Source Tracking <i>Mycobacterium ulcerans</i> Infections in the Ashanti Region, Ghana

<div><p>Although several studies have associated <i>Mycobacterium ulcerans</i> (MU) infection, Buruli ulcer (BU), with slow moving water bodies, there is still no definite mode of transmission. Ecological and transmission studies suggest Variable Number Tandem Repeat (VNTR) typing as a useful tool to differentiate MU strains from other Mycolactone Producing Mycobacteria (MPM). Deciphering the genetic relatedness of clinical and environmental isolates is seminal to determining reservoirs, vectors and transmission routes. In this study, we attempted to source-track MU infections to specific water bodies by matching VNTR profiles of MU in human samples to those in the environment. Environmental samples were collected from 10 water bodies in four BU endemic communities in the Ashanti region, Ghana. Four VNTR loci in MU Agy99 genome, were used to genotype environmental MU ecovars, and those from 14 confirmed BU patients within the same study area. Length polymorphism was confirmed with sequencing. MU was present in the 3 different types of water bodies, but significantly higher in biofilm samples. Four MU genotypes, designated W, X, Y and Z, were typed in both human and environmental samples. Other reported genotypes were only found in water bodies. Animal trapping identified 1 mouse with lesion characteristic of BU, which was confirmed as MU infection. Our findings suggest that patients may have been infected from community associated water bodies. Further, we present evidence that small mammals within endemic communities could be susceptible to MU infections. <i>M. ulcerans</i> transmission could involve several routes where humans have contact with risk environments, which may be further compounded by water bodies acting as vehicles for disseminating strains.</p></div

16S and IS2404 positivity in sampled water bodies and matrices.

<p>Environmental samples were first screened for mycobacterial 16s rRNA positivity. Positive samples were then screened for IS2404. At each water body, biofilm were collected in quintuplicates. All other samples were collected in triplicates. Biofilm were the most positive, (22/38) for 16S and this difference in matrix positivity was statistically significant (P = 0.0007) using the Chi-square contingency table for independence.</p><p>16S and IS2404 positivity in sampled water bodies and matrices.</p

Community-based geographical distribution of MPM genotypes from humans and water bodies.

<p>The map of study communities was drawn using ArcMap 10. Rectangular and circular callouts contain genotypes of MU detected in humans and the environment respectively. Intersection of callouts contain genotypes common to both sources. Red triangle (in Sukuumu) represents MU-positive animal. Genotypes W, X, Y and Z were found both in the environment and human population. The Offin River is represented by blue dotted lines and contact sites on it are represented by green squares. Communities are represented by symbol for a house and other sampled water bodies are represented by colored dots as defined in the legend.</p

16S and IS2404 positivity in sampled water bodies and matrices.

<p>Environmental samples were first screened for mycobacterial 16s rRNA positivity. Positive samples were then screened for IS2404. At each water body, biofilm were collected in quintuplicates. All other samples were collected in triplicates. Biofilm were the most positive, (22/38) for 16S and this difference in matrix positivity was statistically significant (P = 0.0007) using the Chi-square contingency table for independence.</p><p>16S and IS2404 positivity in sampled water bodies and matrices.</p

MU confirmation and VNTR profile of MU detected in human samples.

<p>UA, unassigned. PCR amplification of Locus 19 was unsuccessful for FS3 and FS6. Locus 6 and Locus 19 were the main determinants for a genotype. Patient, FB2 was negative for MU. Pos, positive, Neg = negative, L6 = locus 6, L19 = locus 19.</p><p>MU confirmation and VNTR profile of MU detected in human samples.</p

Sequence confirmation of VNTR repeats and phylogeny of MU isolates.

<p>The evolutionary history was inferred using the UPGMA method [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003437#pntd.0003437.ref039" target="_blank">39</a>]. The optimal tree with the sum of branch length = 1.31639434. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003437#pntd.0003437.ref040" target="_blank">40</a>]. The reference sequences (represented with square) of the MIRU1 (ScoA gene) orthologs, <i>M. marinum</i>, <i>M. liflandii</i> and <i>M. ulcerans</i> were retrieved from GenBank with accession numbers CP000854.1, CP003899.1 and DQ397533.1 respectively. Sequences of human and environmental samples are represented with triangles and circles respectively. Tandem repeats were analysed using Tandem Repeat Finder [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003437#pntd.0003437.ref041" target="_blank">41</a>] and pattern searches in Microsoft Word 2013.</p

Mycobacteria detection in organs of animals with lesions.

<p>Stomach(St), Kidney(K), Small intestine(SI), Spleen(SP), Lesion biopsy(LB), Lesion swab(LS), caecum(CS), lungs(Lu), anal swab(AS). NA, not amplified.</p><p>* Also positive for IS2404 (99% sequence identity to MU Agy99). 16S positivity was used to infer presence of <i>Mycobacterium</i> spp in organs but IS2404 sequencing was used to confirm presence of MU.</p><p>Mycobacteria detection in organs of animals with lesions.</p

Four water bodies that were sampled.

<p>A) Twingun 2 pond at Sukuumu; B) illegal mining activities (galamsey) on the Offin River at Monia-Gyaman; C) Nkotia stream at Sukuumu; D) Bebonu pond at Wromanso.</p