A non-invasive human skin sampling technique for the detection of volatile organic compounds associated with Mycobacterium tuberculosis

Abstract

Tuberculosis (TB) is a contagious, airborne bacterial infection caused by Mycobacterium tuberculosis. It is one of the leading causes of death in our country; South Africa has the third highest number of TB incidents in the world, according to the World Health Organisation (WHO), and it is estimated that 80% of South Africa’s population is infected with TB. Despite medical and technological advances, blood and sputum samples are still the only primary biological materials used in the detection of Tuberculosis by various diagnostic tests. It is common knowledge that certain diseases are associated with biochemical/metabolic changes; therefore, this suggests that biochemicals/metabolites can be used for diagnostic purposes. The lack of screening programmes for early TB detection, and the shortcomings of current diagnostic tools, such as the requirement of skilled professionals due to the complex nature of the tests and the need for good infrastructure, has left a gap for the development of a point-of-care detection tool. Such a tool ideally should test for active TB in settings with limited resources, and it should be specific, sensitive, fast, cost-effective and non-invasive. The availability of such a tool would improve the current diagnostic time, leading to early detection and treatment, thus avoiding the need for later hospitalisation. This study investigated the use of human skin volatile organic compounds (VOCs) as prospective TB diagnostic biomarkers. The use of a non-invasive sampling technique in a non-targeted biochemical screening context was examined with the aim of exploring biochemical differences between healthy and diseased states. The study cohort consisted of test and control groups both containing male and female participants (n=38). The test group was clinically diagnosed with TB (n=15), without complications and they were sampled before the commencement of the TB drug treatment. The control group (n=23) was clinically proven to be non-TB infected and without respiratory complications. Each participant was sampled in duplicate (m= 76). Samples of VOCs from skin emanations of the participants were collected using Polydimethylsiloxane (PDMS) silicone rubber loops, worn as bracelets or a patch, followed by thermal desorption and analysis with comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GCxGC-TOFMS). More than 1550 compounds were detected, of which 1500 compounds were retained after blank/artefact removal/correction. From the 1500 retained compounds, 27 compounds associated with TB were found emanating from skin. Multivariate analysis was conducted to investigate prospective TB diagnostic biomarkers from skin emanation VOCs, using two approaches; targeted analysis where compounds found in this study were compared to those associated with TB in literature and non-targeted analysis which focused on finding novel compounds that might serve as biomarkers. This study discovered, for the first time, six compounds known to be associated with TB (heptanal; octanal; 1-octene; heptane-3-ethyl-2-methyl; acetaldehyde and 1H-indene, 2,3-dihydro-1,1,3-trimethyl-3-phenyl-), and two novel compounds (hexane, 3-methyl- and 2-octylcyclopropene-1-heptanol) emanating from human skin as potential biomarkers. Finally, the diagnostic ability of the VOCs selected by statistical methods was investigated using predictive modelling techniques. Results established a correlation between compounds found in this study and those associated with TB from matrices other than skin as reported in the literature. Predictive modelling was able to distinguish between patients and controls with a sensitivity of 0.848 and a specificity of 0.867 for targeted analysis, and as well as a sensitivity of 0.833 and specificity of 0.875 for non-targeted analysis. These values compare excellently with the sensitivities and specificities of breath tests and the Gene-Xpert (0.796 and 0.903; 0.930 and 0.930 respectively ), thereby forming the basis for the premise that the skin VOC test shows considerable promise and warrants further investigation.