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BACKGROUND: 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. OBJECTIVE: This article provides Australian general practitioners with an overview of BU, including its epidemiology, transmission, clinical features, diagnosis and management. DISCUSSION: 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.

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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.

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INTRODUCTION: 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. STUDY AIMS AND METHODS: 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. DISCUSSION AND 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.

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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.

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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.

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Critical scientific questions remain regarding infection with Mycobacterium ulcerans, the organism responsible for the neglected tropical disease, Buruli ulcer (BU). A controlled human infection model has the potential to accelerate our knowledge of the immunological correlates of disease, to test prophylactic interventions and novel therapeutics. Here we present microbiological evidence supporting M. ulcerans JKD8049 as a suitable human challenge strain. This non-genetically modified Australian isolate is susceptible to clinically relevant antibiotics, can be cultured in animal-free and surfactant-free media, can be enumerated for precise dosing, and has stable viability following cryopreservation. Infectious challenge of humans with JKD8049 is anticipated to imitate natural infection, as M. ulcerans JKD8049 is genetically stable following in vitro passage and produces the key virulence factor, mycolactone. Also reported are considerations for the manufacture, storage, and administration of M. ulcerans JKD8049 for controlled human infection.

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no abstract requested for correspondance items.

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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.

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The classical lineage of Mycobacterium ulcerans is the most prevalent clonal group associated with Buruli ulcer in humans. Its reservoir is strongly associated with the environment. We analyzed together 1,045 isolates collected from 13 countries on two continents to define the evolutionary history and population dynamics of this lineage. We confirm that this lineage spread over 7,000 years from Australia to Africa with the emergence of outbreaks in distinct waves in the 18th and 19th centuries. In sharp contrast with its global spread over the last century, transmission chains are now mostly local, with little or no dissemination between endemic areas. This study provides new insights into the phylogeography and population dynamics of M. ulcerans, highlighting the importance of comparative genomic analyses to improve our understanding of pathogen transmission. IMPORTANCE: Mycobacterium ulcerans is an environmental mycobacterial pathogen that can cause Buruli ulcer, a severe cutaneous infection, mostly spread in Africa and Australia. We conducted a large genomic study of M. ulcerans, combining genomic and evolutionary approaches to decipher its evolutionary history and pattern of spread at different geographic scales. At the scale of villages in an endemic area of Benin, the circulating genotypes have been introduced in recent decades and are not randomly distributed along the river. On a global scale, M. ulcerans has been spreading for much longer, resulting in distinct and compartmentalized endemic foci across Africa and Australia.

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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.

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Buruli ulcer, a chronic subcutaneous infection caused by Mycobacterium ulcerans, is increasing in prevalence in southeastern Australia. Possums are a local wildlife reservoir for M. ulcerans and, although mosquitoes have been implicated in transmission, it remains unclear how humans acquire infection. We conducted extensive field survey analyses of M. ulcerans prevalence among mosquitoes in the Mornington Peninsula region of southeastern Australia. PCR screening of trapped mosquitoes revealed a significant association between M. ulcerans and Aedes notoscriptus. Spatial scanning statistics revealed overlap between clusters of M. ulcerans-positive Ae. notoscriptus, M. ulcerans-positive possum excreta and Buruli ulcer cases, and metabarcoding analyses showed individual mosquitoes had fed on humans and possums. Bacterial genomic analysis confirmed shared single-nucleotide-polymorphism profiles for M. ulcerans detected in mosquitoes, possum excreta and humans. These findings indicate Ae. notoscriptus probably transmit M. ulcerans in southeastern Australia and highlight mosquito control as a Buruli ulcer prevention measure.

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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.

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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.

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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.

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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.

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