a non-fluorescent complex. The nanoprobe's intra-day and inter-day relative standard deviations were 1.4 % and 3.2 %, respectively. This nanoprobe was successfully used to determine vancomycin in the patients receiving this drug collected from the expiratory circuit of the mechanical ventilator.

A smart mask for exhaled breath condensate harvesting and analysis.

Recent respiratory outbreaks have garnered substantial attention, yet most respiratory monitoring remains confined to physical signals. Exhaled breath condensate (EBC) harbors rich molecular information that could unveil diverse insights into an individual's health. Unfortunately, challenges related to sample collection and the lack of on-site analytical tools impede the widespread adoption of EBC analysis. Here, we introduce EBCare, a mask-based device for real-time in situ monitoring of EBC biomarkers. Using a tandem cooling strategy, automated microfluidics, highly selective electrochemical biosensors, and a wireless reading circuit, EBCare enables continuous multimodal monitoring of EBC analytes across real-life indoor and outdoor activities. We validated EBCare's usability in assessing metabolic conditions and respiratory airway inflammation in healthy participants, patients with chronic obstructive pulmonary disease or asthma, and patients after COVID-19 infection.

Hidden in the blow - a matrix to characterise cetaceans’ respiratory microbiome: short-finned pilot whale as case study

Cetaceans are key sentinel species of marine ecosystems and ocean health, being a strategic taxonomic group that evaluates the well-being of aquatic habitats and detects harmful environmental trends. Respiratory diseases are amongst the main causes of death in these animals, so identifying the microbiome community in their exhaled breath condensates (EBC), i.e. blow, has been proposed as a key biomarker for assessing respiratory health. Yet, to characterise microbiomes related to these animals’ respiratory tract and use them as a proxy for health status, it is necessary to develop baseline data on the microorganisms associated with cetaceans. Here, the short-finned pilot whale (SFPW, Globicephala macrorhynchus) was used as a case study to validate the most suitable primer set to explore the prokaryotic diversity of the cetaceans’ respiratory tract. DNA extracted from blow samples (n = 12) of animals off Madeira Island was sequenced to amplify both V3-V4 and V4-V5 hypervariable regions of the 16S rRNA gene, using the same sequencing platform (Illumina MiSeq). Independently of the primer set used, all blows shared Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria phyla in their composition. V3-V4 resulted in a higher diversity of taxa with relative abundance above 1%, whereas the V4-V5 primers captured a higher number of microbial Amplicon Sequence Variants, detecting the rare microbial biosphere with pathogen potential. Additionally, it captured the core microbiome more efficiently. Thus, this study provides a detailed characterisation of SFPW respiratory-associated microbial communities, strengthening the idea of sociality influencing microbiome composition in the respiratory tract. Moreover, it supports the use of blow as a relevant biomarker for the physiological state of the airways in free-ranging cetaceans.

Design of the SPIROMICS Study of Early COPD Progression: SOURCE Study.

The biological mechanisms leading some tobacco-exposed individuals to develop early-stage chronic obstructive pulmonary disease (COPD) are poorly understood. This knowledge gap hampers development of disease-modifying agents for this prevalent condition. Accord-ingly, with National Heart, Lung and Blood Institute support, we initiated the SPIROMICS Study of Early COPD Progression (SOURCE), a multicenter observational cohort study of younger individuals with a history of cigarette smoking and thus at-risk for, or with, early-stage COPD. Our overall objectives are to identify those who will develop COPD earlier in life, characterize them thoroughly, and by contrasting them to those not developing COPD, define mechanisms of disease progression. SOURCE utilizes the established SPIROMICS clinical network. Its goal is to enroll n=649 participants, ages 30-55 years, all races/ethnicities, with ≥10 pack-years cigarette smoking, in either Global Initiative for Chronic Obstructive Lung Disease (GOLD) groups 0-2 or with Preserved Ratio Impaired Spirometry (PRISm); and an additional n=40 never-smoker controls. Participants undergo baseline and three-year follow-up visits, each including high-resolution computed tomography; respiratory oscillometry and spirometry (pre- and post-bronchodilator administration), exhaled breath condensate (baseline only); and extensive biospecimen collection, including sputum induction. Symptoms, interim healthcare utilization, and exacerbations are captured every six months via follow-up phone calls. An embedded bronchoscopy sub-study involving n=100 participants (including all never-smokers) will allow collection of lower airway samples for genetic, epigenetic, genomic, immunological, microbiome, mucin analyses, and basal cell culture. SOURCE should provide novel insights into the natural history of lung disease in younger individuals with a smoking history, and its biological basis.

Breath and Sputum Analyses in Asthmatic Patients: An Overview.

Recent advancements in asthma management include non-invasive methodologies such as sputum analysis, exhaled breath condensate (EBC), and fractional exhaled nitric oxide (FeNO). These techniques offer a means to assess airway inflammation, a critical feature of asthma, without invasive procedures. Sputum analysis provides detailed insights into airway inflammation patterns and cellular composition, guiding personalized treatment strategies. EBC collection, reflecting bronchoalveolar lining fluid composition, provides a non-invasive window into airway physiology. FeNO emerges as a pivotal biomarker, offering insights into eosinophilic airway inflammation and aiding in asthma diagnosis, treatment monitoring, and the prediction of exacerbation risks. Despite inherent limitations, each method offers valuable tools for a more comprehensive assessment of asthma. Combining these techniques with traditional methods like spirometry may lead to more personalized treatment plans and improved patient outcomes. Future research is crucial to refine protocols, validate biomarkers, and establish comprehensive guidelines in order to enhance asthma management with tailored therapeutic strategies and improved patient outcomes.

Cannabinoids detected in exhaled breath condensate after cannabis use

Cannabinoids can be detected in breath after cannabis use, but different breath matrices need to be explored as studies to date with filter-based devices that collect breath aerosols have not demonstrated that breath-based measurements can reliably identify recent cannabis use. Exhaled breath condensate (EBC) is an unexplored aqueous breath matrix that contains condensed volatile compounds and water vapor in addition to aerosols. EBC was collected from participants both before and at two time points (0.7 ± 0.2 h and 1.7 ± 0.3 h) after observed cannabis use. Eleven different cannabinoids were monitored with liquid chromatography tandem mass spectrometry. Five different cannabinoids, including Δ9-tetrahydrocannabinol (THC), were detected in EBC collected from cannabis users. THC was detected in some EBC samples before cannabis use, despite the requested abstinence period. THC was detected in all EBC samples collected at 0.7 h post use and decreased for all participants at 1.7 h. Non-THC cannabinoids were only detected after cannabis use. THC concentrations in EBC samples collected at 0.7 h showed no trend with sample metrics like mass or number of breaths. EBC sampling devices deserve further investigation with respect to modes of cannabis use (e.g, edibles), post use time points, and optimization of cannabinoid recovery.

Extraction and characterization of exosomes from the exhaled breath condensate and sputum of lung cancer patients and vulnerable tobacco consumers—potential noninvasive diagnostic biomarker source

Noninvasive sample sources of exosomes, such as exhaled breath and sputum, which are in close proximity to the tumor microenvironment and may contain biomarkers indicative of lung cancer, are far more permissive than invasive sample sources for biomarker screening. Standardized exosome extraction and characterization approaches for low-volume noninvasive samples are critically needed. We isolated and characterized exhaled breath condensate (EBC) and sputum exosomes from healthy nonsmokers (n = 30), tobacco smokers (n = 30), and lung cancer patients (n = 40) and correlated the findings with invasive sample sources. EBC samples were collected by using commercially available R-Tubes. To collect sputum samples the participants were directed to take deep breaths, hold their breath, and cough in a collection container. Dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy were used to evaluate the exosome morphology. Protein isolation, western blotting, exosome quantification via EXOCET, and Fourier transform infrared spectroscopy were performed for molecular characterization. Exosomes were successfully isolated from EBC and sputum samples, and their yields were adequate and sufficiently pure for subsequent downstream processing and characterization. The exosomes were confirmed based on their size, shape, and surface marker expression. Remarkably, cancer exosomes were the largest in size not only in the plasma subgroups, but also in the EBC (p < 0.05) and sputum (p = 0.0036) subgroups, according to our findings. A significant difference in exosome concentrations were observed between the control sub-groups (p < 0.05). Our research confirmed that exosomes can be extracted from noninvasive sources, such as EBC and sputum, to investigate lung cancer diagnostic biomarkers for research, clinical, and early detection in smokers.

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.

In Situ Collection and SERS Detection of Nitrite in Exhalations on Facemasks Based on Wettability Differences.

As one of the common carriers of biological information, along with human urine specimens and blood, exhaled breath condensate (EBC) carries reliable and rich information about the body's metabolism to track human physiological normal/abnormal states and environmental exposures. What is more, EBC has gained extensive attention because of the convenient and nondestructive sampling. Facemasks, which act as a physical filter barrier between human exhaled breath and inhaled substances from the external environment, are safe, noninvasive, and economic devices for direct sampling of human exhaled breath and inhaled substances. Inspired by the ability of fog collection of Namib desert beetle, a strategy for in situ collecting and detecting EBC with surface-enhanced Raman scattering is illustrated. Based on the intrinsic and unique wettability differences between the squares and the surrounding area of the pattern on facemasks, the hydrophilic squares can capture exhaled droplets and spontaneously enrich the analytes and silver nanocubes (AgNCs), resulting in good repeatability in situ detection. Using R6G as the probe molecule, the minimal detectable concentration can reach as low as 10-16 M, and the relative standard deviation is less than 7%. This proves that this strategy can achieve high detection sensitivity and high detection repeatability. Meanwhile, this strategy is applicable for portable nitrite analysis in EBC and may provide an inspiration for monitoring other biomarkers in EBC.