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Background The diagnosis of the neglected tropical skin and soft tissue disease Buruli ulcer (BU) is made on clinical and epidemiological grounds, after which treatment with BU-specific antibiotics is initiated empirically. Given the current decline in BU incidence, clinical expertise in the recognition of BU is likely to wane and laboratory confirmation of BU becomes increasingly important. We therefore aimed to determine the diagnostic accuracy of clinical signs and microbiological tests in patients presenting with lesions clinically compatible with BU. Methods A total of 227 consecutive patients were recruited in southern Benin and evaluated by clinical diagnosis, direct smear examination (DSE), polymerase chain reaction (PCR), culture, and histopathology. In the absence of a gold standard, the final diagnosis in each patient was made using an expert panel approach. We estimated the accuracy of each test in comparison to the final diagnosis and evaluated the performance of 3 diagnostic algorithms. Results Among the 205 patients with complete data, the attending clinicians recognized BU with a sensitivity of 92% (95% confidence interval [CI], 85%–96%), which was higher than the sensitivity of any of the laboratory tests. However, 14% (95% CI, 7%–24%) of patients not suspected to have BU at diagnosis were classified as BU by the expert panel. The specificities of all diagnostics were high (≥91%). All diagnostic algorithms had similar performances. Conclusions A broader clinical suspicion should be recommended to reduce missed BU diagnoses. Taking into consideration diagnostic accuracy, time to results, cost-effectiveness, and clinical generalizability, a stepwise diagnostic approach reserving PCR to DSE-negative patients performed best. [ABSTRACT FROM AUTHOR]

Background. Basidiobolomycosis is a rare subcutaneous mycosis, which can be mistaken for several other diseases, such as soft tissue tumors, lymphoma, or Buruli ulcer in the preulcerative stage. Microbiological confirmation by PCR for Basidiobolus ranarum and culture yield the most specific diagnosis, yet they are not widely available in endemic areas and with varying sensitivity. A combination of histopathological findings, namely, granulomatous inflammation with giant cells, septate hyphal fragments, and the Splendore-Hoeppli phenomenon, can confirm basidiobolomycosis in patients presenting with painless, hard induration of soft tissue. Case Presentations. We report on three patients misdiagnosed as suffering from Buruli ulcer, who did not respond to Buruli treatment. Histopathological review of the tissue sections from these patients suggests basidiobolomycosis. All patients had been lost to follow-up, and none received antifungal therapy. On visiting the patients at their homes, two were reported to have died of unknown causes. The third patient was found alive and well and had experienced local spontaneous healing. Conclusion. Basidiobolomycosis is a rare subcutaneous fungal disease mimicking preulcerative Buruli ulcer. We stress the importance of the early recognition by clinicians and pathologists of this treatable disease, so patients can timely receive antifungal therapy. [ABSTRACT FROM AUTHOR]

Buruli ulcer or Mycobacterium ulcerans disease occurs mainly in areas in proximity to standing or slowly running freshwater, habitats in which free-living amoebae occur. For this reason, a possible link between the habitat of M. ulcerans and free-living amoebae was investigated. Free-living amoebae and mycobacteria were isolated from water and biofllm specimens taken from protected and unprotected sources of water in villages known to have either high or low endemicity for Buruli ulcer in Benin. Amoebae were isolated from 78.8% of samples. A greater proportion of water bodies in areas of high endemicity had amoebae than in areas of low endemicity (83.3% versus 66.7%). Protected sources of water were significantly more likely to contain amoebae in areas of high endemicity than in areas of low endemicity (88.0% versus 11.1%). Several pathogenic free-living amoebae and mycobacteria were isolated. However, no M. ulcerans was isolated and no specimen was positive for IS2404 PCR. Our results show that the study area has a water hygiene problem, which is greater in areas of high Buruli ulcer endemicity than in areas of low endemicity. Our observations indicate that additional studies are required to explore the possible link between free-living amoebae and mycobacteria. [ABSTRACT FROM AUTHOR]

Background: Increased availability of Next Generation Sequencing (NGS) techniques allows, for the first time, to distinguish relapses from reinfections in patients with multiple Buruli ulcer (BU) episodes., Methodology: We compared the number and location of single nucleotide polymorphisms (SNPs) identified by genomic screening between four pairs of Mycobacterium ulcerans isolates collected at the time of first diagnosis and at recurrence, derived from a collection of almost 5000 well characterized clinical samples from one BU treatment center in Benin., Principal Findings: The findings suggest that after surgical treatment-without antibiotics-the second episodes were due to relapse rather than reinfection. Since specific antibiotics were introduced for the treatment of BU, the one patient with a culture available from both disease episodes had M. ulcerans isolates with a genomic distance of 20 SNPs, suggesting the patient was most likely reinfected rather than having a relapse., Conclusions: To our knowledge, this study is the first to study recurrences in M. ulcerans using NGS, and to identify exogenous reinfection as causing a recurrence of BU. The occurrence of reinfection highlights the contribution of ongoing exposure to M. ulcerans to disease recurrence, and has implications for vaccine development.

Invasive punch or incisional skin biopsy specimens are currently employed for the bacteriological confirmation of the clinical diagnosis of Buruli ulcer (BU), a cutaneous infectious disease caused by Mycobacterium ulcerans. The efficacy of fine-needle aspirates (FNA) using fine-gauge needles (23G by 25 mm) for the laboratory confirmation of BU was compared with that of skin tissue fragments obtained in parallel by excision or punch biopsy. In three BU treatment centers in Benin, both types of diagnostic material were obtained from 33 clinically suspected cases of BU and subjected to the same laboratory analyses: i.e., direct smear examination, IS2404 PCR, and in vitro culture. Twenty-three patients, demonstrating 17 ulcerative and 6 nonulcerative lesions, were positive by at least two tests and were therefore confirmed to have active BU. A total of 68 aspirates and 68 parallel tissue specimens were available from these confirmed patients. When comparing the sensitivities of the three confirmation tests between FNA and tissue specimens, the latter yielded more positive results, but only for PCR was this significant. When only nonulcerative BU lesions were considered, however, the sensitivities of the confirmation tests using FNA and tissue specimens were not significantly different. Our results show that the minimally invasive FNA technique offers enough sensitivity to be used for the diagnosis of BU in nonulcerative lesions.

Tissue specimens collected from patients with clinically suspected Buruli ulcer treated in two Buruli ulcer treatment centers in Benin between 1998 and 2004 were placed in semisolid transport medium and transported at ambient temperature for microbiological analysis at the Institute of Tropical Medicine in Antwerp, Belgium. The impact of the delay before microbiological analysis on primary culture of Mycobacterium ulcerans was investigated. The length of storage in semisolid transport medium varied from 6 days to 26 weeks. Of the 1,273 tissue fragments positive for M. ulcerans DNA by an IS2404-specific PCR, 576 (45.2%) yielded positive culture results. The sensitivity of direct smear examination was 64.6% (822/1,273 tissue fragments). The median time required to obtain a positive culture result was 11 weeks. Positive cultures were obtained even from samples kept for more than 2 months at ambient temperatures. Moreover, there was no reduction in the viability of M. ulcerans, as detected by culture, when specimens remained in semisolid transport medium for long periods of time (up to 26 weeks). We can conclude that the method with semisolid transport medium is very robust for clinical specimens from patients with Buruli ulcer that, due to circumstances, cannot be analyzed in a timely manner. This transport medium is thus very useful for the confirmation of a diagnosis of Buruli ulcer with specimens collected in the field.