Chief Investigator: Dr Thomas Goddard

Funding Amount: $32,000

Recipient: Women’s and Children’s Health Network

Overview:

CF is the most common fatal genetic condition in our community. Children die from respiratory disease caused by recurrent & chronic lung infections. Currently, sputum or broncho-alveolar lavage samples are cultured for several days before lung infection is diagnosed & treated. Because children cannot produce sputum on demand they can require an anaesthetic with a small camera inserted into their lungs to obtain the sample. However, if infections are detectable in the breath, diagnosis and monitoring is far simpler, cheaper and faster, enabling prompt treatment and prevention of disease.

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Research Outcomes:

Researchers: Dr Thomas Goddard, Dr James Martin, Dr Roger Yazbek, Ms Michelle Parsons, Associate Professor David Parsons

Research Completed: 2021

Research Findings:

175 exhaled breath samples were collected from 75 patients with cystic fibrosis (CF), both with – and without Allergic Bronchopulmonary Aspergillosis (ABPA). These breath samples were paired with exhaled breath condensate (EBC), sputum culture +/- bronchoalveolar lavage (BAL or ‘lung washout’) culture, lung function testing, and blood markers for CF and ABPA (where clinically applicable).

The breath samples were divided in the laboratory. The first part was analysed using selected-ion flow-tube mass spectrometry (SIFT-MS), an instrument designed to directly analyse gas samples for exhaled volatile organic compounds (VOCs) or breath ‘flavours’. The remainder of each sample was collected onto columns for gas-chromatography mass spectrometry (GC-MS) and sent to Netherlands for further analysis. GC-MS is a better validated method of analysis but it is not designed for directly processing gaseous breath samples and is therefore more cumbersome.

Although breath can be analysed by SIFT-MS directly as it is exhaled by the subject, for infection control reasons this was not the method used in this study. Rather, CF patients exhaled into specially designed single-use receptacles (SKC FlexFoil Plus®) that were used to store and transport the breath samples to the laboratory. Validation studies were undertaken to analyse the stability of breath samples looking at the effects of time, temperature, and transport. Comparison was made with other scientific and food-grade commercially available receptacles (e.g. Tedlar and Scholle IPN) and determined that FlexFoil Plus® receptacles had the most stable performance across these variables (for up to 6 hours).

The sputum and BAL samples were immediately frozen at -80°C. A portion of these specimens were sent for standard clinical assessment (using microscopy, bacterial and fungal cultures, and antibiotic sensitivity analysis). The remainder was thawed and prepared for comparison using whole genome sequencing (WGS) analysis. This technique has been used to validate the results as it allows for more detailed analysis of all the bacteria and fungi (including Aspergillus) in the specimens and ensures that pathogens (including viruses) are not missed. This is important as breath analysis is very sensitive and may detect very low levels of infection or inflammation in the lungs. If the pathogen is not detected on standard clinical assessment of the sputum or BAL sample (e.g. falsely negative), then there is a risk that the breath results will appear incongruous. Specific probes were developed to use flow cytometry to quantify (and not just identify presence or absence) the specific pathogens. This quantification method was adopted to allow for comparison with the quantification of VOCs in the exhaled breath. The WGS was only completed once COVID-19 restrictions on research were lifted and analysis of this data and comparison with breath data is ongoing.

Although EBC has been collected and processed, it is yet to be analysed. EBC is the moisture component of breath and is a surrogate for fluid lining the airways of the lungs (airway lining fluid). The EBC contains inflammatory markers which can be indicative of infection and inflammation in the cystic fibrosis lung. The results from the EBC will be compared with the blood markers of CF and ABPA already obtained to determine whether EBC can assist in diagnosis and treatment response. Comparison can also be made with the exhaled VOCs to determine whether there is correlation between the two techniques or additional clinical utility in combining them. There is a clear difference in the VOC breath profiles between CF patients who have received intravenous antibiotic therapy compared to those who have received oral/inhaled antibiotics and/or those who have not recently received antibiotics. SIFT-MS analysis of exhaled VOCs also allows for prediction of cigarette smoke exposure (e.g. ‘second-hand smoking’) with a high degree of sensitivity and specificity. In addition, VOC profiles appear to correlate with other markers of ABPA and allow a rapid non-invasive method of screening the presence (or absence) of this condition. Data comparisons of the results from SIFT-MS analysis with the GC-MS analysis (performed in Netherlands) are underway and will be used to validate the SIFT-MS data (or highlight any discrepancies).

Key Outcomes: Cystic fibrosis (CF) patients can be differentiated based on exhaled breath flavours into those receiving antibiotic therapy from those who are not receiving antibiotics. Other breath flavours allow for detection of cigarette smoke exposure (e.g. ‘second-hand smoking’) in CF children with a high degree of accuracy. Breath flavours appear to match other markers of specific diseases infecting the CF lung and allow rapid non-invasive screening of patients, potentially improving diagnosis and treatment outcomes.

Research Papers:

Published:

1.  Dharmawardana N., Goddard T., Woods C., et al. Development of a non-invasive exhaled breath test for the diagnosis of head and neck cancer. Br J Cancer 2020; https://doi.org/10.1038/s41416-020-01051-9.

2.  Goddard T., Yazbek R., Dharmawardana N., et al. Exposure to cigarette smoke in a cystic fibrosis cohort – distinctive volatile organic compound profiles. European Cystic Fibrosis Conference; June 2019, Liverpool, UK.

3.  Goddard T., Yazbek R., Dharmawardana N., et al. Longitudinal analysis of fasting hydrogen and methane production in cystic fibrosis (CF) patients receiving intravenous antibiotic (IV-A), oral antibiotic (O-A), and inhaled antibiotic (I-A) therapy. IABR Breath Summit; June 2018, Maastricht, Netherlands.

4.  Goddard T., Yazbek R., Dharmawardana N., et al. Longitudinal analysis of fasting hydrogen and methane production in cystic fibrosis (CF) patients receiving intravenous antibiotic (IV-A), oral antibiotic (O-A), and inhaled antibiotic (I-A) therapy. Florey Postgraduate Research Conference; September 2018, Adelaide, Australia.

5.  Goddard T., Dharmawardana N., Martin, J., et al. Longitudinal analysis of background room air VOCs in a new hospital. Presented at IABR Breath Summit; June 2018, Maastricht, Netherlands.

In preparation/Under review:

1.  Goddard T., Yazbek R., Parsons D., et al. Comparison of commercially available receptacles for exhaled breath storage, transport, and analysis.

2.  Goddard T., Dharmawardana N., Parsons D., et al. Diagnostic implications of longitudinal background room air VOC analysis across different metropolitan hospitals.

3.  Goddard T., Yazbek R., Dharmawardana N., et al. Exhaled breath analysis of short-chain fatty acid, hydrogen and methane production in cystic fibrosis patients – the effects of antibiotic therapy.

4.  Goddard T., Dharmawardana N., Yazbek R., et al. Exhaled breath analysis correlates with clinical markers of ABPA and treatment response in cystic fibrosis patients. 5.             Goddard T., Dallinga J., Yazbek R., et al. A comparison of SIFT-MS and GCxGC-MS analysis of cystic fibrosis exhaled breath.

Related Publications:

Future Outcomes: