High-mortality epizootic Mycobacterium ulcerans ecovar Liflandii in a colony of Zaire Dwarf Clawed Frogs (Hymenochirus boettgeri).

Mycobacterium ulcerans ecovar Liflandii (MuLiflandii) was identified as the causative agent of mycobacteriosis in a research colony of Zaire dwarf clawed frogs (Hymenochirus boettgeri) at the University of Michigan. Clinical presentation included lethargy, generalized septicemia, cutaneous granulomas, coelomic effusion, and acute mortality. Identification of the mycobacterial species was based on molecular, microbiological, and histopathologic characteristics. These findings indicate that MuLiflandii is a primary cause of morbidity and mortality in Zaire dwarf clawed frogs and should be considered in the differential diagnosis of sepsis and coelomic effusion in amphibians. Mycobacterial speciation is important given the variability in pathogenesis within the family Mycobacteriaceae and the implications for both animal and human health as potential zoonoses. The Zaire dwarf clawed frog is a species common in the pet trade, and these findings provide consideration for this pathogen as a potentially important public health concern. This is the first report of MuLiflandii infection in the genus Hymenochirus and illustrates the diagnostic challenges of differentiating among both mycolactone-producing mycobacteria and Mycobacterium marinum. Furthermore, we demonstrate the utility of environmental sampling for this pathogen within the tank system, suggesting this mode of sampling could replace the need for direct frog surveillance.

Statistical modeling based on structured surveys of Australian native possum excreta harboring Mycobacterium ulcerans predicts Buruli ulcer occurrence in humans.

Background: Buruli ulcer (BU) is a neglected tropical disease caused by infection of subcutaneous tissue with Mycobacterium ulcerans. BU is commonly reported across rural regions of Central and West Africa but has been increasing dramatically in temperate southeast Australia around the major metropolitan city of Melbourne, with most disease transmission occurring in the summer months. Previous research has shown that Australian native possums are reservoirs of M. ulcerans and that they shed the bacteria in their fecal material (excreta). Field surveys show that locales where possums harbor M. ulcerans overlap with human cases of BU, raising the possibility of using possum excreta surveys to predict the risk of disease occurrence in humans. Methods: We thus established a highly structured 12 month possum excreta surveillance program across an area of 350 km2 in the Mornington Peninsula area 70 km south of Melbourne, Australia. The primary objective of our study was to assess using statistical modeling if M. ulcerans surveillance of possum excreta provided useful information for predicting future human BU case locations. Results: Over two sampling campaigns in summer and winter, we collected 2,282 possum excreta specimens of which 11% were PCR positive for M. ulcerans-specific DNA. Using the spatial scanning statistical tool SaTScan, we observed non-random, co-correlated clustering of both M. ulcerans positive possum excreta and human BU cases. We next trained a statistical model with the Mornington Peninsula excreta survey data to predict the future likelihood of human BU cases occurring in the region. By observing where human BU cases subsequently occurred, we show that the excreta model performance was superior to a null model trained using the previous year's human BU case incidence data (AUC 0.66 vs 0.55). We then used data unseen by the excreta-informed model from a new survey of 661 possum excreta specimens in Geelong, a geographically separate BU endemic area to the southwest of Melbourne, to prospectively predict the location of human BU cases in that region. As for the Mornington Peninsula, the excreta-based BU prediction model outperformed the null model (AUC 0.75 vs 0.50) and pinpointed specific locations in Geelong where interventions could be deployed to interrupt disease spread. Conclusions: This study highlights the One Health nature of BU by confirming a quantitative relationship between possum excreta shedding of M. ulcerans and humans developing BU. The excreta survey-informed modeling we have described will be a powerful tool for the efficient targeting of public health responses to stop BU. Funding: This research was supported by the National Health and Medical Research Council of Australia and the Victorian Government Department of Health (GNT1152807 and GNT1196396).

Environmental risk factors associated with the presence of Mycobacterium ulcerans in Victoria, Australia.

In recent years reported cases of Buruli ulcer, caused by Mycobacterium ulcerans, have increased substantially in Victoria, Australia, with the epidemic also expanding geographically. To develop an understanding of how M. ulcerans circulates in the environment and transmits to humans we analyzed environmental samples collected from 115 properties of recent Buruli ulcer cases and from 115 postcode-matched control properties, for the presence of M. ulcerans. Environmental factors associated with increased odds of M. ulcerans presence at a property included certain native plant species and native vegetation in general, more alkaline soil, lower altitude, the presence of common ringtail possums (Pseudocheirus peregrinus) and overhead powerlines. However, only overhead powerlines and the absence of the native plant Melaleuca lanceolata were associated with Buruli ulcer case properties. Samples positive for M. ulcerans were more likely to be found at case properties and were associated with detections of M. ulcerans in ringtail possum feces, supporting the hypothesis that M. ulcerans is zoonotic, with ringtail possums the strongest reservoir host candidate. However, the disparity in environmental risk factors associated with M. ulcerans positive properties versus case properties indicates the involvement of human behavior or the influence of other environmental factors in disease acquisition that requires further study.

Molecular and epidemiological characterization of recurrent Mycobacterium ulcerans infections in Benin.

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.

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.

Evaluation of different DNA extraction methods and loop-mediated isothermal amplification primers for the detection of Mycobacterium ulcerans in clinical specimens.

BACKGROUND: Early diagnosis and treatment of Buruli ulcer is critical in order to avoid the debilitating effects of the disease. In this regard, the development of new diagnostic and point of care tools is encouraged. The loop-mediated isothermal amplification for the detection of Mycobacterium ulcerans represents one of the new tools with a good potential of being developed into a point of care test. There is however the need to standardize the assays, reduce sample preparation times, improve the detection/visualization system and optimize them for high-throughput screening, adaptable to low resourced laboratories. METHODS: In this study, we assessed two DNA extraction protocols (modified Boom and EasyNAT methods), three previously published LAMP primer sets (BURULI, MU 2404 and BU-LAMP), and compared the sensitivity and specificity of LAMP assays on three DNA amplification platforms. RESULTS: Our results show that Buruli ulcer diagnosis using primers targeting IS2404 for the LAMP method is sensitive (73.75-91.49%), depending on the DNA extraction method used. Even though the modified Boom DNA extraction method provided the best results, its instrumentation requirement prevent it from being field applicable. The EasyNAT method on the other hand is simpler and may represent the best method for DNA extraction in less resourced settings. CONCLUSIONS: For further work on the development and use of LAMP tests for Buruli diagnosis, it is recommended that the BURULI sets of primers be used, as these yielded the best results in terms of sensitivity (87.50-91.49%) and specificity (89.23-100%), depending on the DNA extraction methods used.

Buruli ulcer: a new case definition for Victoria.

ABSTRACT: Laboratory-confirmed infection with Mycobacterium ulcerans is currently notifiable to health departments in several jurisdictions. Accurate surveillance is imperative to understanding current and emerging areas of endemicity and to facilitate research into a neglected tropical disease with poorly-understood transmission dynamics. The state of Victoria currently reports some of the highest numbers of M. ulcerans cases in the world each year, with 340 cases notified in 2018 (an incidence of 5.5 per 100,000 population). In May 2019, a group of clinical, laboratory and public health experts met to discuss a new case definition for the surveillance of M. ulcerans disease in Victoria, incorporating clinical and epidemiological elements. The new case definition supports important public health messaging and actions for residents and visitors to popular tourist areas in Victoria.

Translocating Mycobacterium ulcerans: An experimental model.

Mycobacterium ulcerans is a non-tuberculous environmental mycobacterium responsible for extensive cutaneous and subcutaneous ulcers in mammals, known as Buruli ulcer in humans. M. ulcerans has seldom been detected in the faeces of mammals and has not been detected in human faeces. Nevertheless, the detection and isolation of M. ulcerans in animal faeces does not fit with the current epidemiological schemes for the disease. Here, using an experimental model in which rats were fed with 109 colony-forming units of M. ulcerans, we detected M. ulcerans DNA in the faeces of challenged rats for two weeks and along their digestive tract for 10 days. M. ulcerans DNA was further detected in the lymphatic system including in the cervical and axillary lymph nodes and the spleen, but not in any other tissue including healthy and broken skin, 10 days post-challenge. These observations indicate that in some herbivorous mammals, M. ulcerans contamination by the digestive route may precede translocation and limited contamination of the lymphatic tissues without systemic infection. These herbivorous mammals may be sources of M. ulcerans for exposed populations but are unlikely to be reservoirs for the pathogen.

Introduction of Mycobacterium ulcerans disease in the Bankim Health District of Cameroon follows damming of the Mapé River.

Buruli ulcer (BU) is an emerging ulcerative skin disease caused by infection with Mycobacterium ulcerans. Efforts to control its spread have been hampered by our limited understanding of M. ulcerans reservoirs and transmission, and the factors leading to the emergence of BU disease in a particular region. In this report we investigate an anecdotal link between damming the Mapé River in Cameroon and the emergence of BU in the Health Districts bordering Lake Bankim, the impoundment created by the Mapé dam. We used bacterial population genomics and molecular dating to find compelling support for a 2000 M. ulcerans introduction event that followed about 10 years after the filling of the newly created impoundment in 1988. We compared the genomic reconstructions with high-resolution satellite imagery to investigate what major environmental alterations might have driven the emergence of the new focus.

Screening anti-infectious molecules against Mycobacterium ulcerans: A step towards decontaminating environmental specimens.

Mycobacterium ulcerans, a non-tuberculous mycobacterium responsible for Buruli ulcer, resides in poorly defined environmental niches in the vicinity of stagnant water. Very few isolates have been confirmed. With a view to culturing M. ulcerans from such contaminated environmental specimens, we tested the in vitro susceptibility of the M. ulcerans CU001 strain co-cultivated with XTC cells to anti-infectious molecules registered in the French pharmacopoeia. We used a standardised concentration to identify molecules that were inactive against M. ulcerans and which could be incorporated into a decontaminating solution. Of 116 tested molecules, 64 (55.1%) molecules were ineffective against M. ulcerans CU001. These included 34 (29.3%) antibiotics, 14 (12%) antivirals, eight (6.8%) antiparasitics, and eight (6.8%) antifungals. This left 52 molecules which were active against M. ulcerans CU001. Three of the inactive antimicrobial molecules (oxytetracycline, polymyxin E and voriconazole) were then selected to prepare a decontamination solution which was shown to respect M. ulcerans CU001 viability. These three antimicrobials could be incorporated into a decontamination solution to potentially isolate and culture M. ulcerans from environmental samples.

Histological and quantitative polymerase chain reaction-based analysis of Buruli ulcer using mapping biopsy method.

BACKGROUND: In Japan, Buruli ulcer cases are often advanced, requiring surgical treatment. However, extensive debridement is often difficult because of cosmetic and functional sequelae. Moreover, the lesions are complicated and composed of edematous erythema, necrotic ulcer, and erythematous skin lesions caused by a paradoxical reaction, which also make it difficult to perform adequate debridement. METHODOLOGY/PRINCIPAL FINDINGS: We performed quantitative polymerase chain reaction (PCR) analysis for IS2404 using 29 samples taken from mapping biopsy. We evaluated the relationship among mycobacterial burden, histopathological findings, and clinical outcomes using 83 tissue samples taken from mapping biopsy and debrided Buruli ulcer. On quantitative PCR, the Cp values of IS2404 amplification were substantially different in each site. The major histological findings could be divided into massive subcutaneous necrosis with scant inflammatory cell infiltration and dense inflammatory cell infiltration. Of the 84 sites, 34 were subjected to repeated histological evaluations. In these sites, histological necrosis did not disappear over time despite standard antibiotic treatment. In contrast, the ulcers were cured and no recurrences were observed without resecting the 11 biopsied sites that lacked histological necrosis. Although quantitative PCR revealed that a lower Cp value of IS2404 was associated with histological massive necrosis, sites that showed lower Cp values clinically did not always need debridement. CONCLUSION/SIGNIFICANCE: Our descriptive study revealed that the histological findings and amounts of mycobacterial DNA differed according to the sites despite being found in one lesion. Our results showed that the need for surgical debridement in each site was correlated with histological necrosis without inflammatory cell infiltration, as the inflammation is supposed to represent an active host immune response rather than mycobacterial burden. We suggest that the debridement of lesions with histological necrosis in mapping biopsy may be useful for Japanese cases with unsuccessful standard antibiotic treatment to achieve sufficient clinical improvement.

Development of an ELISA for the quantification of mycolactone, the cytotoxic macrolide toxin of Mycobacterium ulcerans.

Mycolactones, macrolide cytotoxins, are key virulence factors of Mycobacterium ulcerans, the etiological agent of the chronic necrotizing skin disease Buruli ulcer. There is urgent need for a simple point-of-care laboratory test for Buruli ulcer and mycolactone represents a promising target for the development of an immunological assay. However, for a long time, all efforts to generate mycolactone-specific antibodies have failed. By using a protein conjugate of a truncated non-toxic synthetic mycolactone derivative, we recently described generation of a set of mycolactone-specific monoclonal antibodies. Using the first mycolactone-specific monoclonal antibodies that we have described before, we were able to develop an antigen competition assay that detects mycolactones. By the systematic selection of a capturing antibody and a reporter molecule, and the optimization of assay conditions, we developed an ELISA that detects common natural variants of mycolactone with a limit of detection in the low nanomolar range. The mycolactone-specific ELISA described here will be a very useful tool for research on the biology of this macrolide toxin. After conversion into a simple point-of-care test format, the competition assay may have great potential as laboratory assay for both the diagnosis of Buruli ulcer and for the monitoring of treatment efficacy.

Tracing Mycobacterium ulcerans along an alimentary chain in Côte d'Ivoire: A one health perspective.

BACKGROUND: Mycobacterium ulcerans is an environmental mycobacterium responsible for an opportunistic, noncontagious tropical infection named Buruli ulcer that necrotizes the skin and the subcutaneous tissues. M. ulcerans is thought to penetrate through breached skin after contact with contaminated wetland environments, yet the exact biotopes where M. ulcerans occurs remain elusive, hence obscuring the epidemiological chain of transmission of this opportunistic pathogen. METHODOLOGY/PRINCIPAL FINDINGS: Polymerase chain reaction investigations detected M. ulcerans in 39/46 (84.7%) rhizosphere specimens collected in 13 Buruli ulcer-endemic areas in Côte d'Ivoire and 3/20 (15%) specimens collected in a nonendemic area (P = 5.73.E-7); only 3/63 (4.7%) sediment specimens from sediment surrounding the rhizospheres were positive in endemic area (P = 6.51.E-12). High-throughput sequencing further detected three PCR-positive plants, Croton hirtus, Corton kongensis and Oriza sativa var. japonica (rice), in the rectal content of two M. ulcerans-positive wild Thryonomys swinderianus grasscutters that were hunted in Buruli ulcer-endemic areas, while no PCR-positive plants were detected in the rectal content of two negative control animals that were farmed in a nonendemic area. CONCLUSIONS/SIGNIFICANCE: Our data suggest an alimentary chain of transmission of M. ulcerans from plants to T. swinderianus grasscutters and people that utilize T. swinderianus as bush meat in Buruli ulcer-endemic areas in Côte d'Ivoire. Guidance to adopt protective measures and avoid any direct contact with potentially contaminated rhizospheres and with grasscutter intestinal content when preparing the animals for cooking should be established for at-risk populations.

Pharmacologic management of Mycobacterium ulcerans infection.

INTRODUCTION: Pharmacological treatment of Buruli ulcer (Mycobacterium ulcerans infection; BU) is highly effective, as shown in two randomized trials in Africa. AREAS COVERED: We review BU drug treatment - in vitro, in vivo and clinical trials (PubMed: '(Buruli OR (Mycobacterium AND ulcerans)) AND (treatment OR therapy).' We also highlight the pathogenesis of M. ulcerans infection that is dominated by mycolactone, a secreted exotoxin, that causes skin and soft tissue necrosis, and impaired immune response and tissue repair. Healing is slow, due to the delayed wash-out of mycolactone. An array of repurposed tuberculosis and leprosy drugs appears effective in vitro and in animal models. In clinical trials and observational studies, only rifamycins (notably, rifampicin), macrolides (notably, clarithromycin), aminoglycosides (notably, streptomycin) and fluoroquinolones (notably, moxifloxacin, and ciprofloxacin) have been tested. EXPERT OPINION: A combination of rifampicin and clarithromycin is highly effective but lesions still take a long time to heal. Novel drugs like telacebec have the potential to reduce treatment duration but this drug may remain unaffordable in low-resourced settings. Research should address ulcer treatment in general; essays to measure mycolactone over time hold promise to use as a readout for studies to compare drug treatment schedules for larger lesions of Buruli ulcer.

Cell-mediated and serology-based tests for Mycobacterium ulcerans disease: A systematic review and meta-analysis.

Buruli ulcer (BU) is a subcutaneous necrotic infection of the skin caused by Mycobacterium ulcerans. It is the third most common human mycobacterial disease after tuberculosis (TB) and leprosy. The available methods for detection of the bacilli in lesions are microscopic detection, isolation and cultivation of the bacterium, histopathology, and polymerase chain reaction (PCR). These methods, although approved by the World Health Organization (WHO), have infrastructural and resource challenges in medical centres and cell-mediated immunity (CMI) and/or serology-based tests have been suggested as easier and more appropriate for accurate assessment of the disease, especially in remote or underdeveloped areas. This study systematically reviewed and conducted a meta-analysis for all research aimed at developing cell-mediated immunity (CMI) and/or serology-based tests for M. ulcerans disease. Information for this review was searched through PubMed and Web of Science databases and identified up to June 2019. References from relevant articles and reports from the WHO Annual Meeting of the Global Buruli Ulcer Initiative were also used. Twelve studies beginning in 1952, that attempted to develop CMI and/or serology-based tests for the disease were identified. These studies addressed issues of specificity and sensitivity in context of antigen composition as well as study heterogeneity and bias. The two main types of antigenic preparations considered were pathogen-derived and recombinant protein preparations. There was slight difference in test performance when M. ulcerans recombinant proteins [positivity: 67.5%; 32.5%] or pathogen-derived [positivity: 76.0%; 24.0%] preparations were used as test antigens among BU patients. However, pathogen-derived preparations were better at differentiating between patients and control groups [odds ratio (OR) of 27.92, 95%CI: 5.05-154.28]. This was followed by tests with the recombinant proteins [OR = 1.23, 95%CI: 0.27-5.62]. Overall, study heterogeneity index, I2 was 92.4% (p = 0.000). It is apparent from this review that standardisation is needed in any future CMI and/or serology-based tests used for M. ulcerans disease.

Stable and Local Reservoirs of Mycobacterium ulcerans Inferred from the Nonrandom Distribution of Bacterial Genotypes, Benin.

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.

Emerging human infectious diseases of aquatic origin: a comparative biogeographic approach using Bayesian spatial modelling.

BACKGROUND: With the increase in unprecedented and unpredictable disease outbreaks due to human-driven environmental changes in recent years, we need new analytical tools to map and predict the spatial distribution of emerging infectious diseases and identify the biogeographic drivers underpinning their emergence. The aim of the study was to identify and compare the local and global biogeographic predictors such as landscape and climate that determine the spatial structure of leptospirosis and Buruli Ulcer (BU). METHODS: We obtained 232 hospital-confirmed leptospirosis (2007-2017) cases and 236 BU cases (1969-2017) in French Guiana. We performed non-spatial and spatial Bayesian regression modeling with landscape and climate predictor variables to characterize the spatial structure and the environmental drivers influencing the distribution of the two diseases. RESULTS: Our results show that the distribution of both diseases is spatially dependent on environmental predictors such as elevation, topological wetness index, proximity to cropland and increasing minimum temperature at the month of potential infection. However, the spatial structure of the two diseases caused by bacterial pathogens occupying similar aquatic niche was different. Leptospirosis was widely distributed across the territory while BU was restricted to the coastal riverbeds. CONCLUSIONS: Our study shows that a biogeographic approach is an effective tool to identify, compare and predict the geographic distribution of emerging diseases at an ecological scale which are spatially dependent to environmental factors such as topography, land cover and climate.