A Concise Review of Exhaled Breath Testing for Respiratory Clinicians and Researchers

Abstract

Exhaled breath contains an extensive reservoir of biomolecules. The collection of exhaled breath is noninvasive and low risk. Therefore, its testing is an appealing strategy for the discovery of biomarkers of respiratory diseases. In this concise review, we summarize the evidence of exhaled breath tests for airways diseases and respiratory infections. An overview of breath collection methods in both individuals who are spontaneously breathing and those receiving mechanical ventilation is outlined. We also highlight the challenges in exhaled breath testing and areas for future research.

Introduction

The use of distinctive breath odors to identify diseases has been described since Hippocrates but became more extensively researched in the past two decades.^1,2 Exhaled breath gives access to a vast number of biomolecules that arise directly from the respiratory and conducting airways, which are potentially reflective of both local and systemic metabolism; therefore, it is an ideal medium for biomarker discovery.³ Exhaled breath testing is noninvasive and feasible, and, therefore, is an attractive and rapidly evolving field in clinical medicine and translational research. As such, exhaled biomarkers have been evaluated in virtually every respiratory disease.¹

The biomarkers in exhaled breath can be classified into (1) inorganic gaseous compounds, such as carbon dioxide and nitric oxide; (2) volatile organic compounds, which are carbon-based molecules with low molecular weight and are responsible for the sense of smell; (3) water-soluble biomolecules dissolved in exhaled water vapor; (4) aerosolized droplets and particles that arise from airway lining fluid; and (5) the temperature of exhaled breath (Fig. 1). The list of biomarkers discovered within these 5 categories contains thousands of compounds, the majority of which are volatile organic compounds.⁴ The size of this list and the fact that newly discovered compounds continue to emerge frequently makes a comprehensive list impossible to provide. However, a new project, dubbed the Human Breath Atlas, aims to serve as reference that will “map each molecule in our breath to health status.”⁵

Fig. 1.

An illustration of the fractions of exhaled breath with examples biomarkers found in each fraction. With permission.

Here, we summarize the evidence of exhaled breath testing in airway diseases and respiratory infections; we pay particular attention to breath collection methods in both individuals who are spontaneously breathing and those receiving mechanical ventilation. We also highlight the challenges in exhaled breath testing and areas for future research. This concise review is designed to be a resource for respiratory clinicians and translational researchers.

Exhaled Breath Testing

Because exhaled breath directly contacts the airways, its testing is an appealing strategy for the discovery of biomarkers of respiratory diseases. Exhaled nitric oxide, produced by the airway epithelial cells in response to the up-regulated interleukin 13 activities in T helper cell type 2 high asthma, is a pivotal example of a biomarker with known underlying mechanisms that has resulted in the development of several exhaled breath tests.⁶ Fraction of exhaled nitric oxide is now a widely available point-of-care single-breath test used as a surrogate biomarker for eosinophilic airway inflammation and is clinically implicated in both asthma diagnosis and management (Table 1).⁷

Table 1.

Summary of Exhaled Breath Testing in Respiratory Diseases

The “breathprint” exhibited from exhaled volatile organic compounds is far more complex. A typical breath sample contains hundreds to thousands of volatile organic compounds⁸ that originate from several sources, such as the external environment, diet, host metabolism, or non-host metabolism (eg, from microbials or pathogens); the source(s) from which volatile organic compounds originate is often not mutually exclusive and is often unclear.⁹ The comprehensive analysis of breath biomolecules is termed as “breathomics,” an advancing branch of metabolomics.⁹ To date, results of studies have found that the exhaled volatile organic compounds can be useful in the diagnosis and management of airways diseases and respiratory infections. For example, volatile organic compound signatures can discriminate patients with airways diseases from healthy controls^9,10; their clinical applications in phenotyping, drug monitoring, and prediction of exacerbations show promising results in both adults and children with asthma.¹¹ volatile organic compound–based breath analysis may also be useful for a COVID-19 diagnosis as well as invasive aspergillosis.^12,13 Exhaled volatile organic compounds can discriminate patients suspected of ventilator-associated pneumonia with confirmed infections from those with negative cultures¹⁴ and, therefore, may be a potentially useful tool to guide anti-microbial treatment.

Other exhaled breath tests, such as particles in exhaled air and compounds within exhaled-breath condensate (EBC), have also been validated as biomarkers of respiratory diseases (Table 1). EBC contains exhaled water vapor and aerosolized droplets. The water vapor fraction makes up the majority of EBC and contains numerous water-soluble compounds, such as salts, acids, nitrite, nitrate, glutathione, isoprostanes, and hydrogen peroxide, whereas the aerosol fraction includes insoluble and larger compounds, including viral particles, cytokines, and surfactants.¹⁵ Particles in exhaled air testing allows access to larger non-volatile biomolecules through specific breathing maneuvers that dislodge airway lining fluid during repeated closing and reopening of distal airways.¹⁶ These particles typically contain viral particles, immunoglobulins, albumin, surfactant proteins, and inflammatory cytokines.

EBC and particles in exhaled air may be noninvasive alternatives to bronchoscopy and bronchoalveolar lavage because they contain samples of airway lining fluid.¹⁷ Although EBC has demonstrated potential clinical applications in chronic respiratory diseases such as laryngopharyngeal reflux, asthma, COPD, cystic fibrosis, primary ciliary dyskinesia, and lung cancer,^15,18,19 a number of small proof-of-concept studies have shown the potentials of particles in exhaled air as an exhaled breath test for small airway diseases.¹⁶ Finally, exhaled breath temperature is thought to be correlated with airway hypervascularization and inflammation, giving it potential as a biomarker for inflammatory airways diseases (Table 1).²⁰

How Is Exhaled Breath Collected?

In Individuals Who Are Spontaneously Breathing

Exhaled breath can be collected noninvasively by a variety of different methods, each of which is dependent on the biomarkers of interest and analytical methods used. The required equipment is usually commercially available (Table 1). For individuals who are conscious, tidal breathing over a few minutes is generally required (often through a mouthpiece) for the collection of volatile organic compounds (by using bags, containers or adsorbent traps), EBC (into cold condensate chambers), and exhaled breath temperature (onto thermal sensors).^3,15,20 Examples of exhaled breath collection from individuals who are spontaneously breathing are displayed in Figure 2. It is also possible to collect exhaled volatile organic compound samples by using a face mask that covers both the nose and mouth, which may be more tolerated by individuals who find breathing through a mouthpiece challenging, such as in very young children and patients with acute breathlessness or stroke.²¹

Fig. 2.

Collection of exhaled breath from individuals who are spontaneously breathing. A: Tedlar bags can be used to collect exhaled gas or volatile organic compound (VOC). B: The RTube (Respiratory Research, Inc, Austin, Texas) can be used to collect exhaled breath condensate (EBC) or VOCs. C: The Inflammacheck (Exhalation Medical Technology, Cambridge, United Kingdom) can be used for point-of-care breath collection and analysis of EBC. Although these devices are commercially available and standardized, similar homemade devices are also frequently used to collect exhaled breath biomarkers. D-F: Examples of patient interfaces during collection are shown in. Of note, each collection device can be used with a mask instead of a mouthpiece if a seal is maintained between the mask and the patient’s face.

In contrast, the measurements of fraction of exhaled nitric oxide and the sampling of particles in exhaled air rely on specific breathing techniques through the mouth.^6,17 Sample collection by targeting specific fractions of the breathing cycle (such as end-expiration, which reflects a larger fraction of alveolar air) is also possible through tracking of the respiration.²² It is worth noting that a well-validated breath test is the use of nasal nitric oxide levels to diagnose primary ciliary dyskinesia. This test involves aspiration of gas from the sinonasal passages during a breath-hold, as opposed to an active exhalation, but is still included in many guidelines for exhaled breath testing.²³

In Patients on Mechanical Ventilation

Exhaled breath can be collected during mechanical ventilation. This collection occurs either in line with the ventilator circuit or directly from the exhaust port of the ventilator.^19,24-26 When collecting from the exhaust port, only volatile organic compound and exhaled water vapor biomarkers will be obtained because other biomarkers will be either lost in the ventilator circuit or trapped in expiratory filters. When collecting in line, collection is either passive or active: for passive collection, a trap is inserted into the expiratory limb of the ventilator for the duration of collection and then is removed^19,24; for active collection, the sample is aspirated from the expiratory limb (Fig. 3).¹⁹ Of note, during active in-line collection, samples can be coordinated with capnography to allow for sampling during a specific breath fraction, such as the end-tidal fraction.²²

Fig. 3.

In-line collection of exhaled breath during mechanical ventilation. A: Passive in-line exhaled breath condensate (EBC) collection requires placement of a condenser and collection device in line with the expiratory limb of the ventilator circuit; both insertion and removal of this device involves a brief interruption of mechanical ventilation (unless it is inserted before initiation of mechanical ventilation); collection units should be sterilized before clinical use. B: Active in-line breath collection requires placement of a sampling adapter in the expiratory limb of the ventilator; exhaled gas, condensate, or volatile organic compound (VOC) can then be actively aspirated by syringe or vacuum pump, similar to sidestream capnography; the removal of breath from the exhalation limb can affect the accuracy of ventilator measurements (eg, display a falsely low exhaled tidal volume), particularly when large amounts of breath are aspirated in a short period of time.

Clinical Concerns During Breath Collection

Although collection of breath is noninvasive and relatively safe, risks still exist with this procedure, both for the clinician and the patient, that should be addressed. When collecting breath from a patient who is spontaneously breathing, hyperventilation may occur. Also, some patients may find breathing techniques uncomfortable.^15,19 Warning a patient before collection and coaching throughout the procedure may reduce the likelihood of these events occurring.

Although still relatively safe, collection of breath during mechanical ventilation carries more risks than collection from patients who are spontaneously breathing.²⁴ When connecting a device to the exhaust port or in line with the expiratory limb of a ventilator, obstruction may occur. This can cause apnea, volutrauma, barotrauma, or, ultimately, a pneumothorax. During passive in-line collection, the ventilator circuit and ventilation itself must be interrupted during insertion and removal of the collection device, which can cause alveolar de-recruitment and hypoxia. During active in-line collection, some exhaled volume is removed (the exhaled breath sample); this can alter minute ventilation and exhaled tidal volume values reported by the ventilator. A skilled respiratory clinician who is familiar with the breath collection procedure should be performing it or be present during collection, and collection from patients who may decompensate from a brief interruption in ventilation should be avoided unless breath collection is necessary.^19,24

Particles in exhaled air and the aerosol fraction of EBC can carry bacterial and viral particles from patients with respiratory infections. Universal precautions should be taken when collecting and processing breath samples; similarly, droplet or airborne precautions should be taken when collecting and processing breath samples from patients suspected of having droplet- or airborne-transmitted respiratory infections.

The Challenges and the Future

Exhaled breath testing offers a promising approach for biomarker discoveries. However, most of the techniques are still at their infancy. The notable gaps in knowledge include the lack of (1) standardization of sampling techniques; (2) validated pipelines in sample processing, analytical methods, accurate quantification, and identification of compounds; (3) understanding of the source of detected compounds and appropriate adjustment for confounding factors; and (4) external validation of findings.^10,15 Furthermore, because highly complex data sets are generated (that often contain a vast number of compounds from a much smaller sample size), statistical analysis is either based on multivariate machine learning algorithms or by looking at the compound of interest in serial univariate analyses; often both approaches are adopted, but inconsistencies in analysis remain among published work.¹⁰ There is also a considerable lack of longitudinal and population studies to establish reliable discriminative abilities and reference ranges.

Summary

Development of more rapid, reliable, point-of-care tests for breath biomarkers is necessary to make exhaled breath testing more useful for respiratory clinicians and patients. Further advances in exhaled breath testing require the continuous interdisciplinary collaborations among clinicians, analytical chemists, scientists, bioinformatics specialists, and industry. Future research should prioritize the unmet needs in the field, including the standardization of collection and analytical methods.