FVB/N Mice Spontaneously Heal Ulcerative Lesions Induced by Mycobacterium ulcerans and Switch M. ulcerans into a Low Mycolactone Producer.

Buruli ulcer, a debilitating disease, is caused by Mycobacterium ulcerans. The incidence of this neglected tropical disease is steadily increasing. As a rule, without treatment, skin ulcers occur and a lengthy healing process may be observed associated with severe functional disabilities. Mouse models are already available to study establishment of lesions or evaluation of therapy but a lack of a suitable animal model, mimicking all clinical stages, in particular the healing process, remains an obstacle to understand the pathophysiology of M. ulcerans infection. M. ulcerans was s.c. inoculated in three consanguine mouse strains, that is, BALB/c and C57BL/6, classically used to study mycobacterial infection, and FVB/N. Strikingly, FVB/N mice, although as sensitive as all other mouse strains with respect to M. ulcerans infection, presented a spontaneous healing after the ulcerative phase despite stable bacterial load, and mycolactone toxin was not detected in the healed tissues. The spontaneous healing process was accompanied by an activation of the innate immune system. The adaptive response initiated by FVB/N mice was not involved in the healing process and did not confer protection against M. ulcerans. Our work highlights the importance of innate immune responses to control M. ulcerans infection. This in vivo model of M. ulcerans infection now paves the way for new avenues of research toward the elucidation of critical stages of this disease, such as the characterization of the regulation of mycolactone production, a better understanding of the pathophysiology of M. ulcerans infection, and the development of new therapeutic strategies.

Mycolactone-mediated neurite degeneration and functional effects in cultured human and rat DRG neurons: Mechanisms underlying hypoalgesia in Buruli ulcer.

BACKGROUND: Mycolactone is a polyketide toxin secreted by the mycobacterium Mycobacterium ulcerans, responsible for the extensive hypoalgesic skin lesions characteristic of patients with Buruli ulcer. A recent pre-clinical study proposed that mycolactone may produce analgesia via activation of the angiotensin II type 2 receptor (AT2R). In contrast, AT2R antagonist EMA401 has shown analgesic efficacy in animal models and clinical trials for neuropathic pain. We therefore investigated the morphological and functional effects of mycolactone in cultured human and rat dorsal root ganglia (DRG) neurons and the role of AT2R using EMA401. Primary sensory neurons were prepared from avulsed cervical human DRG and rat DRG; 24 h after plating, neurons were incubated for 24 to 96 h with synthetic mycolactone A/B, followed by immunostaining with antibodies to PGP9.5, Gap43, ß tubulin, or Mitotracker dye staining. Acute functional effects were examined by measuring capsaicin responses with calcium imaging in DRG neuronal cultures treated with mycolactone. RESULTS: Morphological effects: Mycolactone-treated cultures showed dramatically reduced numbers of surviving neurons and non-neuronal cells, reduced Gap43 and ß tubulin expression, degenerating neurites and reduced cell body diameter, compared with controls. Dose-related reduction of neurite length was observed in mycolactone-treated cultures. Mitochondria were distributed throughout the length of neurites and soma of control neurons, but clustered in the neurites and soma of mycolactone-treated neurons. Functional effects: Mycolactone-treated human and rat DRG neurons showed dose-related inhibition of capsaicin responses, which were reversed by calcineurin inhibitor cyclosporine and phosphodiesterase inhibitor 3-isobutyl-1-Methylxanthine, indicating involvement of cAMP/ATP reduction. The morphological and functional effects of mycolactone were not altered by Angiotensin II or AT2R antagonist EMA401. CONCLUSION: Mycolactone induces toxic effects in DRG neurons, leading to impaired nociceptor function, neurite degeneration, and cell death, resembling the cutaneous hypoalgesia and nerve damage in individuals with M. Ulcerans infection.

[Community-based approaches in the fight against Buruli ulcer : review of the literature]. / Approches communautaires dans la lutte contre l'ulcère de Buruli : revue de la littérature.

Buruli ulcer (BU) is an infectious skin disease caused by Mycobacterium ulcerans. It mainly affects poor communities living close to bodies of water. In the absence of early treatment, this "neglected" disease can cause lasting deformities and may require limb amputation. It is reported in 34 countries and is the third most common mycobacterial disease in immunocompetent patients. Considerable progress has been made in treatment and prevention. The Cotonou Declaration (2009) describes the recommended control strategies. Although effective, current control strategies are limited because they do not take into account all the factors that influence emergence, prevention and cure of the disease. The control of Buruli ulcer mainly depends on intervention on social, cultural and psychosocial factors that influence preventive and self-care behaviour. The health promotion approach requires collaboration with populations in order to perform simultaneous actions on BU factors in the community setting. Although effective on many health problems, health promotion is not applied in the fight against BU due to the absence of action on all factors such as poverty. This article presents a review of the literature on BU strategies and community approaches. 407 relevant articles published in 1998-2013 period were examined. Eleven programmes are based on a top-down approach, which does not include populations in decision-making processes, unlike the bottom-up participatory approaches recommended in health promotion. Three health promotion programmes and 6 community-based participatory approaches were identified and examined. Community participation and empowerment constitute the basis for a community approach in the fight against Buruli ulcer.

Illness meanings and experiences for pre-ulcer and ulcer conditions of Buruli ulcer in the Ga-West and Ga-South Municipalities of Ghana.

BACKGROUND: Ghana is a Buruli ulcer (BU) endemic country yet there is paucity of socio-cultural research on BU. Examining distinctive experiences and meanings for pre-ulcers and ulcers of BU may clarify the disease burden, illness experience and local perceptions of causes and spread, and environmental features of BU, which are useful to guide public health programmes and future research. This study aimed to explain local meanings and experiences of BU for persons with pre-ulcers and ulcers in the Ga-West and Ga-South municipalities in Accra. METHODS: Semi-structured interviews based on the Explanatory Model Interview Catalogue framework were administered to 181 respondents comprising 15 respondents with pre-ulcers and 166 respondents with ulcers. The Wilcoxon rank-sum test was used to compare categories of illness experiences (PD) and perceived causes (PC) among respondents with pre-ulcer and ulcer conditions. The Fisher's exact test was used to compare the most troubling PD and the most important PC variables. Qualitative phenomenological analysis of respondents' narratives clarified illness experiences and meanings with reference to PC and PD variables. RESULTS: Families of respondents with pre-ulcers and the respondents themselves were often anxious about disease progression, while families of respondents with ulcers, who had to give care, worried about income loss and disruption of school attendance. Respondents with pre-ulcers frequently reported swimming in ponds and rivers as a perceived cause and considered it as the most important PC (53.3%). Respondents with ulcers frequently attributed their BU illness to witchcraft (64.5%) and respondents who claimed they had no water contact, questioned the credibility of health messages CONCLUSIONS: Affected persons with pre-ulcers are likely to delay treatment because of social and financial constraints and the absence of pain. Scepticism on the role of water in disease contagion and prolonged healing is perceived to make ideas of witchcraft as a PC more credible, among respondents with ulcers. Health messages should address issues of locally perceived risk and vulnerability. Guided by study findings, further research on the role of environmental, socio-cultural and genetic factors in BU contagion, is also needed to clarify and formulate health messages and strengthen public health initiatives.

Mycobacterium ulcerans infections cause progressive muscle atrophy and dysfunction, and mycolactone impairs satellite cell proliferation.

Clinical observations from Buruli ulcer (BU) patients in West Africa suggest that severe Mycobacterium ulcerans infections can cause skeletal muscle contracture and atrophy leading to significant impairment in function. In the present study, male mice C57BL/6 were subcutaneously injected with M. ulcerans in proximity to the right biceps muscle, avoiding direct physical contact between the infectious agent and the skeletal muscle. The histological, morphological, and functional properties of the muscles were assessed at different times after the injection. On day 42 postinjection, the isometric tetanic force and the cross-sectional area of the myofibers were reduced by 31% and 29%, respectively, in the proximate-infected muscles relative to the control muscles. The necrotic areas of the proximate-infected muscles had spread to 7% of the total area by day 42 postinjection. However, the number of central nucleated fibers and myogenic regulatory factors (MyoD and myogenin) remained stable and low. Furthermore, Pax-7 expression did not increase significantly in mycolactone-injected muscles, indicating that the satellite cell proliferation is abrogated by the toxin. In addition, the fibrotic area increased progressively during the infection. Lastly, muscle-specific RING finger protein 1 (MuRF-1) and atrogin-1/muscle atrophy F-box protein (atrogin-1/MAFbx), two muscle-specific E3 ubiquitin ligases, were upregulated in the presence of M. ulcerans. These findings confirmed that skeletal muscle is affected in our model of subcutaneous infection with M. ulcerans and that a better understanding of muscle contractures and weakness is essential to develop a therapy to minimize loss of function and promote the autonomy of BU patients.

Individual and clinical variables associated with the risk of Buruli ulcer acquisition: A systematic review and meta-analysis.

BACKGROUND: Buruli ulcer (BU) is a necrotizing skin disease, caused by Mycobacterium ulcerans, with poorly understood acquisition risk factors. This review aims at evaluating the importance of individual-sex, age, family ties with history of BU, gene variants-and clinical-Bacillus Calmette-Guérin (BCG) immunization, Human Immunodeficiency Virus (HIV) infection-variables in this process. METHODS: A systematic review was performed considering the following databases: ClinicalTrials.gov, Cochrane Controlled Register of Trials (CENTRAL), Current Contents Connect, Embase, MEDLINE, SciELO, Scopus and Web of Science. Eligible studies were critically appraised with The Joanna Briggs Institute checklists and heterogeneity was assessed with Cochran Q-test and I2 statistic. Published demographic data was descriptively analysed and clinical data pooled within random-effects modelling for meta-analysis. RESULTS: A total of 29 studies were included in the systematic review. Two randomized controlled trials (RCTs) and 21 case-control studies were selected for meta-analysis. Studies show that BU mainly affects age extremes, more preponderately males among children. Data pooled from RCTs do not reveal BCG to be protective against BU (odds ratio (OR) = 0.63; 95% CI = 0.38-1.05; I2 = 56%), a finding case-control studies appear to corroborate. HIV infection (OR = 6.80; 95% CI = 2.33-19.85; I2 = 0%) and SLC11A1 rs17235409 A allele (OR = 1.86; 95% CI = 1.25-2.77; I2 = 0%) are associated with increased prevalence of the disease. No definite conclusions can be drawn regarding the influence of previous family history of BU. DISCUSSION: While available evidence warrants further robustness, these results have direct implications on current interventions and future research programs, and foster the development of more cost-effective preventive and screening measures. REGISTRATION: The study was registered at PROSPERO with number CRD42019123611.

Mycolactone is responsible for the painlessness of Mycobacterium ulcerans infection (buruli ulcer) in a murine study.

Buruli ulcer is a chronic skin disease caused by Mycobacterium ulcerans, which produces a toxic lipid mycolactone. Despite the extensive necrosis and tissue damage, the lesions are painless. This absence of pain prevents patients from seeking early treatment and, as a result, many patients experience severe sequelae, including limb amputation. We have reported that mice inoculated with M. ulcerans show loss of pain sensation and nerve degeneration. However, the molecules responsible for the nerve damage have not been identified. In order to clarify whether mycolactone alone can induce nerve damage, mycolactone A/B was injected to footpads of BALB/c mice. A total of 100 microg of mycolactone induced footpad swelling, redness, and erosion. The von Frey sensory test showed hyperesthesia on day 7, recovery on day 21, and hypoesthesia on day 28. Histologically, the footpads showed epidermal erosion, moderate stromal edema, and moderate neutrophilic infiltration up to day 14, which gradually resolved. Nerve bundles showed intraneural hemorrhage, neutrophilic infiltration, and loss of Schwann cell nuclei on days 7 and 14. Ultrastructurally, vacuolar change of myelin started on day 14 and gradually subsided by day 42, but the density of myelinated fibers remained low. This study demonstrated that initial hyperesthesia is followed by sensory recovery and final hypoesthesia. Our present study suggests that mycolactone directly damages nerves and is responsible for the absence of pain characteristic of Buruli ulcer. Furthermore, mice injected with 200 microg of mycolactone showed pulmonary hemorrhage. This is the first study to demonstrate the systemic effects of mycolactone.

Assessing and managing wounds of Buruli ulcer patients at the primary and secondary health care levels in Ghana.

BACKGROUND: Beyond Mycobacterium ulcerans-specific therapy, sound general wound management is required for successful management of Buruli ulcer (BU) patients which places them among the large and diverse group of patients in poor countries with a broken skin barrier. METHODS: Clinically BU suspicious patients were enrolled between October 2013 and August 2015 at a primary health care (PHC) center and a municipal hospital, secondary health care (SHC) center in Ghana. All patients were IS2404 PCR tested and divided into IS2404 PCR positive and negative groups. The course of wound healing was prospectively investigated including predictors of wound closure and assessment of infrastructure, supply and health staff performance. RESULTS: 53 IS2404 PCR positive patients-31 at the PHC center and 22 at the SHC center were enrolled-and additionally, 80 clinically BU suspicious, IS2404 PCR negative patients at the PHC center. The majority of the skin ulcers at the PHC center closed, without the need for surgical intervention (86.7%) compared to 40% at the SHC center, where the majority required split-skin grafting (75%) or excision (12.5%). Only 9% of wounds at the PHC center, but 50% at the SHC center were complicated by bacterial infection. The majority of patients, 54.8% at the PHC center and 68.4% at the SHC center, experienced wound pain, mostly severe and associated with wound dressing. Failure of ulcers to heal was reliably predicted by wound area reduction between week 2 and 4 after initiation of treatment in 75% at the PHC center, and 90% at the SHC center. Obvious reasons for arrested wound healing or deterioration of wound were missed additional severe pathology; at the PHC center (chronic osteomyelitis, chronic lymphedema, squamous cell carcinoma) and at the SHC center (malignant ulceration, chronic lymphedema) in addition to hygiene and wound care deficiencies. When clinically suspicious, but IS2404 PCR negative patients were recaptured in the community, 76/77 (98.7%) of analyzed wounds were either completely closed (85.7%) or almost closed (13%). Five percent were found to have important missed severe pathology (chronic osteomyelitis, ossified fibroma and suspected malignancy). CONCLUSION: The wounds of most BU patients attending the primary health care level can be adequately managed. Additionally, the patients are closer to their families and means of livelihood. Non-healing wounds can be predicted by wound area reduction between 2 to 4 weeks after initiation of treatment. Patients with clinically BU suspicious, but PCR negative ulcers need to be followed up to capture missed diagnoses.

Pain Associated with Wound Care Treatment among Buruli Ulcer Patients from Ghana and Benin.

Buruli ulcer (BU) is a necrotizing skin disease caused by Mycobacterium ulcerans. People living in remote areas in tropical Sub Saharan Africa are mostly affected. Wound care is an important component of BU management; this often needs to be extended for months after the initial antibiotic treatment. BU is reported in the literature as being painless, however clinical observations revealed that some patients experienced pain during wound care. This was the first study on pain intensity during and after wound care in BU patients and factors associated with pain. In Ghana and Benin, 52 BU patients above 5 years of age and their relatives were included between December 2012 and May 2014. Information on pain intensity during and after wound care was obtained during two consecutive weeks using the Wong-Baker Pain Scale. Median pain intensity during wound care was in the lower range (Mdn = 2, CV = 1), but severe pain (score > 6) was reported in nearly 30% of the patients. Nevertheless, only one patient received pain medication. Pain declined over time to low scores 2 hours after treatment. Factors associated with higher self-reported pain scores were; male gender, fear prior to treatment, pain during the night prior to treatment, and pain caused by cleaning the wound. The general idea that BU is painless is incorrect for the wound care procedure. This procedural pain deserves attention and appropriate intervention.

Persisting social participation restrictions among former Buruli ulcer patients in Ghana and Benin.

BACKGROUND: Buruli ulcer may induce severe disabilities impacting on a person's well-being and quality of life. Information about long-term disabilities and participation restrictions is scanty. The objective of this study was to gain insight into participation restrictions among former Buruli ulcer patients in Ghana and Benin. METHODS: In this cross-sectional study, former Buruli ulcer patients were interviewed using the Participation Scale, the Buruli Ulcer Functional Limitation Score to measure functional limitations, and the Explanatory Model Interview Catalogue to measure perceived stigma. Healthy community controls were also interviewed using the Participation Scale. Trained native interviewers conducted the interviews. Former Buruli ulcer patients were eligible for inclusion if they had been treated between 2005 and 2011, had ended treatment at least 3 months before the interview, and were at least 15 years of age. RESULTS: In total, 143 former Buruli ulcer patients and 106 community controls from Ghana and Benin were included in the study. Participation restrictions were experienced by 67 former patients (median score, 30, IQR; 23;43) while 76 participated in social life without problems (median score 5, IQR; 2;9). Most restrictions encountered related to employment. Linear regression showed being female, perceived stigma, functional limitations, and larger lesions (category II) as predictors of more participation restrictions. CONCLUSION: Persisting participation restrictions were experienced by former BU patients in Ghana and Benin. Most important predictors of participation restrictions were being female, perceived stigma, functional limitations and larger lesions.

The impact of community health workers (CHWs) on Buruli ulcer in sub-Saharan Africa: a systematic review.

UNLABELLED: Buruli ulcer (BU) is a cutaneous neglected tropical disease caused by Mycobacterium ulcerans. Participation of Community Health Workers (CHWs) is an integral part of the management of BU, yet their impact has not been systematically evaluated in sub-Saharan Africa. METHODS: Our objectives were to summarize the evidence on the impact of CHWs on the control of BU in sub-Saharan Africa by looking at their recruitment, training, non-governmental support and performance. We searched the following electronic databases from January 1998 to July 2012: Medline, EMBASE (Excerpta Medica Database), The Cochrane Library, Google Scholar, CINAHL (Cumulative Index to Nursing and Allied Health Literature), WHOLIS (World Health Organization Library Database), LILACS (Latin American and Caribbean Literature on Health Sciences) and contacted experts in the field. There were no restrictions to language or publication status. All study designs that could provide the information we sought were eligible, provided the studies were conducted in sub-Saharan Africa. Critical appraisal of all identified citations was done independently by two authors to establish the possible relevance of the articles for inclusion in the review. Of 195 hits, 17 papers met the inclusion criteria. For the management of Buruli Ulcer, CHWs are often recruited from the communities they will serve. Communities play a role in CHW selection. Larger numbers of CHWs are needed in order to improve the detection and management of cases. One of the major obstacles to the control of BU is inadequate and poorly- equipped health facilities in the affected areas. Evidence from this review suggests that CHW programmes can have large impacts on the control of BU in sub-Saharan Africa. Large-scale rigorous studies, including RCTs, are needed to assess whether the CHWs programs promote equity and access.

Functional limitations after surgical or antibiotic treatment for Buruli ulcer in Benin.

Almost half of patients have functional limitations after treatment of Buruli ulcer disease. Antibiotic treatment (along with surgery) was introduced in the National Program for Buruli ulcer in Benin in 2005. The aim of this study was to compare functional limitations in patients who were treated by antibiotics, surgery, or both, using a validated questionnaire. One hundred seventy-nine former patients in Lalo, Benin were retrieved and interviewed in their village. Hospital records were used to gather data about size of lesion at presentation and treatment provided. No significant differences in resulting functional limitations were found between the different treatments. Larger lesions (> 15 cm cross-sectional diameter) at presentation; lesions on a joint, muscular atrophy, and amputation were all associated with a higher risk for functional limitations. Advantages of antibiotic treatment may involve other domains, like costs of treatment or a change in help-seeking behavior.

Limited repair and structural damages displayed by skeletal muscles loaded with mycolactone.

Mycolactone produced by Mycobacterium ulcerans is the toxin responsible for most of the pathology in Buruli ulcer, the cutaneous signature of a complex disease. Although mycolactone cytopathicity is well described in various in vitro and in vivo models, the effect of this molecule on mammalian skeletal muscles has not been addressed. This is particularly surprising since muscle damage is characteristic of severe Buruli ulcer. We have thus investigated the impact of mycolactone on the mouse soleus muscle during degenerative and regenerative phases. Mice were intramuscularly injected with 300 microg of mycolactone and soleus muscles assessed histologically, biochemically and functionally at 7 and 42 days post-injection. Our results show that mycolactone induces local acute and chronic inflammatory responses which are respectively associated with a 65% and 68% decrease in maximal isometric force production (P(0)) relative to sham injections. In addition, muscle stiffness and total hydroxyproline content rose by 46% and 134% at day 42 relative to sham injections indicating an extensive fibrotic process in injured soleus muscles. Histological observations demonstrate significant muscle necrosis and atrophy with limited signs of regeneration. Together, our data indicate that mycolactone not only induces muscle damage but also prevents muscle regeneration to occur. These results may help to explain why patients with Buruli ulcer, experience muscle weakness and contracture.