Discovery of Buruli Ulcer Infection Associated Metabolic Markers

Abstract

Buruli ulcer (BU), is a severe slow progressing necrotizing skin disease, caused by the environmental pathogen Mycobacterium ulcerans (MU). The diagnosis of the disease can be carried out in four main ways. These include microscopy which is preceded by Ziehl-Neelsen stain, PCR amplification of the IS2404 insertion sequence, histopathology and culture. The limitations of BU diagnosis include microscopy which is not sensitive and specific, culture which takes 8-12 weeks for a growth to be observed and PCR which requires skilled labour and is very expensive. Early diagnostic techniques are urgently needed for this disease at areas and communities where they are most endemic since the disease can be treated if it is diagnosed early. The aim of this study was to discover Buruli ulcer infection associated metabolic markers using a metabolomics approach. Wound swabs from 47 participants were taken and BU cases were confirmed with microscopy using Ziel-Neelson staining and PCR. Metabolites were extracted from the wound swabs with chloroform, methanol and water and derivatized for Gas Chromatography-Mass Spectrometry to determine the different metabolites of patients with or without the infection. After DNA extraction and PCR 65% of the cases were found to be positive for mycobacterial DNA, 34% of the cases were also found to be positive for M. ulcerans and 8.5% were found to be positive for H. ducreyi. Seventeen metabolites were identified, and palmitate was found to be significantly higher when the cases were compared to the controls. Palmitate and stearate were found to show significance between the cases that were on treatment and those that were not on treatment. For all cases that were positive for mycobacteria they had significantly higher levels of pentadecanoate and heptadecanoate. Finally, cases positive for H. ducreyi and had higher levels of cervonate (docosahexaenoic acid) and palmitate. Unique metabolites specific to the BU cases were found and these can be confirmed as biomarkers and utilized in the development of a possible diagnostic for this Neglected Tropical Disease.