Occurrence and Distribution of Mycobacterium Ulcerans along the Densu and Offin River Basins of Ghana
Date
2017-06
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Publisher
University Of Ghana
Abstract
Background: Buruli ulcer (BU), caused by Mycobacterium ulcerans (MU), is the third most important mycobacterial disease after tuberculosis and leprosy. Many features of MU ecology, including distribution within the environment, reservoir hosts and niche preference are still an enigma. The exact route of its transmission is also not known, although it has been identified as an environmental pathogen. A major drawback is the difficulty to isolate viable MU from the environment due to the slow growth rate and the complex mix of fast growing bacteria and fungi that outgrow culture media. This thesis sought to detect and isolate M. ulcerans from the environment.
Methodology: Samples were collected from ten communities along the Offin and Densu river bodies at different seasons. Following DNA extraction, the presence of MU was determined by gene targets, IS2404, IS2606 and the Ketoreductase (KR) gene. Samples were first analysed for IS2404, after which IS2404 positive samples with CT ≤ 35 were multiplexed for the MU specific markers IS2606 and KR gene. Four decontamination solutions and supplemented media were optimized after which the best were used for MU culture. The isolates were identified on the basis of specific genetic sequences, including heat shock protein (hsp) 65, IS2404, IS2606, rpoB, and the KR gene. Presumptive BU cases from active case search and passive reports were collected and analysed by IS2404 PCR. A case control study was further conducted to assess potential environmental and behavioural risk factors of BU.
Results: One thousand six hundred environmental samples were analyzed along the Densu (434; 27%) and Offin (1166; 73%) river basins and MU DNA was detected in 139 (9%) of the combined samples. The positivity of MU DNA along the Densu river basin
was 89/434 (20.5%) whilst that of the Offin river basin was 50/1166 (4.3%). The DNA was detected mainly in snails 5/6 (83%), moss 8/40 (20%), soil 55/586 (9%) and vegetation 55/675 (8%). The proportion of MU positive samples recorded was higher during the months with higher rainfall levels (126/1175 [11%]) than during the dry season months (13/425 [3%]). Among the methods evaluated, decontamination with 1 M NaOH followed by 5% oxalic acid gave the highest rate of recovery of mycobacteria (50.0%) and the lowest rate of contamination (15.6%). The cultivation medium that supported the highest rate of recovery of mycobacteria was polymyxin B-amphotericin B-nalidixic acidtrimethoprim-azlocillin–supplemented medium (34.4%), followed by isoniazid-supplemented medium (28.1%). Among the 139 samples cultivated in the main analysis, 58 (41.7%) yielded mycobacterial growth, 70 (50.4%) had no growth, and 11 (7.9%) had all inoculated tubes contaminated. A total of 25 different mycobacterial species were identified. Fifteen species (60%) were slowl growing (e.g., Mycobacterium ulcerans, Mycobacterium avium, and Mycobacterium malmoense), and 10 (40%) were rapidl growing (e.g., Mycobacterium chelonae, and Mycobacterium abscessus). The occurrence of mycobacterial species in the various environmental samples analysed was as follows: soil, 16 species (43.2%); vegetation, 14 species (38.0%); water, 3 species (8.0%); moss, 2 species (5.4%); snail, 1 species (2.7%); fungi, 1 species (2.7%). Out of 84 presumptive BU cases analyzed, 32 (38.1%) were laboratory confirmed by IS2404 PCR; 24 (75%) were from Densu, and 8 (25%) from Offin. A total of 176 cases and 176 controls were enrolled in the case-control study. Multivariate conditional logistic regression analysis identified farming in swampy areas (OR = 4.10, 95% CI = 3.82-7.18), farming while wearing short clothing (OR = 1734.1, 95% CI = 68.1-44120.9), insect bite M(OR = 988.3, 95% CI = 31.4-31115.6) and application of leaves on wounds (OR = 6.23, 95% CI = 4.74-18.11) as potential risk factors. Farming in long clothing (OR = 0.000, 95% CI = 0.00-0.14), washing wound with water and soap (OR = 0.37, 95% CI = 0.29-0.98) and application of adhesive bandage on wounds (OR = 0.31, 95% CI = 0.15-0.82) were found to be protective against BU infection.
Conclusion: The study confirmed the abundance of M. ulcerans in the environment which is influenced by seasonality. This study established an efficient and effective in-house decontamination procedure (NaOH-OA) and cultivation media that are supplemented with various antimicrobials. This study is the first to report on the in vitro isolation of M. ulcerans from environmental samples. Lastly, the study identified potential risk and protective factors for BU along the Densu river basin which can be integrated in BU control measures in Ghana.
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Keywords
Mycobacterium Ulcerans, Densu and Offin River Basins, Buruli ulcer (BU),