Volatile organic compound detection of Buruli ulcer disease: Headspace analysis of Mycobacterium ulcerans and used gauzes of Buruli-compatible ulcers

Abstract

Diagnosing Buruli ulcer (BU) is complicated by limited access to the sensitive IS2404 qPCR. Experienced clinicians report a distinct odour of Buruli ulcers. We explored the potential of headspace analysis by thermal desorption-gas chromatography-mass spectrometry to detect volatile organic compounds (VOCs) from Mycobacterium ulcerans both in vitro and clinically. This study was conducted in two phases: a discovery and validation phase. During the discovery phase, VOCs that enable identification of M. ulcerans cultures were determined. During the validation phase, these VOCs were evaluated in clinical samples for which we used gauzes from patients with skin ulcerations in the Democratic Republic of Congo. Seven M. ulcerans headspace samples were compared with four from sterile growth medium and laboratory environmental air. The univariate analysis resulted in the selection of 24 retained VOC fragments and a perfect differentiation between cultures and controls. Sixteen of 24 fragments were identified, resulting in eleven unique compounds, mainly alkanes. Methylcyclohexane was the best performing compound. Based on these 24 fragments, headspace samples originating from gauzes of 50 open skin lesions (12 qPCR positive and 38 negative) were analysed and an AUC of 0.740 (95%-CI 0.583-0.897) was obtained. As this is an experimental study, future research has to confirm whether the identified compounds can serve as novel biomarkers.

Fig 1. Set-up for headspace sampling of M. ulcerans cultures.

For the sampling of the headspace of the gauzes (clinical samples), the same materials were used, but instead of a bacterial culture, a used gauze was put in the bottle. TDT = thermal desorption tube. Filter = carbon filter to clean inflow of air.

Fig 2. Flowchart of data analyses.

Orange: discovery phase (in vitro, M. ulcerans cultures). Blue: validation phase (clinical, gauzes).

Fig 3. SPLS-DA plot of in vitro samples based on 24 fragments that were retained after univariate analysis.

Fig 4

a. AUROC curve of SPLS-DA analysis with 24 retained compounds. b. AUROC curve of SPLS-DA analysis with best performing compound, methylcyclohexane.

Fig 5. Excerpt from a chromatogram of M. ulcerans culture headspace, indicating the fragment at 444 seconds that was identified as methylcyclohexane.