Genetic Diversity of PCR-Positive, Culture-Negative and Culture-Positive Mycobacterium ulcerans Isolated from Buruli Ulcer Patients in Ghana.

Culture of Mycobacterium ulcerans from Buruli ulcer patients has very low sensitivity. Thus confirmation of M. ulcerans infection is primarily based on PCR directed against IS2404. In this study we compare the genotypes obtained by variable number of tandem repeat analysis of DNA from IS2404-PCR positive cultures with that obtained from IS2404 positive, culture-negative tissue. A significantly greater genetic heterogeneity was found among culture-negative samples compared with that found in cultured strains but a single genotype is over-represented in both sample sets. This study provides evidence that both the focal location of bacteria in a lesion as well as differences in the ability to culture a particular genotype may underlie the low sensitivity of culture. Though preliminary, data from this work also suggests that mycobacteria previously associated with fish disease (M. pseudoshottsii) may be pathogenic for humans

Sero-Epidemiology as a Tool to Screen Populations for Exposure to Mycobacterium ulcerans

Sero-epidemiological analyses revealed that a higher proportion of sera from individuals living in the Buruli ulcer (BU) endemic Densu River Valley of Ghana contain Mycobacterium ulcerans 18 kDa small heat shock protein (shsp)-specific IgG than sera from inhabitants of the Volta Region, which was regarded so far as BU non-endemic. However, follow-up studies in the Volta Region showed that the individual with the highest anti-18 kDa shsp-specific serum IgG titer of all participants from the Volta Region had a BU lesion. Identification of more BU patients in the Volta Region by subsequent active case search demonstrated that sero-epidemiology can help identify low endemicity areas. Endemic and non-endemic communities along the Densu River Valley differed neither in sero-prevalence nor in positivity of environmental samples in PCR targeting M. ulcerans genomic and plasmid DNA sequences. A lower risk of developing M. ulcerans disease in the non-endemic communities may either be related to host factors or a lower virulence of local M. ulcerans strains

Single Nucleotide Polymorphism Typing of Mycobacterium ulcerans Reveals Focal Transmission of Buruli Ulcer in a Highly Endemic Region of Ghana

Buruli ulcer (BU) is an emerging necrotizing disease of the skin and subcutaneous tissue caused by Mycobacterium ulcerans. While proximity to stagnant or slow flowing water bodies is a risk factor for acquiring BU, the epidemiology and mode of M. ulcerans transmission is poorly understood. Here we have used high-throughput DNA sequencing and comparisons of the genomes of seven M. ulcerans isolates that appeared monomorphic by existing typing methods. We identified a limited number of single nucleotide polymorphisms (SNPs) and developed a real-time PCR SNP typing method based on these differences. We then investigated clinical isolates of M. ulcerans on which we had detailed information concerning patient location and time of diagnosis. Within the Densu river basin of Ghana we observed dominance of one clonal complex and local clustering of some of the variants belonging to this complex. These results reveal focal transmission and demonstrate, that micro-epidemiological analyses by SNP typing has great potential to help us understand how M. ulcerans is transmitted

Genomic Diversity and Evolution of Mycobacterium ulcerans Revealed by Next-Generation Sequencing

Mycobacterium ulcerans is the causative agent of Buruli ulcer, the third most common mycobacterial disease after tuberculosis and leprosy. It is an emerging infectious disease that afflicts mainly children and youths in West Africa. Little is known about the evolution and transmission mode of M. ulcerans, partially due to the lack of known genetic polymorphisms among isolates, limiting the application of genetic epidemiology. To systematically profile single nucleotide polymorphisms (SNPs), we sequenced the genomes of three M. ulcerans strains using 454 and Solexa technologies. Comparison with the reference genome of the Ghanaian classical lineage isolate Agy99 revealed 26,564 SNPs in a Japanese strain representing the ancestral lineage. Only 173 SNPs were found when comparing Agy99 with two other Ghanaian isolates, which belong to the two other types previously distinguished in Ghana by variable number tandem repeat typing. We further analyzed a collection of Ghanaian strains using the SNPs discovered. With 68 SNP loci, we were able to differentiate 54 strains into 13 distinct SNP haplotypes. The average SNP nucleotide diversity was low (average 0.06–0.09 across 68 SNP loci), and 96% of the SNP locus pairs were in complete linkage disequilibrium. We estimated that the divergence of the M. ulcerans Ghanaian clade from the Japanese strain occurred 394 to 529 thousand years ago. The Ghanaian subtypes diverged about 1000 to 3000 years ago, or even much more recently, because we found evidence that they evolved significantly faster than average. Our results offer significant insight into the evolution of M. ulcerans and provide a comprehensive report on genetic diversity within a highly clonal M. ulcerans population from a Buruli ulcer endemic region, which can facilitate further epidemiological studies of this pathogen through the development of high-resolution tools

Overview: mycolactone, the macrolide toxin of Mycobacterium ulcerans

The acquisition by a Mycobacterium marinum-like progenitor of a plasmid encoding enzymes for the biosynthesis of the highly potent macrolide toxin mycolactone has set off the evolution of M. ulcerans toward a new mycobacterial species. While the selective advantage of producing mycolactone for survival in environmental niche(s) of the pathogen is unclear, there is no doubt that the cytotoxic, immunomodulatory, and analgesic properties of mycolactone are key for the establishment and progression of M. ulcerans infections in the host. Improved procedures for the isolation, handling, and detection of the amphiphilic and light-sensitive toxin have facilitated studies to unravel molecular mechanisms of mycolactone action on host cells in vitro and on cellular and immune responses in animal models. The pivotal role of mycolactone in the pathology of Buruli ulcer and the fact that the toxin has not been associated with other pathogens make it an ideal target for therapeutics/vaccines aiming at mycolactone neutralization and for the development of assays for the diagnosis of the disease

Epidemiology and disease burden of Buruli ulcer: a review

Katharina Röltgen,1,2 Gerd Pluschke1,2 1Molecular Immunology, Swiss Tropical and Public Health Institute, 2University of Basel, Basel, Switzerland Abstract: Buruli ulcer (BU) is a neglected tropical skin disease caused by Mycobacterium ulcerans. Infection foci occur mainly in remote, rural areas of Central and West Africa, but also in Australia and Papua New Guinea. In addition, infections caused by M. ulcerans strains of a different lineage are sporadically reported from scattered foci in Asia and the Americas. While in the past decade more than 42,000 BU cases have been reported worldwide, an assessment of the actual global disease burden is complicated by the remoteness of affected populations and a lack of data on the incidence of BU in a number of countries, from which cases have been historically reported. Moreover, as BU patients present with diverse clinical manifestations ranging from relatively unspecific nodules, plaques, or edema to necrotic, ulcerative lesions, differential diagnosis is manifold and thus clinical misclassification may occur. Since to date reservoirs and transmission pathways of M. ulcerans remain equivocal, early diagnosis and treatment of patients are key determinants to control the disease. Particularly in view of the apparent decline in BU incidence in regions of West Africa, awareness and knowledge of BU in endemic regions must be retained to ensure a continuous monitoring and control. An integrated approach for the control of tropical skin diseases should be considered to cope with this difficult task. This review article aims at providing an overview of the current global burden of BU and summarizes the state of knowledge on the various epidemiological aspects of this enigmatic disease. Keywords: neglected tropical disease, chronic skin ulcers, Mycobacterium ulcerans, geographically confined infection foc

Overview: development of drugs against Mycobacterium ulcerans

For many years, wide margin surgical excision of Buruli ulcer lesions has been the main approach for the treatment of Mycobacterium ulcerans disease. The WHO now recommends an eight-week course of oral antibiotics with a combination of rifampicin and clarithromycin in Africa. However, disease management is complicated by social stigma, lack of awareness, and limited access to healthcare facilities, resulting in underreporting and frequently late initiation of medical treatment. Inadequate initial treatment can drive permanent disabilities and also limited compliance to the eight-week therapy is a limitation. Therefore, search for a faster and more simple treatment modality is ongoing, focusing primarily on the testing of new tuberculosis drug candidates for the treatment of M. ulcerans disease