Buruli Ulcer Treatment Research Articles

Optimizing β-lactam-containing antibiotic combination therapy for the treatment of Buruli ulcer.

The current treatment for Buruli ulcer is based on empirical evidence of efficacy. However, there is an opportunity for shortening its duration and improving response rates. Evolving understanding of the pharmacokinetic-pharmacodynamic relationships provides the basis for a stronger dose rationale for antibiotics. In conjunction with modelling and simulation, it is possible to identify dosing regimens with the highest probability of target attainment (PTA). This investigation aims to: (i) assess the dose rationale for a new combination therapy including amoxicillin/clavulanic acid (AMX/CLV) currently in clinical trials; and (ii) compare its performance with alternative dosing regimens including rifampicin, clarithromycin and AMX/CLV. In vitro estimates of the minimum inhibitory (MIC) concentration were selected as a measure of the antibacterial activity of different drug combinations. Clinical trial simulations were used to characterize the concentration vs. time profiles of rifampicin, clarithromycin and amoxicillin in a virtual cohort of adult and paediatric patients, considering the effect of baseline covariates on disposition parameters and interindividual variability in exposure. The PTA of each regimen was then assessed using different thresholds of the time above MIC. A weight-banded dosing regimen including 150-600 mg rifampicin once daily, 250-1000 mg clarithromycin and AMX/CLV 22.5mg/kg /1000 mg twice daily ensures higher PTA than the standard of care with AMX/CLV 45 mg/kg/2000 mg once daily. The higher PTA values support the proposed 4-drug combination (rifampicin, clarithromycin, AMX/CLV) currently under clinical investigation. Our findings also suggest that higher rifampicin doses might contribute to enhanced treatment efficacy.

Exploring the efficacy of negative pressure wound therapy in the management of mycobacterium ulcerans wounds: a comprehensive literature review

Mycobacterium ulcerans (M Ulcerans) infection leads to the debilitating Buruli ulcer (BU), characterized by necrotizing skin and soft tissue lesions. Conventional treatment primarily focuses on an antibiotic regimen, but wound management remains paramount to patient recovery. This literature review aims to evaluate the efficacy and benefits of Vacuum Assisted Dressing (VAC) in the treatment and management of BU wounds. A systematic literature search was undertaken using databases such as PubMed, Cinahl, Cochrane database, Joanna Briggs Institute, Medline, Internurse, Nursing & Allied Health database, and Scopus search from January 1995 to December 2023. The key search terms included “Mycobacterium ulcerans”, OR “Buruli ulcer”, AND “vacuum assisted dressing”, “vacuum assisted therapy”, “Vacuum Assisted Dressing”, “negative pressure wound therapy”, and “Negative Pressure Wound Therapy (NPWT)”. The exclusion criteria were animal studies and studies not in the English language. The current literature emphasizes the importance of antibiotic treatment for BU and highlights the skin and soft tissue damage that results in open, infected wounds. However, there is a notable lack of quantitative data on the efficacy of NPWT for treating BU wounds. Early evidence indicates that NPWT might accelerate wound healing, decrease secondary infections, and enhance wound bed readiness for grafting or secondary healing. While more comprehensive quantitative studies are warranted, NPWT emerges as a promising adjunctive therapy in the holistic management of BU wounds, offering benefits that may improve patient outcomes and reduce morbidity.

Evaluation of an electricity-independent method for IS2404 Loop-mediated isothermal amplification (LAMP) diagnosis of Buruli ulcer in resource-limited settings.

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. 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. 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. 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.

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

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. 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. 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.

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

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

Repurposing of 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.

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.

What about Current Diversity of Mycolactone-Producing Mycobacteria? Implication for the Diagnosis and Treatment of Buruli Ulcer.

The identification of an emerging pathogen in humans can remain difficult by conventional methods such as enrichment culture assays that remain highly selective, require appropriate medium and cannot avoid misidentifications, or serological tests that use surrogate antigens and are often hampered by the level of detectable antibodies. Although not originally designed for this purpose, the implementation of polymerase-chain-reaction (PCR) has resulted in an increasing number of diagnostic tests for many diseases. However, the design of specific molecular assays relies on the availability and reliability of published genetic sequences for the target pathogens as well as enough knowledge on the genetic diversity of species and/or variants giving rise to the same disease symptoms. Usually designed for clinical isolates, molecular tests are often not suitable for environmental samples in which the target DNA is mixed with a mixture of environmental DNA. A key challenge of such molecular assays is thus to ensure high specificity of the target genetic markers when focusing on clinical and environmental samples in order to follow the dynamics of disease transmission and emergence in humans. Here we focus on the Buruli ulcer (BU), a human necrotizing skin disease mainly affecting tropical and subtropical areas, commonly admitted to be caused by Mycobacterium ulcerans worldwide although other mycolactone-producing mycobacteria and even mycobacterium species were found associated with BU or BU-like cases. By revisiting the literature, we show that many studies have used non-specific molecular markers (IS2404, IS2606, KR-B) to identify M. ulcerans from clinical and environmental samples and propose that all mycolactone-producing mycobacteria should be definitively considered as variants from the same group rather than different species. Importantly, we provide evidence that the diversity of mycolactone-producing mycobacteria variants as well as mycobacterium species potentially involved in BU or BU-like skin ulcerations might have been underestimated. We also suggest that the specific variants/species involved in each BU or BU-like case should be carefully identified during the diagnosis phase, either via the key to genetic identification proposed here or by broader metabarcoding approaches, in order to guide the medical community in the choice for the most appropriate antibiotic therapy.

An Overview of 10 Years of Activity of a Molecular Laboratory for Buruli Ulcer Diagnosis at a Field Hospital in Benin.

Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. Early diagnosis is crucial to prevent morbidity. In November 2012, a field laboratory fully equipped for the rapid on-site quantitative PCR (qPCR) diagnosis of M. ulcerans was established at the Buruli ulcer treatment center (CDTLUB) center in Pobè Benin, a region where BU is endemic. We describe its first 10 years of activity and its gradual evolution into an expert laboratory for BU diagnosis. From 2012 to 2022, the laboratory analyzed 3,018 samples from patients attending consultations for suspected BU at the CDTLUB in Pobè. Ziehl-Neelsen staining and qPCR targeting the IS2404 sequence were performed. Since 2019, the laboratory has also received and analyzed 570 samples from other centers. The laboratory confirmed the diagnosis of BU by qPCR for 39.7% samples: M. ulcerans DNA was detected in 34.7% of swabs, 47.2% of all fine needle aspiration samples (FNA) and 44.6% of all skin biopsy specimens. Positive Ziehl-Neelsen staining results were obtained for 19.0% samples. Bacterial load, estimated by qPCR, was significantly greater for the Ziehl-Neelsen-positive samples than for Ziehl-Neelsen-negative samples, and detection rates were highest for FNA samples. Overall, 26.3% of the samples received from other centers were positive for BU. Most of these samples were sent by the CDTLUBs of Lalo, Allada, and Zagnanado, Benin. The establishment of the laboratory in the CDTLUB of Pobè has been a huge success. Optimal patient care depends on the close proximity of a molecular biology structure to BU treatment centers. Finally, FNA should be promoted among caregivers. IMPORTANCE Here, we describe the first 10 years of activity at a field laboratory established at the Buruli ulcer treatment center (CDTLUB) in Pobè, Benin, a country in which Mycobacterium ulcerans is endemic. Between 2012 and 2022, the laboratory analyzed 3,018 samples from patients consulting the CDTLUB of Pobè with a suspected clinical BU. Ziehl-Neelsen staining and qPCR targeting the IS2404 sequence were performed. In total, 39.7% of samples tested positive by qPCR and 19.0% tested positive by Ziehl-Neelsen staining. Detection rates were highest for FNA samples, and the bacterial loads estimated by qPCR were significantly higher for Ziehl-Neelsen-positive samples than for Ziehl-Neelsen-negative samples. Since 2019, the laboratory has also analyzed 570 samples received from outside the CDTLUB of Pobè, 26.3% of which were positive for BU. Most of these samples were sent by the CDTLUBs of Lalo, Allada, and Zagnanado in Benin. The establishment of the laboratory in the CDTLUB of Pobè has been a huge success, with major benefits for both the medical staff and patients. Our findings illustrate that the usefulness and feasibility of having a diagnostic center in rural Africa, where the disease is endemic, is a key part of optimal patient care, and that FNA should be promoted to increase detection rates.

Buruli-RifDACC: Evaluation of the efficacy and cost-effectiveness of high-dose versus standard-dose rifampicin on outcomes in Mycobacteriumulcerans disease, a protocol for a randomised controlled trial in Ghana.

Buruli ulcer (BU) can lead to disfiguring ulcers and permanent disability. The 2030 World Health Organization (WHO) road map for Neglected Tropical Diseases (NTDs) calls for major scaling up in diagnosis and management to eliminate disability due to the disease. Current treatment for BU is with daily oral rifampicin (10mg/kg dose) and clarithromycin (15mg/kg dose) for eight weeks, combined with standard gauze wound dressings. Dialkylcarbamoyl chloride (DACC)-coated dressings have been shown to irreversibly bind bacteria on wound surfaces resulting in their removal when dressings are changed. This trial aims to determine whether combining a high-dose oral rifampicin regimen with DACC dressings can improve the rate of wound healing relative to standard-dose oral rifampicin combined with DACC dressings. This is an individual, multi-centre Phase 3 randomised controlled trial, which will be conducted in three clinical sites in Ghana. The primary outcome measure will be the mean time to clearance of viable mycobacteria. Cost and health-related quality of life data will be collected, and a cost-effectiveness analysis will be performed. The findings from this trial could lead to a change in how BU is treated. A shorter but more efficacious regimen would lead to improved treatment outcomes and potentially substantial financial and economic savings. Pan African Clinical Trials Repository (registration number; PACTR202011867644311). Registered on 30 th November 2020.

Buruli-RifDACC: Evaluation of the efficacy and cost-effectiveness of high-dose versus standard-dose rifampicin on outcomes in Mycobacterium ulcerans disease, a protocol for a randomised controlled trial in Ghana [version 1; peer review: 2 approved

Buruli ulcer (BU) can lead to disfiguring ulcers and permanent disability. The 2030 World Health Organization (WHO) road map for Neglected Tropical Diseases (NTDs) calls for major scaling up in diagnosis and management to eliminate disability due to the disease. Current treatment for BU is with daily oral rifampicin (10mg/kg dose) and clarithromycin (15mg/kg dose) for eight weeks, combined with standard gauze wound dressings. Dialkylcarbamoyl chloride (DACC)-coated dressings have been shown to irreversibly bind bacteria on wound surfaces resulting in their removal when dressings are changed. This trial aims to determine whether combining a high-dose oral rifampicin regimen with DACC dressings can improve the rate of wound healing relative to standard-dose oral rifampicin combined with DACC dressings. This is an individual, multi-centre Phase 3 randomised controlled trial, which will be conducted in three clinical sites in Ghana. The primary outcome measure will be the mean time to clearance of viable mycobacteria. Cost and health-related quality of life data will be collected, and a cost-effectiveness analysis will be performed. The findings from this trial could lead to a change in how BU is treated. A shorter but more efficacious regimen would lead to improved treatment outcomes and potentially substantial financial and economic savings.

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

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

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Kinetic studies of the interaction between  γ-Butyrolactone and one of the phytomedecines such as Mathesia used for Buruli ulcer treatment were investigated by conductimetry. γ-Butyrolactone which is the simplest form of lactone was used as probe in order to fully understand how a cyclic ester, such as lactone, could behave in a basic environment. It was observed that for the Mathesia solution at pH 11 the minimal apparent rate constant was around 3.54636 compare to sodium hydroxyde solution at pH 12 where the minimal apparent rate constant was around 3.12295. The apparent rate constants of successive diluted solutions of Phytomedicine Mathesia show up the dilution factor 1: 4. A plot of apparent rate constant versus dilution factor gave a curve with a correlation coefficient of 0.99831. Â

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The study aimed to characterize the influence of Mansonella perstans co-infection on immunity. This research was carried out in November, 2021 in the neighboring villages of Saint Camille de Davougon in the Abomey Commune. Blood samples were collected from both genders of all ages. Buruli ulcer was diagnosed by polymerase chain reaction (PCR), using specific IS2404 repeat sequence for Mycobacterium ulcerans. Microfilaria detection was done with the help of the counting chamber technique. Using 100 white blood cells, the proportion of eosinophils was determined. Minividas® Biomerieux was used to quantify the IgE titers in the patients' serum using an immuno-enzymatic sandwich enzyme linked immunosorbent assay (ELISA). This study shows an overall co-infection of Buruli ulcers with M. perstans as 23.86%. In the age group of fewer than 16 years, the co-infection prevalence was 28.57% (n=6), while in patients of age greater than 16, the co-infection prevalence was 71.42% (n=15). The Buruli ulcer patients co-infected with M. perstans had the highest median eosinophil level, whereas the control group had the lowest. The lowest median total IgE titer was observed in the control, while the maximum was observed in the Buruli ulcer patients co-infected with M. perstans. This study concludes that Buruli ulcer co-infection with M. perstans prevails in some rural villages in Benin. Therefore, this pathogen must be considered in the treatment of Buruli ulcer. The findings imply that IgE could be used to assess the patient's immunological response during Buruli ulcer and M. perstans. Key words: Mansonella perstans, co-infection, immunity, Buruli ulcer.

The One That Got Away: How Macrophage-Derived IL-1β Escapes the Mycolactone-Dependent Sec61 Blockade in Buruli Ulcer.

Buruli ulcer (BU), caused by Mycobacterium ulcerans, is a devastating necrotizing skin disease. Key to its pathogenesis is mycolactone, the exotoxin virulence factor that is both immunosuppressive and cytotoxic. The discovery that the essential Sec61 translocon is the major cellular target of mycolactone explains much of the disease pathology, including the immune blockade. Sec61 inhibition leads to a loss in production of nearly all cytokines from monocytes, macrophages, dendritic cells and T cells, as well as antigen presentation pathway proteins and costimulatory molecules. However, there has long been evidence that the immune system is not completely incapable of responding to M. ulcerans infection. In particular, IL-1β was recently shown to be present in BU lesions, and to be induced from M. ulcerans-exposed macrophages in a mycolactone-dependent manner. This has important implications for our understanding of BU, showing that mycolactone can act as the “second signal” for IL-1β production without inhibiting the pathways of unconventional secretion it uses for cellular release. In this Perspective article, we validate and discuss this recent advance, which is entirely in-line with our understanding of mycolactone’s inhibition of the Sec61 translocon. However, we also show that the IL-1 receptor, which uses the conventional secretory pathway, is sensitive to mycolactone blockade at Sec61. Hence, a more complete understanding of the mechanisms regulating IL-1β function in skin tissue, including the transient intra-macrophage stage of M. ulcerans infection, is urgently needed to uncover the double-edged sword of IL-1β in BU pathogenesis, treatment and wound healing.

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

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

Hemostatic potential of total aqueous extract of Sacoglottis gabonensis (Baille) Urban (Humiriaceae) stem bark in Wistar rats pretreated with warfarin

Background: Sacoglottis gabonensis is a medicinal plant used in traditional treatment of Buruli ulcer and leprosy in Côte d'Ivoire. A study conducted on the healing potential of this herb on induced wounds showed good results. The objective of this study is to evaluate the hemostatic potential of the total aqueous extract of Sacoglottis gabonensis (TAESg) in rats after a pretreatment with warfarin. Methods: Thus, 42 rats were evenly distributed into seven groups of six rats each. The rats in group 1 did not receive treatment whereas those in groups 2, 3, 4, 5, 6 and 7 pretreated with warfarin received by oral route, distilled water, 100 mg/kg body weight (bw) of Vitamin K1 and doses of 3.5; 17.5; 35 and 350 mg/kg bw of TAESg, respectively. Blood samples were collected from the rats’ retro-orbital sinus before the experiment, after induction the blood hypocoagulation with warfarin and after the treatments with vitamin K or the extract in orderto determine hemostatic parameters such as the prothrombin time, the activated partial thromboplastin time and the International Normalized Ratio, the fibrinogen level, the calcium level and the thrombocyte level. Results: The results showed disturbances in the hemostatic parameters of warfarin-induced hypocoagulability rats. The treatments with TAESg significantly normalized these parameters by reducing the prothrombin time, the activated partial thromboplastin time and the INR and by increasing fibrinogen levels. However, the levels of calcium and thrombocytes which were increased after the administration of warfarin did not experience any significant change after the treatments with TAESg or Vitamin K, depending on the group. Conclusion: TAESg has some hemostatic properties similar to those of vitamin K. Key words: hypocoagulation, hemostatic parameters, rats, Sacoglottis gabonensis.

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

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

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

BackgroundEarly 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.MethodsIn 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.ResultsOur 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.ConclusionsFor 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.

Natural antioxidants attenuate mycolactone toxicity to RAW 264.7 macrophages.

Mycobacterium ulcerans produces a macrolide exotoxin, mycolactone which suppresses immune cells activity, is toxic to most cells and the key virulence factor in the pathogenesis of Buruli ulcer disease. Mycolactone is reported to mediate the production of reactive oxygen species in keratinocytes; cells that play critical role in wound healing. Increased levels of reactive oxygen species have been shown to disrupt the well-ordered process of wound repair; hence, the function of wound-healing cells such as macrophages, keratinocytes, and fibroblast could be impaired in the presence of the reactive oxygen species mediator, mycolactone. To ensure regeneration of tissues in chronic ulcers, with proper and timely healing of the wounds, natural antioxidants that can combat the effects of induced reactive oxygen species in wound-healing cells ought to be investigated. Reactive oxygen species activity was determined in mycolactone-treated RAW 264.7 macrophages and the scavenging ability of the antioxidants (ascorbic acid, gallic acid, and green tea kombucha) against mycolactone-induced reactive oxygen species (superoxide anions) was assessed using fluorescein probe (DCF-DA) and nitroblue tetrazolium dye. Cytotoxicity of the antioxidants, mycolactone, and the protective effect of the antioxidants on the cells upon treatment with mycolactone were determined using the Alamar blue assay. The expression levels of endogenous antioxidant enzyme genes (superoxide dismutase, catalase, and glutathione peroxidase) in response to mycolactone-mediated reactive oxygen species were determined using RT-qPCR. Mycolactone induced the production of reactive oxygen species in RAW 264.7 macrophages, and the resulting superoxide anions were scavenged by some of the antioxidants. The selected endogenous antioxidant enzyme genes in the macrophages were upregulated in the presence of the antioxidants and mycolactone. The exogenously supplied ascorbic acid and green tea kombucha offered moderate protection to the macrophages against the toxicity of mycolactone. We conclude that the results provide insights into alternate and adjunct therapeutic approaches in Buruli ulcer treatment, which could significantly attenuate the toxicity of the pathogenic factor; mycolactone.