Breath Diagnostics Using Chromatography for COVID-19 and Triage Applications

Professor Paul Thomas from the Centre for Analytical Science at Loughborough University in Leicestershire, UK, discusses the role of a rapid breath diagnostic method for COVID-19 using gas chromatography–ion mobility spectrometry (GC–IMS) and the wider impact and realities of field breath testing and analysis through the European Toxi-Triage project.

Q. Following the emergence of the COVID-19 pandemic your research focus has switched to the development of a breath-based diagnostic method (1). What benefits would a diagnostic method based on breath analysis have over the current standard of a RT-qPCR test?

A: I don’t think there is a single silver bullet test that is perfect for every eventuality. In developing breath tests for COVID-19, we are seeking to augment capability and capacity rather than provide something better than other test methods. The most powerful attributes for breath testing are the speed of analysis (less than one minute), flexibility, and the fact that breath testing does not encounter sampling artefacts to the same extent as nasal pharyngeal swabbing. Nasal pharyngeal swabs are problematic in that they are hit or miss—often miss.

A breath sample does not rely on a hit or miss sample as it takes a systemic sample that is representative of the whole patient. This is especially important when caring for or testing children and other vulnerable people.

Furthermore, the biomarker panel can be continuously refined and adjusted as the disease evolves and our understanding develops with it. There are other attributes of breath testing that go beyond a single positive negative result. I am exploring the proposition that different patterns of derangement across multiple systems and organs will manifest themselves within the exhaled volatalome. If this is demonstrated we should be able to go beyond infection identification and stage the severity of infection. Diseases are often staged to denote their progression and severity. It is a classification system that uses diagnostic findings to produce clusters of patients who require similar treatment and have similar expected outcomes (2).

Q. Two feasibility studies were performed to investigate the potential of breath analysis using gas chromatography–ion mobility spectrometry (GC–IMS) to diagnose COVID-19 (1). What did the results of these studies indicate?

A: Using a Haldane breath sampler with a sample loop injection onto a GC–IMS system, we were able to identify participants with COVID-19 from an acutely ill cohort with respiratory disease at hospital emergency departments. Our AUROC was ca. 0.9. AUROC stands for area under the receiver operator characteristic. AUROC is a widely used measure of the reliability or quality of a classification test, and is a plot of test sensitivity vs. one minus test-specificity. A value of 1 is perfection, 100% true positive and 100% true negative.

I think that as instruments become ever more analytically sensitive, future discussions about limits of detection are likely to involve the receiver operator characteristic and AUROC values. The panel of biomarkers we isolated were consistent with multi‑system derangement and indicated ketosis, inflammation, and gastrointestinal effect.

Q. What compounds distinguished COVID-19 from other similar respiratory infections?

A: For ketosis, acetone, acetone/2-butanone cluster, and 2-butanone were present. For inflammation, ethanal, propanal, octanal, and heptanal. For gastrointestinal effect, methanol monomer and methanol dimer.

This is still very much a work in progress, and with GC–mass spectrometry (MS) data coming online all the time I’m anticipating refinement of our pilot panel of volatile organic compounds (VOCs).

Q. What current or future research are you undertaking related to COVID-19 and what are you hoping to achieve?

A: Our partnership has grown their work to include clinics in the tropics (Sri Lanka) and communities in the mountains of British Columbia in Canada. From tropical heat and humidity to the biting winter cold of the Canadian mountains, we have been seeking to verify and refine our pilot studies. The data from these studies are being worked on and we hope to share our findings as soon as we can.

My hope and intent is to describe a reliable breath test for COVID-19. Looking ahead, I am interested in detection of the disease during its latent/incubation phase when no symptoms are apparent.

Q. Are you aware of any other research involving chromatography and COVID-19?

A: There has been a lot of activity within
the global breath research community addressing COVID-19 testing and diagnosis, and there is not space here to provide a comprehensive, systematic, and unbiased overview.

However, I have been really impressed by the agile and effective development of sensor array techniques, electronic noses if you like. Although I don’t know what the specific molecules are that the tests are based on, the fact is they do appear to work and they are really fast (3).

My friends and partners at Leicester University have undertaken a GC–MS-based study and are looking at the longer term as well. I think their recent paper is really useful (4).

I’m collaborating with British Columbia Cancer Centre—cancer patients are particularly vulnerable to COVID-19—and they are a team to watch as they are combining clinical- and community-based cohorts across different platforms, both GC–IMS and GC–MS. They are led by Professor Renelle Myers.

Another team I am watching is led by Dr Amalia Berna at The Children’s Hospital of Philadelphia. They are using GC–MS and their focus is on children, which, I think, is really important (5).

Q. Have you any advice to separation scientists using IMS for the first time? Is IMS hyphenated to chromatography becoming more commonly used? If so, in what ways and which application areas?

A: I’ve worked with and studied IMS since the 1980s and the last few years have been really wonderful with more and more applications for IMS being reported. I’m going to offer four pieces of advice to a first‑time IMS user (see https://www.isims.info).

First, read and study Ion Mobility Spectrometry by G.A. Eiceman, Z. Karpas, and Herbert H. Hill, Jr. (6).

Second, drift tube temperature, pressure, and water concentration are the priority for maintaining reproducible IMS performance. Ensure your quality control and statistical process control are monitoring these at all times.

Third, the ionization process lies at the heart of this technique. In the same way that separation scientists pay close attention to the stationary phase, you will need to understand the ionization properties of your analytes under the ionization regime you are using. This is particularly important if you have coeluting compounds or significant matrix artefacts that might sequester charge and suppress your signal.

And my fourth piece of advice would be to attend the International Society of Ion Mobility Spectrometry annual meeting. In my experience there is a no more efficient way of learning about this technique. Networking with a global community of experts, who absolutely want to see any newcomer to the field do well and generate useful data and outcomes, is a must.

Q. You have a great deal of experience in the field of breath testing and analysis and work as part of the European-funded project Toxi-Triage. Could you tell us a little bit about the project and what it aims to do?

A: I was the project coordinator for Toxi-Triage. Eighteen partners from seven European countries developed complementary technologies that were integrated to provide situational awareness at the level of an individual casualty. We were able to show how to combine drone, detector, informatics, artificial intelligence (AI), toxicology, and breath-testing techniques, and how such capabilities could be used to protect emergency service personnel and reduce mortality in mass casualty events. We were able to tag and trace every individual on-scene and provide a continuous update on their clinical and care-state along with a traceable timeline of everyone who had cared for them and where they had been treated (7).

Q. Why is accurate and rapid chemical, biological, radiological, and nuclear (CBRN) diagnosis necessary in the modern world and what are the challenges and realities in tackling these threats?

A:Where to begin? Emerging diseases have been a constant threat in recent times: HIV, SARS, MERS, Ebola, Zika, and now SARS‑COV2. There is no evidence to suggest that this procession of threats will abate and diminish. In fact, the reverse seems more likely; we need flexible and effective bio-surveillance.

The explosion of the Delta variant across India was a mass casualty incident in every sense of the word, demonstrating how CBRN responses go much wider than responses to terrorist outrages; although there is no doubt that, given the ways and means, there are many groups prepared to use CBRN agents on unprotected civilians and critical infrastructure. Indeed, some states have normalized the use of CBRN, particularly C agents in overt and covert ways.

In mass casualty CBRN events, hospital casualty surge protection and hospital staff protection are vital to maintain health care services and service continuity. Agile technologies that are able to detect and identify emerging threats deliver such capability. Metabolomic-based approaches can do this for a wide range of agents. Separation science combined with a range of detector technologies is well suited to this role in the medium term. You cannot repurpose a lateral flow assay from viral detection to chemical exposure or radiation injury, but you can repurpose casualty screening with the right breath biomarkers.

The rate-determining step at the moment is building the biomarker evidence base and integrating the appropriate machine learning into a networked system so that disease/detection algorithms are developed semi‑autonomously as a threat emerges.

An important health impact from pesticides needs addressing. Far too many young adults across the world take their lives by drinking pesticides, and knowledge of what pesticide and formulation would help clinicians improve survival rates. Our collaboration with Peradeniya University and the University of Edinburgh has shown how breath and saliva testing might be a useful approach (8).

Q. You mention the biomarker evidence base as being a rate-limiting step in the process. What are the challenges and techniques being used, and what is needed to address this bottleneck?

A: Breath research and analysis really need to standardize sampling, analytical workflows, and data pipelines. In recent years, the research and development effort has been really focused on exploring and defining the optimum techniques and methods to use. The breath research community really needs to consolidate their approaches and start generating digital biobanks of breath data. Once this has been accomplished, I think we will start to move much more rapidly.

References

1. D.M. Ruszkiewicz et al., EClinicalMedicine 29, 100609 (2020) https://doi.org/10.1016/j.eclinm.2020.100609
2. https://www.jefferson.edu/academics/colleges-schools-institutes/skmc/research/research-medical-education/disease-staging
3. A.G.W.E. Wintjens et al., Surg. Endosc. (2020) https://doi.org/10.1007/s00464-020-08169-0
4. W. Ibrahim et al., ERJ Open Research 7, 00139-2021 (2021) https://doi.org/10.1183/23120541.00139-2021
5. A.Z. Berna et al., medRxiv (2020) https://doi.org/10.1101/2020.12.04.20230755
6. G.A. Eiceman, Z. Karpas, and Herbert H. Hill, Jr., Ion Mobility Spectrometry 3rd Edition (CRC Press, 2016).
7. www.TOXI-triage.EU
8. Publication forthcoming

Paul Thomas studied the detection of trace volatile organic compounds for his Ph.D. at the University of Manchester Institute of Science and Technology (UMIST). After a short stint with the Atomic Energy Authority Safety and Reliability Directorate, he was awarded the Thorn Security endowed lectureship at UMIST and has subsequently studied and researched the analysis of trace complex VOC mixtures in complex samples. He is a past president of the International Society of Ion Mobility Spectrometry, current Honorary Secretary to the International Association of Breath Research, and Chair of Analytical Science at Loughborough University.

E-mail: c.l.p.thomas@lboro.ac.uk
Website: www.lboro.ac.uk/science/research/centreforanalyticalscience/