Changes in the proteomics of exhaled breath condensate under the influence of inhaled hydrogen in patients with post-COVID syndrome.

Aim. To study the effect of inhalation therapy with an active hydrogen (AH) on the protein composition of exhaled breath condensate (EBC) in patients with post-COVID syndrome (PCS). Material and methods. This randomized controlled parallel prospective study included 60 patients after coronavirus disease 2019 (COVID-19) with PCS during the recovery period and clinical manifestations of chronic fatigue syndrome who received standard therapy according to the protocol for managing patients with chronic fatigue syndrome (CFS). The patients were divided into 2 groups: group 1 (main) - 30 people who received standard therapy and AH inhalations (SUISONIA, Japan) for 10 days, and group 2 (control) - 30 medical workers who received only standard therapy. Patients in both groups were comparable in sex and mean age. All participants in the study were sampled with EBC on days 1 and 10. Samples were subjected to tryptic digestion and high-performance liquid chromatography combined with tandem mass spectrometry analysis using a nanoflow chromatograph (Dionex 3000) in tandem with a high-resolution time-of-flight mass spectrometer (timsTOF Pro). Results. A total of 478 proteins and 1350 peptides were identified using high resolution mass spectrometry. The number of proteins in samples after AH therapy, on average, is 12% more than before treatment. An analysis of the distribution of proteins in different groups of patients showed that only half of these proteins (112) are common for all groups of samples and are detected in EBC before, after, and regardless of hydrogen therapy. In addition to the qualitative difference in the EBC protein compositions in different groups, quantitative changes in the concentration of 36 proteins (mainly structural and protective) were also revealed, which together made it possible to reliably distinguish between subgroups before and after treatment. It is worth noting that among these proteins there are participants of blood coagulation (alpha-1-antitrypsin), chemokine- and cytokine-mediated inflammation, and a number of signaling pathways (cytoplasmic actin 2), response to oxidative stress (thioredoxin), glycolysis (glyceraldehyde-3- phosphate dehydrogenase), etc. Conclusion. The use of hydrogen therapy can contribute to the switching of a number of physiological processes, which may affect the success of recovery in PCS patients. In particular, the obtained results indicate the activation of aerobic synthesis of adenosine triphosphate in mitochondria by hydrogen therapy, which correlates well with the decrease in the blood lactate level detected by laboratory studies. At the same time, this therapy can inhibit pro-inflammatory activity, negatively affecting the coagulation and signaling pathways of integrins and apoptosis, and, in addition, activate protective pathways, tricarboxylic acid cycle, FAS signaling, and purine metabolism, which may be essential for effective recovery after COVID-19.Copyright © 2023 Vserossiiskoe Obshchestvo Kardiologov. All rights reserved.

Evaluation of lung inflammation using Exhaled breath condensate in patients with post-COVID syndrome

Introduction: The term "post-COVID syndrome" encompasses a wide range of clinical conditions following SARSCOV2 infection. Whether post-COVID syndrome can be associated with a prolonged inflammatory and immune response is still unknown. Exhaled Breath Condensate (EBC) pH has been recognized as a robust marker of lung inflammation in various diseases (Kharitonov et al. Chest 2006;130(5):1541-46). However, evidences on the role of EBC pH in diagnosing lung inflammation in post-COVID syndrome are still lacking. Aims and objectives: We aimed to investigate EBC pH in patients suffering from post-COVID syndrome. Method(s): We enrolled 10 patients hospitalized with acute respiratory failure and COVID-19 pneumonia. We performed a complete follow up after 3 months (T1) and 6 months (T2) from discharge. Each visit included routine blood tests, arterial blood gas analysis, 6 minute walking test and body plethysmography. Finally, bronchial and alveolar EBC pH were collected at the end of each visit. Result(s): Alveolar EBC pH was significantly lower at T1 compared with T2 samples (p= 0.0007). Moreover, in T1 analysis, we found a less acid pH in bronchial EBC compared to the alveolar one (p=0.003). Alveolar and bronchial EBC did not differ at T2, as well as bronchial EBC from T1 to T2. Serum inflammatory biomarkers did not differ from T1 to T2 analysis. Finally, alveolar EBC was directly correlated with Neutrophil-Lymphocyte ratio (R=0.71, p=0.02). Conclusion(s): Alveolar EBC pH is a useful non-invasive tool to characterize and monitor lung inflammation in patients with post-COVID syndrome. Furthermore, no other serum biomarker seems to be sensitive enough to identify residual phlogosis after COVID-19 disease.

SARS-CoV-2 load in exhaled end-tidal breath fine aerosol condensates among acutely symptomatic COVID-19 cases

SARS-CoV-2 infectious virions have been reported in exhaled breath, but their source remains elusive: breath sampling systems used to date do not separate breath aerosols by size, fail to prevent salivary/fomite contamination, or aerosol size evolution before sample capture. We hypothesised that sampling end-tidal, oral exhaled breath condensate (EBC), after separating large droplets by inertial impaction 4cm from the lips, would quantify viral loads in distal lung-derived fine aerosols (FA). We used a collector (PBM-HALE ) that captures mechanically aerosolised viruses to sample adult participants for <30 min under informed consent;cases symptomatic for <5 days (n=30) or >5 days (n=12), positive by nasopharyngeal swab RT-PCR (Ct>=13.1), were sampled in clinical triage 'red zones', or COVID-19 wards with no mechanical ventilation or open windows. Salivary alpha amylase activity (Salimetrics LLC), or SARS-CoV-2 viral load (VIASURE SARS-CoV-2 (ORF1ab and N gene)) after QIAsymhpony DSP midi extraction, was quantified in 0.2mL FA EBC fractions. No salivary alpha amylase activity was detected in healthy participant FA EBC (>1:1,750 dilution of paired saliva vs assay detection limit (n=300)). No SARS-CoV-2 RNA was detected in FA EBC (1.18mL +/- 0.32 total volume) among any COVID-19 cases (Aug 2020-Jan 2022) at limits of detection of 120 genomes/mL FA EBC or 4.72 genomes/min exhalation. No pre-extraction spike-in control reaction inhibition was observed. No ambient contamination of the alveolar FA EBC was detected with this sampling device. The alveolar fraction of orally exhaled tidal breath lacks detectable SARS-CoV-2 viral load.

Exhaled breath condensate as bioanalyte: from collection considerations to biomarker sensing.

Since the SARS-CoV-2 pandemic, the potential of exhaled breath (EB) to provide valuable information and insight into the health status of a person has been revisited. Mass spectrometry (MS) has gained increasing attention as a powerful analytical tool for clinical diagnostics of exhaled breath aerosols (EBA) and exhaled breath condensates (EBC) due to its high sensitivity and specificity. Although MS will continue to play an important role in biomarker discovery in EB, its use in clinical setting is rather limited. EB analysis is moving toward online sampling with portable, room temperature operable, and inexpensive point-of-care devices capable of real-time measurements. This transition is happening due to the availability of highly performing biosensors and the use of wearable EB collection tools, mostly in the form of face masks. This feature article will outline the last developments in the field, notably the novel ways of EBA and EBC collection and the analytical aspects of the collected samples. The inherit non-invasive character of the sample collection approach might open new doors for efficient ways for a fast, non-invasive, and better diagnosis.

Potential Use of Exhaled Breath Condensate for Diagnosis of SARS-CoV-2 Infections: A Systematic Review and Meta-Analysis.

BACKGROUND: Reverse-transcriptase polymerase chain reaction (RT-qPCR) assays performed on respiratory samples collected through nasal swabs still represent the gold standard for COVID-19 diagnosis. Alternative methods to this invasive and time-consuming options are still being inquired, including the collection of airways lining fluids through exhaled breath condensate (EBC). MATERIALS AND METHODS: We performed a systematic review and meta-analysis in order to explore the reliability of EBC as a way to collect respiratory specimens for RT-qPCR for diagnosis of COVID-19. RESULTS: A total of 4 studies (205 specimens), were ultimately collected, with a pooled sensitivity of 69.5% (95%CI 26.8-93.4), and a pooled specificity of 98.3% (95%CI 87.8-99.8), associated with high heterogeneity and scarce diagnostic agreement with the gold standard represented by nasal swabs (Cohen's kappa = 0.585). DISCUSSION: Even though non-invasive options for diagnosis of COVID-19 are still necessary, EBC-based RT-qPCR showed scarce diagnostic performances, ultimately impairing its implementation in real-world settings. However, as few studies have been carried out to date, and the studies included in the present review are characterized by low numbers and low sample power, further research are requested to fully characterize the actual reliability of EBC-based RT-qPCR in the diagnosis of COVID-19.

Exhaled Breath Condensate Study for Biomarkers Discovery

Biomarkers seem to play an important role in understanding various diseases’ nature, course and management, including respiratory ones. Yet, discovering verifiable and validated ones, that are useful in pulmonology, is challenging and constant. A special body specimen that has been characterized as a matrix of biomarkers, is the exhaled breath condensate (EBC). It is a fluid resulting from freezing the exhaled air. Water is its main constituent. The rest is a rich mix of water-soluble volatile compounds and aerosol droplets of airway lining fluid. The droplets carry non-volatile organic compounds. Their concentration is very small and the techniques applied to measure it are very accurate and sensitive. The content of the exhaled breath condensate reflects important processes taking place in the lungs, such as inflammation and oxidative stress, which are the basis of respiratory diseases’ pathophysiology. It seems that it has a role in diagnosis, monitoring, stratification and therapy of respiratory diseases, including COVID19. This paper presents information on exhaled breath condensate and highlights its importance as a potential source of biomarkers. © 2022, Springer Nature Switzerland AG.

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.

Implementation of a harmonized approach for monitoring exposure to engineered and incidental nanoparticles and their potential health effects: First results from the EU-LIFE project NanoExplore

Introduction: We aimed at validating a harmonized protocol for monitoring occupational exposure to engineered/incidental nanoparticles (EINP) and to assess their health effects. Materials and Methods: A multicentric prospective cohort study was designed involving repeated field campaigns of 4-day exposure monitoring and two biological samplings, at the beginning (T1) and at the end (T2) of working week. To detect a significant difference in effect biomarkers of at least 25%, a sample size of 120 workers (60 exposed, 60 non-exposed) was determined, along with two control groups, internal and external to company. The protocol feasibility was tested in three countries: Switzerland, Spain, and Italy. Number and mass concentration, morphology, size distribution and surface area of EINP were measured, and effect biomarkers (oxidative stress and inflammation) were assessed in exhaled breath condensate (EBC) and/or urine samples. Results: The preliminary results of 42 external controls showed no significant change in effect biomarker levels between T1 and T2, with the exception of malondialdehyde and 8-Isoprostane in EBC. The biomarkers levels were within the ranges reported in healthy adults. The analysis of data and samples collected in 60 exposed workers and 36 internal controls recruited at six EINP-handling facilities are ongoing. Conclusions: These findings confirm the feasibility of the harmonized protocol. For its implementation, a particular effort on organization, coordination and communication between each team was mandatory, particularly during COVID time.

A method to detect and quantify SARS-CoV-2 in nasal exhaled breath

SARS-CoV-2 is assumed to spread through exhaled respiratory droplets and aerosols, but very little research has directly measured the virus in exhaled breath, and our understanding of the relationship between olfactory symptoms of COVID-19 and infectious shedding of the virus is limited. Whether there is any relationship between olfactory dysfunction and viral shedding in nasal exhalate is unknown. Here, we developed a device and a method for collecting and quantifying SARS-CoV-2 RNA in the exhaled breath condensate of COVID-19 patients. Our device is portable, inexpensive and simple enough to use that breath samples can be self-collected by patients in their homes. It has configurations for both oral and nasal breath collection, allowing for comparison of viral loads across the two breathing routes. This device can be used to determine whether there is a difference between oral and nasal shedding of the virus on breath, and to determine whether viral shedding on nasal or oral breath is related to chemosensory symptoms.

Exhaled breath condensate (EBC) for SARS-CoV-2 diagnosis still an open debate.

The real-time PCR (RT-PCR) on nasopharyngeal swabs (NPS) is the gold standard for the diagnosis of SARS-CoV-2. The exhaled breath condensate (EBC) is used to perform collection of biological fluid condensed in a refrigerated device from deep airways' exhaled air. We aimed to verify the presence of SARS-CoV-2 virus in the EBC from patients with confirmed SARS-CoV-2 infection by RT-PCR, and to determine if the EBC may represent a valid alternative to the NPS. Seventeen consecutive patients admitted to the Emergency Department of the Policlinico were enrolled in the present study with RT-PCR, clinical and radiological evidence of SARS-CoV-2. Within 24 h from the NPS collection the EBC collection was performed on SARS-CoV-2 positive patients. Informed written consent was gathered and the Ethic Committee approved the study. The mean age of patients was 60 years (24-92) and 64.7% (11/17) were male. Patient n.9 and n.17 died. All NPS resulted positive for SARS-CoV-2 at RT-PCR. RT-PCR on EBC resulted negative for all but one patients (patient n.12). In this study we did not find any correlation between positive NPS and the EBC in all but one patients enrolled. Based on these data which greatly differ from previous reports on the topic, this study opens several questions related to small differences in the complex process of EBC collection and how EBC could be really standardized for the diagnosis of SARS-CoV-2 infection. Further studies will be warranted to deepen this topic.

Is the composition of exhaled breath condensate a key to explain the course of COVID-19 in children?

INTRODUCTION: The relative resistance of children to severe course of the novel coronavirus infection remains unclear. We hypothesized that there might be a link between this phenomenon and observation from our previous studies concerning an inhibitory or cytotoxic effect of exhaled breath condensate (EBC) on endothelial cell cultures in children. AIM: Since we could not find any data on the similar effect caused by EBC in adults, the aim of our study was to evaluate and compare the biological activity of EBC in adults and children in an experimental in vitro model. Furthermore, in order to identify a putative agent responsible for these properties of EBC in children, we attempted to analyse the composition of selected EBC samples. MATERIAL AND METHODS: The influence of EBC samples on metabolic activity of endothelial cell line C-166 was assessed using colorimetric tetrazolium salt reduction assay (MTT assay). Selected EBC samples were fractionated using size exclusion chromatography and subjected to mass spectrometry analysis. RESULTS: Exhaled breath condensates in healthy children, but not in adults, revealed a cytotoxic effect on in vitro cell cultures. This effect was most significant in condensate fraction, which contained a prominent 4.8 kDa peak in the mass spectra. CONCLUSIONS: Breath condensates of healthy children contain the factor which reveals the inhibitory/cytotoxic effect on endothelial cell cultures. Although the physiological role of this agent remains unclear, its identification may potentially be useful in ongoing research on SARS-CoV-2/COVID-19.

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.

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.

RT-PCR diagnosis of COVID-19 from exhaled breath condensate: a clinical study.

Current diagnostic testing for coronavirus disease 2019 (COVID-19) is based on detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swab samples by reverse transcription polymerase chain reaction (RT-PCR). However, this test is associated with increased risks of viral dissemination and environmental contamination and shows relatively low sensitivity, attributable to technical deficiencies in the sampling method. Given that COVID-19 is transmitted via exhaled aerosols and droplets, and that exhaled breath condensate (EBC) is an established modality for sampling exhaled aerosols, detection of SARS-CoV-2 in EBC offers a promising diagnostic approach. However, current knowledge on the detection and load of the virus in EBC collected from COVID-19 patients remains limited and inconsistent. The objective of the study was to quantify the viral load in EBC collected from COVID-19 patients and to validate the feasibility of SARS-CoV-2 detection from EBC as a diagnostic test for the infection. EBC samples were collected from 48 COVID-19 patients using a collection device, and viral loads were quantified by RT-PCR targeting the E gene. Changes in detection rates and viral loads relative to patient characteristics and days since disease onset were statistically evaluated. Need for mechanical ventilation was significantly associated with higher viral load (p< 0.05). Need for oxygen administration or mechanical ventilation, less than 3 d since onset, and presence of cough or fever were significantly associated with higher detection rates (p< 0.05). Among spontaneously breathing patients, viral load in EBC attenuated exponentially over time. The detection rate was 86% at 2 d since onset and deteriorated thereafter. In mechanically ventilated patients, detection rate and viral load were high regardless of days since onset. These results support the feasibility of using RT-PCR to detect SARS-CoV-2 from EBC for COVID-19 patients within 2 d of symptom onset.

Environmental virus detection associated with asymptomatic SARS-CoV-2-infected individuals with positive anal swabs.

In the fight against the outbreak of COVID-19 in China, we treated some asymptomatic infected individuals. This study aimed to detect pathogens in biological and environmental samples of these asymptomatic infected individuals and analyse their association. Using a cross-sectional study design, we collected biological and environmental samples from 19 patients treated in the isolation ward of Nanjing No.2 Hospital. Biological samples included saliva, pharyngeal swabs, blood, anal swabs, and exhaled breath condensate. Swab samples from the ward environment included inside masks, outside masks, palm swabs, bedside handrails, bedside tables, cell phone screens, toilet cell phone shelves, toilet pads and toilet lids. We also obtained some samples from public areas. We used RT-PCR to detect pathogens and colloidal gold to detect antibodies. As results, 19 asymptomatic infected individuals participated in the survey, with 8 positives for pathogens and 11 positives only for antibodies. Three positive samples were detected from among 96 environmental samples, respectively, from a cell phone surface, a cell phone shelf and a bedside handrail. No positive samples were detected in the exhaled breath condensate in this work. All patients identified pathogens in the environment had positive anal swabs. There was a statistical association between positive anal swabs and positive environmental samples. The association of positive samples from the surrounding of asymptomatically infected patients with positive anal swabs suggested that patients might secrete the virus for a more extended period.