Diagnosis of COVID-19 by exhaled breath analysis using gas chromatography–mass spectrometry

Study design and participants

This was a prospective, real-world observational study conducted in Leicester, United Kingdom. The National Research Ethics Service Committee East Midlands approved the study protocol (REC number: 16/LO/1747). Patients admitted to hospital with suspected COVID-19 infection were approached and provided informed consent. Those who agreed to take part provided a breath sample within 24 h of admission to hospital, and samples were analysed using thermal desorption coupled gas chromatography–mass spectrometry (TD-GC-MS).

All participants underwent-testing for SARS-CoV-2 using PCR of nasopharyngeal swab, as part of the local streaming and clinical care pathways.

Clinical probability

In addition to PCR testing participants were classified by clinical probability of COVID-19 infection. This was with the aim of capturing patients with false-negative PCR swabs or those with delayed presentation who had pneumonitis but were no longer shedding viral load.

We classified the study subjects as high clinical probability of SARS-CoV-2 infection if they fulfilled either two major or one major and three minor diagnostic criteria (table 1).

These criteria were contrived by a panel of senior clinicians, supported by peer-reviewed published evidence [14–20].

Exhaled breath sample collection

Breath sampling was optimised to minimise cross-contamination and infection risk. The breath sampling apparatus included a mouthpiece with integrated HEPA filter (GVS) and a t-piece with directional valves (Intersurgical), connected to a polypropylene shut-off valve with 1/4″ push connections (RS Components) via a 22F-10 mm connector (Intersurgical). The valve was connected on the other side, via a piece of 1/4″ PFA tubing of minimal length, to the large port of a 3 L Tedlar bag with dual stainless-steel fittings with a piece of pre-conditioned silicon tubing surrounding the fitting to ensure a secure connection. The tubing and shut-off valves were pre-conditioned by heating for 4 h at 60°C and the Tedlar bags flushed with nitrogen and vacuum evacuated six times prior to sampling to reduce background VOCs. The pre-assembled, single-use device was handed to the patient at bedside, and patients were asked to perform tidal breathing, opening the shut-off valve when providing the sample and closing it again once the bag was inflated. Average sampling time was less than a minute.

The mouthpiece and all connections above the shut-off valve were disconnected and disposed of on the ward. The remaining bag and valve assembly were wiped down and double bagged before being taken immediately for loading onto sorbent tubes in triplicate (Carbograph 1TD, Markes International Ltd, Llantrisant, UK). The unused fitting on the Tedlar bag was connected to a handheld Elf pump (Escort® Elf, Sigma Aldrich, Dorset, UK) using sealed Tygon tubing, and the sample within the bag drawn through the sorbent tube under negative pressure at a flow rate of 0.5 L·min⁻¹ for 2 min, loading a total of 1 L of breath onto each of the three sorbent tubes. The sorbent tubes were sealed with brass caps (Swagelok) and stored in the fridge for up to 48 h before being transferred to the lab for analysis. Blank samples were taken from Tedlar bags filled with air (BTCA grade, BOC) at the beginning of each sampling week.

Sample analysis

All sample tubes were loaded with internal standard (0.6 μL of 20 μg·mL⁻¹ toluene-d₈, phenanthrene-d₁₀ (Sigma Aldrich) and n-octane-d₁₈ (Cambridge Isotope Laboratories) mixture in methanol (Sigma Aldrich)) in a stream of nitrogen at a flow rate of 100 mL·min⁻¹ for 2 min, purging off the excess solvent.

Reference standards

A 100 μg·mL⁻¹ multi component air standard (p/n 4M7537-U, Sigma Aldrich) was used to monitor instrument performance and for peak identity confirmation and was diluted in methanol to give a final concentration of 10 μg·mL⁻¹. A 1 μL sample of standard was loaded onto sorbent tubes into a stream of N₂ (zero grade, BOC) at 100 mL·min⁻¹ and purged for 1 min, before addition of internal standard as described above. Benzaldehyde, 1-propanol and octanal (Sigma Aldrich) and SPEX Certiprep Can-Terp Mix (Fisher Scientific, Loughborough, UK) standards were diluted in the same way and loaded onto tubes to confirm the identity of additional selected peaks.

A reference solution used to monitor retention behaviour was prepared from a 40 mg·L⁻¹ C₈–C₂₀ saturated alkanes certified reference material (Sigma Aldrich) combined with a 2000 μg·mL⁻¹ aromatics calibration standard (NJDEP EPH 10/08 Rev.2, Thames Restek). The mixture was diluted in methanol (SupraSolv grade, Sigma Aldrich) to give final concentrations of 10 μg·mL⁻¹ and 20 μg·mL⁻¹ for n-alkanes and aromatics, respectively.

TD-GC-MS analysis

GC-MS analysis was carried out on an Agilent 7820A with 5977B MS (Agilent Technologies Ltd, Stockport, UK). A DB-5MS capillary column 60 m×0.25 mm×0.25 μm (from Agilent Technologies) was used with a 1 mL·min⁻¹ column flow rate using helium (N6.0, BOC) as a carrier gas. The GC conditions were as follows: the column starting temperature was 35°C which was then raised to 130°C at 2.8°C·min⁻¹, to 220°C at 4°C·min⁻¹ and then to 320°C at 25°C·min⁻¹, where it was held for a further 10 min.

The mass spectrometer was operated in full scan mode from 40 to 350 amu. The transfer line to the mass spectrometer was heated to 300°C; the source temperature was maintained at 230°C and the quadrupole at 150°C.

The instrument was interfaced with a Markes TD-100 xr thermal desorption unit (Markes International Ltd). Tubes were pre-purged with carrier gas for 1 min at 50 mL·min⁻¹ and then desorbed at 300°C for 5 min with a flow of 50 mL·min⁻¹ onto a “hydrophobic, general purpose” trap (Markes International Ltd) held at −10°C. The trap was then purged for 2 min at 2 mL·min⁻¹ before being heated at the maximum heating rate to 300°C for 5 min, with a spit flow of 2 mL·min⁻¹.

Data analysis

A custom library was built in Unknowns (Agilent Technologies) from random selection of 10 patient samples, covering all patient groups, based on their age, PCR status and clinical probability. From this library a method was built in MassHunter Quantitative analysis (Agilent Technologies) using the base peak as quantifier and two qualifier ions for identity confirmation. All peaks were manually screened to ensure correct integration, and siloxane peaks were removed prior to further analysis. Peaks were removed if <60% of samples did not exceed the mean blank integrated peak area plus three standard deviations. Three patient samples were discarded owing to the high levels of exogenous contamination.

Statistical analysis

Feature selection was performed by applying LASSO regression to the final VOCs peak table with the dependent variable as PCR status first, then clinical probability, extracting a set of relevant features for each model.

A logistic regression model was then fitted with the dependent variable as PCR status first, then clinical probability, with the independent variables set as the respective relevant features selected by the LASSO model above. Partial least squares discriminant analysis (PLS-DA) and principal component analysis (PCA) were then applied to the two sets of features, with the dependent variable as SARS-CoV-2 PCR status and clinical probability, respectively.

All analyses were performed using R 4.0.0 [21]. LASSO regression was performed using glmnet [22], PCA was performed using the mixOmics library [23] and PLS-DA was performed using the ropls library [24].