Breath Analysis: An Emerging Diagnostic Approach

The diagnosis of disease relies on information obtained from a variety of sources, including the physical examination, imaging studies and laboratory tests to evaluate fluid and tissue samples. Further, the experience and diagnostic acumen of the clinician are also critical.

For oral diseases, the clinical examination and imaging are essential and continue to be the basis for arriving at a diagnosis. In addition, tissue samples are evaluated for the diagnosis of mucosal lesions. Analysis of oral fluids [gingival crevicular fluid (GCF) and saliva] has been studied for their diagnostic and predictive relationship to oral diseases, most notably periodontitis.1Taylor JJ, Preshaw PM. Gingival crevicular fluid and saliva. Periodontol 2000. 2016;70(1):7-10 While there is strong evidence that some of these proposed tests for periodontitis (primarily based on analysis of specific inflammatory mediators in GCF and saliva) hold promise for early identification of tissue destruction, these tests have not been widely adopted by the practicing community.

Another potential diagnostic approach for systemic disease identification and management involves breath analysis. The analysis of exhaled breath has been examined for its diagnostic potential. The advantages of using exhaled breath include.2Pham YL, Beauchamp J. Breath biomarkers in diagnostic applications. Molecules. 2021;26(18)

  1. Unlimited availability of samples, collected non-invasively. We breathe approximately 15 times per minute, which changes based on demand. This contrasts with blood and tissue samples, where collection requires an invasive procedure, and can be limited in amount.
  2. Lower cost of collection, as a health care professional is not required for collection. Further, a health care facility is not required, and patient discomfort is minimal or non-existent.
  3. Depending on the collection method, no potentially infectious waste is generated.

These advantages will improve patient acceptance of sample collection. Nevertheless, the analysis of exhaled breath presents challenges.2Pham YL, Beauchamp J. Breath biomarkers in diagnostic applications. Molecules. 2021;26(18) Breath contains a wide variety of substances. One focus has been on metabolomics, which is the study of small (low molecular weight) molecules that are the metabolic products of cellular and tissue function. As such, deviations from what is present in health may be associated with disease. In addition, exhaled breath is a gas. Air contains nearly 80% nitrogen, 21% oxygen and very small amounts of carbon dioxide and water. Exhaled breath contains about the same amount of nitrogen, less oxygen (16.5 %) and more carbon dioxide (4.5%). The change in the oxygen and carbon dioxide concentration reflects the cellular use of oxygen in energy generation, with carbon dioxide as a product of respiration.

The metabolites in breath include hundreds of organic and nonorganic compounds. In addition, if breath is exhaled through the mouth (versus the nose) volatile sulfur compounds (VSC) can be added as a product of oral bacterial metabolism.

While breath analysis has shown promise as an aid in the diagnosis of a limited number of conditions, this discipline has suffered from methodological issues that often characterize new diagnostic approaches. These challenges include:

  1. Identification of appropriate molecules/compounds that are to be analyzed in each disease state.
  2. Development of methods to detect these compounds, which may be in very small concentrations in breath. Ideally such methods would be point-of-care tests, which could be used when the patient is present or even in other non-clinical settings for screening of groups of people.
  3. Standardization of the collection of the breath sample that is required for analysis of the breath constituents of interest. This includes the conditions under which the sample is collected, and the collection device.3Di Gilio A, et al. Breath Analysis: Comparison among Methodological Approaches for Breath Sampling. Molecules. 2020;25(24)

Nevertheless, one needs only to point to the use of breath analysis as a measure of th alcohol concentration in blood, and the analysis of nitric oxide in exhaled breath as a diagnostic test in the evaluation of asthma, as evidence of the potential value of breath analysis in diagnosis. This essay will provide an introduction to breath analysis, with a focus on disorders of interest to oral health professionals.

Examples of Current Use of Breath Analysis

Alcohol breath test
The concentration of ethyl alcohol (EtOH) in blood can be accurately estimated by determining the concentration of EtOH in exhaled breath. This test has been used for more than half a century4Hlastala MP. Paradigm shift for the alcohol breath test. J Forensic Sci. 2010;55(2):451-6 to determine if a person, usually a driver of a vehicle, is operating under the influence of EtOH. This test can used on-site by law enforcement. In general, these tests rely on the fact that ingested EtOH is excreted in exhaled breath, and when exposed to a catalyst in the presence of oxygen, the EtOH is converted to acetic acid, and ultimately to water and carbon dioxide. The energy released as chemical energy (voltage) is then determined, and the greater the energy, the greater the concentration of EtOH (see Figure 1). Infrared-based detection devices are also available, which are based on absorption of the infrared energy by alcohol in the sample.

As is the case for almost all biological samples, test results are subject to differences in how the sample is collected. For assessment of the blood alcohol level by an alcohol breath test, an increase in the exhaled breath volume increases the detected concentration of alcohol. This is expected since EtOH is very soluble in blood.5Anderson JC, Hlastala MP. The alcohol breath test in practice: effects of exhaled volume. J Appl Physiol (1985). 2019;126(6):1630-5 While concern has been raised about the importance of quality assurance for the on-site evaluation of the blood alcohol level,4Hlastala MP. Paradigm shift for the alcohol breath test. J Forensic Sci. 2010;55(2):451-6,6Gullberg RG. Methodology and quality assurance in forensic breath alcohol analysis. Forensic Sci Rev. 2000;12(1-2):49-68 the breath alcohol test has become an essential tool for evaluation and ideally reducing drunk driving. In this context the application of breath analysis is expanding to include identification of both recreational and therapeutic drugs.7Trefz P, et al. Drug detection in breath: non-invasive assessment of illicit or pharmaceutical drugs. J Breath Res. 2017;11(2):024001

Nitric Oxide in Asthma
Asthma is a common, chronic inflammatory disorder of the respiratory tract, with different types recognized based on the clinical and inflammatory characteristics.8Ulrik CS, et al. Fractional exhaled nitric oxide as a determinant for the clinical course of asthma: a systematic review. Eur Clin Respir J. 2021;8(1):1891725 The need for diagnostic tests to assess both the disease status and response to therapy represents a challenge. One type of asthma is characterized by production of inflammatory mediators (cytokines), including interleukin (IL) 4, IL-5 and IL-13. This type of asthma is notable for the presence of eosinophils, a type of acute inflammatory cell similar to neutrophils (that characterize acute inflammatory reactions in the oral cavity and elsewhere). In response to this inflammatory milieu, nitric oxide (NO) is produced by the epithelial cells lining the bronchi and has an important regulatory function in the airway. When asthma is present, levels of NO increase as a result of the action of inflammatory mediators on the cells of the airway. The NO can be measured in exhaled breath and has been shown to increase as the inflammatory response in the bronchial wall increases.8Ulrik CS, et al. Fractional exhaled nitric oxide as a determinant for the clinical course of asthma: a systematic review. Eur Clin Respir J. 2021;8(1):1891725 The NO that is measured is referred to as fractional exhaled NO (FeNO; see Figure 1)

The review by Ulrik et al8Ulrik CS, et al. Fractional exhaled nitric oxide as a determinant for the clinical course of asthma: a systematic review. Eur Clin Respir J. 2021;8(1):1891725 examined three questions regarding the NO breath test. Does the test:

  1. Measure reduced lung function? Conclusion: yes for adults, insufficient data for children.
  2. Identify the risk for exacerbation of asthma? Conclusion: yes for adults with moderate to severe asthma, yes for children.
  3. Identify increased risk for loss of effectiveness of current medications? Conclusion: for adults the literature suggests that it does, but not all studies agree. For children, the data is less clear.

The authors concluded that assessment of NO is a surrogate for inflammation in the airway and has diagnostic and prognostic applications. In the context of this essay, breath analysis of NO is an example of how breath analysis has become a valuable aspect of health care.

For oral health care providers, asthma has been identified as a risk factor for several oral diseases, specifically caries, periodontal disease and Candida infection. The risk is due to a side-effect of the medications used to treat/prevent asthma, which can cause hyposalivation. In turn, these medications can promote an acidic environment in the oral cavity.9Gani F, et al. Oral health in asthmatic patients: a review : asthma and its therapy may impact on oral health. Clin Mol Allergy. 2020;18(1):22 For children, the severity of oral disease (including caries in the primary and permanent dentition, dental erosion and gingival inflammation) has been associated with asthma. The most advanced disease was seen in the most severe asthmatic patients.10Arafa A, et al. Assessment of the oral health status of asthmatic children. Eur J Dent. 2017;11(3):357-63

Figure 1.

Potential Future Applications of Breath Analysis for Systemic Disease

Given the potential advantages of using breath analysis for diagnosis of disease, studies are examining a broader application of breath analysis in health care. Among the disorders for which breath analysis has been considered are lung function, neonatal jaundice, cardiac transplant rejection, Helicobacter pylori infection (associated with gastric ulcers and certain cancers), gastroparesis (delayed stomach emptying), liver function2Pham YL, Beauchamp J. Breath biomarkers in diagnostic applications. Molecules. 2021;26(18) as well as diabetes mellitus.11Wang W, et al. Accuracy of breath test for diabetes mellitus diagnosis: a systematic review and meta-analysis. BMJ Open Diabetes Res Care. 2021;9(1) Two of these conditions (H. pylori infection and diabetes mellitus) are worth further mention, as both have importance for oral health care providers.

Detection of H. pylori via Breath Analysis

H. pylori has been identified as a causative microorganism in gastric ulcers, colorectal adenomas, as well as certain malignancies including stomach cancer, and non-Hodgkins lymphoma. Of note, though controversial, H. pylori has been identified in the oral cavity and has been examined as a possible source of re-infection after the organism has been eliminated from the lower gastrointestinal track.12Yee JK. Helicobacter pylori colonization of the oral cavity: a milestone discovery. World J Gastroenterol. 2016;22(2):641-8 Through this finding has been debated, evidence indicates that the oral biofilm can be a reservoir for H. pylori, and that the presence of this organism in the oral cavity can influence the eradication of the organism from the stomach.12Yee JK. Helicobacter pylori colonization of the oral cavity: a milestone discovery. World J Gastroenterol. 2016;22(2):641-8

H. pylori infection is often determined by endoscopy and sampling of the lesion, with histologic evaluation for presence of the organism. Given the invasive nature of this procedure, non-invasive diagnostic approaches have been sought, and evaluation of urea in breath has been one focus of these investigations.13Best LM, et al. Non-invasive diagnostic tests for Helicobacter pylori infection. Cochrane Database Syst Rev. 2018;3:CD012080 H. pylori produces large amounts of the enzyme urease. When challenged with radioactive urea, evaluation of exhaled radioactive carbon dioxide (here C13O2, a byproduct of urea metabolism) serves as a measure of H. pylori infection. The review of non-invasive testing for H. pylori infection concluded that urea breath tests for H. pylori were more accurate than either blood or stool tests, but also more resource intensive as a radioactive substrate is utilized.13Best LM, et al. Non-invasive diagnostic tests for Helicobacter pylori infection. Cochrane Database Syst Rev. 2018;3:CD012080 Further development of such breath tests for H. pylori is likely to occur, with the goal of making these tests less burdensome and therefore more widely utilized.

Breath Tests for Diabetes Mellitus

Diabetes mellitus (DM) is a very common chronic disease of sugar metabolism, that can result in a variety of clinical manifestations, including retinopathy, nephropathy, cardiovascular disease and poor wound healing. For oral health care providers, DM is recognized as the most important systemic disease risk factor for periodontitis, and has been associated with many other oral changes, including increased risk for root caries, candidiasis, xerostomia and burning mouth syndrome.14Lamster IB. Diabetes Mellitus and Oral Health: An Interprofessional Approach. Ames, Iowa: Wiley Blackwell; 2014. 272 p.

Breath analysis has been examined as a non-invasive means of determining metabolic control for persons with DM. At present, a peripheral blood (laboratory test) or finger stick sample of capillary blood (point of care test) are used to evaluate the level of glucose or glycated hemoglobin. For patients, repeated finger sticks can be difficult over time, and while continuous glucose monitoring is available, an attached sensor is required.

A systematic review of this area of investigation suggested that the concentration of carbon dioxide (CO2) in breath holds the greatest promise as a marker for metabolic control for patients with diabetes.11Wang W, et al. Accuracy of breath test for diabetes mellitus diagnosis: a systematic review and meta-analysis. BMJ Open Diabetes Res Care. 2021;9(1) The major source of CO2 is the oxidation of glucose in cells. Specifically, this assay requires ingestion of radioactive glucose (C13-containing glucose) for analysis and the C13O2 concentration is reduced when diabetes is present. Among the other potential breath markers of metabolic control in patients with DM that have been examined are acetone and isopropanol.

The use of radioactive glucose for this test limits current applications to health care facilities. Nevertheless, the expanding interest in the use of breath tests for common chronic diseases such as DM indicates broad interest in the use of non-invasive breath analysis in health care.

Application of Breath Analysis for Oral Disease

Breath analysis as an adjunct to diagnosis and evaluation of oral diseases offers intriguing possibilities. The oral cavity represents the last anatomical area for addition of biological markers to exhaled breath. Research on breath analysis has examined the relationship markers in breath to periodontal disease and oral squamous cell cancer (OSCC).

Periodontal Disease and Breath Analysis

Halitosis is the most widely recognized aspect of breath analysis that is associated with oral disease. Patients are often seen in the dental office for a complaint of halitosis, and identification of the underlying problem associated with bad breath suggests both a treatment approach and may offer diagnostic possibilities.

Halitosis has been evaluated both qualitatively and quantitatively, and the association of periodontal disease and halitosis has been studied. A scoping review that examined the relationship of periodontitis and oral malodor noted that halitosis is primarily attributable to VSC, which include hydrogen sulfide and methyl mercaptan. These compounds are produced by Gram negative microorganisms that metabolize host cells. The dorsal surface of the tongue contains accumulations of bacteria, desquamated epithelial cells and other components in saliva. The authors indicated the need for a reliable breath test to detect halitosis.15Morita M, Wang HL. Association between oral malodor and adult periodontitis: a review. J Clin Periodontol. 2001;28(9):813-9 A more recent systematic review concluded that there was an association of periodontitis and halitosis, with an impressive odds ratio of 3.16. Since the included studies were cross-sectional, no temporal relationship could be determined.16Silva MF, et al. Is periodontitis associated with halitosis? A systematic review and meta-regression analysis. J Clin Periodontol. 2017;44(10):1003-9

Devices for quantitative assessment of VSC in breath have been developed17Nakhleh MK, et al. Detection of halitosis in breath: between the past, present, and future. Oral Dis. 2018;24(5):685-95 but are not used in clinical practice. It is recognized that halitosis can have negative effects on oral health-related quality of life. A meta-analysis of this relationship indicated a standardized mean difference of 0.51, indicating a 49% reduction in quality of life when halitosis was present.18Schertel Cassiano L, et al. The association between halitosis and oral-health-related quality of life: a systematic review and meta-analysis. J Clin Periodontol. 2021;48(11):1458-69

Oral Squamous Cell Cancer and Breath Analysis

Specifically in regard to oral disease, breath analysis has been most widely studied for its potential as a diagnostic test for oral squamous cell cancer (OSCC).

A review of the use of exhaled breath analysis for the diagnosis of OSCC discussed both the potential makers and aspects of breath collection that represent methodological challenges.19Makitie AA, et al. Exhaled breath analysis in the diagnosis of head and neck cancer. Head Neck. 2020;42(4):787-93 Potential biomarkers were divided into three categories:

  1. Small volatile compounds. Examples include ammonia and formic acid.
  2. Low molecular weight biomolecules. Examples include polypeptides and nucleic acids.
  3. “Other” compounds. Examples include arachidonic acid metabolites and cytokines.

The potential complications and unanswered questions for the application in OSCC include how best to collect the sample, how to store the sample (if the analysis is remote), and potential contamination by saliva and the oral microflora. The use of this approach for both early diagnosis and assessment of the effects of treatment holds promise and has attracted attention considering the poor outcomes associated with delayed diagnosis of OSCC. However, many questions remain before this technique is widely adopted in clinical settings.

As an example, a small study of breath analysis applied to OSCC examined volatile organic compounds (VOC) in exhaled breath20Bouza M, et al. Exhaled breath and oral cavity VOCs as potential biomarkers in oral cancer patients. J Breath Res. 2017;11(1):016015 . Evaluating a small number of OSCC patients and cancer-free controls (26 in each group) a distinct clustering of certain compounds (i.e., benzaldehyde) was associated with OSCC. They also suggested collecting and analyzing air in the immediate vicinity of the suspected lesion. This represents a very interesting approach to evaluation of individual lesions.

Another study examined VOC from the approach of a breath component profile.21Dharmawardana N, et al. Development of a non-invasive exhaled breath test for the diagnosis of head and neck cancer. Br J Cancer. 2020;123(12):1775-81 181 patients with different stages of OSCC were evaluated, and comparison was to individuals without OSCC. The best models had a sensitivity (identifying true positives) of 80% and a specificity (identifying true negatives) of 86%. These values were higher than what could be achieved with a clinical approach (either direct observation or with an endoscope). These same authors published a review on this topic and concluded that this approach to the evaluation of OSCC shows promise, particularly related to a profile of VOC. Methodological issues remain to be defined.22Dharmawardana N, et al. A review of breath analysis techniques in head and neck cancer. Oral Oncol. 2020;104:104654

As this area of research has developed, new approaches have been identified. One study examined the use of machine learning of data from the analysis of VOC and reported accuracy of 86%-90% when identifying OSCC.23Mentel S, et al. Prediction of oral squamous cell carcinoma based on machine learning of breath samples: a prospective controlled study. BMC Oral Health. 2021;21(1):500 Another study reported on the use of a portable device to detect VOC (described as a “electronic nose”). The data was analyzed using an artificial neural network, and diagnostic accuracy was 72%.24van de Goor R, et al. Detecting head and neck squamous carcinoma using a portable handheld electronic nose. Head Neck. 2020;42(9):2555-9


Breath analysis is currently utilized in public health (i.e., alcohol breath test) and health care (i.e., asthma) situations, and this analysis is being evaluated for other common conditions (i.e., H. pylori infection, diabetes mellitus). Use in oral conditions is also under investigation, with the primary focus being diagnosis and management of OSCC. The analysis of exhaled breath offers the advantages of a simple means of collection, and therefore screening of large numbers of individuals at non-clinical and clinical settings. Mechanistically, it is interesting to note that the developed and applied tests (ethyl alcohol, nitic oxide) rely on evaluation of the molecule of interest by using a reaction that results in a measurable amount of energy such as light or electricity (see Figure 1).

There are many biological and methodological issues to be defined, the most important of which is identification of the most appropriate biomarker for each disease. This research task becomes even more complicated when several biomarkers are considered at the same time (i.e., OSCC). There are also issues of sample collection and analysis. Both exhaled breath and local sampling (i.e., at the site of a specific lesion) are being considered. Further, the design of these tests may require a clinical environment (with the sample sent to a laboratory or used bedside/chairside when the patient is in a clinical setting) or be designed for use in a non-clinical environment as a screening tool.

It is important for oral health providers to be aware of new developments in the diagnosis of disease, certainly including oral diseases. Additional findings and new data on breath analysis will be published in the future. It is noteworthy that a journal specifically devoted to breath analysis is available (Journal of Breath Research).

Additional information

On April 14, 2022, the Food and Drug Administration authorized the use of a breath test to detect COVID-19. The test relies on detection of a profile of VOC and utilizes a suitcase-sized detection device that requires a trained technician to operate. The device, manufactured by InspectIR Systems, can evaluate 20 samples per hour (one sample every 3 minutes), and been reported to have a sensitivity of 91% and sensitivity of 99%. Among other applications, this test may be employed as a screening tool at airports.


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