Selecting Potential Breath Biomarkers for Development in Malaria through the Breath Biopsy VOC Atlas®

As of August 2024, we have collated 158 VOCs from 34 research papers on malaria. In this case study we discuss potential VOC biomarkers for malaria and demonstrate how to use the information provided in our VOC Atlas for biomarker development.

The need for novel malaria tests- early detection and identification of asymptomatic subjects

Malaria is a disease caused by Plasmodium parasites, which are transmitted to humans through infected mosquitoes. Once injected into the body from a bite from an infected mosquito, the parasite first enters the liver, where it stays dormant for 10 days (about 1 and a half weeks) to four weeks. Some species, such as P. vivax and P. ovale, can remain dormant in the liver for up to a year [1]. The signs and symptoms of malaria appear when the parasites leave the liver and infect the red blood cells. This is also the stage when malaria can spread to other humans through the bite of an uninfected mosquito, which then becomes infected. Despite entering the bloodstream, chronic infection- more common with p. falciparum in regions with seasonal transmission- is typically asymptomatic [2]. This is a major concern, as asymptomatic individuals may not seek treatment, making it difficult to eliminate malaria when mosquito vectors return. Due to the various lengths of the liver and blood stages in different Plasmodium species’ life cycles, as well as the added complexity of asymptomatic infection during the blood stage, it has been challenging to develop effective malaria tests, treatments, and vaccines. Nearly 290 million people are infected with malaria each year, resulting in more than 400,000 deaths [3]. To prevent disease spread and eliminate malaria, it is crucial to detect the parasites early enough (ideally before entering the bloodstream, especially species that undergo long dormancy in the liver) or to identify asymptomatic individuals for early treatment.

Dr. Audrey John from the Children’s Hospital of Philadelphia recently presented at the Breath Biopsy Conference 2024, on ‘Breath-based Diagnostics of Pediatric Malaria’. You can watch Dr. John’s talk on-demand, as well as the rest of the presentations from the conference here.

Currently, visual examination of parasite-infected red blood cells under a microscope remains the gold standard for confirming malaria [4]. However, asymptomatic individuals might not have their blood tested in the first place. Additionally, those who present symptoms with very low parasite levels may require repeat sampling, as the parasites may be initially undetectable. While alternative options are available, such as detecting specific malaria antigens through blood sampling with Rapid Diagnostic Test (RDT), some less common Plasmodium species still lack sufficient sensitivity data to evaluate the effectiveness of this test [4]. Polymerase chain reaction (PCR) is a useful molecular detection for confirming the species of malarial parasite. However, this method has not been established for malaria infection diagnosis because the results are often not fast enough to obtain. There is still a need for a malaria test that is: 1) rapid, with high sensitivity and specificity for early detection, and 2) low cost, home-based, and ideally non-invasive, allowing people in areas of seasonal transmission to regularly test themselves if they are infected but asymptomatic. The low-cost and portable features for malaria testing are especially crucial in rural and remote regions where there is not enough health infrastructure.

Breath tests are non-invasive, potentially low cost, and suitable for point-of-care application, making them an ideal novel tool for malaria detection. Exhaled breath contains volatile organic compounds (VOCs) that reflect physiological changes in the body. Potential VOC biomarkers for malaria may originate from the Plasmodium parasite (exogenous VOCs), or from the human body’s response to infection (endogenous VOCs). One recent malaria study conducted breath analysis in children and found that selected key VOCs provide high sensitivity for diagnosing malaria [5]. The authors also demonstrated that the model they developed can predict malaria infection status, even when the parasite levels are low. This highlights the potential of breath VOCs as biomarkers for identifying asymptomatic individuals of malaria, enabling early treatment. Because studies on VOC biomarkers have been conducted using different biological matrices and various sampling and analytical methods, a literature review that integrates the findings and assesses whether the VOCs were chemically identified is essential. A comprehensive literature review will help determine which VOCs have been consistently observed, the confidence in their identification, and their connection to pathophysiology.

VOC Atlas- a resource for selecting potential biomarkers for development

We recently conducted a literature review on VOCs associated with malaria, encompassing studies on bacterial culture, animals, and humans. The biological matrices used in these studies included breath and skin. Based on 34 papers, we identified 158 unique compounds altered in malaria. These compounds belong to over 24 different chemical classes, with the top five classes being terpenes (28), aldehydes (19), ketones (16), alkanes (16), and alcohols (14).

The Breath Biopsy VOC Atlas® is a reference database we are developing to support researchers interested in studying breath VOCs across different disease areas. VOCs reported in the literature from various disease areas and biological matrices (including breath, blood, urine, feces, and skin) are compiled in the VOC Atlas, with URLs linking to the original publications. To serve as a source of centralized, robust information, the VOC Atlas includes detectable exhaled breath VOCs from our analytical platform OMNI with confirmed identities. Additionally, we have developed specific metrics to differentiate compounds that are considered on-breath, meaning they are detected in exhaled breath with certain abundance differences from blank samples. We have established on-breath VOCs at baseline by sampling a healthy cohort. The on-breath status of certain compounds, such as those associated with immune response, or those with exogenous origins in infectious diseases, is likely to change as we add data from new studies involving disease cohorts to the database. The features provided by the VOC Atlas will facilitate users in easily accessing information and selecting compounds of interest within specific disease areas for focused research.

Examples of potential compounds in the VOC Atlas for malaria biomarker development

Within the malaria disease area, 80 of the 158 VOCs identified in our literature review can be found in the VOC Atlas. Of these 80 VOCs, 41% (33) are identified as on-breath based on our developed metrics. Eleven of these on-breath compounds (3-carene, alpha-pinene, beta-pinene, limonene, terpinolene, m-cymene, 2-methylbutanoic acid, 3-methylbutanoic acid, butyric acid, hexanal, and isoprene) have appeared in more than one study. Over half of these on-breath compounds are classified as terpenes, and the exact counts of these 6 terpenes found in the literature are shown in Table 1. These terpenes appear in the VOC Atlas as on-breath with the current baseline cohort because they can originate from plant-dietary sources (humans are unable to synthesize terpenes) and are therefore commonly detected with higher levels than blank samples.

In the case of malaria, increased levels of these terpenes are thought to be associated with the Plasmodium parasites as mosquito attractants, as demonstrated by cell culture studies with infection [6, 7]. Whether the altered terpene levels in humans with malaria resulted from the production of terpenes by the parasites requires further investigation. It is also possible that the parasites modulate the CYP450 system, thereby disrupting the biotransformation of terpenes ingested through the diet in the liver. Correlation analysis between liver function clinical metadata and exhaled breath terpene levels could provide further insights. Regardless of the unknown mechanism, terpenes, in the context of malaria are strong potential biomarkers for development.

Aldehydes, as mentioned earlier in this article, are the chemical class with the second-highest counts in our malaria literature search. We identified 11 aldehydes mentioned in more than two studies (Table 1). All of the identified aldehydes are included in the VOC Atlas. Although they are not currently identified as on-breath in the baseline cohort, this status may change as more disease studies are added to the database. Aldehydes are intermediate compounds of lipid peroxidation, which is associated with inflammation. While further research is needed to determine whether aldehyde levels in human breath are altered during parasite dormancy (i.e. P. vivax and P. ovale), their association with inflammation suggests that aldehydes could be potential markers for infection status in malaria.

Table 1. List of the top two chemical classes associated with malaria and compounds reported more than twice in literature.

Conclusion

It should be noted that many of the studies did not validate the compound identities, making a database like the VOC Atlas- where detectable breath compounds are confirmed by reference standards- very helpful for decision-making in biomarker development. As demonstrated above, researchers can select VOCs to focus on based on their needs and the information provided by the VOC Atlas. In the case of malaria, where elimination is more important than merely controlling the disease spread, biomarkers that are Plasmodium species-specific can be particularly advantageous. If detected while the parasites are still in their dormant stage, individuals can be treated before these parasites have a chance to enter the blood stage of their lifecycle. Biomarkers that can identify asymptomatic individuals will also be valuable for early treatment, and the development of point-of-care devices will enable regular self-screening in areas with seasonal transmission.

The VOC Atlas serves as a reliable resource for researchers to search for compounds associated with their disease areas of interest. The database is frequently updated with new compounds that have confirmed identities and assessed on-breath status. Make sure you sign up for access to the Atlas when it becomes available. If you would like to learn more about how we can assist with VOC biomarker discovery and development, please feel free to contact us.

References

1. Escalante, A.A., A.S. Cepeda, and M.A. Pacheco, Why Plasmodium vivax and Plasmodium falciparum are so different? A tale of two clades and their species diversities. Malar J, 2022. 21(1): p. 139. https://doi.org/10.1186/s12936-022-04130-9
2. Zhang, X. and K.W. Deitsch, The mystery of persistent, asymptomatic Plasmodium falciparum infections. Curr Opin Microbiol, 2022. 70: p. 102231. https://doi.org/10.1016/j.mib.2022.102231
3. Mayo Clinic. Malaria. Available from: https://www.mayoclinic.org/diseases-conditions/malaria/symptoms-causes/syc-20351184.
4. CDC. Malaria Diagnostic Tests. Available from: https://www.cdc.gov/malaria/hcp/diagnosis-testing/malaria-diagnostic-tests.html#:~:text=Microscopic%20examination%20of%20blood%20films,species%20to%20ensure%20proper%20treatment.
5. Berna, A.Z., et al., Breath biomarkers of pediatric malaria: reproducibility and response to antimalarial therapy. J Infect Dis, 2024. DOI: 10.1093/infdis/jiae323
6. Kelly, M., et al., Malaria parasites produce volatile mosquito attractants. mBio, 2015. 6(2). doi: 10.1128/mBio.00235-15
7. Emami, S.N., et al., A key malaria metabolite modulates vector blood seeking, feeding, and susceptibility to infection. Science, 2017. 355(6329): p. 1076-1080. DOI: 10.1126/science.aah4563