DEVELOPMENT OF MOLECULAR EPIDEMIOLOGICAL TOOLS FOR BETTER UNDERSTANDING AND CONTROL OF BURULI ULCER

ABSTRACT The limited ability of molecular typing methods to differentiate Mycobacterium ulcerans strains from the same geographic areas, particularly at the local level, hampers our understanding of Buruli ulcer (BU) epidemiology and calls for the search for molecular epidemiological tools with higher discriminatory power. Moreover, the current available laboratory tests for BU diagnosis have various limitations, ranging from low diagnostic sensitivities to financial and technical constraints associated with their use, that drive the search for simpler, cost-effective, rapid and reliable point of care diagnostic assays to facilitate effective control of Buruli ulcer especially in resource-poor countries. This study used two genome-wide approaches to index genetic diversity in M. ulcerans (MU). In a resequencing approach, whole genome sequence of M. ulcerans isolates were compared with a reference sequence for the identification of single nucleotide polymorphic sites. The study characterized whole genome single nucleotide polymorphisms (SNPs) of MU isolates using Ion Torrent and Illumina sequencing technologies and assessed the discriminatory power of variable number of tandem repeat (VNTR), based on newly-described loci, in typing M. ulcerans from Buruli ulcer endemic areas. It also assessed the diagnostic value of the pocket warmer loop-mediated isothermal amplification (pw-LAMP) in comparison with IS2404 PCR and the conventional LAMP for M. ulcerans detection in clinical samples. Whole genome sequencing of MU isolates of different endemic areas in Ghana and Benin discovered a total of 299 SNP positions in comparison with the reference isolate, Mu_Agy99, with genomic regions of high SNP variability. Phylogenetic analysis based on SNP numbers inferred by neighbor-joining method produced five distinct M. ulcerans clusters with high confidence in clustal branches: two for the isolates from the Asante Akim North District and one each for the isolates from other endemic areas studied. Out of the 25 newly-described  VNTR loci tested, DNA amplification was observed in 23 (92%) of them with 15 (60%) VNTR loci amplification in 14 (82%) of the M. ulcerans isolates. The DNA band sizes observed for all loci were estimated to range between 300 bp and 750 bp, but all isolates had similar estimated band sizes for each locus. The results for the pw-LAMP, conventional LAMP and IS2404 PCR were comparable, yielding 90.5% sensitivity for the pw-LAMP and 100% sensitivity for both the conventional LAMP and IS2404 PCR when purified DNA extracts were used with no significant difference (χ² [1] = 0.40, p = 0.580). Specificities recorded for all three assays were 100%. Further genotyping study based on identified VNTR loci in combination with sequencing may provide useful information on the diversity of M. ulcerans and the epidemiology of Buruli ulcer. The pw-LAMP method for M. ulcerans DNA amplification is as efficient as that for IS2404 PCR. The genomic regions of high SNP variability identified in this study may be useful targets for genotyping to differentiate M. ulcerans isolates with unknown SNP characteristics. Analysis of SNPs based on DNA sequencing of whole genomes using Ion Torrent and Illumina sequencing technologies is useful for MU strain discrimination at local level.