Amoxicillin/clavulanate in combination with rifampicin/clarithromycin is bactericidal against Mycobacterium ulcerans.

BACKGROUND: Buruli ulcer (BU) is a skin neglected tropical disease (NTD) caused by Mycobacterium ulcerans. WHO-recommended treatment requires 8-weeks of daily rifampicin (RIF) and clarithromycin (CLA) with wound care. Treatment compliance may be challenging due to socioeconomic determinants. Previous minimum Inhibitory Concentration and checkerboard assays showed that amoxicillin/clavulanate (AMX/CLV) combined with RIF+CLA were synergistic against M. ulcerans. However, in vitro time kill assays (TKA) are a better approach to understand the antimicrobial activity of a drug over time. Colony forming units (CFU) enumeration is the in vitro reference method to measure bacterial load, although this is a time-consuming method due to the slow growth of M. ulcerans. The aim of this study was to assess the in vitro activity of RIF, CLA and AMX/CLV combinations against M. ulcerans clinical isolates by TKA, while comparing four methodologies: CFU enumeration, luminescence by relative light unit (RLU) and optical density (at 600 nm) measurements, and 16S rRNA/IS2404 genes quantification. METHODOLOGY/PRINCIPAL FINDINGS: TKA of RIF, CLA and AMX/CLV alone and in combination were performed against different M. ulcerans clinical isolates. Bacterial loads were quantified with different methodologies after 1, 3, 7, 10, 14, 21 and 28 days of treatment. RIF+AMX/CLV and the triple RIF+CLA+AMX/CLV combinations were bactericidal and more effective in vitro than the currently used RIF+CLA combination to treat BU. All methodologies except IS2404 quantitative PCR provided similar results with a good correlation with CFU enumeration. Measuring luminescence (RLU) was the most cost-effective methodology to quantify M. ulcerans bacterial loads in in vitro TKA. CONCLUSIONS/SIGNIFICANCE: Our study suggests that alternative and faster TKA methodologies can be used in BU research instead of the cumbersome CFU quantification method. These results provide an in vitro microbiological support to of the BLMs4BU clinical trial (NCT05169554, PACTR202209521256638) to shorten BU treatment.

Unlocking Opportunities for Mycobacterium leprae and Mycobacterium ulcerans.

In the recent decade, scientific communities have toiled to tackle the emerging burden of drug-resistant tuberculosis (DR-TB) and rapidly growing opportunistic nontuberculous mycobacteria (NTM). Among these, two neglected mycobacteria species of the Acinetobacter family, Mycobacterium leprae and Mycobacterium ulcerans, are the etiological agents of leprosy and Buruli ulcer infections, respectively, and fall under the broad umbrella of neglected tropical diseases (NTDs). Unfortunately, lackluster drug discovery efforts have been made against these pathogenic bacteria in the recent decade, resulting in the discovery of only a few countable hits and majorly repurposing anti-TB drug candidates such as telacebec (Q203), P218, and TB47 for current therapeutic interventions. Major ignorance in drug candidate identification might aggravate the dramatic consequences of rapidly spreading mycobacterial NTDs in the coming days. Therefore, this Review focuses on an up-to-date account of drug discovery efforts targeting selected druggable targets from both bacilli, including the accompanying challenges that have been identified and are responsible for the slow drug discovery. Furthermore, a succinct discussion of the all-new possibilities that could be alternative solutions to mitigate the neglected mycobacterial NTD burden and subsequently accelerate the drug discovery effort is also included. We anticipate that the state-of-the-art strategies discussed here may attract major attention from the scientific community to navigate and expand the roadmap for the discovery of next-generation therapeutics against these NTDs.

Mycobacterium ulcerans-Bordetella trematum chronic tropical cutaneous ulcer: A four-case series, Côte d'Ivoire.

BACKGROUND: Chronic tropical cutaneous ulcers remain a neglected medical condition in West Africa, particularly Buruli ulcer, which is caused by mycolactone cytotoxin-secreting Mycobacterium ulcerans (M. ulcerans). Medical management of this highly debilitating and necrotising skin infection may be modified by colonisation and co-infection of the ulcer by opportunistic and pathogenic microorganisms, which considerably delays and increases the cost of treatment. METHODOLOGY/PRINCIPAL FINDING: We diagnosed chronic tropical cutaneous ulcers in nine patients in Côte d'Ivoire using M. ulcerans-specific PCRs and culturomics. This revealed M. ulcerans in 7/9 ulcer swabs and 5/9 control swabs as well as an additional 122 bacterial species, 32 of which were specific to ulcers, 61 specifics to the controls, and 29 which were shared, adding 40 bacterial species to those previously reported. Whole genome sequencing of four Bordetella trematum (B. trematum) isolates in four Buruli ulcer swabs and no controls indicated cytolethal distending toxins, as confirmed by cytotoxic assay. CONCLUSIONS/SIGNIFICANCE: In four cases of Buruli ulcer in Côte d'Ivoire, B. trematum was a co-pathogen which was resistant to rifampicin and clarithromycin, unmatching M. ulcerans antibiotic susceptibility profile and counteracting the current treatment of Buruli ulcer in West Africa and Australia. Thus, we report here chronic mixed M. ulcerans-B. trematum chronic tropical ulcer as a specific form of Buruli ulcer in West Africa.

Repurposing of Tuberculosis Drug Candidates for the Treatment of Mycobacterium ulcerans Disease.

Buruli ulcer (BU) is a chronic necrotizing skin disease caused by Mycobacterium ulcerans. Historically, the disease was treated by surgical excision of the skin lesions, until an 8-week combination therapy of rifampicin and streptomycin was introduced in 2004. This treatment modality was effective and reduced recurrence rates. Rifampicin is the most efficacious antibiotic for the treatment of BU and, should rifampicin-resistant M. ulcerans strains emerge, there is currently no replacement for it. As for mycobacterial diseases in general, there is a pressing need for the development of novel, fast-acting drugs. Under market economy conditions, repurposing of new tuberculosis drug candidates is the most promising avenue for alternative BU treatments. Our drug repurposing activities have led to the identification of several actives against M. ulcerans. In particular, the cytochrome bc1 complex inhibitor telacebec (Q203) is a promising drug candidate for the treatment of BU in Africa and Australia. While an active cytochrome-bd oxidase bypass limits the potency of the cytochrome-bc1-specific inhibitor telacebec against M. tuberculosis, classical lineage M. ulcerans strains rely exclusively on cytochrome-bc1 to respire. Hence, telacebec is effective at nanomolar concentration against M. ulcerans, and a high treatment efficacy in an experimental mouse infection model indicates that treatment of BU could be substantially shortened and simplified by telacebec.

Buruli ulcer - A neglected tropical disease in the Barwon region of Victoria, Australia: An emerging public health threat with local and national ramifications.

Mycobacterium ulcerans (MU) is known to be endemic in heavily touristed coastal regions of Victoria and is the cause of Buruli ulcer (BU) disease. The incidence, severity and geographic spread of MU infection/BU disease is increasing, including metropolitan Victorian suburbs. While the specifics of disease transmission and effective prevention strategies remain uncertain, severe complications can be mitigated by health systems that provide vigilant population surveillance to underpin early recognition, early specialist involvement and definitive treatment for the individual. Current theories regarding disease transmission and 'best practice' (or best guess) prevention and mitigation measures are presented herein. Opportunities to improve the health system response to this emerging public health threat are identified. It is incumbent upon all healthcare providers, including ED clinicians, to contribute by familiarising themselves with the established and emerging areas of endemicity of MU infection and the array of BU clinical presentations.

From Bacterial Toxin to Therapeutic Agent: The Unexpected Fate of Mycolactone.

"Recognizing a surprising fact is the first step towards discovery." This famous quote from Louis Pasteur is particularly appropriate to describe what led us to study mycolactone, a lipid toxin produced by the human pathogen Mycobacterium ulcerans. M. ulcerans is the causative agent of Buruli ulcer, a neglected tropical disease manifesting as chronic, necrotic skin lesions with a "surprising" lack of inflammation and pain. Decades after its first description, mycolactone has become much more than a mycobacterial toxin. This uniquely potent inhibitor of the mammalian translocon (Sec61) helped reveal the central importance of Sec61 activity for immune cell functions, the spread of viral particles and, unexpectedly, the viability of certain cancer cells. We report in this review the main discoveries that marked our research into mycolactone, and the medical perspectives they opened up. The story of mycolactone is not over and the applications of Sec61 inhibition may go well beyond immunomodulation, viral infections, and oncology.

Importance of consultations using mobile teams in the screening and treatment of neglected tropical skin diseases in Benin.

CONTEXT: Since 2013, the World Health Organization has recommended integrated control strategies for neglected tropical diseases (NTDs) with skin manifestations. We evaluated the implementation of an integrated approach to the early detection and rapid treatment of skin NTDs based on mobile clinics in the Ouémé and Plateau areas of Benin. METHODS: This descriptive cross-sectional study was performed in Ouémé and Plateau in Benin from 2018 to 2020. Consultations using mobile teams were performed at various sites selected by reasoned choice based on the epidemiological data of the National Program for the Control of Leprosy and Buruli Ulcer. All individuals presenting with a dermatological lesion who voluntarily approached the multidisciplinary management team on the day of consultation were included. The information collected was kept strictly anonymous and was entered into an Excel 2013 spreadsheet and analyzed with Stata 11 software. RESULTS: In total, 5,267 patients with various skin conditions consulted the medical team. The median age of these patients was 14 years (IQR: 7-34 years). We saw 646 (12.3%) patients presenting NTDs with skin manifestations, principally scabies, in 88.4% (571/646), followed by 37 cases of Buruli ulcer (5.8%), 22 cases of leprosy (3.4%), 15 cases of lymphatic filariasis (2.3%) and one case of mycetoma (0.2%). We detected no cases of yaws. CONCLUSION: This sustainable approach could help to decrease the burden of skin NTDs in resource-limited countries.

Changes in Inflammatory Markers in Patients Treated for Buruli Ulcer and Their Ability to Predict Paradoxical Reactions.

Mycobacterium ulcerans causes Buruli ulcer, the third most frequent mycobacterial disease after tuberculosis and leprosy. Transient clinical deteriorations, known as paradoxical reactions (PRs), occur in some patients during or after antibiotic treatment. We investigated the clinical and biological features of PRs in a prospective cohort of 41 patients with Buruli ulcer from Benin. Neutrophil counts decreased from baseline to day 90, and interleukin 6 (IL-6), granulocyte colony-stimulating factor, and vascular endothelial growth factor were the cytokines displaying a significant monthly decrease relative to baseline. PRs occurred in 10 (24%) patients. The baseline biological and clinical characteristics of the patients presenting with PRs did not differ significantly from those of the other patients. However, the patients with PRs had significantly higher IL-6 and tumor necrosis factor alpha (TNF-α) concentrations on days 30, 60, and 90 after the start of antibiotic treatment. The absence of a decrease in IL-6 and TNF-α levels during treatment should alert clinicians to the possibility of PR onset.

Buruli ulcer caused by Mycobacterium ulcerans subsp. shinshuense: A case report.

Buruli ulcer is the third most common mycobacterial infection worldwide and is mainly diagnosed in tropical regions. Globally, this progressive disease is caused by Mycobacterium ulcerans; however, Mycobacterium ulcerans subsp. shinshuense, an Asian variant, has been exclusively identified in Japan. Because of insufficient clinical cases, the clinical features of M. ulcerans subsp. shinshuense-associated Buruli ulcer remain unclear. A 70-year-old Japanese woman presented with erythema on her left backhand. The skin lesion deteriorated without an apparent etiology of inflammation, and she was referred to our hospital 3 months after disease onset. A biopsy specimen was incubated in 2% Ogawa medium at 30 °C. After 66 days, we detected small yellow-pigmented colonies, suggesting scotochromogens. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI Biotyper; Bruker Daltonics, Billerica, MA, USA) indicated that the organism was Mycobacterium pseudoshottsii or Mycobacterium marinum. However, additional PCR testing for the insertion sequence 2404 (IS2404) was positive, suggesting that the pathogen was either M. ulcerans or M. ulcerans subsp. shinshuense. Further examination by 16S rRNA sequencing analysis, focusing on nucleotide positions 492, 1247, 1288, and 1449-1451, we finally identified the organism as M. ulcerans subsp. shinshuense. The patient was successfully treated with 12 weeks of clarithromycin and levofloxacin treatment. Mass spectrometry is the latest microbial diagnostic method; however, it cannot be used to identify M. ulcerans subsp. shinshuense. To accurately detect this enigmatic pathogen and uncover its epidemiology and clinical characteristics in Japan, more accumulation of clinical cases with accurate identification of the causative pathogen is essential.

Therapeutic efficacy of rifalazil (KRM-1648) in a M. ulcerans-induced Buruli ulcer mouse model.

Buruli ulcer (BU) is a skin disease caused by Mycobacterium ulcerans infection that requires long-term antibiotic treatment and/or surgical excision. In this study, we investigated the therapeutic efficacy of the rifamycin derivative, rifalazil (RLZ) (also known as KRM-1648), in an advanced M. ulcerans infection model. Six-week-old female BALB/c mice were infected with 3.25 x 104 colony-forming units (CFU) of M. ulcerans subcutaneously into the bilateral hind footpads. At 33 days post-infection, when the footpads exhibited significant redness and swelling, mice were treated orally with 5 or 10 mg/kg of RLZ for up to 15 weeks. Mice were followed for an additional 15 weeks following treatment cessation. Untreated mice exhibited a progressive increase in footpad redness, swelling, and erosion over time, and all untreated mice reached to endpoint within 5-8 weeks post-bacterial injection. In the RLZ-treated mice, footpad redness and swelling and general condition improved or completely healed, and no recurrence occurred following treatment cessation. After 3 weeks of treatment, the CFU counts from the footpads of recovered RLZ-treated mice showed a 104 decrease compared with those of untreated mice. We observed a further reduction in CFU counts to the detection limit following 6 to 15 weeks of treatment, which did not increase 15 weeks after discontinuing the treatment. Histopathologically, bacteria in the treated mice became fragmented one week after RLZ-treatment. At the final point of the experiment, all the treated mice (5mg/kg/day; n = 6, 10mg/kg/day; n = 7) survived and had no signs of M. ulcerans infection. These results indicate that the rifamycin analogue, RLZ, is efficacious in the treatment of an advanced M. ulcerans infection mouse model.

Membrane Active Immunomodulator As a Novel Therapy for an Infectious Bacterial Disease, Buruli Ulcer.

BACKGROUND/AIM: Mycobacterium ulcerans causes the necrotizing skin disease Buruli ulcer (BU), characterized by the formation of subcutaneous lesions and immunosuppression thought to be mediated by the virulence factor mycolactone. Since early BU lesions are typically painless, patients often seek standard oral antibiotic therapy at the advanced stages when the treatment is less effective. Given that currently there is no curative topical treatment for BU, our objective was to evaluate a plasma membrane fluidizer, diethyl azelate (DEA), as a potential novel topical therapy for BU. MATERIALS AND METHODS: We evaluated the effects of DEA against bacterial extracts and live strains of M. ulcerans ATCC 35840 (mycolactone positive; M+) and ATCC 19423 (mycolactone negative; M-) by measuring cytokine levels in cultured cells and tissue extracts using multiplexed immunoassays and numbers of skin lesions as the endpoints. RESULTS: In vitro, DEA counteracted immunosuppression induced by extract from the M+ strain in the 3-D human skin model (EpiDerm) and in human dendritic cells. In vivo, topical DEA reduced immunosuppressive activities of M+ and M- strains at all stages of BU, including advanced ulcers. DEA also diminished lesion formation and ulceration, accelerated healing of skin lesions and preserved normal immune responsiveness to pathogen-associated molecular pattern receptor agonists in blood of infected animals. CONCLUSION: The efficacy of DEA in BU models is linked to overcoming the immunosuppressive activity of virulence factors produced by M. ulcerans. Thanks to its pluripotent activity, DEA is a promising novel treatment for BU and possibly other pathogenic mycobacteria.

Mycobacterium ulcerans lung infection in an immunocompetent patient.

Mycobacterium ulcerans has been implicated in cutaneous manifestations in humans, causing persistent wounds called Buruli ulcer. However, it has not been associated with pulmonary infections in humans to date. Herein, we report a case of an immunocompetent adult man with no underlying medical problems presenting with dyspnoea and generalised malaise and diagnosed with M. ulcerans lung infection. The patient was prescribed clarithromycin 500 mg two times per day, rifampin 300 mg two tablets daily and moxifloxacin 400 mg daily for 6 months, with complete resolution of his symptoms.

Comparison of 8 weeks standard treatment (rifampicin plus clarithromycin) vs. 4 weeks standard plus amoxicillin/clavulanate treatment [RC8 vs. RCA4] to shorten Buruli ulcer disease therapy (the BLMs4BU trial): study protocol for a randomized controlled multi-centre trial in Benin.

BACKGROUND: Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans that affects skin, soft tissues, and bones, causing long-term morbidity, stigma, and disability. The recommended treatment for BU requires 8 weeks of daily rifampicin and clarithromycin together with wound care, physiotherapy, and sometimes tissue grafting and surgery. Recovery can take up to 1 year, and it may pose an unbearable financial burden to the household. Recent in vitro studies demonstrated that beta-lactams combined with rifampicin and clarithromycin are synergistic against M. ulcerans. Consequently, inclusion of amoxicillin/clavulanate in a triple oral therapy may potentially improve and shorten the healing process. The BLMs4BU trial aims to assess whether co-administration of amoxicillin/clavulanate with rifampicin and clarithromycin could reduce BU treatment from 8 to 4 weeks. METHODS: We propose a randomized, controlled, open-label, parallel-group, non-inferiority phase II, multi-centre trial in Benin with participants stratified according to BU category lesions and randomized to two oral regimens: (i) Standard: rifampicin plus clarithromycin therapy for 8 weeks; and (ii) Investigational: standard plus amoxicillin/clavulanate for 4 weeks. The primary efficacy outcome will be lesion healing without recurrence and without excision surgery 12 months after start of treatment (i.e. cure rate). Seventy clinically diagnosed BU patients will be recruited per arm. Patients will be followed up over 12 months and managed according to standard clinical care procedures. Decision for excision surgery will be delayed to 14 weeks after start of treatment. Two sub-studies will also be performed: a pharmacokinetic and a microbiology study. DISCUSSION: If successful, this study will create a new paradigm for BU treatment, which could inform World Health Organization policy and practice. A shortened, highly effective, all-oral regimen will improve care of BU patients and will lead to a decrease in hospitalization-related expenses and indirect and social costs and improve treatment adherence. This trial may also provide information on treatment shortening strategies for other mycobacterial infections (tuberculosis, leprosy, or non-tuberculous mycobacteria infections). TRIAL REGISTRATION: ClinicalTrials.gov NCT05169554 . Registered on 27 December 2021.

Development of an antibiotics delivery system for topical treatment of the neglected tropical disease Buruli ulcer.

Skin infection by Mycobacterium ulcerans causes Buruli ulcer (BU) disease, a serious condition that significantly impact patient' health and quality of life and can be very difficult to treat. Treatment of BU is based on daily systemic administration of antibiotics for at least 8 weeks and presents drawbacks associated with the mode and duration of drug administration and potential side effects. Thus, new therapeutic strategies are needed to improve the efficacy and modality of BU therapeutics, resulting in a more convenient and safer antibiotic regimen. Hence, we developed a dual delivery system based on poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) microparticles and a gellan gum (GG) hydrogel for delivery of rifampicin (RIF) and streptomycin (STR), two antibiotics used for BU treatment. RIF was successfully loaded into PHBV microparticles, with an encapsulation efficiency of 43%, that also revealed a mean size of 10 µm, spherical form and rough topography. These microparticles were further embedded in a GG hydrogel containing STR. The resultant hydrogel showed a porous microstructure that conferred a high water retention capability (superior to 2000%) and a controlled release of both antibiotics. Also, biological studies revealed antibacterial activity against M. ulcerans, and a good cytocompatibility in a fibroblast cell line. Thus, the proposed drug delivery system can constitute a potential topical approach for treatment of skin ulcers caused by BU disease.

Mycobacterial skin infection.

PURPOSE OF REVIEW: The aim of this article is to review the most recent evidences concerning mycobacterial skin infections, limiting the period of literature research to 2020--2021. RECENT FINDINGS: Mycobacterial skin infections include a heterogeneous group of cutaneous diseases.Cutaneous tuberculosis is usually the result of hematogenous dissemination or spread from underlying foci and it must be distinguished from tuberculids, resulting from the immunological reaction to Mycobacterium tuberculosis antigens. Leprosy prevalence was drastically reduced after introduction of multidrug therapy in the 1980 s, but cases are still reported due to underdiagnosis, and animal and environmental reservoirs. Recent advances concentrate in the diagnostic field. Specific guidelines for the treatment of nontuberculous mycobacteria skin infections are missing and surgical procedures may be required. Prognosis is better as compared to nontuberculous mycobacteria lung disease. Rapid laboratory-confirmed diagnosis of Buruli ulcer may be achieved by the IS2404 PCR. Among new drugs, telacebec is promising in terms of potency, shorter duration and tolerability in animal studies. A clinical trial in humans is planned. SUMMARY: Mycobacterial cutaneous lesions are nonpathognomonic and clinical suspicion must be confirmed by culture or molecular detection. Long-course multidrug treatment is required based on susceptibility tests. Surgical intervention may also be required. Rehabilitation and psychosocial support reduce long-term physical and mental consequences mostly in Buruli ulcer and leprosy.

Efficacy of an Acid-Oxidizing Solution against Mycobacterium ulcerans.

For the treatment of chronic wounds, acid-oxidizing solutions (AOSs) with broad-spectrum microbicidal activity without disturbing granulation tissue formation have been developed. We found AOSs to efficiently kill Mycobacterium ulcerans, the causative agent of Buruli ulcer, which is able to survive harsh decontamination treatments. Topical AOS treatment of Buruli ulcer lesions may support the recommended antibiotic therapy (oral rifampin and clarithromycin), prevent contamination of the environment by the mycobacteria, and control secondary infections, which are a prevalent wound management problem in resource-poor settings where Buruli ulcer is endemic.

Overview: Mycolactone , the Macrolide Toxin of Mycobacterium ulcerans.

The acquisition by a Mycobacterium marinum-like progenitor of a plasmid encoding enzymes for the biosynthesis of the highly potent macrolide toxin mycolactone has set off the evolution of M. ulcerans toward a new mycobacterial species. While the selective advantage of producing mycolactone for survival in environmental niche(s) of the pathogen is unclear, there is no doubt that the cytotoxic, immunomodulatory, and analgesic properties of mycolactone are key for the establishment and progression of M. ulcerans infections in the host. Improved procedures for the isolation, handling, and detection of the amphiphilic and light-sensitive toxin have facilitated studies to unravel molecular mechanisms of mycolactone action on host cells in vitro and on cellular and immune responses in animal models. The pivotal role of mycolactone in the pathology of Buruli ulcer and the fact that the toxin has not been associated with other pathogens make it an ideal target for therapeutics/vaccines aiming at mycolactone neutralization and for the development of assays for the diagnosis of the disease.

Overview: Development of Drugs Against Mycobacterium ulcerans.

For many years, wide margin surgical excision of Buruli ulcer lesions has been the main approach for the treatment of Mycobacterium ulcerans disease. The WHO now recommends an eight-week course of oral antibiotics with a combination of rifampicin and clarithromycin in Africa. However, disease management is complicated by social stigma, lack of awareness, and limited access to healthcare facilities, resulting in underreporting and frequently late initiation of medical treatment. Inadequate initial treatment can drive permanent disabilities and also limited compliance to the eight-week therapy is a limitation. Therefore, search for a faster and more simple treatment modality is ongoing, focusing primarily on the testing of new tuberculosis drug candidates for the treatment of M. ulcerans disease.

Drug Efficacy Testing in the Mouse Footpad Model of Buruli Ulcer.

Great progress has been made in understanding the pathogenesis and treatment of Buruli ulcer over the last 20 years. The rediscovery of the mouse footpad model of the disease with translation to clinical practice has changed treatment of this infectious disease, caused by Mycobacterium ulcerans, from surgery and skin grafting to the administration of antibiotics for 8 weeks or less with superior cure rates. Here we describe the development and enhancement of the mouse model during the last two decades.

Chronic wounds in Sierra Leone: Searching for Buruli ulcer, a NTD caused by Mycobacterium ulcerans, at Masanga Hospital.

BACKGROUND: Chronic wounds pose a significant healthcare burden in low- and middle-income countries. Buruli ulcer (BU), caused by Mycobacterium ulcerans infection, causes wounds with high morbidity and financial burden. Although highly endemic in West and Central Africa, the presence of BU in Sierra Leone is not well described. This study aimed to confirm or exclude BU in suspected cases of chronic wounds presenting to Masanga Hospital, Sierra Leone. METHODOLOGY: Demographics, baseline clinical data, and quality of life scores were collected from patients with wounds suspected to be BU. Wound tissue samples were acquired and transported to the Swiss Tropical and Public Health Institute, Switzerland, for analysis to detect Mycobacterium ulcerans using qPCR, microscopic smear examination, and histopathology, as per World Health Organization (WHO) recommendations. FINDINGS: Twenty-one participants with wounds suspected to be BU were enrolled over 4-weeks (Feb-March 2019). Participants were predominantly young working males (62% male, 38% female, mean 35yrs, 90% employed in an occupation or as a student) with large, single, ulcerating wounds (mean diameter 9.4cm, 86% single wound) exclusively of the lower limbs (60% foot, 40% lower leg) present for a mean 15 months. The majority reported frequent exposure to water outdoors (76%). Self-reports of over-the-counter antibiotic use prior to presentation was high (81%), as was history of trauma (38%) and surgical interventions prior to enrolment (48%). Regarding laboratory investigation, all samples were negative for BU by microscopy, histopathology, and qPCR. Histopathology analysis revealed heavy bacterial load in many of the samples. The study had excellent participant recruitment, however follow-up proved difficult. CONCLUSIONS: BU was not confirmed as a cause of chronic ulceration in our cohort of suspected cases, as judged by laboratory analysis according to WHO standards. This does not exclude the presence of BU in the region, and the definitive cause of these treatment-resistance chronic wounds is uncertain.