Development of Tools for the Control of Mycobacterium ulcerans Disease (Buruli Ulcer)

Buruli ulcer (BU) – or Mycobacterium ulcerans disease – is a neglected tropical disease endemic in over 30 countries worldwide in tropical and subtropical regions. BU manifests as chronic ulcerated or non-ulcerated lesions in skin and soft tissues, with permanent disabilities and disfigurements being typical outcomes of the disease in low-resource endemic areas. The current BU control strategy is contingent upon the diagnosis and treatment of every case. No reliable preventive measures are currently available. However, BU control is hampered by the difficulties inherent in the real-world application of available diagnostics, and by the lengthy antibiotic and surgical interventions necessary especially for severe disease, which limit patient adherence and increase the risk of relapses. In this thesis, we have developed a variety of tools that could potentially ameliorate some of the challenges affecting BU control efforts. The immunoassays described are highly sensitive, being able to detect 1 – 2 ng of mycolactone, and could potentially become BU-specific diagnostics, since mycolactone is unique to M. ulcerans. The mycolactone competitive ELISA could detect the toxin in laboratory samples such as extracts from M. ulcerans cultures and tissues from M. ulcerans-infected mice. It has also proven capable of detecting mycolactone in clinical samples, in limited pilot studies. This assay is currently being converted into a lateral flow format, which could then be amenable to point-of-care diagnosis. The mycolactone capture ELISA is the first report of such an assay for the hapten-like mycolactone molecule. Similar to the competitive assay, the mycolactone capture assay is highly specific and sensitive, and could detect the toxin in a variety of samples, including clinical samples. Further optimisation of this assay, and ultimately conversion into a lateral flow format, is envisaged. Additionally, our PMA-qPCR method represents a culture-independent means of differentiating live and dead M. ulcerans cells. As an extension of the diagnostic gold standard IS2404 qPCR, this PMA-qPCR method could be directly suitable for applications where live/dead discrimination is necessitated but for which routine microbial culture is infeasible. New treatment options will help in BU control by easing treatment (thus improving patient adherence), and would reduce the overreliance on rifampicin – the only highly active antibiotic against M. ulcerans in clinical use. We took advantage of drug discovery efforts for other diseases by identifying scaffolds that could be repurposed for BU treatment. Two topical agents developed for chronic wound management, and compounds from the tuberculosis drug development pipeline, were assessed for efficacy against M. ulcerans. The topical agents – a wound care disinfectant and a bismuth-based antimicrobial agent – are already marketed for the management of chronic wounds of other aetiologies, and could therefore be applied to BU management in a relatively straightforward manner. Scaffolds with antituberculosis activity – which included arylvinylpiperazine amides, quinazoline amines, pyrrolopyridine diones, and quinolone carboxamides – were active against M. ulcerans in nanogram to low microgram concentrations. Further optimisation, including in vivo assessment in a BU mouse model, will help guide the repurposing of these scaffolds into novel BU treatment options. BU prevention by breaking transmission chains is currently not feasible because these transmission routes are still unknown. Vaccination could be one option for BU prevention, and is currently an actively researched field. We assessed the utility of three mycolactone-based vaccine candidates and found that potent mycolactone-neutralising monoclonal antibodies could be reproducibly generated using PG-203, a protein-conjugated non-toxic derivative of mycolactone comprising the invariant parts of the mycolactone molecule. PG-203, therefore, could serve as the basis for the development of a toxoid vaccine against BU. Altogether, the tools developed in the framework of this thesis could facilitate BU control by simplifying diagnosis and treatment of the disease, and contributing to the development of a vaccine against BU