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This study reports the first successful application of real-time PCR for the detection of Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU), in Ghana, a BU-endemic country. Environmental samples and organs of small mammals were analyzed. The real-time PCR assays confirmed the presence of M. ulcerans in a water sample collected in a BU-endemic village in the Ashanti Region. [ABSTRACT FROM AUTHOR]

IntroductionBuruli ulcer (BU) caused by Mycobacterium ulcerans (MU) is a devastating necrotic skin disease. PCR, recommended for confirmation of BU by WHO, requires an adequately equipped laboratory, therefore often delaying timely diagnosis and treatment of BU patients in remote settings. Loop-mediated isothermal amplification (LAMP) is a PCR-based protocol for isothermal amplification of DNA that has been suggested for diagnosis of BU in low-resource settings.Study aims and methodsThis is an exploratory diagnostic test evaluation study, with an embedded qualitative sub-study. Its aims are two-fold: First, to evaluate a simple rapid syringe-based DNA extraction method (SM) in comparison with a more elaborate conventional DNA extraction method (CM), followed by a LAMP assay targeting IS2404 for the detection of MU, either using a commercially available pocket warmer (pw) or a heat block (hb) for incubation. Second, to complement this by exploring the diagnostic workflow for BU at a community-based health centre in an endemic area in rural Ghana as an example of a potential target setting, using interviews with researchers and health care workers (HCWs). Diagnostic test evaluation results are discussed in relation to the requirements of a target product profile (TPP) for BU diagnosis and the target setting.ResultsA protocol using SM for DNA extraction followed by IS2404 PCR (IS2404 PCRSM) was able to identify MU DNA in 73 out of 83 BU clinical specimens submitted for diagnosis. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of IS2404 PCRSM were 90.12%, 100%, 100% and 65.21% respectively, as compared to the reference standard IS2404 PCR in combination with a standard extraction protocol for mycobacterial DNA. Evaluation of the LAMP assay on 64 SM DNA extracts showed a sensitivity, specificity, PPV and NPV of 83.6%, 100%, 100% and 50%, respectively, using either pocket warmer (pwLAMPSM) or heat block (hbLAMPSM) for incubation of the reaction, as compared to the same reference standard. The limit of detection of pwLAMPSM was found to be 30 copies of the IS2404 target. Interview findings explored barriers to BU diagnosis and treatment, including perceptions of the disease, costs, and availability of transport. Participants confirmed that a diagnosis at the PoC, in addition to screening based on clinical criteria, would be advantageous in order to prevent delays and loss to follow-up.Discussion and conclusionsThe high diagnostic and analytic accuracy of the pwLAMP, evaluated by us in combination with a syringe-based DNA extraction method, supports its potential use for the rapid detection of MU in suspected BU samples at the community or primary health care level without reliable electricity supply. Further optimization needs include a lysis buffer, evaluation directly at the PoC and/or other sites, assessing staff training requirements and quality control.

Clostridium perfringens can cause a plethora of diseases in humans and animals, making it a growing public health concern. Insight into C. perfringens genomic studies has enhanced our understanding of the diversity and evolution of this bacterium. However, most of these studies were centered on clinical strains from human and animal hosts, with limited data from environmental sources and developing countries. Here, we present genomic and phenotypic insights into C. perfringens strains from hospital wastewater samples from Ghana. Antibiotic susceptibility testing, toxinotyping, and whole-genome sequencing were performed on the seven C. perfringens isolates. The strains were susceptible to all antibiotics screened except for three strains that were resistant to clindamycin. The majority of these strains were type F strains, characterized by a chromosomally encoded cpe gene, whereas the remaining strains were classified as type A. The cpe gene is flanked upstream by IS1469 and downstream by IS1470. The genotype for the type F strains was ST721, whereas the type A strains were assigned to ST722, ST143, and ST211. Our type F strains clustered with other strains all within phylogroup I, which are mostly associated with foodborne illnesses, whereas the type A strains were grouped within phylogroup III. This study provides the first genomic characterization of C. perfringens from West Africa and contributes to further understanding of genomic diversity in C. perfringens. Moreover, the possible disseminated situation of C. perfringens in the environment highlights the need to monitor this bacterium in clinical specimens in Ghana. IMPORTANCE Clostridium perfringens causes gas gangrene and food poisoning in humans, and monitoring this bacterium is important for public health. Although whole-genome sequencing is useful to comprehensively understand the virulence, resistome, and global genetic relatedness of bacteria, limited genomic data from environmental sources and developing countries hamper our understanding of the richness of the intrinsic genomic diversity of this pathogen. Here, we successfully accumulated the genetic data on C. perfringens strains isolated from hospital effluent and provided the first evidence that predicted pathogenic C. perfringens may be disseminated in the clinical environment in Ghana. Our findings suggest the importance of risk assessment in the environment as well as the clinical setting to mitigate the potential outbreak of C. perfringens food poisoning in Ghana. [ABSTRACT FROM AUTHOR]

ABSTRACTThe spread of hypervirulent (hv) and carbapenem-/multidrug-resistant Klebsiella pneumoniae is an emerging problem in healthcare settings. The New Delhi metallo-β-lactamase-1 (blaNDM-1) is found in Enterobacteriaceae including K. pneumoniae. The blaNDM-1 is capable of hydrolyzing β-lactam antibiotics which are used for treatment of severe infections caused by multidrug-resistant Gram-negative bacteria. This is associated with the unacceptably high mortality rate in immunocompromised burn injury patients. This study reports on the characterization of blaNDM-1 gene and virulence factors in hv carbapenem-/multidrug-resistant K. pneumoniae ST147 in the burns unit of a tertiary teaching hospital during routine surveillance. Two K. pneumoniae strains were obtained from wounds of burn-infected patients from May 2020 to July 2021. The hypervirulence genes and genetic context of the blaNDM-1 gene and mobile genetic elements potentially involved in the transposition of the gene were analyzed. We identified a conserved genetic background and an IS26 and open reading frame flanking the blaNDM-1 gene that could suggest its involvement in the mobilization of the gene. The plasmid harbored additional antibiotic resistance predicted regions that were responsible for resistance to almost all the routinely used antibiotics. To ensure the identification of potential outbreak strains during routine surveillance, investigations on resistance genes and their environment in relation to evolution are necessary for molecular epidemiology.IMPORTANCEData obtained from this study will aid in the prompt identification of disease outbreaks including evolving resistance and virulence of the outbreak bacteria. This will help establish and implement antimicrobial stewardship programs and infection prevention protocols in fragile health systems in countries with limited resources. Integration of molecular surveillance and translation of whole-genome sequencing in routine diagnosis will provide valuable data for control of infection. This study reports for the first time a high-risk clone K. pneumoniae ST147 with hypervirulence and multidrug-resistance features in Ghana.

Classical methods of treatment of polychlorinated biphenyls (PCBs) in environmental matrices such as soil encapsulation, high-temperature heating, thermal desorption, and chemical decontamination, have been costly, and they leave behind problems of environmental footprints. They must therefore give way to bioremediation techniques which are less costly and result in eco-friendly products. The aim of the study was to assess the suitability of PCB degrading bacteria (using published data) from their environmental matrix for use in environmental bioremediation. Initial isolation of the bacteria was obtained by culturing ash samples taken from the combustion chamber of an MP400 Incinerator of infectious waste at Komfo Anokye Teaching hospital (KATH) in Kumasi, Ghana, and composite soil samples taken from a transformer-servicing site at an electricity substation in Sunyani, Ghana. Test for thermotolerance was carried out by the culturing of bacteria from the soil samples and exposing them to higher temperatures (55 °C and 65 °C) to assess their resilience in an incubator for 48 h. Growing cultures were sub-cultured, after which a cocktail of bacteria isolate characterization assays was carried out on the growing isolates. These included biochemical tests on the bacterial suspensions using the analytical profile index (API 20E) for examining Enterobacteriaceae and other non-fastidious bacteria. This was followed by MALDI-ToF-MS analyses of the bacterial strains to profile and image their proteins. A molecular step was needed which was the conventional polymerase chain reaction (PCR) method, using specific primer pairs, to amplify and accentuate sections of the bacterial DNA extracts. Results of the study showed a confirmed sample site in Ghana for harvesting bacteria capable of degrading polychlorinated biphenyls (PCBs). MALDI-ToF-MS analyses revealed the possible species as Pseudomonas aeruginosa and Bacillus cereus. The conventional PCR and gel electrophoresis steps confirmed the presence of bph genes encoding the 2,3-dioxygenase enzymes in bacteria with PCB-degrading ability as documented in published data. The serial dilution of the bacterial suspensions was modified from 10−7 to 10−5 which was enough for distinct colonies to be counted, and it was a novel contribution to environmental microbiology. The confirmation of Pseudomonas and Bacillus species isolated from a transformer oil-impacted soil in this study shows promise in growing these strains in various environmental media such as in a gaseous medium to mineralize gas-phase PCBs from combustion smoke or in bottom ash residues to degrade solid-phase PCBs. It can also be applied in bioremediation of waste liquid systems containing PCBs.

IntroductionExtended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae are pathogens of significant public health interest for which new antibiotics are urgently needed.AimTo determine the prevalence of ESBLs in E. coli and K. pneumoniae isolates from patients attending the Tamale Teaching Hospital (TTH) in Ghana.MethodologyThe study was a cross-sectional study involving convenience sampling of E. coli and K. pneumoniae isolates from consenting patients' clinical specimens, between April and June 2015. Antimicrobial susceptibility test was performed, and ESBL-producer phenotypes were further screened for BlaTEM, BlaSHV, and BlaCTX-M genes. Patients' clinical data were additionally collected using a structured questionnaire.ResultsOf the 150 non-duplicate E. coli and K. pneumoniae isolates identified, 140 were confirmed as E. coli (84%, n = 117) and K. pneumoniae (16%, n = 23). Of these, sixty-two (44%) [E. coli (84%; n = 52); K. pneumoniae (16%; n = 10)] phenotypically expressed ESBLs. The proportion of ESBL-producing isolates was higher in adults (15-65 years) than in neonates (< 28 days) (p = 0.14). Most of the isolates showed a high percentage resistance to ampicillin (96%) and tetracycline (89%), but a relatively lower resistance to amikacin (36%). No isolate was resistant to meropenem. More ESBL producers were multidrug resistant compared to non-ESBL-producers [23% (14/62) versus 18% (14/78); p = 0.573]. Overall, 74% (n = 46) of the ESBL genotypes expressed BlaCTX-M-1 genes, followed by 63% (n = 39) BlaTEM, and 16% (n = 10) BlaSHV. The study showed a high prevalence of ESBL-positive E. coli and K. pneumoniae, mostly CTX-M-1 producers at TTH.ConclusionRoutine laboratory ESBL screening is warranted to inform patient management.

OBJECTIVE: To investigate the microbial quality of foods sold on streets of Accra and factors predisposing to their contamination. METHODS: Structured questionnaires were used to collect data from 117 street vendors on their vital statistics, personal hygiene, food hygiene and knowledge of foodborne illness. Standard methods were used for the enumeration, isolation, and identification of bacteria. FINDINGS: Most vendors were educated and exhibited good hygiene behaviour. Diarrhoea was defined as the passage of > or = 3 stools per day) by 110 vendors (94.0%), but none associated diarrhoea with bloody stools; only 21 (17.9%) associated diarrhoea with germs. The surroundings of the vending sites were clean, but four sites (3.4%) were classified as very dirty. The cooking of food well in advance of consumption, exposure of food to flies, and working with food at ground level and by hand were likely risk factors for contamination. Examinations were made of 511 menu items, classified as breakfast/snack foods, main dishes, soups and sauces, and cold dishes. Mesophilic bacteria were detected in 356 foods (69.7%): 28 contained Bacillus cereus (5.5%), 163 contained Staphylococcus aureus (31.9%) and 172 contained Enterobacteriaceae (33.7%). The microbial quality of most of the foods was within the acceptable limits but samples of salads, macaroni, fufu, omo tuo and red pepper had unacceptable levels of contamination. Shigella sonnei and enteroaggregative Escherichia coli were isolated from macaroni, rice, and tomato stew, and Salmonella arizonae from light soup. CONCLUSION: Street foods can be sources of enteropathogens. Vendors should therefore receive education in food hygiene. Special attention should be given to the causes of diarrhoea, the transmission of diarrhoeal pathogens, the handling of equipment and cooked food, hand-washing practices and environmental hygiene.