Back-to-Africa introductions of Mycobacterium tuberculosis as the main cause of tuberculosis in Dar es Salaam, Tanzania.

In settings with high tuberculosis (TB) endemicity, distinct genotypes of the Mycobacterium tuberculosis complex (MTBC) often differ in prevalence. However, the factors leading to these differences remain poorly understood. Here we studied the MTBC population in Dar es Salaam, Tanzania over a six-year period, using 1,082 unique patient-derived MTBC whole-genome sequences (WGS) and associated clinical data. We show that the TB epidemic in Dar es Salaam is dominated by multiple MTBC genotypes introduced to Tanzania from different parts of the world during the last 300 years. The most common MTBC genotypes deriving from these introductions exhibited differences in transmission rates and in the duration of the infectious period, but little differences in overall fitness, as measured by the effective reproductive number. Moreover, measures of disease severity and bacterial load indicated no differences in virulence between these genotypes during active TB. Instead, the combination of an early introduction and a high transmission rate accounted for the high prevalence of L3.1.1, the most dominant MTBC genotype in this setting. Yet, a longer co-existence with the host population did not always result in a higher transmission rate, suggesting that distinct life-history traits have evolved in the different MTBC genotypes. Taken together, our results point to bacterial factors as important determinants of the TB epidemic in Dar es Salaam.

SARS-CoV-2 Molecular Evolutionary Dynamics in the Greater Accra Region, Ghana.

To assess dynamics of SARS-CoV-2 in Greater Accra Region, Ghana, we analyzed SARS-CoV-2 genomic sequences from persons in the community and returning from international travel. The Accra Metropolitan District was a major origin of virus spread to other districts and should be a primary focus for interventions against future infectious disease outbreaks.

Seasonal Pattern of Mycobacterium ulcerans, the Causative Agent of Buruli Ulcer, in the Environment in Ghana.

This study aimed to contribute to the understanding of Mycobacterium ulcerans (MU) ecology by analysing both clinical and environmental samples collected from ten communities along two major river basins (Offin and Densu) associated with Buruli ulcer (BU) at different seasons. We collected clinical samples from presumptive BU cases and environmental samples from ten communities. Following DNA extraction, clinical samples were confirmed by IS2404 PCR and environmental samples were confirmed by targeting MU-specific genes, IS2404, IS2606 and the ketoreductase (KR) using real-time PCR. Environmental samples were first analysed for IS2404; after which, IS2404-positive samples were multiplexed for the IS2606 and KR gene. Our findings indicate an overall decline in BU incidence along both river basins, although incidence at Densu outweighs that of Offin. Overall, 1600 environmental samples were screened along Densu (434, 27 %) and Offin (1166, 73 %) and MU was detected in 139 (9 %) of the combined samples. The positivity of MU along the Densu River basin was 89/434 (20.5 %), whilst that of the Offin River basin was 50/1166 (4.3 %). The DNA was detected mainly in snails (5/6, 83 %), moss (8/40, 20 %), soil (55/586, 9 %) and vegetation (55/675, 8 %). The proportion of MU positive samples recorded was higher during the months with higher rainfall levels (126/1175, 11 %) than during the dry season months (13/425, 3 %). This study indicates for the first time that there is a seasonal pattern in the presence of MU in the environment, which may be related to recent rainfall or water in the soil.

The Biology and Epidemiology of Mycobacterium africanum.

West Africa is the only region in the world where six out of seven mycobacterial lineages of human importance are endemic. In particular, two evolutionary ancient lineages, Mycobacterium africanum West Africa 1 (MTBC Lineage 5) and M. africanum West Africa 2 (MTBC Lineage 6) are of interest as they cause up to 40% of all pulmonary TB cases in some West African countries. Although these M. africanum lineages are closely related to M. tuberculosis sensu stricto lineages, they differ significantly in respect to biology, epidemiology and in their potential to cause disease in humans. Most importantly the M. africanum lineages are exclusive to West Africa. Although the exact mechanisms underlying this geographical restriction are still not understood, it is increasingly suspected that this is due to an adaptation of the bacteria to West African host populations. In this chapter, we summarize the geographical distribution of the M. africanum lineages within the region, describe biological and clinical differences and the consequent implications for TB control in West Africa. We also try to shed light on the geographical restriction, based on recently published analyses on whole genomes of M. africanum isolates.

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

UNLABELLED: 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.

Molecular epidemiology of Mycobacterium africanum in Ghana.

BACKGROUND: Mycobacterium africanum comprises two phylogenetic lineages within the M. tuberculosis complex (MTBC) and is an important cause of human tuberculosis (TB) in West Africa. The reasons for this geographic restriction of M. africanum remain unclear. Here, we performed a prospective study to explore associations between the characteristics of TB patients and the MTBC lineages circulating in Ghana. METHOD: We genotyped 1,211 MTBC isolates recovered from pulmonary TB patients recruited between 2012 and 2014 using single nucleotide polymorphism typing and spoligotyping. Associations between patient and pathogen variables were assessed using univariate and multivariate logistic regression. RESULTS: Of the 1,211 MTBC isolates analysed, 71.9 % (871) belonged to Lineage 4; 12.6 % (152) to Lineage 5 (also known as M. africanum West-Africa 1), 9.2 % (112) to Lineage 6 (also known as M. africanum West-Africa 2) and 0.6 % (7) to Mycobacterium bovis. Univariate analysis revealed that Lineage 6 strains were less likely to be isoniazid resistant compared to other strains (odds ratio = 0.25, 95 % confidence interval (CI): 0.05-0.77, P < 0.01). Multivariate analysis showed that Lineage 5 was significantly more common in patients from the Ewe ethnic group (adjusted odds ratio (adjOR): 2.79; 95 % CI: 1.47-5.29, P < 0.001) and Lineage 6 more likely to be found among HIV-co-infected TB patients (adjOR = 2.2; 95 % confidence interval (CI: 1.32-3.7, P < 0.001). CONCLUSION: Our findings confirm the importance of M. africanum in Ghana and highlight the need to differentiate between Lineage 5 and Lineage 6, as these lineages differ in associated patient variables.

In Vitro Activity of Selected West African Medicinal Plants against Mycobacterium ulcerans Disease.

Buruli ulcer (BU) is the third most prevalent mycobacteriosis, after tuberculosis and leprosy. The currently recommended combination of rifampicin-streptomycin suffers from side effects and poor compliance, which leads to reliance on local herbal remedies. The objective of this study was to investigate the antimycobacterial properties and toxicity of selected medicinal plants. Sixty-five extracts from 27 plant species were screened against Mycobacterium ulcerans and Mycobacterium smegmatis, using the Resazurin Microtiter Assay (REMA). The cytotoxicity of promising extracts was assayed on normal Chang liver cells by an MTT assay. Twenty five extracts showed activity with minimal inhibitory concentration (MIC) values ranging from 16 µg/mL to 250 µg/mL against M. smegmatis, while 17 showed activity against M. ulcerans with MIC values ranging from 125 µg/mL to 250 µg/mL. In most of the cases, plant extracts with antimycobacterial activity showed no cytotoxicity on normal human liver cells. Exception were Carica papaya, Cleistopholis patens, and Polyalthia suaveolens with 50% cell cytotoxic concentrations (CC50) ranging from 3.8 to 223 µg/mL. These preliminary results support the use of some West African plants in the treatment of Buruli ulcer. Meanwhile, further studies are required to isolate and characterize the active ingredients in the extracts.

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.

Primary cultivation: factors affecting contamination and Mycobacterium ulcerans growth after long turnover time of clinical specimens.

BACKGROUND: While cultivation of pathogens represents a foundational diagnostic approach in the study of infectious diseases, its value for the confirmation of clinical diagnosis of Buruli ulcer is limited by the fact that colonies of Mycobacterium ulcerans appear only after about eight weeks of incubation at 30°C. However, for molecular epidemiological and drug sensitivity studies, primary isolation of M. ulcerans remains an essential tool. Since for most of the remote Buruli ulcer endemic regions of Africa cultivation laboratories are not easily accessible, samples from lesions often have to be stored for extended periods of time prior to processing. The objective of the current study therefore was to determine which transport medium, decontamination method or other factors decrease the contamination rate and increase the chance of primary isolation of M. ulcerans bacilli after long turnover time. METHODS: Swab and fine needle aspirate (FNA) samples for the primary cultivation were collected from clinically confirmed Buruli ulcer patients in the Mapé Basin of Cameroon. The samples were either stored in the semi-solid transport media 7H9 or Amies or dry for extended period of time prior to processing. In the laboratory, four decontamination methods and two inoculation media were evaluated and statistical methods applied to identify factors that decrease culture contamination and factors that increase the probability of M. ulcerans recovery. RESULTS: The analysis showed: i) that the use of moist transport media significantly increased the recovery rate of M. ulcerans compared to samples kept dry; ii) that the choice of the decontamination method had no significant effect on the chance of M. ulcerans isolation; and iii) that Löwenstein-Jensen supplemented with antibiotics as inoculation medium yielded the best results. We further found that, ten extra days between sampling and inoculation lead to a relative decrease in the isolation rate of M. ulcerans by nearly 20%. Finally, collection and processing of multiple samples per patient was found to significantly increase the M. ulcerans isolation rate. CONCLUSIONS: Based on our analysis we suggest a procedure suitable for the primary isolation of M. ulcerans strains from patients following long delay between sample collection and processing to establish a M. ulcerans strain collection for research purposes.

Tuberculosis and diabetes mellitus: The complexity of the comorbid interactions.

The double burden of tuberculosis (TB) and diabetes mellitus (DM) represents a major public health challenge that demands urgent and integrated approaches. The interplay between these two chronic conditions presents unique clinical and public health management challenges, as well as social and economic implications. We explored the bidirectional relationship between TB and DM, emphasizing how DM increases susceptibility to TB and complicates its management, while TB may exacerbate glycaemic control in diabetic patients. This review underscores the challenges associated with the management of both diseases, obstacles in screening TB patients for DM and TB preventive therapy for DM since inadequate glycaemic control can impact treatment outcomes. Several studies have investigated the disease interplay; however, the results have been equivocal, and this may be exerting negative impacts on the disease prevention and treatment. TB-diabetes comorbidity has been linked to poor treatment outcomes whereas TB prevention in people with DM at present is a dilemma. In addition to highlighting how urgent it is to address this comorbidity, this review offers a road map for better prevention, treatment, and control of several factors underlying the TB-diabetes syndemic interaction.

Stigma experiences, effects and coping among individuals affected by Buruli ulcer and yaws in Ghana.

BACKGROUND: Stigma related to skin neglected tropical diseases like Buruli ulcer (BU) and yaws has remained underexplored and existing studies are limited to individual diseases despite the WHO call for integration in disease management. Within two districts in central Ghana, we explored stigma associated with BU and yaws to understand overlaps and disease-specific nuances to help guide integrated interventions. METHODOLOGY/PRINCIPAL FINDINGS: In-depth interviews were conducted with 31 current or formerly affected individuals to assess the experiences, effects and coping strategies adopted to manage disease related stigma. Data were analysed along broad themes based on the sociological construct of macro and micro interaction and Goffman's treatise on stigma. Disapproving community labels fueled by misconceptions were noted among BU participants which contributed to macro stigma experiences, including exclusion, discrimination and avoidance. In contrast, a high level of social acceptance was reported among yaws participants although some micro-level stigma (anticipated, felt and self-stigma) were noted by individuals with both diseases. While younger participants experienced name-calling and use of derogatory words to address affected body parts, older participants and caregivers discussed the pain of public staring. Stigma experiences had negative consequences on psychosocial well-being, schooling, and social relations, particularly for BU affected people. Problem-focused strategies including confrontation, selective disclosure and concealment as well as emotion-focused strategies (religious coping and self-isolation) were noted. CONCLUSIONS AND SIGNIFICANCE: The types and levels of stigma varied for BU and yaws. Stigma experiences also differed for adults and children in this setting and these differences should be accounted for in integrated interventions for these skin NTDs. School health programs need to prioritize educating school teachers about skin NTDs and the negative impact of stigma on the wellbeing of children.

Opinion review of drug resistant tuberculosis in West Africa: tackling the challenges for effective control.

Drug-resistant (DR) tuberculosis (TB) is a major public health concern globally, complicating TB control and management efforts. West Africa has historically faced difficulty in combating DR-TB due to limited diagnostic skills, insufficient access to excellent healthcare, and ineffective healthcare systems. This has aided in the emergence and dissemination of DR Mycobacterium tuberculosis complex (MTBC) strains in the region. In the past, DR-TB patients faced insufficient resources, fragmented efforts, and suboptimal treatment outcomes. However, current efforts to combat DR-TB in the region are promising. These efforts include strengthening diagnostic capacities, improving access to quality healthcare services, and implementing evidence-based treatment regimens for DR-TB. Additionally, many West African National TB control programs are collaborating with international partners to scale up laboratory infrastructure, enhance surveillance systems, and promote infection control measures. Moreso, novel TB drugs and regimens, such as bedaquiline and delamanid, are being introduced to improve treatment outcomes for DR-TB cases. Despite these obstacles, there is optimism for the future of DR-TB control in West Africa. Investments are being made to improve healthcare systems, expand laboratory capacity, and support TB research and innovation. West African institutions are now supporting knowledge sharing, capacity building, and resource mobilization through collaborative initiatives such as the West African Network for TB, AIDS, and Malaria (WANETAM), the West African Health Organization (WAHO), and other regional or global partners. These efforts hold promise for improved diagnostics, optimized treatment regimens, and provide better patient outcomes in the future where drug-resistant TB in WA can be effectively controlled, reducing the burden of the disease, and improving the health outcomes of affected individuals.

Improving experiences of neglected tropical diseases of the skin: Mixed methods formative research for development of a complex intervention in Atwima Mponua District, Ghana.

Integrated approaches to managing co-endemic neglected tropical diseases (NTDs) of the skin within primary healthcare services are complex and require tailoring to local contexts. We describe formative research in Atwima Mponua District in Ghana's Ashanti Region designed to inform the development of a sustainable intervention to improve access to skin NTD care. We employed a convergent, parallel, mixed-methods design, collecting data from February 2021 to February 2022. We quantitatively assessed service readiness using a standardised checklist and reviewed outpatient department registers and condition-specific case records in all government health facilities in the district. Alongside a review of policy documents, we conducted 49 interviews and 7 focus group discussions with purposively selected affected persons, caregivers, community members, health workers, and policy-makers to understand skin NTD care-seeking practices and the policy landscape. Outside the district hospital, skin NTD reporting rates in the surveyed facilities were low; supply chains for skin NTD diagnostics, consumables, and medicines had gaps; and health worker knowledge of skin NTDs was limited. Affected people described fragmented care, provided mostly by hospitals (often outside the district) or traditional healers, resulting in challenges obtaining timely diagnosis and treatment and high care-seeking costs. Affected people experienced stigma, although the extent to which stigma influenced care-seeking behaviour was unclear. National actors were more optimistic than district-level actors about local resource availability for skin NTD care and were sceptical of including traditional healers in interventions. Our findings indicate that improvement of the care cascade for affected individuals to reduce the clinical, economic, and psychosocial impact of skin NTDs is likely to require a complementary set of interventions. These findings have informed the design of a strategy to support high-quality, integrated, decentralised care for skin NTDs in Atwima Mponua, which will be assessed through a multidisciplinary evaluation.

Development of an integrated and decentralised skin health strategy to improve experiences of skin neglected tropical diseases and other skin conditions in Atwima Mponua District, Ghana.

Integrated strategies are recommended to tackle neglected tropical diseases of the skin (skin NTDs), which pose a substantial health and economic burden in many countries, including Ghana. We describe the development of an integrated and decentralised skin health strategy designed to improve experiences of skin NTDs in Atwima Mponua district in Ashanti Region. A multidisciplinary research team led an iterative process to develop an overall strategy and specific interventions, based on a theory of change informed by formative research conducted in Atwima Mponua district. The process involved preparatory work, four co-development workshops (August 2021 to November 2022), collaborative working groups to operationalise intervention components, and obtaining ethical approval. Stakeholders including affected individuals, caregivers, other community members and actors from different levels of the health system participated in co-development activities. We consulted these stakeholders at each stage of the research process, including discussion of study findings, development of our theory of change, identifying implementable solutions to identified challenges, and protocol development. Participants determined that the intervention should broadly address wounds and other skin conditions, rather than only skin NTDs, and should avoid reliance on non-governmental organisations and research teams to ensure sustainable implementation by district health teams and transferability elsewhere. The overall strategy was designed to focus on a decentralised model of care for skin conditions, while including other interventions to support a self-care delivery pathway, community engagement, and referral. Our theory of change describes the pathways through which these interventions are expected to achieve the strategy's aim, the assumptions, and problems addressed. This complex intervention strategy has been designed to respond to the local context, while maximising transferability to ensure wider relevance. Implementation is expected to begin in 2023.

Enhancing Buruli ulcer control in Ghana through social interventions: a case study from the Obom sub-district.

BACKGROUND: Buruli ulcer is considered a re-emerging disease in West Africa where it has suffered neglect over the years, though children below the age of 16 years are the worst affected in most endemic regions. Due to delayed health seeking, the disease leads to disabilities resulting from amputation and loss of vital organs like the eye leading to school dropout and other social and economic consequences for the affected family. Early treatment with antibiotics is effective; however, this involves daily oral and intramuscular injection at distant health facilities for 56 days making it a challenge among poor rural folks living on daily subsistence work. The mode of transmission of Buruli ulcer is not known and there is no effective preventive vaccine for Buruli ulcer. Thus the only effective control tool is early case detection and treatment to reduce morbidity and associated disabilities that occurs as a result of late treatment. It is therefore essential to implement interventions that remove impediments that limit early case detection; access to early effective treatment and this paper reports one such effort where the feasibility of social interventions to enhance Buruli ulcer control was assessed. METHODS: This was a qualitative study using in-depth interviews to generate information to ascertain the benefit or otherwise of the intervention implemented. Clinical records of patients to generate data to determine the feasibility and effectiveness of social interventions in the fight against Buruli ulcer was examined. In all, 56 in-depth interviews (28 at baseline and 28 at evaluation) were conducted for this report. RESULTS: At full implementation, treatment default and dropout reduced significantly from 58.8% and 52.9% at baseline to 1.5% and 1.5% respectively. The number of early case detection went up significantly. Affected families were happy with social interventions such as provision of transportation and breakfast to patients on daily basis. Families were happy with the outpatient services provided under the intervention where no patient was admitted into the hospital. CONCLUSION: The study showed that with a little more investment in early case detection, diagnosis and treatment, coupled with free transportation and breakfast for patients, most of the cases could be treated effectively with the available antibiotics to avoid disability and complications from the disease.

Macrophage susceptibility to infection by Ghanaian Mycobacterium tuberculosis complex lineages 4 and 5 varies with self-reported ethnicity.

Background: The epidemiology of Mycobacterium tuberculosis complex (MTBC) lineage 5 (L5) infections in Ghana revealed a significantly increased prevalence in Ewes compared to other self-reported ethnic groups. In that context, we sought to investigate the early phase of tuberculosis (TB) infection using ex vivo infection of macrophages derived from the blood of Ewe and Akan ethnic group volunteers with MTBC L4 and L5 strains. Methods: The study participants consisted of 16 controls, among which self-reported Akan and Ewe ethnicity was equally represented, as well as 20 cured TB cases consisting of 11 Akans and 9 Ewes. Peripheral blood mononuclear cells were isolated from both healthy controls and cured TB cases. CD14+ monocytes were isolated and differentiated into monocyte-derived macrophages (MDMs) before infection with L4 or L5 endemic strains. The bacterial load was assessed after 2 hours (uptake) as well as 3 and 7 days post-infection. Results: We observed a higher capacity of MDMs from Ewes to phagocytose L4 strains (p < 0.001), translating into a higher bacillary load on day 7 (p < 0.001) compared to L5, despite the higher replication rate of L5 in Ewe MDMs (fold change: 1.4 vs. 1.2, p = 0.03) among the controls. On the contrary, within macrophages from Akans, we observed a significantly higher phagocytic uptake of L5 (p < 0.001) compared to L4, also translating into a higher load on day 7 (p = 0.04). However, the replication rate of L4 in Akan MDMs was higher than that of L5 (fold change: L4 = 1.2, L4 = 1.1, p = 0.04). Although there was no significant difference in the uptake of L4 and L5 among cured TB cases, there was a higher bacterial load of both L4 (p = 0.02) and L5 (p = 0.02) on day 7 in Ewe MDMs. Conclusion: Our results suggest that host ethnicity (driven by host genetic diversity), MTBC genetic diversity, and individual TB infection history are all acting together to modulate the outcome of macrophage infections by MTBC.

On the origin of Mycobacterium ulcerans, the causative agent of Buruli ulcer.

BACKGROUND: Mycobacterium ulcerans is an unusual bacterial pathogen with elusive origins. While closely related to the aquatic dwelling M. marinum, M. ulcerans has evolved the ability to produce the immunosuppressive polyketide toxin mycolactone and cause the neglected tropical disease Buruli ulcer. Other mycolactone-producing mycobacteria (MPM) have been identified in fish and frogs and given distinct species designations (M. pseudoshottsii, M. shinshuense, M. liflandii and M. marinum), however the evolution of M. ulcerans and its relationship to other MPM has not been defined. Here we report the comparative analysis of whole genome sequences from 30 MPM and five M. marinum. RESULTS: A high-resolution phylogeny based on genome-wide single nucleotide polymorphisms (SNPs) showed that M. ulcerans and all other MPM represent a single clonal group that evolved from a common M. marinum progenitor. The emergence of the MPM was driven by the acquisition of the pMUM plasmid encoding genes for the biosynthesis of mycolactones. This change was accompanied by the loss of at least 185 genes, with a significant overrepresentation of genes associated with cell wall functions. Cell wall associated genes also showed evidence of substantial adaptive selection, suggesting cell wall remodeling has been critical for the survival of MPM. Fine-grain analysis of the MPM complex revealed at least three distinct lineages, one of which comprised a highly clonal group, responsible for Buruli ulcer in Africa and Australia. This indicates relatively recent transfer of M. ulcerans between these continents, which represent the vast majority of the global Buruli ulcer burden. Our data provide SNPs and gene sequences that can differentiate M. ulcerans lineages, suitable for use in the diagnosis and surveillance of Buruli ulcer. CONCLUSIONS: M. ulcerans and all mycolactone-producing mycobacteria are specialized variants of a common Mycobacterium marinum progenitor that have adapted to live in restricted environments. Examination of genes lost or retained and now under selective pressure suggests these environments might be aerobic, and extracellular, where slow growth, production of an immune suppressor, cell wall remodeling, loss or modification of cell wall antigens, and biofilm-forming ability provide a survival advantage. These insights will guide our efforts to find the elusive reservoir(s) of M. ulcerans and to understand transmission of Buruli ulcer.

Rapid and sensitive detection of Mycobacterium ulcerans by use of a loop-mediated isothermal amplification test.

This work reports the design and evaluation of a rapid loop-mediated isothermal amplification test for detecting Mycobacterium ulcerans DNA based on the multicopy insertion sequence IS2404. The test is robust and specific with a detection limit equivalent to 20 copies of the target sequence (0.01 to 0.1 genome). The test has potential for the diagnosis of Buruli ulcer under field conditions.

Epidemiological aspects of non-human antibiotic usage and resistance: implications for the control of antibiotic resistance in Ghana.

OBJECTIVES: To provide insights into the epidemiology of antibiotic usage in animal husbandry in Ghana and its effect on resistance. METHODS: Three hundred and ninety-five randomly sampled commercial livestock keepers who practised intensive or extensive farming were interviewed about their antibiotic usage practices using a structured questionnaire. Escherichia coli isolated from stool specimens of farmers and their animals were tested against eight antibiotics using the Kirby Bauer method. RESULTS: Ninety-eight percent (387) of the farmers used antibiotics on animals and the main purpose was to prevent infections in animals; 41% applied antibiotics monthly. The overall prevalence of multiple drug resistance among the E. coli isolates was 91.6%; rates in human and animal isolates were 70.6% and 97.7%, respectively. The prevalence of resistance in animal isolates to the various drugs ranged from 60.8% (amikacin) to 95.7% (ampicillin); the prevalence of resistance in human isolates to the drugs ranged from 2% (cefuroxime) to 94.1% (gentamicin). Animal E. coli isolates showed higher resistance than that of human isolates for five of eight drugs tested. CONCLUSION: It is concluded that antibiotic usage in animal husbandry in Ghana is more driven by the interest of livestock keepers to prevent and treat animal infections than growth enhancement. Both animal and human E. coli showed high levels of antibiotic resistance, although resistance of animal isolates appeared to be higher than that of humans. There is the need for the development of an antibiotic-resistance management programme in Ghana that will focus simultaneously on human and animal use of antibiotics.