Evaluation of an electricity-independent method for IS2404 Loop-mediated isothermal amplification (LAMP) diagnosis of Buruli ulcer in resource-limited settings.

INTRODUCTION: Buruli ulcer (BU) caused by Mycobacterium ulcerans (MU) is a devastating necrotic skin disease. PCR, recommended for confirmation of BU by WHO, requires an adequately equipped laboratory, therefore often delaying timely diagnosis and treatment of BU patients in remote settings. Loop-mediated isothermal amplification (LAMP) is a PCR-based protocol for isothermal amplification of DNA that has been suggested for diagnosis of BU in low-resource settings. STUDY AIMS AND METHODS: This is an exploratory diagnostic test evaluation study, with an embedded qualitative sub-study. Its aims are two-fold: First, to evaluate a simple rapid syringe-based DNA extraction method (SM) in comparison with a more elaborate conventional DNA extraction method (CM), followed by a LAMP assay targeting IS2404 for the detection of MU, either using a commercially available pocket warmer (pw) or a heat block (hb) for incubation. Second, to complement this by exploring the diagnostic workflow for BU at a community-based health centre in an endemic area in rural Ghana as an example of a potential target setting, using interviews with researchers and health care workers (HCWs). Diagnostic test evaluation results are discussed in relation to the requirements of a target product profile (TPP) for BU diagnosis and the target setting. RESULTS: A protocol using SM for DNA extraction followed by IS2404 PCR (IS2404 PCRSM) was able to identify MU DNA in 73 out of 83 BU clinical specimens submitted for diagnosis. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of IS2404 PCRSM were 90.12%, 100%, 100% and 65.21% respectively, as compared to the reference standard IS2404 PCR in combination with a standard extraction protocol for mycobacterial DNA. Evaluation of the LAMP assay on 64 SM DNA extracts showed a sensitivity, specificity, PPV and NPV of 83.6%, 100%, 100% and 50%, respectively, using either pocket warmer (pwLAMPSM) or heat block (hbLAMPSM) for incubation of the reaction, as compared to the same reference standard. The limit of detection of pwLAMPSM was found to be 30 copies of the IS2404 target. Interview findings explored barriers to BU diagnosis and treatment, including perceptions of the disease, costs, and availability of transport. Participants confirmed that a diagnosis at the PoC, in addition to screening based on clinical criteria, would be advantageous in order to prevent delays and loss to follow-up. DISCUSSION AND CONCLUSIONS: The high diagnostic and analytic accuracy of the pwLAMP, evaluated by us in combination with a syringe-based DNA extraction method, supports its potential use for the rapid detection of MU in suspected BU samples at the community or primary health care level without reliable electricity supply. Further optimization needs include a lysis buffer, evaluation directly at the PoC and/or other sites, assessing staff training requirements and quality control.

Snapshot fecal survey of domestic animals in rural Ghana for Mycobacterium ulcerans.

Identifying the source reservoirs of Mycobacterium ulcerans is key to understanding the mode of transmission of this pathogen and controlling the spread of Buruli ulcer (BU). In Australia, the native possum can harbor M. ulcerans in its gastrointestinal tract and shed high concentrations of the bacteria in its feces. To date, an analogous animal reservoir in Africa has not been identified. Here we tested the hypothesis that common domestic animals in BU endemic villages of Ghana are reservoir species analogous to the Australian possum. Using linear-transects at 10-meter intervals, we performed systematic fecal surveys across four BU endemic villages and one non-endemic village in the Asante Akim North District of Ghana. One hundred and eighty fecal specimens from a single survey event were collected and analyzed by qPCR for the M. ulcerans diagnostic DNA targets IS2404 and KR-B. Positive and negative controls performed as expected but all 180 test samples were negative. This structured snapshot survey suggests that common domestic animals living in and around humans do not shed M. ulcerans in their feces. We conclude that, unlike the Australian native possum, domestic animals in rural Ghana are unlikely to be major reservoirs of M. ulcerans.

Whole genome comparisons suggest random distribution of Mycobacterium ulcerans genotypes in a Buruli ulcer endemic region of Ghana.

Efforts to control the spread of Buruli ulcer--an emerging ulcerative skin infection caused by Mycobacterium ulcerans--have been hampered by our poor understanding of reservoirs and transmission. To help address this issue, we compared whole genomes from 18 clinical M. ulcerans isolates from a 30 km2 region within the Asante Akim North District, Ashanti region, Ghana, with 15 other M. ulcerans isolates from elsewhere in Ghana and the surrounding countries of Ivory Coast, Togo, Benin and Nigeria. Contrary to our expectations of finding minor DNA sequence variations among isolates representing a single M. ulcerans circulating genotype, we found instead two distinct genotypes. One genotype was closely related to isolates from neighbouring regions of Amansie West and Densu, consistent with the predicted local endemic clone, but the second genotype (separated by 138 single nucleotide polymorphisms [SNPs] from other Ghanaian strains) most closely matched M. ulcerans from Nigeria, suggesting another introduction of M. ulcerans to Ghana, perhaps from that country. Both the exotic genotype and the local Ghanaian genotype displayed highly restricted intra-strain genetic variation, with less than 50 SNP differences across a 5.2 Mbp core genome within each genotype. Interestingly, there was no discernible spatial clustering of genotypes at the local village scale. Interviews revealed no obvious epidemiological links among BU patients who had been infected with identical M. ulcerans genotypes but lived in geographically separate villages. We conclude that M. ulcerans is spread widely across the region, with multiple genotypes present in any one area. These data give us new perspectives on the behaviour of possible reservoirs and subsequent transmission mechanisms of M. ulcerans. These observations also show for the first time that M. ulcerans can be mobilized, introduced to a new area and then spread within a population. Potential reservoirs of M. ulcerans thus might include humans, or perhaps M. ulcerans-infected animals such as livestock that move regularly between countries.