Bacterial diversity in Buruli ulcer lesions in Ghana.

Background: Previous studies have demonstrated secondary microbial infection of Buruli ulcer (BUD) lesions before, during and after treatment. However, there is limited data on the bacterial diversity across treatment and their influence on clinical outcome. The present study aimed to investigate the relationship between bacterial diversity within BUD lesions and clinical outcome in affected individuals. Methods: We investigated the bacterial diversity within lesions of individuals with PCR confirmed BUD from 5 endemic districts within central Ghana. Samples were collected longitudinally from lesions over treatment period. Microbiological analyses including isolation of bacteria, and species identification were performed using the VITEK 2 compact. Results: Out of 36 participants included, 80.5 % presented with ulcers on the lower limbs. Higher bacterial diversity was observed in ulcers compared to other clinical forms of BUD. There was a significant association between bacterial diversity and clinical outcome (p = 0.002). ESBL producing bacteria and MRSA were isolated in slow healing BUD lesions. Conclusion: Higher diversity of secondary organisms colonizing BUD lesions may have an impact on clinical outcome in affected individuals. There is the need for the development of treatment guidelines for simultaneous management of M. ulcerans and other potential pathogens within lesions to improve clinical outcome.

Mosquitoes as Vectors of Mycobacterium ulcerans Based on Analysis of Notifications of Alphavirus Infection and Buruli Ulcer, Victoria, Australia.

Alphavirus infections are transmitted by mosquitoes, but the mode of transmission for Mycobacterium ulcerans, which causes Buruli ulcer, is contested. Using notification data for Victoria, Australia, during 2017-2022, adjusted for incubation period, we show close alignment between alphavirus and Buruli ulcer seasons, supporting the hypothesis of mosquito transmission of M. ulcerans.

Buruli ulcer, tuberculosis and leprosy: Exploring the One Health dimensions of three most prevalent mycobacterial diseases: A narrative review.

ΟBJECTIVES: Although Buruli ulcer, tuberculosis, and leprosy are the three most common mycobacterial diseases, One Health dimensions of these infections remain poorly understood. This narrative review aims at exploring the scientific literature with respect to the presence of animal reservoir(s) and other environmental sources for the pathogens of these infections, their role in transmission to humans and the research on/practical implementation of One Health relevant control efforts. METHODS: The literature review was conducted using the online databases PubMed, Scopus, ProQuest and Google Scholar, reviewing articles that were written in English in the last 15 years. Grey literature, published by intergovernmental agencies, was also reviewed. RESULTS: For the pathogen of Buruli ulcer, evidence suggests possums as a possible animal reservoir and thus having an active role in disease transmission to humans. Cattle and some wildlife species are deemed as established animal reservoirs for tuberculosis pathogens, with a non-negligible proportion of infections in humans being of zoonotic origin. Armadillos constitute an established animal reservoir for leprosy pathogens with the transmission of the disease from armadillos to humans being deemed possible. Lentic environments, soil and other aquatic sources may represent further abiotic reservoirs for viable Buruli ulcer and leprosy pathogens infecting humans. Ongoing investigation and implementation of public health measures, targeting (sapro)zoonotic transmission can be found in all three diseases. CONCLUSION: Buruli ulcer, tuberculosis and leprosy exhibit important yet still poorly understood One Health aspects. Despite the microbiological affinity of the respective causative mycobacteria, considerable differences in their animal reservoirs, potential environmental sources and modes of zoonotic transmission are being observed. Whether these differences reflect actual variations between these diseases or rather knowledge gaps remains unclear. For improved disease control, further investigation of zoonotic aspects of all three diseases and formulation of One Health relevant interventions is urgently needed.

Severe cases of Buruli ulcer (infection with Mycobacterium ulcerans) in common ringtail possums in Victoria adversely affect animal welfare.

Buruli ulcer is a chronic ulcerative disease of the skin and subcutaneous tissues caused by infection with Mycobacterium ulcerans. Although Australian possums are known to be susceptible to Buruli ulcer, many aspects of the disease in possums, including welfare impacts, remain largely unreported. Severe clinical Buruli ulcer was identified in four common ringtail possums (Pseudocheirus peregrinus) from Melbourne, Victoria. All four possums were euthanased due to the presence of deep ulcerative lesions on paws, with extensive tissue necrosis that exposed bones and tendons in three cases. Histologically, there was severe ulcerative necrotising pyogranulomatous dermatitis, panniculitis and myositis, with intralesional acid-fast bacteria. M. ulcerans was detected by real-time PCR in all swabs, tissues and faeces collected from all four cases. Buruli ulcer may be an important and under-recognised cause of poor possum welfare in endemic areas. The physical impacts of the severe cutaneous lesions, especially those extending to underlying bones and joints, would have directly impaired the mobility of these possums, affecting navigation of their natural environments and expression of natural behaviours including foraging and socialising. Systemic distribution of M. ulcerans throughout all major internal organs, as observed here, may further impact the health and fitness of infected possums. Faecal shedding of M. ulcerans in all four cases supports the role of possums as zoonotic reservoirs. Further research is needed to investigate the epidemiology, pathogenesis and welfare impacts of Buruli ulcer in possums and to inform the design of interventions that may protect their health and welfare.

A Mycobacterium ulcerans vaccine pilot trial using an accurate low-dose challenge.

A Mycobacterium ulcerans human challenge model has the potential to fundamentally advance our understanding of early human immune responses to infection, while rapidly evaluating vaccines and other therapeutic interventions. Here, using a murine tail infection model, we tested a very well-characterized working cell bank of the proposed challenge isolate M. ulcerans JKD8049 in naïve and Mycobacterium bovis bacille Calmette-Guérin (BCG)-vaccinated BALB/c mice. All 10 naïve mice were successfully infected with 20 colony-forming units (CFU) of M. ulcerans [95% confidence interval (CI) 17-22 CFU] with a mean time to visible lesion of 86 days (95% CI 79-92 days). In the 10 vaccinated mice, there was a significant delay in the mean time to lesion compared to the naïve controls of 24 days (P = 0.0003), but all mice eventually developed ulcerative lesions. This study informs a future human infection model by demonstrating the successful application of the challenge agent in this in vivo model and highlights both the promise and the problems with trying to induce protective immunity against M. ulcerans. IMPORTANCE: In preparation for its proposed use in a controlled human infection model (CHIM), this study reports the successful infection of BALB/c mice using a carefully characterized, low-dose inoculum of Mycobacterium ulcerans JKD8049 (our proposed CHIM strain). We also demonstrate that Mycobacterium bovis bacille Calmette-Guérin delays the onset of disease but cannot alter the course of illness once a lesion becomes apparent. We also validate the findings of previous low-dose challenges that used less accurate methods to determine the inoculum, but our presented methodology is practical, accurate, and anticipated to be reproducible.

Multisensory medical illustrations of Buruli ulcer for improved disease detection, help seeking behaviour and adherence to treatment.

Buruli ulcer (BU) is a skin infection caused by Mycobacterium ulcerans and a neglected tropical disease of the skin (skin NTD). Antibiotic treatments are available but, to be effective in the absence of surgery, BU must be detected at its earliest stages (an innocuous-looking lump under the skin) and adherence to prescribed drugs must be high. This study aimed to develop multisensory medical illustrations of BU to support communication with at-risk communities. We used a Think Aloud method to explore community health workers' (n = 6) experiences of BU with a focus on the role of their five senses, since these non-medical disease experts are familiar with the day-to-day challenges presented by BU. Thematic analysis of the transcripts identified three key themes relating to 'Detection,' 'Help Seeking,' and 'Adherence' with a transcending theme 'Senses as key facilitators of health care'. New medical illustrations, for which we coin the phrase "5D illustrations" (signifying the contribution of the five senses) were then developed to reflect these themes. The senses therefore facilitated an enriched narrative enabling the production of relevant and useful visuals for health communication. The medical artist community could utilise sensory experiences to create dynamic medical illustrations for use in practice.

Extensive buruli ulcer in a patient returning from Mali and Senegal.

no abstract requested for correspondance items.

Implementation of an integrated control programme for neglected tropical diseases of the skin in Ghana: The essential role of the laboratory.

Introduction: In this study, we report on findings from approaches used, the outcomes and the lessons learnt from the laboratory support provided for integrated control of skin NTDs including Buruli ulcer (BU), and yaws in seven selected districts in Ghana. Methods: Actions implemented from July 2018 to October 2022 included; training district-level health workers on specimen collection, storage, and transport to laboratories, integrated case searches, continual monitoring and supervision for trained health workers, laboratory confirmation of BU and yaws samples and providing results of the analysed samples to guide decision making. Descriptive analysis of data was performed. Results: A total of 18,683 (including suspected BU 976; suspected yaws 10,995) individuals were screened for BU and yaws. Of 976 suspected BU cases, 16.8% [median (IQR) age 24 (12.0-37.8) years] were confirmed positive by IS2404 PCR; BU mostly presented as ulcers (78.7%); category I (37.2%) and category II (36%). 480 individuals (4.4%) had DPP positive yaws. Multiplex PCR analysis of 75 selected DPP positive cases identified; 7 DPP positive yaws cases as Treponema pallidum, 28 as Haemophilus ducreyi and 7 as Treponema pallidum/Haemophilus ducreyi coinfection. Laboratory results were sent to the districts within a median (IQR) of 5 (3 - 9) days. Conclusion: The implementation of integrated diagnostic confirmation for skin NTDs is feasible with provision of timely results within a week. Multiplex diagnostic tools differentiated Treponema pallidum and Haemophilus ducreyi. There is a need to sustain active case search activities, enhance health worker training, and improve laboratory confirmation of cases as part of the overall strategy for the integrated control of skin neglected tropical diseases.

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.

Pseudogenomic insights into the evolution of Mycobacterium ulcerans.

BACKGROUND: Buruli ulcer (BU) disease, caused by Mycobacterium ulcerans (MU), and characterized by necrotic ulcers is still a health problem in Africa and Australia. The genome of the bacterium has several pseudogenes due to recent evolutionary events and environmental pressures. Pseudogenes are genetic elements regarded as nonessential in bacteria, however, they are less studied due to limited available tools to provide understanding of their evolution and roles in MU pathogenicity. RESULTS: This study developed a bioinformatic pipeline to profile the pseudogenomes of sequenced MU clinical isolates from different countries. One hundred and seventy-two MU genomes analyzed revealed that pseudogenomes of African strains corresponded to the two African lineages 1 and 2. Pseudogenomes were lineage and location specific and African lineage 1 was further divided into A and B. Lineage 2 had less relaxation in positive selection than lineage 1 which may signify different evolutionary points. Based on the Gil-Latorre model, African MU strains may be in the latter stages of evolutionary adaption and are adapting to an environment rich in metabolic resources with a lower temperature and decreased UV radiation. The environment fosters oxidative metabolism and MU may be less reliant on some secondary metabolites. In-house pseudogenomes from Ghana and Cote d'Ivoire were different from other African strains, however, they were identified as African strains. CONCLUSION: Our bioinformatic pipeline provides pseudogenomic insights to complement other whole genome analyses, providing a better view of the evolution of the genome of MU and suggest an adaptation model which is important in understanding transmission. MU pseudogene profiles vary based on lineage and country, and an apparent reduction in insertion sequences used for the detection of MU which may adversely affect the sensitivity of diagnosis.

SIGNIFICANCE: Prevention and treatment of Buruli ulcer is still a problem but large whole genome datasets on M. ulcerans are readily available. However, genomic studies fail to thoroughly investigate pseudogenes to probe evolutionary changes in the bacteria, and this can be attributed to the lack of bioinformatic tools. This work studied pseudogenes in Mycobacterium ulcerans (MU) to understand its adapted niche and evolutionary differences across African strains. Our results posit an MU niche-adapted model important in understanding transmission. Also, MU pseudogene profiles vary based on lineage and country, suggesting their influence on pseudogenization patterns in the genome. We further identify a reduction in insertion sequences that are used for the detection of the bacteria which may affect the sensitivity of diagnosis.

Clinical and microbiological predictors of healing in Buruli ulcer disease.

Introduction: Wound measurements are relevant in monitoring the rate of healing (RoH) and may predict time to healing. Predicting the time to healing can help improve the management of Buruli ulcer. We examine three methods for the determination of RoH and their use as predictors of time to healing. Methods: Lesion measurements of Buruli ulcer patients treated from 2007 to 2022 were obtained with acetate sheet tracings (2D) or Aranz software (3D) fortnightly. RoH was determined using the absolute area, percentage area reduction and linear methods at 4 weeks post onset of antibiotic treatment. Predicted time to healing was compared to the actual healing time. Baseline characteristics were assessed for associations with healing. Results: All three methods for calculating the RoH significantly distinguished between fast and slow healers (p < 0.0001). The predicted healing time using the linear method was comparable to the actual healing time for fast healers (p = 0.34). The RoH was influenced by the form of lesion, with plaques [OR 2.19 5 %CI (1.2-3.6), p = 0.009], and oedemas [OR 8.5; 95 %CI (1.9--36.9), p = 0.004] being associated with delayed healing. The proportion of patients with paradoxical reactions 16 % vs 3 %, p < 0.0001), higher baseline bacterial load (75/104;72 % vs 21/47;45 %, p = 0.001) and delayed clearance of viable organisms (71/104;68 % vs 9/47;19 %, p < 0.0001) was higher in the slow healers than the fast healers. Conclusion: Predicted healing rates were comparatively lower for slow healers than fast healers. Baseline characteristics associated with healing can be explored for an improved disease management plan to reduce patient and caregiver anxiety.

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.

Multi-epitope vaccine candidates based on mycobacterial membrane protein large (MmpL) proteins against Mycobacterium ulcerans.

Buruli ulcer (BU) is a neglected tropical disease. It is caused by the bacterium Mycobacterium ulcerans and is characterized by skin lesions. Several studies were performed testing the Bacillus Calmette-Guérin (BCG) vaccine in human and animal models and M. ulcerans-specific vaccines in animal models. However, there are currently no clinically accepted vaccines to prevent M. ulcerans infection. The aim of this study was to identify T-cell and B-cell epitopes from the mycobacterial membrane protein large (MmpL) proteins of M. ulcerans. These epitopes were analysed for properties including antigenicity, immunogenicity, non-allergenicity, non-toxicity, population coverage and the potential to induce cytokines. The final 8 CD8+, 12 CD4+ T-cell and 5 B-cell epitopes were antigenic, non-allergenic and non-toxic. The estimated global population coverage of the CD8+ and CD4+ epitopes was 97.71%. These epitopes were used to construct five multi-epitope vaccine constructs with different adjuvants and linker combinations. The constructs underwent further structural analyses and refinement. The constructs were then docked with Toll-like receptors. Three of the successfully docked complexes were structurally analysed. Two of the docked complexes successfully underwent molecular dynamics simulations (MDS) and post-MDS analysis. The complexes generated were found to be stable. However, experimental validation of the complexes is required.

Qualitative study of the stigma associated with the lived experiences of Buruli ulcer disease among patients in Nigeria.

BACKGROUND: Buruli ulcer disease (BUD) is a neglected tropical disease of the skin, subcutaneous tissue and bone caused by Mycobacterium ulcerans, with various disabling and stigmatising sequelae. Patients face a range of adverse impacts owing to the disease as most are unable to relate effectively with their communities, battling stigma and fear of discrimination. This study aimed to explore the stigma associated with the lived experiences of BUD in patients in Nigeria. METHODS: This was a cross-sectional qualitative study that employed a focus group discussion (FGD) and key informant interviews in Enugu State, Nigeria. A random sampling of communities was followed by a purposive selection of FGD participants and key informants. A total of six key informants and eight FGD participants took part in the study. Data were collected over 6 wk from December 2022 to February 2023. The NVivo application was used to analyse transcripts and compute themes of quotes. RESULTS: Patients experienced stigma owing to the condition. The most reported experiences by participants were shame and culpability, isolation and social exclusion, including maltreatment from family members. CONCLUSIONS: Identifying the stigma experienced by patients owing to BUD helps in the provision of psychosocial management services and support for patients.

Repurposing of statins for Buruli Ulcer treatment: antimicrobial activity against Mycobacterium ulcerans.

Mycobacterium ulcerans causes Buruli Ulcer, a neglected infectious skin disease that typically progresses from an early non-ulcerative lesion to an ulcer with undermined edges. If not promptly treated, these lesions can lead to severe disfigurement and disability. The standard antibiotic regimen for Buruli Ulcer treatment has been oral rifampicin combined with intramuscular streptomycin administered daily for 8 weeks. However, there has been a recent shift toward replacing streptomycin with oral clarithromycin. Despite the advantages of this antibiotic regimen, it is limited by low compliance, associated side effects, and refractory efficacy for severe ulcerative lesions. Therefore, new drug candidates with a safer pharmacological spectrum and easier mode of administration are needed. Statins are lipid-lowering drugs broadly used for dyslipidemia treatment but have also been reported to have several pleiotropic effects, including antimicrobial activity against fungi, parasites, and bacteria. In the present study, we tested the susceptibility of M. ulcerans to several statins, namely atorvastatin, simvastatin, lovastatin and fluvastatin. Using broth microdilution assays and cultures of M. ulcerans-infected macrophages, we found that atorvastatin, simvastatin and fluvastatin had antimicrobial activity against M. ulcerans. Furthermore, when using the in vitro checkerboard assay, the combinatory additive effect of atorvastatin and fluvastatin with the standard antibiotics used for Buruli Ulcer treatment highlighted the potential of statins as adjuvant drugs. In conclusion, statins hold promise as potential treatment options for Buruli Ulcer. Further studies are necessary to validate their effectiveness and understand the mechanism of action of statins against M. ulcerans.

A Bibliometric Study on Buruli Ulcer Based on the Web of Science Database.

Buruli ulcer is caused by Mycobacterium ulcerans, a skin infection that occurs mostly in people living in the developing economies of Africa and is considered a neglected tropical disease by the World Health Organization (WHO). Left untreated, it can lead to chronic wounds and loss of limbs. This disease is one of the target diseases of the WHO, and there are very limited bibliometric studies published on this subject. Also, no similar study using the Web of Science Core Collection was found in the available literature. The aim of this study was to evaluate the bibliometric analysis of the literature on Buruli ulcers. For data visualization and analysis, the open-source visualization program Biblioshiny (version 2.0) was used. Although most publications are from Ghana, the United States, and European countries have also made significant contributions. The number of publications has increased especially since 2016. The most preferred keywords in the publications were treatment, diagnosis, and transmission routes. This is the first bibliometric analysis that examines the trend of scientific publications on Buruli ulcer that have been indexed in the Web of Science. Our findings have the potential to be used by academics to improve their research.

Recent Advances in the Management Strategies for Buruli Ulcers.

Buruli ulcer (BU) is a bacterial skin infection that is caused by Mycobacterium ulcerans and mainly affects people who reside in the rural areas of Africa and in suburban and beach resort communities in Australia. The infection typically begins as a painless papule or nodule that gradually develops into a large ulcer that can cause substantial impairment, damaging soft tissues and even bones. Early detection and immediate treatment are crucial to preventing further tissue damage and any potential complications, although it is worth noting that access to proper therapeutic resources can be limited in certain areas. The most commonly used antibiotics for treating BU are rifampicin, streptomycin, and clarithromycin; efforts have recently been made to introduce new treatments that increase the effectiveness and adherence to therapy. This article presents the latest research and management strategies regarding BU, providing an updated and intriguing perspective on this topic.

Comprehensive Case-Control Study of Protective and Risk Factors for Buruli Ulcer, Southeastern Australia.

To examine protective and risk factors for Buruli ulcer (BU), we conducted a case-control study of 245 adult BU cases and 481 postcode-matched controls across BU-endemic areas of Victoria, Australia. We calculated age- and sex-adjusted odds ratios for socio-environmental, host, and behavioral factors associated with BU by using conditional logistic regression. Odds of BU were >2-fold for persons with diabetes mellitus and persons working outdoors who had soil contact in BU-endemic areas (compared with indoor work) but were lower among persons who had bacillus Calmette-Guérin vaccinations. BU was associated with increasing numbers of possums and with ponds and bore water use at residences. Using insect repellent, covering arms and legs outdoors, and immediately washing wounds were protective; undertaking multiple protective behaviors was associated with the lowest odds of BU. Skin hygiene/protection behaviors and previous bacillus Calmette-Guérin vaccination might provide protection against BU in BU-endemic areas.