While the average molecular diagnostics (MDx) lab may encounter a range of sample types in its specimen stream, it’s a safe generalization that most labs see the bulk of their specimens in a few common types such as whole blood, serum, various swabs, and the like. For a particular medical presentation and diagnostic test requirement, there is usually a preferred specimen type, which is a compromise among three factors: sensitivity in context (higher is better), collection complexity and cost (lower is better), and invasiveness (lower is better). Perhaps not surprisingly, when the requirement is to balance the three factors, methods which are toward the far extreme of any one factor are not commonly chosen. This holds true for this month’s Primer topic—molecular diagnosis of expelled breath—but as we’ll see, it has been the focus of some work and indeed shows promise for use in some applications.
Breath as an analyte
That expelled breath can be obtained readily and non-invasively is an immediate observation; and that it can be used as a diagnostic specimen is common knowledge (albeit probably in the context of the “breathalyzer” test for determination of blood alcohol content). In the clinical chemistry lab, exhaled breath as a specimen for the detection of volatile organic compounds (VOCs) has been examined in multiple studies and has shown some utility in monitoring everything from liver function to inflammatory diseases, although this has been challenged by technical difficulties in normalization and standardization.
In such applications, the analytes are naturally volatile small molecules. But what about using exhaled breath for the detection of DNA or RNA—either from pathogens, or endogenous? It’s a big step from a few molecules of ethanol or acetone escaping in breath to enormous macromolecules like DNA or RNA or even entire cells. Nature does remind us, though, that in fact this happens (and with some efficiency) in every respiratory infection season, where aerosol transmission of bacteria and viruses takes center stage. Strictly speaking, much of this is the result of coughing or sneezing (which cause much more forceful air expulsion than normal breathing) and works through the expulsion of fomites (tiny droplets), but the precedent is there.
Lest the reader have sudden and awkward visions of a large plastic bag inflated with breath, and somehow trying to get their polymerase chain reaction (PCR) tube inside the bag and wave it about to introduce sample to their reaction, we should at this stage clarify that the sample format commonly employed in breath MDx applications is actually an expelled breath condensate (EBC). As the name implies, this is both concentrated from breath (thereby assisting with the extreme sensitivity requirement of the approach) and liquid in nature, allowing for its handling and introduction to reactions by micropipettor, much as with any other more usual sample type. According to a review by Hunt,1 EBC consists primarily of variable-sized droplets of airway lining fluid, water condensing out of vapor phase, and water soluble volatiles.
The first of these is our likely source of MDx substrates, acting much as fomites do. Variations in the contribution from condensing vapor phase water are highly variable but are reported to range from 20 to 30,000 times that of the alveolar lining fluid, making for challenges in normalization and standardization as alluded to above. While this can make quantitative MDx analysis challenging, it leaves room for applications where purely qualitative detection can be relevant. Actual sample collection is done through the use of various commercially available devices,2 and is well tolerated with a 10-minute breath sample yielding one ml to two ml of EBC.1
One of the first applications where MDx has looked to expelled breath as a sample type has been in the diagnosis of lung cancer. If one imagines that a lung tumor has exposed surface within the alveoli, and that tumor cells might slough off, then it’s not unreasonable to expect that these cells might be detectable at a very low frequency in expelled breath. Compared to a traditional lung biopsy, the advantages in collection complexity and invasiveness balance the requirement of extreme sensitivity which may be met through molecular methods. An early and widely cited examination of this is the study by Gessner and colleagues,3 who examined EBC from patients with non small-cell lung carcinoma (NSCLC) and matched healthy controls for beta-actin (cellular DNA control marker) and sequenced particular exons of the p53 tumor suppressor for oncogenic mutations. Nearly two-thirds of samples in this study were found to contain detectable amplifiable host DNA, and p53 mutations were observed in a number of the NSCLC patient samples (although oddly, when compared to invasive biopsy specimens on the same cases, the specific mutations noted were discordant, suggesting heterogeneity in the tumor cells).
Mutations in the mitochondrial DNA, particularly the D-loop region, are also associated with lung cancer. Ai and coworkers4 sequenced D-loop DNA from EBC samples taken from NSCLC patients and controls. Utilizing a long period (20-minute) EBC collection method along evaporative concentration and a commercial DNA extraction methodology, this study achieved mtDNA suitable for sequence analysis from all samples and demonstrated significant elevations in D-loop mutation rates in the NSCLC sample compared to controls.
Still within the NSCLC context, EBC has also been examined as a potential specimen for examination of epigenetic markers linked with cancer. A study by Xiao and associates5 looked for evidence of abnormal methylation of the P16 promotor region with a methylation-specific real-time PCR method, and compared results of tumor biopsy, blood plasma, and EBC from NSCLC cases against controls. While EBC showed the lowest sensitivity of the three sample types (40 percent, compared to 87 percent for tumor biopsy and 50 percent for plasma), it’s a good proof-of-principle result which suggests further
development may be warranted.
Lest we think that MDx of exhaled breath is limited to DNA, a study by Mehta et al6 examined mRNA expression ratios of two markers (GATA6 and NKX2-1) in EBC from lung cancer and control populations. Utilizing a real-time qRT-PCR approach, this study claimed an impressive 98 percent sensitivity and 90 percent specificity in detecting lung cancer.
Markers of infectious disease
Another clinical picture in which one might expect to find characteristic molecular markers in the lungs (and thus in EBC) is, of course, infectious disease. While the most obvious examples would be acute respiratory infections, we’ll segue into these applications for breath MDx through yet another NSCLC-related study, by Carpagnano and colleagues.7 They examined EBC of NSCLC patients and controls for human papillomavirus (HPV) DNA. While no HPV DNA was found in exhaled breath of the controls, it was detected in just over 16 percent of NSCLC samples, suggesting at least a correlation with some cases—and demonstrating detection of infectious agents in exhaled breath.
Finally, just to prove that EBC isn’t the only way to sample exhaled breath for infectious agents, there’s the work of Mitchell et. al.8 They took the simple approach of examining disposable spirometer mouthpiece filters as their breath sampling system. Using RT-PCR, samples were screened for common acute respiratory viruses including rhinovirus, RSV A/B, influenza A/B, parainfluenza viruses 1, 2, and 3, and human metapneumovirus. Detection results compared favorably with more traditional (and invasive) sampling methods including nasal washing and bronchoalveolar lavage (BAL).
In some diagnostic settings (mostly for infectious diseases), it may be of concern that an expelled breath sample could be contaminated with upper airway or salivary contributions; a detected organism might well be considered pathogenic when it originates in the lower airway but less likely so from the upper airway. These concerns, of course, can also apply with traditional collection methods like BAL. As a partial defense against this complication, most commercial EBC collection systems have a salivary trap system which reduces or eliminates salivary contributions to the sample. Combined with methodological approaches, salivary contributions are estimated at less than 1/10 000 of EBC content based on salivary amylase levels.2 Interpretation of the molecular results in full clinical context remains essential, however.
In non-molecular applications, expelled breath has been used as a clinical specimen for more than 30 years. As further molecular applications on this easily obtained sample type are validated, it may start to become a more common specimen type in a laboratory near you.
- Hunt J. Exhaled breath condensate—an overview. Immunol Allergy Clin North Am. 2007;27(4): 587–v.
- Konstantinidi EM, Lappas AS, Tzortzi AS, Behrakis PK. Exhaled breath condensate: technical and diagnostic aspects. The Scientific World Journal. 2015:435160. doi:10.1155/2015/435160.
- Gessner C, Kuhn H, Toepfer K, Hammerschmidt S, Schauer J, Wirtz H. Detection of p53 gene mutations in exhaled breath condensate of non-small cell lung cancer patients. Lung Cancer. 2004;43(2):215-222.
- Ai SSY, Hsu K, Herbert C, et al. Mitochondrial DNA mutations in exhaled breath condensate of patients with lung cancer. Respiratory Medicine. 2013;107(6) 911–918.
- Xiao P, Chen JR, Zhou F, et al. Methylation of P16 in exhaled breath condensate for diagnosis of non-small cell lung cancer. Lung Cancer. 2014;83(1):56-60.
- Mehta A, Cordero J, Dobersch S, et al. Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate. EMBO Mol Med. 2016;8(12):1380-1389.
- Carpagnano GE, Koutelou A, Natalicchio MI, et al. HPV in exhaled breath condensate of lung cancer patients. Br J Cancer. 2011;105(8):1183-1190.
- Mitchell AB, Mourad B, Tovey E, et al. Spirometry filters can be used to detect exhaled respiratory viruses. J Breath Res. 2016;10(4):046002.
John Brunstein, PhD, is a member of the MLO Editorial Advisory Board. He serves as President and Chief Science Officer for British Columbia-based PathoID, Inc., which provides consulting for development and validation of molecular assays.