In Australia, native possums are a major wildlife reservoir for Mycobacterium ulcerans, the causative agent of the neglected tropical skin disease Buruli ulcer (BU). Large-scale possum excreta surveys that use PCR to detect M. ulcerans in 100-1,000 s of excreta specimens are an important tool that can inform geospatial modeling and predict locations of future human BU risk. However, the significant expense of commercial kits used to extract DNA from specimens is a major barrier to routine implementation. Here, we developed a low-cost method for DNA extraction from possum excreta, possum tissue, and pure mycobacterial cultures, using a guanidinium isothiocyanate lysis solution and paramagnetic beads. In a 96-well plate format for high-throughput processing, the paramagnetic bead DNA extraction method was threefold less sensitive but only 1/6 the cost of a commonly used commercial kit. Applied to tissue swabs, the method was fourfold more sensitive and 1/5 the cost of a commercial kit. When used for preparing DNA from pure mycobacterial cultures, the method yielded purified genomic DNA with quality metrics comparable to more lengthy techniques. Our paramagnetic bead method is an economical means to undertake large-scale M. ulcerans environmental surveillance that will directly inform efforts to halt the spread of BU in Victoria, Australia, with potential for applicability in other endemic countries.
OBJECTIVE: Buruli ulcer (BU) is a neglected tropical skin disease, with an incidence that has dramatically increased in temperate southeastern Australia over the last decade. In southeastern Australia, BU is a zoonosis with native possums the major wildlife reservoir of the causative pathogen, Mycobacterium ulcerans. Infected possums shed M. ulcerans in their excreta, and excreta surveys using PCR to screen for the presence of pathogen DNA are a powerful means to predict future areas of Buruli ulcer risk for humans. However, excreta surveys across large geographic areas require testing of many thousands of samples. The cost of commercial DNA extraction reagents used for preparing samples for PCR testing can thus become prohibitive to effective surveillance. Here, we describe a simple, low-cost method for extracting DNA from possum excreta using paramagnetic beads. The method is versatile and adaptable to a variety of other sample types including swabs collected from possum tissues and pure cultures of mycobacteria.

Buruli ulcer (BU) is caused by Mycobacterium ulcerans, an environmental pathogen that causes severe skin and soft-tissue necrosis. In Australia, cases of BU are acquired in endemic regions, which include Victoria and Far North Queensland, but those who have visited these regions can present to health practitioners anywhere.
This article provides Australian general practitioners with an overview of BU, including its epidemiology, transmission, clinical features, diagnosis and management.
BU can manifest as an ulcer or as a non-ulcerated skin lesion, such as a plaque, nodule or oedema. Diagnosis can be achieved with a dedicated Mycobacterium ulcerans polymerase chain reaction (PCR) test performed on a wound swab. Swabs on non-ulcerated disease have a high false negative rate, and a PCR test should be performed on a tissue biopsy to confirm disease. Most cases are managed with prolonged antibiotic therapy - commonly a combination of oral rifampicin and clarithromycin or fluroquinolone (moxifloxacin or ciprofloxacin) - and wound dressings.

Clarithromycin extended-release (CLA-ER) was used as companion drug to rifampicin (RIF) for Mycobacterium ulcerans infection in the intervention arm of a WHO drug trial. RIF enhances CYP3A4 metabolism, thereby reducing CLA serum concentrations, and RIF concentrations might be increased by CLA co-administration. We studied the pharmacokinetics of CLA-ER at a daily dose of 15 mg/kg combined with RIF at a dose of 10 mg/kg in a subset of trial participants, and compared these to previously obtained pharmacokinetic data. Serial dried blood spot samples were obtained over a period of ten hours, and analyzed by LC-MS/MS in 30 study participants-20 in the RIF-CLA study arm, and 10 in the RIF-streptomycin study arm. Median CLA Cmax was 0.4 mg/L-and median AUC 3.9 mg*h/L, following 15 mg/kg CLA-ER. Compared to standard CLA dosed at 7.5 mg/kg previously, CLA-ER resulted in a non-significant 58% decrease in Cmax and a non-significant 30% increase in AUC. CLA co-administration did not alter RIF Cmax or AUC. Treatment was successful in all study participants. No effect of CLA co-administration on RIF pharmacokinetics was observed. Based on our serum concentration studies, the benefits CLA-ER over CLA immediate release are unclear.

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.

UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.

BACKGROUND: 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.
UNASSIGNED: 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.
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.

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.

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.
CONCLUSIONS: 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.

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.
OBJECTIVE: 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.

BACKGROUND: Skin neglected tropical diseases including leprosy and Buruli ulcer (BU)are a group of stigmatizing and disability-inducing conditions and these aspects of the diseases could lead to poor mental health. The study was designed to assess the burden of poor mental health and wellbeing among persons affected by leprosy or BU in Nigeria.
METHODS: A community based cross-sectional study design was employed. The study involved persons affected by leprosy or BU. Ten local government areas with the highest number of notified leprosy or BU cases between 2014 and 2018 in southern Nigeria were purposively selected. Information were obtained using Patient Health Questionnaire-9 (PHQ-9), Generalized Anxiety Disorders-7 (GAD-7), Warwick-Edinburgh Mental Well-being Scale (WEMWBS) and OSLO Social Support Scale. Outcome measure was poor mental health/wellbeing and was determined by proportion of respondents who had depressive symptoms, anxiety disorder and poor mental wellbeing.
RESULTS: A total of 635 persons affected by leprosy or BU participated in the study. The mean age of respondents was 43.8±17.0 years and highest proportion, 22.2% were in age group, 40-49 years. Majority of respondents, 50.7% were males. A higher proportion of respondents, 89.9% had depressive symptoms, 79.4% had anxiety disorders and 66.1% had poor mental wellbeing. Majority, 57.2% had poor mental health/wellbeing. Among the respondents, there was a strong positive correlation between depression and anxiety scores, (r = 0.772, p<0.001). There was a weak negative correlation between depression score and WEMWBS score, (r = -0.457, p<0.001); anxiety score and WEMWBS score, (r = -0.483, p<0.001). Predictors of poor mental health/wellbeing included having no formal education, (AOR = 1.9, 95%CI: 1.1-3.3), being unemployed, (AOR = 3.4, 95%CI: 2.2-5.3), being affected by leprosy, (AOR = 0.2, 95%CI: 0.1-0.4) and having poor social support, (AOR = 6.6, 95%CI: 3.7-11.8).
CONCLUSIONS: The burden of poor mental health/wellbeing among persons affected by leprosy or BU is very high. There is need to include mental health interventions in the management of persons affected with leprosy or BU. Equally important is finding a feasible, cost-effective and sustainable approach to delivering mental health care for persons affected with leprosy or BU at the community level. Improving educational status and social support of persons affected by leprosy or BU are essential. Engaging them in productive activities will be of essence.