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

The profile of risk factors and in-patient outcomes of stroke in Kumasi, Ghana

Background: Stroke is an emerging public health challenge in Ghana requiring urgent attention for its control. Because some of the risk factors for stroke are modifiable, characterisation of these risk factors in theGhanaian population as well as outcomes of stroke are urgently needed to guide policy for non-communicable diseases. We therefore conducted this study to evaluate the frequencies of the traditional risk factors and outcomes of stroke at the main tertiary referral centre in the middle belt of Ghana in a prospective observational study.Methods and results: Patients with a clinical diagnosis of stroke were consecutively recruited and vascular risk factors were assessed as well as  markers of severity of stroke and in-patient treatment outcomes. 265patients were recruited, 56.6% were females and mean ± SD age of 64.6 ± 14.54 years. 85%, 73% and 58% of patients had systemic arterial  hypertension, physical inactivity and obesity respectively as common riskfactors. We identified that patients with stroke had a median of 3 traditional risk factors, were unaware of the presence of these risk factors or were poorly controlled if known. Stroke was associated with a high inpatient case fatality rate of 43% principally among patients with haemorrhagic stroke.Conclusions: Our findings indicate that urgent concerted efforts are required to improve public awareness and management of the prevailing risk factors of stroke in Ghana.Key words: Stroke, risk factors, mortality, Ghan

Mycolactone Diffuses into the Peripheral Blood of Buruli Ulcer Patients - Implications for Diagnosis and Disease Monitoring.

BACKGROUND: Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU), is unique among human pathogens in its capacity to produce a polyketide-derived macrolide called mycolactone, making this molecule an attractive candidate target for diagnosis and disease monitoring. Whether mycolactone diffuses from ulcerated lesions in clinically accessible samples and is modulated by antibiotic therapy remained to be established. METHODOLOGY/PRINCIPAL FINDING: Peripheral blood and ulcer exudates were sampled from patients at various stages of antibiotic therapy in Ghana and Ivory Coast. Total lipids were extracted from serum, white cell pellets and ulcer exudates with organic solvents. The presence of mycolactone in these extracts was then analyzed by a recently published, field-friendly method using thin layer chromatography and fluorescence detection. This approach did not allow us to detect mycolactone accurately, because of a high background due to co-extracted human lipids. We thus used a previously established approach based on high performance liquid chromatography coupled to mass spectrometry. By this means, we could identify structurally intact mycolactone in ulcer exudates and serum of patients, and evaluate the impact of antibiotic treatment on the concentration of mycolactone. CONCLUSIONS/SIGNIFICANCE: Our study provides the proof of concept that assays based on mycolactone detection in serum and ulcer exudates can form the basis of BU diagnostic tests. However, the identification of mycolactone required a technology that is not compatible with field conditions and point-of-care assays for mycolactone detection remain to be worked out. Notably, we found mycolactone in ulcer exudates harvested at the end of antibiotic therapy, suggesting that the toxin is eliminated by BU patients at a slow rate. Our results also indicated that mycolactone titres in the serum may reflect a positive response to antibiotics, a possibility that it will be interesting to examine further through longitudinal studies

Highly prevalent hyperuricaemia is associated with adverse clinical outcomes among Ghanaian stroke patients: An observational prospective study

Background: Although a direct causal relationship between hyperuricaemia and stroke continues to be debated, strong associations between serum uric acid (SUA) and cerebrovascular disease exist. Very few studies have been conducted to evaluate the frequency and association between this potentially modifiable biomarker of vascular risk and stroke in sub-Saharan Africa. Therefore the aim of this study was to examine the association between hyperuricaemia and the traditional risk factors and the outcomes of stroke in Ghanaian patients.Methods: In this prospective observational study, 147 patients presenting with stroke at a tertiary referral centre in Ghana were consecutively recruited. Patients were screened for vascular risk factors and SUA concentrations measured after an overnight fast. Associations between hyperuricaemia and stroke outcomes were analysed using Kaplan-Meier and Cox proportional hazards regression analysis.Results: The frequency of hyperuricaemia among Ghanaian stroke patients was 46.3%. Non-significant associations were observed between hyperuricaemia and the traditional risk factors of stroke. SUA concentration was positively correlated with stroke severity and associated with early mortality after an acute stroke with unadjusted hazards ratio of 2.3 (1.4 - 4.2, p=0.001). A potent and independent dose-response association between increasing SUA concentration and hazard of mortality was found on Cox proportional hazards regression, aHR (95% CI) of 1.65 (1.14-2.39), p=0.009 for each 100μmol/l increase in SUA.Conclusions: Hyperuricaemia is highly frequent and associated with adverse functional outcomes among Ghanaian stroke patients. Further studies are warranted to determine whether reducing SUA levels after a stroke would be beneficial within our setting.Key words: Hyperuricaemia, stroke, mortality, Ghan

Detection of viable Mycobacterium ulcerans in clinical samples by a novel combined 16S rRNA reverse transcriptase/IS2404 real-time qPCR assay.

Detection of Viable <em>Mycobacterium ulcerans</em> in Clinical Samples by a Novel Combined 16S rRNA Reverse Transcriptase/IS<em>2404</em> Real-Time qPCR Assa

Kinetics of mycolactone in human subcutaneous tissue during antibiotic therapy for Mycobacterium ulcerans disease.

BACKGROUND: Mycobacterium ulcerans (M. ulcerans) causes a devastating necrotising infection of skin tissue leading to progressive ulceration. M. ulcerans is the only human pathogen that secretes mycolactone, a polyketide molecule with potent cytotoxic and immunomodulatory properties. These unique features make mycolactone an attractive biomarker for M. ulcerans disease. We sought to measure the concentration of mycolactone within lesions of patients with Buruli ulcer before, during and after antibiotic treatment to evaluate its association with the clinical and bacteriological response to therapy. METHODS: Biopsies of M. ulcerans infected skin lesions were obtained from patients before, during and after antibiotic therapy. Lipids were extracted from the biopsies and concentration of mycolactone was assayed by mass spectrometry and a cytotoxicity assay and correlated with clinical and bacteriological response to therapy. RESULTS: Baseline concentration of mycolactone measured by mass spectrometry predicted time to complete healing of small nodules and ulcers. Even though intra-lesional concentrations of mycolactone declined with antibiotic treatment, the toxin was still present after antibiotic treatment for 6 weeks and also 4 weeks after the end of treatment for 8 weeks in a subgroup of patients with slowly healing lesions. Additionally viable bacilli were detected in a proportion of these slowly healing lesions during and after treatment. CONCLUSIONS: Our findings indicate that baseline intra-lesional mycolactone concentration and its kinetics with antibiotic therapy are important prognostic determinants of clinical and bacteriological response to antibiotic treatment for Mycobacterium ulcerans disease. Mycolactone may be a useful biomarker with potential utility in optimising antibiotic therapy

Liver stiffness and virologic outcomes after introducing tenofovir as part of antiretroviral therapy in lamivudine-experienced adults with HIV and hepatitis B virus (HBV) co-infection in Ghana: four-year follow up of the prospective HEPIK cohort

Introduction Until recently lamivudine was the only available agent to treat hepatitis B in the context of HIV infection in sub‐Saharan Africa. Tenofovir is gradually becoming available although access remains far from universal. Long‐term outcomes of introducing tenofovir as part of antiretroviral therapy (ART) in subjects previously extensively exposed to lamivudine as the sole HBV‐active agent in the region are unknown. Methods We report from a prospective cohort of HIV/HBV co‐infected adults attending for HIV care in Kumasi, Ghana, where HBsAg prevalence is 14%. HBsAg‐positive subjects were invited to attend for transient elastography (TE) and blood sampling before the introduction of tenofovir (TO) as part of ART, and within 1 year (T1) and 4 years (T2) of starting tenofovir. Adherence and alcohol consumption were determined by a questionnaire‐based interview. Results Overall 178 patients underwent evaluation at T0/T1, of whom 98 (55%) also attended for assessment at T2. Remaining patients were lost to follow up (50; 28%); had died (10; 6%); declined to attend (17; 10%); or were excluded due to pregnancy (2; 1 %) or invalid TE (1; 1 %). Of the 98 subjects, 94 had started tenofovir‐based ART and had received tenofovir for median 4 years (IQR 3.8, 4.1), while continuing previous lamivudine (Table 1). By multivariable linear regression, female gender, no history of alcohol excess, and higher HBV DNA level, higher liver stiffness, and lower platelet count at T0/T1 were significant predictors of decreasing liver stiffness between TO/1 and T2. No treatment‐emergent resistance mutations in HBV polymerase were observed by Sanger sequencing among subjects with HBV DNA>100 lU/ml at T2; one subject showed M204V+V173L+L180M at both TO and T2. Conclusions This is the first report of the long‐term impact on liver stiffness and virologic parameters of introducing tenofovir as part of ART in extensively lamivudine exposed HIV/HBV co‐infected patients in sub‐Saharan Africa. Significant reductions in liver stiffness and improved HBV control were observed at four years

Microbiological, histological, immunological, and toxin response to antibiotic treatment in the mouse model of Mycobacterium ulcerans disease.

Mycobacterium ulcerans infection causes a neglected tropical disease known as Buruli ulcer that is now found in poor rural areas of West Africa in numbers that sometimes exceed those reported for another significant mycobacterial disease, leprosy, caused by M. leprae. Unique among mycobacterial diseases, M. ulcerans produces a plasmid-encoded toxin called mycolactone (ML), which is the principal virulence factor and destroys fat cells in subcutaneous tissue. Disease is typically first manifested by the appearance of a nodule that eventually ulcerates and the lesions may continue to spread over limbs or occasionally the trunk. The current standard treatment is 8 weeks of daily rifampin and injections of streptomycin (RS). The treatment kills bacilli and wounds gradually heal. Whether RS treatment actually stops mycolactone production before killing bacilli has been suggested by histopathological analyses of patient lesions. Using a mouse footpad model of M. ulcerans infection where the time of infection and development of lesions can be followed in a controlled manner before and after antibiotic treatment, we have evaluated the progress of infection by assessing bacterial numbers, mycolactone production, the immune response, and lesion histopathology at regular intervals after infection and after antibiotic therapy. We found that RS treatment rapidly reduced gross lesions, bacterial numbers, and ML production as assessed by cytotoxicity assays and mass spectrometric analysis. Histopathological analysis revealed that RS treatment maintained the association of the bacilli with (or within) host cells where they were destroyed whereas lack of treatment resulted in extracellular infection, destruction of host cells, and ultimately lesion ulceration. We propose that RS treatment promotes healing in the host by blocking mycolactone production, which favors the survival of host cells, and by killing M. ulcerans bacilli