1. Volatile Biomarkers of Clostridium difficile infection Dr Christopher Walton 3rd Biomarkers in Diagnostics Conference, Dublin 9 th – 10 th October 2014

2. Clostridium difficile Asymptomatic carriage to severe diarrhoea and colitis – significant associated mortality Most common cause of infectious diarrhoea in hospitals Common factors –Antibiotic exposure –Immunocompromised (e.g. transplant recipients) –Advanced age Increasingly recognised in other groups: IBD, children Early diagnosis: selection of therapy, prevention of spread, effective use of resources Electron micrograph of cultured C. diff bacilli

3. Hypothesis – C. diff and VOCs Bacteria grown in culture emit volatile organic compounds (VOCs) into the surrounding air Different species produce distinct VOC “fingerprints” – known since 1980s Anecdotal evidence of odour associated with C diff diarrhoea Thirty potential VOC biomarkers identified from literature search –Earliest paper published 1970 Current tests are effective but are laboratory – based. Can we devise a method of getting a result in minutes at point-of-care?

4. VOC sampling Headspace sampling system captures VOCs onto sampling tube Constant airflow, constant temperature Diffusive and active processes in sample Rate of emission (not just concentration) Analysis by TD-GC-ToFMS

5. Analysis Using frozen samples –All referred for C diff testing at Guy’s & St. Thomas’ Hospital –53 negative –53 positive (enzymatic & molecular tests) Each Sample produces >100 different VOCs Analytical method separates components chromatographically Identifies and quantifies individual compounds by mass spectrometry Representative chromatograms Entire chromatogram Expanded timescale

6. Potential biomarkers From initial list of thirty, most compounds eliminated: –No significant difference between C. diff positive and negative –Common in the environment (e.g. decane) –Not detectable in a high proportion of samples ROC curves constructed for eight remaining Area Under the ROC Curve Test Result Variable(s)Area Asymptotic 95% Confidence Interval Lower Bound Upper Bound 1-Propanol0.750.650.84 Butanal, 3-methyl-0.840.760.91 Propanoic acid, ethyl ester0.800.720.89 Isovaleric acid0.700.600.80 4-Heptanone0.710.620.81 Phenol, 4-methyl-0.810.730.90 Indole0.850.770.93 Hexanoic acid0.730.630.83

7. para-cresol (4-methylphenol) Produced by Clostridium, not by other species C. diff tolerates higher concentrations than other bacteria Hypervirulent strains tolerate even higher concentrations Possible “composite” measure of bacterial activity and virulence Comparable performance to some enzymatic tests Emission rate ng.l -1.min -1

8. Prospects for point-of-care diagnosis Absorption spectrum of para-cresol gives ability to distinguish from other compounds, especially water and carbon dioxide Interface of sample handling system to novel optical cell based on hollow silica waveguide –Low volume, long path length –Rapid measurement, low limit of detection Prototype system operational - methane from ruminant faeces H2OH2O H2OH2O CH 4 H2OH2O

9. Conclusions We have developed a headspace sampling system capable of capturing VOCs emitted by faecal samples Rate of emission of individual VOCs can be determined Eight VOCs were identified which provided good discrimination between samples from patients diagnosed as either negative or positive for Clostridium difficile infection by current laboratory methods 4-methylphenol (para-cresol) is of interest for point-of-care application Faecal VOC analysis has potential as a novel diagnostic approach in gastrointestinal infection