Samuel Yaw Aboagye

Mycobacterium africanum is associated with patient ethnicity in Ghana.

Mycobacterium africanum is a member of the Mycobacterium tuberculosis complex (MTBC) and an important cause of human tuberculosis in West Africa that is rarely observed elsewhere. Here we genotyped 613 MTBC clinical isolates from Ghana, and searched for associations between the different phylogenetic lineages of MTBC and patient variables. We found that 17.1% (105/613) of the MTBC isolates belonged to M. africanum, with the remaining belonging to M. tuberculosis sensu stricto. No M. bovis was identified in this sample. M. africanum was significantly more common in tuberculosis patients belonging to the Ewe ethnic group (adjusted odds ratio: 3.02; 95% confidence interval: 1.67–5.47, p<0.001). Stratifying our analysis by the two phylogenetic lineages of M. africanum (i.e. MTBC Lineages 5 and 6) revealed that this association was mainly driven by Lineage 5 (also known as M. africanum West Africa 1). Our findings suggest interactions between the genetic diversity of MTBC and human diversity, and offer a possible explanation for the geographical restriction of M. africanum to parts of West Africa.

Late Onset of the Serological Response against the 18 kDa Small Heat Shock Protein of Mycobacterium ulcerans in Children

A previous survey for clinical cases of Buruli ulcer (BU) in the Mapé Basin of Cameroon suggested that, compared to older age groups, very young children may be less exposed to Mycobacterium ulcerans. Here we determined serum IgG titres against the 18 kDa small heat shock protein (shsp) of M. ulcerans in 875 individuals living in the BU endemic river basins of the Mapé in Cameroon and the Densu in Ghana. While none of the sera collected from children below the age of four contained significant amounts of 18 kDa shsp specific antibodies, the majority of sera had high IgG titres against the Plasmodium falciparum merozoite surface protein 1 (MSP-1). These data suggest that exposure to M. ulcerans increases at an age which coincides with the children moving further away from their homes and having more intense environmental contact, including exposure to water bodies at the periphery of their villages.

Isolation of Nontuberculous Mycobacteria from the Environment of Ghanian Communities Where Buruli Ulcer Is Endemic.

ABSTRACT This study aimed to isolate nontuberculous mycobacterial species from environmental samples obtained from some selected communities in Ghana. To optimize decontamination, spiked environmental samples were used to evaluate four decontamination solutions and supplemented media, after which the best decontamination solution and media were used for the actual analysis. The isolates obtained were identified on the basis of specific genetic sequences, including heat shock protein 65, IS2404, IS2606, rpoB, and the ketoreductase gene, as needed. Among the methods evaluated, decontamination with 1 M NaOH followed by 5% oxalic acid gave the highest rate of recovery of mycobacteria (50.0%) and the lowest rate of contamination (15.6%). The cultivation medium that supported the highest rate of recovery of mycobacteria was polymyxin B-amphotericin B-nalidixic acid-trimethoprim-azlocillin–supplemented medium (34.4%), followed by isoniazid-supplemented medium (28.1%). Among the 139 samples cultivated in the main analysis, 58 (41.7%) yielded mycobacterial growth, 70 (50.4%) had no growth, and 11 (7.9%) had all inoculated tubes contaminated. A total of 25 different mycobacterial species were identified. Fifteen species (60%) were slowly growing (e.g., Mycobacterium ulcerans, Mycobacterium avium, Mycobacterium mantenii, and Mycobacterium malmoense), and 10 (40%) were rapidly growing (e.g., Mycobacterium chelonae, Mycobacterium fortuitum, and Mycobacterium abscessus). The occurrence of mycobacterial species in the various environmental samples analyzed was as follows: soil, 16 species (43.2%); vegetation, 14 species (38.0%); water, 3 species (8.0%); moss, 2 species (5.4%); snail, 1 species (2.7%); fungi, 1 species (2.7%). This study is the first to report on the isolation of M. ulcerans and other medically relevant nontuberculous mycobacteria from different environmental sources in Ghana. IMPORTANCE Diseases caused by mycobacterial species other than those that cause tuberculosis and leprosy are increasing. Control is difficult because the current understanding of how the organisms are spread and where they live in the environment is limited, although this information is needed to design preventive measures. Growing these organisms from the environment is also difficult, because the culture medium becomes overgrown with other bacteria that also live in the environment, such as in soil and water. We aimed to improve the methods for growing these organisms from environmental sources, such as soil and water samples, for better understanding of important mycobacterial ecology.

Clinical and Laboratory Diagnosis of Buruli Ulcer Disease: A Systematic Review

Background. Buruli ulcer (BU) is a necrotizing cutaneous infection caused by Mycobacterium ulcerans. Early diagnosis is crucial to prevent morbid effects and misuse of drugs. We review developments in laboratory diagnosis of BU, discuss limitations of available diagnostic methods, and give a perspective on the potential of using aptamers as point-of-care. Methods. Information for this review was searched through PubMed, web of knowledge, and identified data up to December 2015. References from relevant articles and reports from WHO Annual Meeting of the Global Buruli Ulcer initiative were also used. Finally, 59 articles were used. Results. The main laboratory methods for BU diagnosis are microscopy, culture, PCR, and histopathology. Microscopy and PCR are used routinely for diagnosis. PCR targeting IS2404 is the gold standard for laboratory confirmation. Culture remains the only method that detects viable bacilli, used for diagnosing relapse and accrued isolates for epidemiological investigation as well as monitoring drug resistance. Laboratory confirmation is done at centers distant from endemic communities reducing confirmation to a quality assurance. Conclusions. Current efforts aimed at developing point-of-care diagnostics are saddled with major drawbacks; we, however, postulate that selection of aptamers against MU target can be used as point of care.

Possible healthcare-associated transmission as a cause of secondary infection and population structure of Staphylococcus aureus isolates from two wound treatment centres in Ghana

We have previously shown that secondary infections of Buruli ulcer wounds were frequently caused by Staphylococcus aureus. To gain understanding into possible routes of secondary infection, we characterized S. aureus isolates from patient lesions and surrounding environments across two Ghanaian health centres. One hundred and one S. aureus isolates were isolated from wounds (n = 93, 92.1%) and the hospital environment (n = 8, 7.9%) and characterized by the spa gene, mecA and the Panton–Valentine leucocidin toxin followed by spa sequencing and whole genome sequencing of a subset of 49 isolates. Spa typing and sequencing of the spa gene from 91 isolates identified 29 different spa types with t355 (ST152), t186 (ST88), and t346 dominating. Although many distinct strains were isolated from both health centres, genotype clustering was identified within centres. In addition, we identified a cluster consisting of isolates from a healthcare worker, patients dressed that same day and forceps used for dressing, pointing to possible healthcare-associated transmission. These clusters were confirmed by phylogenomic analysis. Twenty-four (22.8%) isolates were identified as methicillin-resistant S. aureus and lukFS genes encoding Panton–Valentine leucocidin were identified in 67 (63.8%) of the isolates. Phenotype screening showed widespread resistance to tetracycline, erythromycin, rifampicin, amikacin and streptomycin. Genomics confirmed the widespread presence of antibiotic resistance genes to β-lactams, chloramphenicol, trimethoprim, quinolone, streptomycin and tetracycline. Our findings indicate that the healthcare environment probably contributes to the superinfection of Buruli ulcer wounds and calls for improved training in wound management and infection control techniques.

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.

Susceptibility to mycobacterium ulcerans disease (buruli ulcer) is associated with IFNG and iNOS gene polymorphisms

Buruli ulcer (BU) is a chronic necrotizing disease of the skin and subcutaneous fat tissue. The causative agent, Mycobacterium ulcerans, produces mycolactone, a macrolide toxin, which causes apoptosis of mammalian cells. Only a small proportion of individuals exposed to M. ulcerans develop clinical disease, as surrounding macrophages may control the infection by bacterial killing at an early stage, while mycolactone concentration is still low. Otherwise, bacterial multiplication leads to in higher concentrations of mycolactone, with formation of necrotizing lesions that are no more accessible to immune cells. By typing a cohort of 96 Ghanaian BU patients and 384 endemic controls without BU, we show an association between BU and single nucleotide polymorphisms (SNPs) in iNOS (rs9282799) and IFNG (rs2069705). Both polymorphisms influence promoter activity in vitro. A previously reported SNP in SLC11A1 (NRAMP, rs17235409) tended to be associated with BU. Altogether, these data reflect the importance of IFNG signaling in early defense against M. ulcerans infection.

Detection and characterization of drug-resistant conferring genes in Mycobacterium tuberculosis complex strains: A prospective study in two distant regions of Ghana

Summary We spoligotyped and screened 1490 clinical Mycobacterium tuberculosis complex strains from Northern and Greater Accra regions of Ghana against INH and RIF using the microplate alamar blue phenotypic assay. Specific drug resistance associated genetic elements of drug resistant strains were analyzed for mutations. A total of 111 (7.5%), 10 (0.7%) and 40 (2.6%) were mono-resistant to INH, RIF, and MDR, respectively. We found the Ghana spoligotype to be associated with drug resistance (INH: 22.1%; p = 0.0000, RIF: 6.2%; p = 0.0103, MDR: 4.6%; p = 0.0240) as compared to the Cameroon spoligotype (INH: 6.7%, RIF: 2.4%, MDR: 1.6%). The propensity for an isolate to harbour katG S315T mutation was higher in M. tuberculosis (75.8%) than Mycobacterium africanum (51.7%) (p = 0.0000) whereas the opposite was true for inhApro mutations; MAF (48.3%) compared to MTBSS (26.7%) (p = 0.0419). We identified possible novel compensatory INH resistance mutations in inhA (G204D) and ahpCpro (-88G/A and -142G/A) and a novel ndh mutation K32R. We detected two possible rpoC mutations (G332R and V483G), which occurred independently with rpoB S450L, respectively. The study provides the first evidence that associate the Ghana spoligotype with DR-TB and calls for further genome analyses for proper classification of this spoligotype and to explore for fitness implications and mechanisms underlying this observation.

Challenges Associated with Management of Buruli Ulcer/Human Immunodeficiency Virus Coinfection in a Treatment Center in Ghana: A Case Series Study

The synergy between Mycobacterium tuberculosis infection and human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome is well established but not so in Buruli ulcer (BU). We screened confirmed BU cases for HIV infection and followed seven BU/HIV-coinfected patients. Management of BU/HIV was based on the World Health Organization guidelines and patient condition. The HIV positivity among BU patients (8.2%; 11/134) was higher compared with that of general patients attending the facility (4.8%; 718/14,863; P = 0.07) and that of pregnant women alone (2.5%; 279/11,125; P = 0.001). All seven BU/HIV-coinfected cases enrolled in the study presented with very large (category III) lesions with four having multiple lesions compared with 54.5% of category III lesions among HIV-negative BU patients. During the recommended BU treatment with streptomycin and rifampicin (SR) all patients developed immune infiltrates including CD4 T cells in their lesions. However, one patient who received antiretroviral therapy (ART) 1 week after beginning SR treatment developed four additional lesions during antibiotic treatment, while two out of the four who did not receive ART died. Further evidence is required to ascertain the most appropriate time to commence ART in relation to SR treatment to minimize paradoxical reactions.

Environmental and Behavioral Drivers of Buruli Ulcer Disease in Selected Communities Along the Densu River Basin of Ghana: A Case-Control Study

The exact route of transmission of Mycobacterium ulcerans (MU) (causative agent of Buruli ulcer [BU]), risk factors, and reservoir hosts are not clearly known, although it has been identified as an environmental pathogen. This study assessed potential environmental and behavioral risk factors that influence BU infections. We conducted a case-control study where cases were matched by their demographic characteristics and place of residence. A structured questionnaire was administered to solicit information on the environmental and behavioral factors of participants that may expose them to infection. A total of 176 cases and 176 controls were enrolled into the study. Multivariate conditional logistic regression analysis identified farming in swampy areas (odds ratio [OR] = 4.10, 95% confidence interval [CI] = 3.82–7.18), farming while wearing short clothing (OR = 1,734.1, 95% CI = 68.1–44,120.9), insect bite (OR = 988.3, 95% CI = 31.4–31,115.6), and application of leaves on wounds (OR = 6.23, 95% CI = 4.74–18.11) as potential risk factors. Farming in long clothing (OR = 0.000, 95% CI = 0.00–0.14), washing wound with water and soap (OR = 0.37, 95% CI = 0.29–0.98), and application of adhesive bandage on wounds (OR = 0.31, 95% CI = 0.15–0.82) were found to be protective against BU infection. In the absence of the exact MU transmission mechanisms, education of public in BU-endemic zones on the use of protective clothing during farming activities to limit exposure of the skin and proper wound care management would be essential in the fight against BU.