Identification of specific metabolites in mycolactone producing mycobacteria and Buruli ulcer infection: diagnostic biomarkers through metabolomic

Buruli ulcer is a severe, slow progressing skin infection which destroys tissue and is caused by bacteria found in the environment called Mycobacterium ulcerans. The disease is classified as a neglected tropical disease despite being described in over 30 countries including Ghana, Togo, Benin, Côte d'Ivoire, Cameroon, Mexico, Papua New Guinea, South East Asia and Australia, and mostly affects individuals in rural communities living in infected areas. The World Health Organisation's Global Buruli Ulcer Initiative has identified the development of simple diagnostic tools as one of the priority areas in the control of the disease. Metabolomics is a growing field in analytical science and biology which seeks to identify novel biomarkers that can distinguish between diseased and non-diseased individuals. In a pilot study, funded under the Cambridge-Africa Partnership for Research Excellence (CAPREx), we sought to determine the best types of sample for the identification of metabolites from the bacteria that can be found only in Buruli ulcer patients. We collected tissue biopsy, swabs and fine needle aspirates from 28 Buruli ulcer confirmed patents and 21 patients with tropical ulcers that were not Buruli ulcer. The samples were pre-processed at the Department of Biochemistry, Cell and Molecular Biology at the University of Ghana, to extract the required components for gas chromatography-mass spectrometry (GC-MS) at the University of Cambridge. We identified tissue biopsy and fine needle aspirates as the best specimen for the identification of metabolites in preference to lesion swabs. Interestingly, metabolites identified in both groups of patients included cadaverine, putrescine, pinitol, palmitate, naphthalene, chlopyrifos and oxaspiro. The former two metabolites are interesting because they classify all the samples as containing degenerating tissue. The fatty acid palmitate is a common metabolite present in human tissue. Pinitol and the later three identifies metabolites are chemical residues that may have been present in the treatment poultice used as unorthodox remedies by the patients.
In this proposal, we are seeking to further this pilot study, building on our observations and collecting the tissue biopsies and fine needle aspirates from a larger patient sample group to allow us to perform a more detailed analysis of the compounds found within the bacteria and also within infected tissue. We also intend to expand the scope of the biological samples from affected individuals by including urine samples in our analysis which may provide a more convenient biofluid to sample. To gain more insight into the novelty of the biomarkers we identify, we will include another phase of experimentation which involves the characterization of the compounds found in M. ulcerans and other similar bacteria strains that infect people in Ghana including M. pseudishottsii, M. liflandii and M. marinum DL. Data generated from this project will be important in the identification of new diagnostics and also give us insight to the biology of the mycobacterium during infection which may lead to better prospects for future treatments.

Buruli ulcer (BU) is a severe, slow progressing necrotizing skin infection caused by the environmental mycobacterium, Mycobacterium ulcerans. The disease is classified as a neglected tropical disease despite being described in over 30 countries including Ghana, Togo, Benin, Côte d'Ivoire, Cameroon, Mexico, Papua New Guinea, South East Asia and Australia, mostly affecting individuals in rural communities living in endemic areas. BU is characterized by nodule, papule and plaque formation which ultimately develops into large painless ulcers with undermined edges. The WHO's Global BU Initiative has identified the development of simple diagnostic tools as one of the priority areas to control BU. In a pilot study, funded under the Cambridge-Africa Partnership for Research Excellence, we sought to determine the best sample matrix for the identification of metabolites from M. ulcerans that can be found only in BU patients using gas chromatography mass spectrometry based metabolomics. We identified tissue biopsy and fine needle aspirates as the best specimens for the identification of metabolites in preference to lesion swabs. Metabolites identified in both groups of patients included cadaverine and putrescine, associated with degenerating tissue. In this proposal, we are seeking to further this pilot study, building on our observations and collecting the tissue biopsies and fine needle aspirates from a larger patient sample cohort to allow us to perform a more detailed analysis of the metabolome of the host and mycobacterium BU. To gain more insight into the novelty of the biomarkers we identify, we will include another phase of experimentation which involves the characterization of the lipid and aqueous metabolome of M. ulcerans and other mycolactone producing mycobacteria including M. pseudishottsii, M. liflandii and M. marinum DL. This project aims to identify novel biomarkers for disease diagnosis and give insight to the biology of the mycobacterium during infection.

The merging of data, local collaboration in endemic locations and interdisciplinary expertise provides an opportune time for our proposed research plan which involves the identification of metabolic markers in mycolactone producing mycobacteria as well as Buruli ulcer infected individuals for the identification of biomarkers that can be used in the development of a field diagnostic for point-of-care use. In Ghana women and children are mostly affected with the disease. Buruli is a major cause of morbidity in patients who have undergone successful healing.The stigma associated with the disease makes it difficult for patients, especially women and children, to reintegrate into society. Results of this work will lead to conclusions and recommendations that can be developed into policy in concert with local collaborators from community, regional, national, and international BU control programs and ultimately the WHO. The project will produce high quality scientific publications for peer reviewed journals.In accordance with MRC policies we will make all our data available to the scientific community through the MetaboLights repository for metabolomics data. In addition to increase the impact of this research we will hold one academic workshop in Ghana for microbiologists and scientists interested in metabolomics. We also intend to give talks at field stations to explain our research to those suffering from Buruli ulcers. Prof. Griffin and Dr. Mosi will also present their work to the Cambridge in Africa network in Cambridge which was the premier platform for forging the initial collaboration between the two investigators. In the long term, we intend to apply for more funding to develop the potential biomarkers into a rapid diagnostic test through various avenues including the development of monoclonal antiboides. This will be of great economic importance both to our respective universities and interested biotech companies. We will develop any intellectual property in conjunction with our universities and the MRC. More importantly, our research will contribute significantly to reduce the treatment burden of Buruli ulcer by identifying and diagnosis infection early and at the point of care.