Genetic Diversity of Staphylococcus aureus in Buruli Ulcer

Background

Buruli ulcer (BU) is a necrotizing skin disease caused by Mycobacterium ulcerans. Previous studies have shown that wounds of BU patients are colonized with M. ulcerans and several other microorganisms, including Staphylococcus aureus, which may interfere with wound healing. The present study was therefore aimed at investigating the diversity and topography of S. aureus colonizing BU patients during treatment.

Methodology

We investigated the presence, diversity, and spatio-temporal distribution of S. aureus in 30 confirmed BU patients from Ghana during treatment. S. aureus was isolated from nose and wound swabs, and by replica plating of wound dressings collected bi-weekly from patients. S. aureus isolates were characterized by multiple-locus variable number tandem repeat fingerprinting (MLVF) and spa-typing, and antibiotic susceptibility was tested.

Principal Findings

Nineteen (63%) of the 30 BU patients tested positive for S. aureus at least once during the sampling period, yielding 407 S. aureus isolates. Detailed analysis of 91 isolates grouped these isolates into 13 MLVF clusters and 13 spa-types. Five (26%) S. aureus-positive BU patients carried the same S. aureus genotype in their anterior nares and wounds. S. aureus isolates from the wounds of seven (37%) patients were distributed over two different MLVF clusters. Wounds of three (16%) patients were colonized with isolates belonging to two different genotypes at the same time, and five (26%) patients were colonized with different S. aureus types over time. Five (17%) of the 30 included BU patients tested positive for methicillin-resistant S. aureus (MRSA).

Conclusion/Significance

The present study showed that the wounds of many BU patients were contaminated with S. aureus, and that many BU patients from the different communities carried the same S. aureus genotype during treatment. This calls for improved wound care and hygiene.     

Treatment Outcome of Patients with Buruli Ulcer Disease in Togo

Background

Following introduction of antimycobacterial treatment of Buruli ulcer disease (BUD), several clinical studies evaluated treatment outcomes of BUD patients, in particular healing times, secondary lesions and functional limitations. Whereas recurrences were rarely observed, paradoxical reactions and functional limitations frequently occurred. Although systematic BUD control in Togo was established as early as 2007, treatment outcome has not been reviewed to date. Therefore, a pilot project on post-treatment follow-up of BUD patients in Togo aimed to evaluate treatment outcomes and to provide recommendations for optimization of treatment success.

Methodology/Principal Findings

Out of 199 laboratory confirmed BUD patients, 129 could be enrolled in the study. The lesions of 109 patients (84.5%) were completely healed without any complications, 5 patients (3.9%) had secondary lesions and 15 patients (11.6%) had functional limitations. Edema, category III ulcers >15cm, healing times >180 days and a limitation of movement at time of discharge constituted the main risk factors significantly associated with BUD related functional limitations (P<0.01). Review of all BUD related documentation revealed major shortcomings, in particular concerning medical records on adjuvant surgical and physiotherapeutic treatment.

Conclusions/Significance

This study presents the first systematic analysis of treatment outcome of BUD patients from Togo. Median times to healing and the absence of recurrences were in line with findings reported by other investigators. The percentage of functional limitations of 11.6% was lower than in other studies, and edema, category III ulcers, healing time >180 days and limitation of movement at discharge constituted the main risk factors for functional limitations in Togolese BUD patients. Standardized treatment plans, patient assessment and follow-up, as well as improved management of medical records are recommended to allow for intensified monitoring of disease progression and healing process, to facilitate implementation of therapeutic measures and to optimize treatment success.     

Vaccination with the Surface Proteins MUL_2232 and MUL_3720 of Mycobacterium ulcerans Induces Antibodies but Fails to Provide Protection against Buruli Ulcer

Background

Buruli ulcer, caused by infection with Mycobacterium ulcerans, is a chronic ulcerative neglected tropical disease of the skin and subcutaneous tissue that is most prevalent in West African countries. M. ulcerans produces a cytotoxic macrolide exotoxin called mycolactone, which causes extensive necrosis of infected subcutaneous tissue and the development of characteristic ulcerative lesions with undermined edges. While cellular immune responses are expected to play a key role against early intracellular stages of M. ulcerans in macrophages, antibody mediated protection might be of major relevance against advanced stages, where bacilli are predominantly found as extracellular clusters.

Methodology/Principal Findings

To assess whether vaccine induced antibodies against surface antigens of M. ulcerans can protect against Buruli ulcer we formulated two surface vaccine candidate antigens, MUL_2232 and MUL_3720, as recombinant proteins with the synthetic Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion. The candidate vaccines elicited strong antibody responses without a strong bias towards a T_H1 type cellular response, as indicated by the IgG2a to IgG1 ratio. Despite the cross-reactivity of the induced antibodies with the native antigens, no significant protection was observed against progression of an experimental M. ulcerans infection in a mouse footpad challenge model.

Conclusions

Even though vaccine-induced antibodies have the potential to opsonise the extracellular bacilli they do not have a protective effect since infiltrating phagocytes might be killed by mycolactone before reaching the bacteria, as indicated by lack of viable infiltrates in the necrotic infection foci.     

Immunohistochemical Monitoring of Wound Healing in Antibiotic Treated Buruli Ulcer Patients

Background

While traditionally surgery has dominated the clinical management of Buruli ulcer (BU), the introduction of the combination chemotherapy with oral rifampicin and intramuscular streptomycin greatly improved treatment and reduced recurrence rates. However management of the often extensive lesions after successful specific therapy has remained a challenge, in particular in rural areas of the African countries which carry the highest burden of disease. For reasons not fully understood, wound healing is delayed in a proportion of antibiotic treated BU patients. Therefore, we have performed immunohistochemical investigations to identify markers which may be suitable to monitor wound healing progression.

Methodology/Principal findings

Tissue specimens from eight BU patients with plaque lesions collected before, during and after chemotherapy were analyzed by immunohistochemistry for the presence of a set of markers associated with connective tissue neo-formation, tissue remodeling and epidermal activation. Several target proteins turned out to be suitable to monitor wound healing. While α-smooth muscle actin positive myofibroblasts were not found in untreated lesions, they emerged during the healing process. These cells produced abundant extracellular matrix proteins, such as pro-collagen 1 and tenascin and were found in fibronectin rich areas. After antibiotic treatment many cells, including myofibroblasts, revealed an activated phenotype as they showed ribosomal protein S6 phosphorylation, a marker for translation initiation. In addition, healing wounds revealed dermal tissue remodeling by apoptosis, and showed increased cytokeratin 16 expression in the epidermis.

Conclusion/Significance

We have identified a set of markers that allow monitoring wound healing in antibiotic treated BU lesions by immunohistochemistry. Studies with this marker panel may help to better understand disturbances responsible for wound healing delays observed in some BU patients.     

Selamectin Is the Avermectin with the Best Potential for Buruli Ulcer Treatment

A comprehensive analysis was done to evaluate the potential use of anti-parasitic macrocyclic lactones (including avermectins and milbemycins) for Buruli ulcer (BU) therapy. A panel containing nearly all macrocyclic lactones used in human or in veterinary medicine was analyzed for activity in vitro against clinical isolates of Mycobacterium ulcerans. Milbemycin oxime and selamectin were the most active drugs against M. ulcerans with MIC values from 2 to 8 μg/mL and 2 to 4 μg/mL, respectively. In contrast, ivermectin and moxidectin, which are both in clinical use, showed no significant activity (MIC> 32 μg/mL). Time-kill kinetic assays showed bactericidal activity of selamectin and in vitro pharmacodynamic studies demonstrated exposure-dependent activity. These data together with analyses of published pharmacokinetic information strongly suggest that selamectin is the most promising macrocyclic lactone for BU treatment.     

Delphinidin Reduces Cell Proliferation and Induces Apoptosis of Non-Small-Cell Lung Cancer Cells by Targeting EGFR/VEGFR2 Signaling Pathways

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) have emerged as two effective clinical targets for non-small-cell lung cancer (NSCLC). In the present study, we found that delphinidin, an anthocyanidin, present in pigmented fruits and vegetables, is a potent inhibitor of both EGFR and VEGFR2 in NSCLC cells that overexpress EGFR/VEGFR2. Using these cells, we next determined the effects of delphinidin on cell growth and apoptosis in vitro and on tumor growth and angiogenesis in vivo. Delphinidin (5-60 µM) treatment of NSCLC cells inhibited the activation of PI3K, and phosphorylation of AKT and MAPKs. Additionally, treatment of NSCLC cells with delphinidin resulted in inhibition of cell growth without having significant toxic effects on normal human bronchial epithelial cells. Specifically, treatment of NCI-H441 and SK-MES-1 cells with delphindin (5-60 µM) resulted in (i) cleavage of PARP protein, (ii) activation of caspase-3 and -9, (iii) downregulation of anti-apoptotic proteins (Bcl2, Bcl-xL and Mcl-1), (iv) upregulation of pro-apoptotic proteins (Bax and Bak), and (v) decreased expression of PCNA and cyclin D1. Furthermore, in athymic nude mice subcutaneously implanted with human NSCLC cells, delphinidin treatment caused a (i) significant inhibition of tumor growth, (ii) decrease in the expression of markers for cell proliferation (Ki67 and PCNA) and angiogenesis (CD31 and VEGF), and (iii) induction of apoptosis, when compared with control mice. Based on these observations, we suggest that delphinidin, alone or as an adjuvant to current therapies, could be used for the management of NSCLC, especially those that overexpress EGFR and VEGFR2.     

Buruli Ulcer Disease and Its Association with Land Cover in Southwestern Ghana

BackgroundBuruli ulcer (BU), one of 17 neglected tropical diseases, is a debilitating skin and soft tissue infection caused by Mycobacterium ulcerans. In tropical Africa, changes in land use and proximity to water have been associated with the disease. This study presents the first analysis of BU at the village level in southwestern Ghana, where prevalence rates are among the highest globally, and explores fine and medium-scale associations with land cover by comparing patterns both within BU clusters and surrounding landscapes.Conclusions/SignificanceOur study highlights the importance of multiple land use changes along the Offin River, particularly mining and agriculture, which might be associated with BU disease in southwestern Ghana. Our study is the first to use both medium- and high-resolution imagery to assess these changes. We also show that older populations (≥ 60 y) appear to be at higher risk of BU disease than children, once BU data were weighted by population age structures.     

Family Relationship,Water Contact and Occurrence of Buruli Ulcer in Benin

Background

Mycobacterium ulcerans disease (Buruli ulcer) is the most widespread mycobacterial disease in the world after leprosy and tuberculosis. How M. ulcerans is introduced into the skin of humans remains unclear, but it appears that individuals living in the same environment may have different susceptibilities.

Objectives

This study aims to determine whether frequent contacts with natural water sources, family relationship or the practice of consanguineous marriages are associated with the occurrence of Buruli ulcer (BU).

Design

Case control study.

Setting

Department of Atlantique, Benin.

Subjects

BU-confirmed cases that were diagnosed and followed up at the BU detection and treatment center (CDTUB) of Allada (Department of the Atlantique, Benin) during the period from January 1st, 2006, to June 30th, 2008, with three matched controls (persons who had no signs or symptoms of active or inactive BU) for age, gender and village of residence per case.

Main Outcomes Measured

Contact with natural water sources, BU history in the family and the practice of consanguineous marriages.

Results

A total of 416 participants were included in this study, including 104 cases and 312 controls. BU history in the family (p<0.001), adjusted by daily contact with a natural water source (p = 0.007), was significantly associated with higher odds of having BU (OR; 95% CI = 5.5; 3.0–10.0). The practice of consanguineous marriage was not associated with the occurrence of BU (p = 0.40). Mendelian disorders could explain this finding, which may influence individual susceptibility by impairing immunity.

Conclusion

This study suggests that a combination of genetic factors and behavioral risk factors may increase the susceptibility for developing BU.     

Spatial Analysis of Anthropogenic Landscape Disturbance and Buruli Ulcer Disease in Benin

Background

Land use and land cover (LULC) change is one anthropogenic disturbance linked to infectious disease emergence. Current research has focused largely on wildlife and vector-borne zoonotic diseases, neglecting to investigate landscape disturbance and environmental bacterial infections. One example is Buruli ulcer (BU) disease, a necrotizing skin disease caused by the environmental pathogen Mycobacterium ulcerans (MU). Empirical and anecdotal observations have linked BU incidence to landscape disturbance, but potential relationships have not been quantified as they relate to land cover configurations.

Methodology/Principal Findings

A landscape ecological approach utilizing Bayesian hierarchical models with spatial random effects was used to test study hypotheses that land cover configurations indicative of anthropogenic disturbance were related to Buruli ulcer (BU) disease in southern Benin, and that a spatial structure existed for drivers of BU case distribution in the region. A final objective was to generate a continuous, risk map across the study region. Results suggested that villages surrounded by naturally shaped, or undisturbed rather than disturbed, wetland patches at a distance within 1200m were at a higher risk for BU, and study outcomes supported the hypothesis that a spatial structure exists for the drivers behind BU risk in the region. The risk surface corresponded to known BU endemicity in Benin and identified moderate risk areas within the boundary of Togo.

Conclusions/Significance

This study was a first attempt to link land cover configurations representative of anthropogenic disturbances to BU prevalence. Study results identified several significant variables, including the presence of natural wetland areas, warranting future investigations into these factors at additional spatial and temporal scales. A major contribution of this study included the incorporation of a spatial modeling component that predicted BU rates to new locations without strong knowledge of environmental factors contributing to disease distribution.     

Buruli Ulcer in Cameroon: The Development and Impact of the National Control Programme

BackgroundCameroon is endemic for Buruli ulcer (BU) and organised institutional BU control began in 2002. The objective was to describe the evolution, achievements and challenges of the national BU control programme (NBUCP) and to make suggestions for scaling up the programme.MethodsWe analysed collated data on BU from 2001 to 2014 and reviewed activity reports NBUCP in Cameroon. Case-detection rates and key BU control indicators were calculated and plotted on a time scale to determine trends in performance. A linear regression analysis of BU detection rate from 2005–2014 was done. The regression coefficient was tested statistically for the significance in variation of BU detection rate.Conclusion/SignificanceAlthough institutional BU control Cameroon only began 30 years after the first cases were reported in 1969, a number of milestones have been attained. These would serve as stepping stones for charting the way forward and improving upon control activities in the country if the major challenge of resource allocation is dealt with.     

Psychometric Properties of the Participation Scale among Former Buruli Ulcer Patients in Ghana and Benin

Background

Buruli ulcer is a stigmatising disease treated with antibiotics and wound care, and sometimes surgical intervention is necessary. Permanent limitations in daily activities are a common long term consequence. It is unknown to what extent patients perceive problems in participation in social activities. The psychometric properties of the Participation Scale used in other disabling diseases, such as leprosy, was assessed for use in former Buruli ulcer patients.

Methods

Former Buruli ulcer patients in Ghana and Benin, their relatives, and healthy community controls were interviewed using the Participation Scale, Buruli Ulcer Functional Limitation Score, and the Explanatory Model Interview Catalogue to measure stigma. The Participation Scale was tested for the following psychometric properties: discrimination, floor and ceiling effects, internal consistency, inter-item correlation, item-total correlation and construct validity.

Results

In total 386 participants (143 former Buruli ulcer patients with their relatives (137) and 106 community controls) were included in the study. The Participation Scale displayed good discrimination between former Buruli ulcer patients and healthy community controls. No floor and ceiling effects were found. Internal consistency (Cronbach''s alpha) was 0.88. In Ghana, mean inter-item correlation of 0.29 and item-total correlations ranging from 0.10 to 0.69 were found while in Benin, a mean inter-item correlation of 0.28 was reported with item-total correlations ranging from −0.08 to 0.79. With respect to construct validity, 4 out of 6 hypotheses were not rejected, though correlations between various constructs differed between countries.

Conclusion

The results indicate the Participation Scale has acceptable psychometric properties and can be used for Buruli ulcer patients in Ghana and Benin. Future studies can use this Participation Scale to evaluate the long term restrictions in participation in daily social activities of former BU patients.     

Climate and Landscape Factors Associated with Buruli Ulcer Incidence in Victoria,Australia

Background

Buruli ulcer (BU), caused by Mycobacterium ulcerans (M. ulcerans), is a necrotizing skin disease found in more than 30 countries worldwide. BU incidence is highest in West Africa; however, cases have substantially increased in coastal regions of southern Australia over the past 30 years. Although the mode of transmission remains uncertain, the spatial pattern of BU emergence in recent years seems to suggest that there is an environmental niche for M. ulcerans and BU prevalence.

Methodology/Principal Findings

Network analysis was applied to BU cases in Victoria, Australia, from 1981–2008. Results revealed a non-random spatio-temporal pattern at the regional scale as well as a stable and efficient BU disease network, indicating that deterministic factors influence the occurrence of this disease. Monthly BU incidence reported by locality was analyzed with landscape and climate data using a multilevel Poisson regression approach. The results suggest the highest BU risk areas occur at low elevations with forested land cover, similar to previous studies of BU risk in West Africa. Additionally, climate conditions as far as 1.5 years in advance appear to impact disease incidence. Warmer and wetter conditions 18–19 months prior to case emergence, followed by a dry period approximately 5 months prior to case emergence seem to favor the occurrence of BU.

Conclusions/Significance

The BU network structure in Victoria, Australia, suggests external environmental factors favor M. ulcerans transmission and, therefore, BU incidence. A unique combination of environmental conditions, including land cover type, temperature and a wet-dry sequence, may produce habitat characteristics that support M. ulcerans transmission and BU prevalence. These findings imply that future BU research efforts on transmission mechanisms should focus on potential vectors/reservoirs found in those environmental niches. Further, this study is the first to quantitatively estimate environmental lag times associated with BU outbreaks, providing insights for future transmission investigations.     

Combined Inflammatory and Metabolic Defects Reflected by Reduced Serum Protein Levels in Patients with Buruli Ulcer Disease

Buruli ulcer is a skin disease caused by Mycobacterium ulcerans that is spreading in tropical countries, with major public health and economic implications in West Africa. Multi-analyte profiling of serum proteins in patients and endemic controls revealed that Buruli ulcer disease down-regulates the circulating levels of a large array of inflammatory mediators, without impacting on the leukocyte composition of peripheral blood. Notably, several proteins contributing to acute phase reaction, lipid metabolism, coagulation and tissue remodelling were also impacted. Their down-regulation was selective and persisted after the elimination of bacteria with antibiotic therapy. It involved proteins with various functions and origins, suggesting that M. ulcerans infection causes global and chronic defects in the host''s protein metabolism. Accordingly, patients had reduced levels of total serum proteins and blood urea, in the absence of signs of malnutrition, or functional failure of liver or kidney. Interestingly, slow healers had deeper metabolic and coagulation defects at the start of antibiotic therapy. In addition to providing novel insight into Buruli ulcer pathogenesis, our study therefore identifies a unique proteomic signature for this disease.     

Potential Wildlife Sentinels for Monitoring the Endemic Spread of Human Buruli Ulcer in South-East Australia

The last 20 years has seen a significant series of outbreaks of Buruli/Bairnsdale Ulcer (BU), caused by Mycobacterium ulcerans, in temperate south-eastern Australia (state of Victoria). Here, the prevailing view of M. ulcerans as an aquatic pathogen has been questioned by recent research identifying native wildlife as potential terrestrial reservoirs of infection; specifically, tree-dwelling common ringtail and brushtail possums. In that previous work, sampling of environmental possum faeces detected a high prevalence of M. ulcerans DNA in established endemic areas for human BU on the Bellarine Peninsula, compared with non-endemic areas. Here, we report research from an emergent BU focus recently identified on the Mornington Peninsula, confirming associations between human BU and the presence of the aetiological agent in possum faeces, detected by real-time PCR targeting M. ulcerans IS2404, IS2606 and KR. Mycobacterium ulcerans DNA was detected in 20/216 (9.3%) ground collected ringtail possum faecal samples and 4/6 (66.6%) brushtail possum faecal samples. The distribution of the PCR positive possum faecal samples and human BU cases was highly focal: there was a significant non-random cluster of 16 M. ulcerans positive possum faecal sample points detected by spatial scan statistics (P<0.0001) within a circle of radius 0.42 km, within which were located the addresses of 6/12 human cases reported from the area to date; moreover, the highest sample PCR signal strength (equivalent to ≥10⁶ organisms per gram of faeces) was found in a sample point located within this cluster radius. Corresponding faecal samples collected from closely adjacent BU-free areas were predominantly negative. Possums may be useful sentinels to predict endemic spread of human BU in Victoria, for public health planning. Further research is needed to establish whether spatial associations represent evidence of direct or indirect transmission between possums and humans, and the mechanism by which this may occur.     

Persisting Social Participation Restrictions among Former Buruli Ulcer Patients in Ghana and Benin

BackgroundBuruli 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.MethodsIn 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.ResultsIn 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.ConclusionPersisting 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.     

Perceptions on the Effectiveness of Treatment and the Timeline of Buruli Ulcer Influence Pre-Hospital Delay Reported by Healthy Individuals

Background

Delay in seeking treatment at the hospital is a major challenge in current Buruli ulcer control; it is associated with severe sequelae and functional limitations. Choosing alternative treatment and psychological, social and practical factors appear to influence delay. Objectives were to determine potential predictors for pre-hospital delay with Leventhal''s commonsense model of illness representations, and to explore whether the type of available dominant treatment modality influenced individuals'' perceptions about BU, and therefore, influenced pre-hospital delay.

Methodology

130 healthy individuals aged >18 years, living in BU-endemic areas in Benin without any history of BU were included in this cross-sectional study. Sixty four participants from areas where surgery was the dominant treatment and sixty six participants from areas where antibiotic treatment was the dominant treatment modality were recruited. Using a semi-structured interview we measured illness perceptions (IPQ-R), knowledge about BU, background variables and estimated pre-hospital delay.

Principal Findings

The individual characteristics ‘effectiveness of treatment’ and ‘timeline acute-chronic’ showed the strongest association with pre-hospital delay. No differences were found between regions where surgery was the dominant treatment and regions where antibiotics were the dominant treatment modality.

Conclusions

Individual characteristics, not anticipated treatment modality appeared predictors of pre-hospital delay.