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Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. Early diagnosis is crucial to prevent morbidity. In November 2012, a field laboratory fully equipped for the rapid on-site quantitative PCR (qPCR) diagnosis of M. ulcerans was established at the Buruli ulcer treatment center (CDTLUB) center in Pobè Benin, a region where BU is endemic. We describe its first 10 years of activity and its gradual evolution into an expert laboratory for BU diagnosis. From 2012 to 2022, the laboratory analyzed 3,018 samples from patients attending consultations for suspected BU at the CDTLUB in Pobè. Ziehl-Neelsen staining and qPCR targeting the IS2404 sequence were performed. Since 2019, the laboratory has also received and analyzed 570 samples from other centers. The laboratory confirmed the diagnosis of BU by qPCR for 39.7% samples: M. ulcerans DNA was detected in 34.7% of swabs, 47.2% of all fine needle aspiration samples (FNA) and 44.6% of all skin biopsy specimens. Positive Ziehl-Neelsen staining results were obtained for 19.0% samples. Bacterial load, estimated by qPCR, was significantly greater for the Ziehl-Neelsen-positive samples than for Ziehl-Neelsen-negative samples, and detection rates were highest for FNA samples. Overall, 26.3% of the samples received from other centers were positive for BU. Most of these samples were sent by the CDTLUBs of Lalo, Allada, and Zagnanado, Benin. The establishment of the laboratory in the CDTLUB of Pobè has been a huge success. Optimal patient care depends on the close proximity of a molecular biology structure to BU treatment centers. Finally, FNA should be promoted among caregivers. IMPORTANCE Here, we describe the first 10 years of activity at a field laboratory established at the Buruli ulcer treatment center (CDTLUB) in Pobè, Benin, a country in which Mycobacterium ulcerans is endemic. Between 2012 and 2022, the laboratory analyzed 3,018 samples from patients consulting the CDTLUB of Pobè with a suspected clinical BU. Ziehl-Neelsen staining and qPCR targeting the IS2404 sequence were performed. In total, 39.7% of samples tested positive by qPCR and 19.0% tested positive by Ziehl-Neelsen staining. Detection rates were highest for FNA samples, and the bacterial loads estimated by qPCR were significantly higher for Ziehl-Neelsen-positive samples than for Ziehl-Neelsen-negative samples. Since 2019, the laboratory has also analyzed 570 samples received from outside the CDTLUB of Pobè, 26.3% of which were positive for BU. Most of these samples were sent by the CDTLUBs of Lalo, Allada, and Zagnanado in Benin. The establishment of the laboratory in the CDTLUB of Pobè has been a huge success, with major benefits for both the medical staff and patients. Our findings illustrate that the usefulness and feasibility of having a diagnostic center in rural Africa, where the disease is endemic, is a key part of optimal patient care, and that FNA should be promoted to increase detection rates., Competing Interests: The authors declare no conflict of interest.

Background: Buruli ulcer is a neglected tropical disease caused by Mycobacterium ulcerans, an environmental mycobacterium. Although transmission of M. ulcerans remains poorly understood, the main identified risk factor for acquiring Buruli ulcer is living in proximity of potentially contaminated water sources. Knowledge about the clinical features of Buruli ulcer and its physiopathology is increasing, but little is known about recurrence due to reinfection., Methodology/principal Findings: We describe two patients with Buruli ulcer recurrence due to reinfection with M. ulcerans, as demonstrated by comparisons of DNA from the strains isolated at the time of the first diagnosis and at recurrence. Based on the spatial distribution of M. ulcerans genotypes in this region and a detailed study of the behavior of these two patients with respect to sources of water as well as water bodies and streams, we formulated hypotheses concerning the sites at which they may have been contaminated., Conclusions/significance: Second episodes of Buruli ulcer may occur through reinfection, relapse or a paradoxical reaction. We formally demonstrated that the recurrence in these two patients was due to reinfection. Based on the sites at which the patients reported engaging in activities relating to water, we were able to identify possible sites of contamination. Our findings indicate that the non-random distribution of M. ulcerans genotypes in this region may provide useful information about activities at risk., Competing Interests: The authors have declared that no competing interests exist.

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a neglected tropical disease found in rural areas of West and Central Africa. Despite the ongoing efforts to tackle Buruli ulcer epidemics, the environmental reservoir of its pathogen remains elusive, underscoring the need for new approaches to improving disease prevention and management. In our study, we implemented a local-scale spatial clustering model and deciphered the genetic diversity of the bacteria in a small area of Benin where Buruli ulcer is endemic. Using 179 strain samples from West Africa, we conducted a phylogeographic analysis combining whole-genome sequencing with spatial scan statistics. The 8 distinct genotypes we identified were by no means randomly spread over the studied area. Instead, they were divided into 3 different geographic clusters, associated with landscape characteristics. Our results highlight the ability of M. ulcerans to evolve independently and differentially depending on location in a specific ecologic reservoir.