Mycobacterium ulcerans persistence at a village water source of Buruli ulcer patients

Abstract

Buruli ulcer (BU), a neglected tropical disease of the skin and subcutaneous tissue, is caused by Mycobacterium ulcerans and is the third most common mycobacterial disease after tuberculosis and leprosy. While there is a strong association of the occurrence of the disease with stagnant or slow flowing water bodies, the exact mode of transmission of BU is not clear. M. ulcerans has emerged from the environmental fish pathogen M. marinum by acquisition of a virulence plasmid encoding the enzymes required for the production of the cytotoxic macrolide toxin mycolactone, which is a key factor in the pathogenesis of BU. Comparative genomic studies have further shown extensive pseudogene formation and downsizing of the M. ulcerans genome, indicative for an adaptation to a more stable ecological niche. This has raised the question whether this pathogen is still present in water-associated environmental reservoirs. Here we show persistence of M. ulcerans specific DNA sequences over a period of more than two years at a water contact location of BU patients in an endemic village of Cameroon. At defined positions in a shallow water hole used by the villagers for washing and bathing, detritus remained consistently positive for M. ulcerans DNA. The observed mean real-time PCR Ct difference of 1.45 between the insertion sequences IS2606 and IS2404 indicated that lineage 3 M. ulcerans, which cause human disease, persisted in this environment after successful treatment of all local patients. Underwater decaying organic matter may therefore represent a reservoir of M. ulcerans for direct infection of skin lesions or vector-associated transmission.

Figure 1. Environmental contact network of laboratory confirmed BU patients from the southern Mapé Basin.

Panel A and B (detailed view of the village of Mbandji 2) show the houses where the 46 laboratory confirmed BU patients in our study lived (black points), the farm(s) where they worked (green points) and the locations where they obtained their water (blue points) during the year before the onset of BU symptoms. The home of each patient is connected with their farm(s) as applicable. Homes of 17 of the 21 non-participating laboratory confirmed BU patients were also mapped and are shown in grey. At the farms and water contact locations, soil (n = 171), plant (n = 153) and water (n = 109) samples were collected. Furthermore, in Mbandji 2 (B), animal faecal samples were collected around patients' houses (brown points). All samples were tested for the presence of M. ulcerans DNA and three village water locations were found to be positive (red points; VW12, VW31 and VW54). Further, at location F07 a positive duck faecal sample (red point) was collected. Photographs of locations VW31, VW54 and F07 are shown in C, D and E, respectively. Finally, Panel B also shows a negative water contact location (VW13) which was studied in detail.

Figure 2. Water contact locations in Mbandji 2 which were investigated in detail.

Based on the high case number and the identification of two environmental locations which were positive for M. ulcerans DNA, water contact locations in Mbandji 2 were analysed in detail. The town is located between the Mapé Dam and the Mbam River (A). Panel B shows the locations of the homes of the 6 patients from Mbandji 2 in our study (black points) and each of the homes is connected with the village water location(s) used by the respective patient. Faecal sampling sites are also shown (brown points). Locations which tested positive for M. ulcerans DNA are highlighted in red (B). A positive (VW12) as well as a close by negative (VW13) village water locations were studied in more detail. Images are based on a 0.5 m resolution WorldView-2 image take on March 12th 2011.

Figure 3. Alterations of the environment at locations VW12 and WV13 at the sampling time points.

Photographs of locations VW12 (A) and VW13 (B) are shown at selected environmental sampling time points.

Figure 4. Persistence of M. ulcerans at a village water location of BU patients.

Panel A shows a diagram of the water hole at VW12A from where samples were collected at eight time points over a period of 27.4 months. Soil sampling sites are shown as brown crosses, water sampling sites as blue crosses and plant sampling sites as green crosses. Table S1 shows how many samples were collected at each sampling site and each time point. All samples were tested for the presence of M. ulcerans DNA by real-time PCR. At 7 sampling time points, M. ulcerans real-time PCR positive samples were identified at VW12 (B with positive sampling sites identified by the larger coloured circles and C). Panel C (line colours correspond to the circle colours in panel B) shows the rate of positivity of the collected sample replicates as well as the average Ct value for the IS2404 real-time PCR performed on the positive samples. Finally, panel C shows the number of active BU cases in the village of Mbandji 2 (black line) and the number of active BU cases using VW12 (red line) at the environmental sampling time points.

Figure 5. In-depth analysis of soil surrounding the log at VW12.

Panel A shows the nature of the soil on the right and the left hand side of the log. To better understand positivity of samples at location VW12, we performed sampling all along the log on either side (B; brown crosses indicate sampling sites). Selected sampling sites were re-sampled over the next 12 days as indicated in panel C. Panel C further shows the rate of positivity among the replicates collected at each sampling time point and the average Ct value of the IS2404 real-time PCR performed.