Volatile Biomarkers Exclusive to Human Microbial Pathogens

The application of breath analysis in infectious disease research has significant potential for the development of rapid, non-invasive diagnostic tools for transmission tracking, and better management of infectious diseases. Infectious diseases can produce unique patterns of VOCs in the breath in two ways. Firstly, VOCs can be products of pathogenic metabolism (except viruses), and therefore can diffuse from the site of infection in the body through to the blood to the lungs, and subsequently be detected in the breath (1). Secondly, pathogens can alter the breath VOC composition of the host by affecting the host metabolism, for example, by triggering an immune response (2).

A schematic indicating the different origins of VOCs in the breath associated with pathogens, from metabolism or from the host response. Some examples of the different infections that have been associated with breath VOC changes are shown.

The detection of Helicobacter pylori (H. pylori) infection is already conducted using a breath test, which involves the ingestion of 13C-urea, and analysis of breath for CO2 as a product of urea metabolism. This test is rapid, non-invasive, and highly accurate. It is possible that breath tests can be developed for other infectious diseases, and could potentially be developed into rapid point-of-care (PoC) devices for infection control regimes. This is especially relevant for remote areas, and those with poor health infrastructure that cannot support widespread expensive laboratory-based techniques. In this blog, we will go through a few examples of microorganism-associated VOCs with the potential to be validated biomarkers for disease diagnosis via breath analysis.

Aspergillosis

Aspergillus fumigatus is the most common fungal species responsible for invasive aspergillosis. Whilst most individuals who inhale Aspergillus spores are unaffected, those who are immunocompromised, or with underlying respiratory conditions can face critical illness with a mortality rate of between 40% and 90% (3). As A. fumigatus is a fungal pathogen, it has complex metabolic processes. This is evidenced by the finding that A. fumigatus can synthesize at least 226 secondary metabolic products, many of which have antibiotic, phytotoxic, or cytotoxic properties that influence the interactions between the fungi and the host’s body (4). Many studies have identified specific VOCs that are commonly released in high concentrations from in vitro cultures of A. fumigatus.

In 2017, Gerritsen et al compared in vitro headspaces to breath samples of patients with invasive Aspergillosis. VOCs metabolites released by A. fumigatus isolated directly from patients included 1,3-pentadiene, 2-methylfuran, UIC, and 2-ethyl-1-hexanol with other VOCs including 2-methyl-2-propanol and 2-methyl-2-butanol in A. niger. Breath samples contained 1,3-pentadiene in all of the patients and 2-ethyl-1-hexanol. 2-methyl-1-propanol was found in all but one (5).

The VOC 2-pentyl furan has been specifically associated with A. fumigatus in studies looking at both headspace cultures and human subject breath samples (6,7). 2-pentyl furan was found in the breath of all cystic fibrosis (CF) patients with colonization by A. fumigatus, in 3 of 7 CF patients without evidence of the fungal infection and was absent in healthy volunteers (8). Compounds produced by A. fumigatus changed depending on the environment, for example with the presence of drugs or higher iron concentrations (9). Compounds always found at high concentrations included α-pinene, camphene, and limonene, as well as sesquiterpenes, identified as α-bergamotene and β-trans-bergamotene.

Camphene, α- and β-pinene, limonene, as well as the sesquiterpene compounds α- and β-trans-bergamotene have also been seen in other studies, and are therefore highly associated with the metabolic processes of A. fumigatus (10). These are all examples of terpenes, a diverse chemical class that plants, fungi, and microbes can produce. It may be the case that unique signatures of these VOCs could reliably indicate the presence of A. fumigatus, therefore paving the way for an accurate diagnostic breath test for aspergillosis to be developed.

Tuberculosis

TB is a disease caused by a bacterial infection that can affect any part of the body, most commonly the lungs. The bacteria responsible is Mycobacteria Tuberculosis, and is transmitted from person to person via airborne respiratory droplets, generated through the coughing and sneezing of individuals with TB in their lungs. Transmission occurs when these droplets are inhaled into the lungs (11). As VOCs that originate or are elevated from a tuberculosis infection can readily enter our lungs and be exhaled from the body in breath, breath analysis presents a powerful platform from which to develop novel biomarkers and diagnostic technology.

We have recently conducted a literature review of 25 papers, and identified 192 breath VOCs that are altered in TB infection. These compounds belong to over 30 different chemical classes, with the top 10 classes being alkanes and their derivatives, alcohols, carboxylic acids, aldehydes, aromatics and their derivatives, ketones, aromatic hydrocarbons, benzenes, furans, and terpenes. Of particular interest is 2-Butanone as it has been shown to increase in abundance in M. Tuberculosis in vitro cultures, and in human breath in those with TB (12–14).

Further research could perform a targeted study design of 2-butanone in exhaled breath of TB-infected versus non-infected subjects to validate the translatability between in vitro and human clinical studies. Also, absolute quantification of 2-butanone levels in subjects with latent and active TB, as well as correlating with M. Tuberculosis load, can facilitate the development of this compound as a biomarker.

Malaria

Malaria is a life-threatening infectious disease caused by five parasite species in the genus Plasmodium, which can infect humans through mosquito bites. The lifecycle of these parasites is complex, with multiple different stages, making the development of biomarkers and treatments for malaria challenging. These different species of plasmodium have distinct metabolic profiles, meaning that they may produce different VOC patterns in the breath. Detection of unique patterns of VOCs through breath analysis therefore has the potential for not only the detection of malaria, but also the stratification of malaria infection by species.

Through a recent literature review, we have identified 158 VOCs associated with malaria from 34 papers. A significant number of these again were terpenes, including alpha-pinene and 3-carene. Increased levels of these terpenes are thought to be generated by the Plasmodium parasites to act as volatile chemoattractants of mosquitos, as demonstrated by cell culture studies with infection (15,16). Other compounds included methyl undecane, dimethyl decane, trimethyl hexane, nonanal, isoprene, and tridecane, which when combined were found to be able to diagnose malaria with 83% accuracy (17).

These six compounds can be found within our Breath Biopsy VOC Atlas®, a reference library that provides high-confidence identification of compounds for improved ability to develop VOC biomarkers for breath tests for clinical breath tests. With further characterization and validation of these VOCs, they could be utilized in the development of specific diagnostic tests for malaria.

How can Owlstone Medical support your infectious disease research?

We are the leaders in breath analysis, and we can help you include breath as an innovative sampling medium in your research. Our Breath Biopsy® OMNI service can provide everything you might need to start or continue using breath analysis, and you can receive expert advice from our team of scientists. Many of the VOCs mentioned that are associated with infectious diseases are included in our VOC Atlas. You can cross-check your data against our VOC Atlas to better understand the levels of VOCs in different populations, and see what mechanistic associations in the literature have been made to more confidently validate your breath biomarkers.

The Bill & Melinda Gates Foundation has recently committed to a $5 million equity investment and initial $1.5 million grant funding to Owlstone Medical to support the development of breath-based diagnostic solutions to improve outcomes in the developing world. With this investment, Owlstone is developing new cost-effective detection technologies for VOCs) that could serve as markers of diseases that disproportionately affect the developing world, including TB and HIV detection. This opens up the possibility of using breath-based testing could be deployed for rapid screening and earlier diagnosis. This data will be made available in the future through our Breath Biopsy VOC Atlas®, a reference database of identified and quantified volatile organic compounds (VOCs) found in exhaled breath.

References

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