Search

Objective: We conducted a cross-sectional study in the five administrative regions of Northern Ghana to determine the diversity of Mycobacterium tuberculosis complex (MTBC) sub/lineages and their susceptibility to isoniazid (INH) and rifampicin (RIF). Methods: Sputum specimens were collected and cultured from 566 pulmonary tuberculosis patients reporting to 17 health facilities from 2015 to 2019. Mycobacterial isolates obtained from solid cultures were confirmed as members of the MTBC by PCR amplification of IS6110 and rpoß and assigned lineages and sub-lineages using spoligotyping. Results: Of 294 mycobacterial isolates recovered, MTBC species identified were: M. tuberculosis sensu stricto (Mtbss) 241 (82.0%), M. africanum 41 (13.9%) and M. bovis four (1.4%) with eight (2.7%) unidentified. The human-adapted lineages (L) identified (N=279) were L1 (8/279, 2.9%), L2 (15/279, 5.4%), L3 (7/279, 2.5%), L4 (208/279, 74.5%), L5 (13/279, 4.7%) and L6 (28/279, 10.0%) with three unidentified lineages. Among the 208 L4, the dominant sub-lineages in the region were the Cameroon 120/208 (57.7%) and Ghana 50/208 (24.0%). We found 4.4% (13/294) and 0.7% (2/294) of the patients infected with MTBC isolates resistant to INH only and RIF only, respectively, with 2.4% (7/294) being infected with MDR strains. Whereas L6 was associated with the elderly, we identified that the Ghana sub-lineage of L4 was associated with both INH and MDR (p

Background: Sputum culture is limited to centralized facilities. Thus, samples require transportation from peripheral laboratories to these facilities, compromising specimen quality since it is difficult to maintain cold chain. We evaluated OMNIgene SPUTUM Reagent (OMS) for transporting sputum samples for tuberculosis (TB) testing. The study was carried out at Noguchi Memorial Institute for Medical Research using sputa from Korle Bu Teaching Hospital and La General Hospital in Ghana. Methods: In a laboratory-based controlled experiment (CE), sputum contaminants were determined on blood agar before treatment with OMS and N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH). TB testing included smear microscopy, culture, and Xpert MTB/RIF. Afterward, two peripheral laboratories were trained to transport sputum samples with OMS without cold chain. Positivity, negativity, and contamination rates were compared between both methods using Chi-square and Fisher's exact tests. Cohen's Kappa was also used to determine agreements. Results: Among 104 sputum samples analyzed in the CE, 93 (89.4%) had bacterial growth on blood agar before decontamination, while 6 (5.8%) and 5 (4.8%) contaminated after NALC-NaOH and OMS treatment, respectively. Contamination was high with NALC-NaOH (12.8%) than OMS (4.3%) on Lowenstein–Jensen media (P < 0.001), but mycobacterial positivity was comparable: NALC-NaOH of 74.5% and OMS of 78.7%. Smear positivity after NALC-NaOH treatment was 89.4% and OMS was 75.9% (P = 0.491). All except one of the samples tested positive by Xpert MTB/RIF after both treatment. Sixteen samples were evaluated in the field experiment and 81.3% yielded positive culture, and no contamination on LJ was observed. Conclusion: Our findings indicate that OMS works well as a transport and decontaminating reagent of samples for TB testing.

Cholera remains a major global public health threat and continuous emergence of new Vibrio cholerae strains is of major concern. We conducted a molecular epidemiological study to detect virulence markers and antimicrobial resistance patterns of V. cholerae isolates obtained from the 2012-2015 cholera outbreaks in Ghana. Archived clinical isolates obtained from the 2012, 2014 and 2015 cholera outbreaks in Ghana were revived by culture and subjected to microscopy, biochemical identification, serotyping, antibiotic susceptibility testing, molecular detection of distinct virulence factors and Multi-Locus Variable-Number of Tandem-Repeat Analysis (MLVA). Of 277 isolates analysed, 168 (60.6%) were confirmed to be V. cholerae and 109 (39.4%) isolates constituted other bacteria (Escherichia coli, Aeromonas sobria, Pseudomonas aeruginosa, Enterobacter cloacae and Enterococci faecalis). Serotyping the V. cholerae isolates identified 151 (89.9%) as Ogawa, 3 (1.8%) as Inaba and 14 (8.3%) as non-O1/O139 serogroup. The O1 serogroup isolates (154/168, 91.7%) carried the cholera toxin ctxB gene as detected by PCR. Additional virulence genes detected include zot, tcpA, ace, rtxC, toxR, rtxA, tcpP, hlyA and tagA. The most common and rare virulence factors detected among the isolates were rtxC (165 isolates) and tcpP (50 isolates) respectively. All isolates from 2014 and 2015 were multidrug resistant against the selected antibiotics. MLVA differentiated the isolates into 2 large unique clones A and B, with each predominating in a particular year. Spatial analysis showed clustering of most isolates at Ablekuma sub-district. Identification of several virulence genes among the two different genotypes of V. cholerae isolates and resistance to first- and second-line antibiotics, calls for scaleup of preventive strategies to reduce transmission, and strengthening of public health laboratories for rapid antimicrobial susceptibility testing to guide accurate treatment. Our findings support the current WHO licensed cholera vaccines which include both O1 Inaba and Ogawa serotypes.

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, IS 2404 , IS 2606 , 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.

BackgroundDiabetes Mellitus (DM) is a known risk factor for tuberculosis (TB) but little is known on TB-Diabetes Mellitus (TBDM) co-morbidity in Sub-Saharan Africa.MethodsConsecutive TB cases registered at a tertiary facility in Ghana were recruited from September 2012 to April 2016 and screened for DM using random blood glucose and glycated hemoglobin (HbA1c) level. TB patients were tested for other clinical parameters including HIV co-infection and TB lesion location. Mycobacterial isolates obtained from collected sputum samples were characterized by standard methods. Associations between TBDM patients' epidemiological as well as microbiological variables were assessed.ResultsThe prevalence of DM at time of diagnosis among 2990 enrolled TB cases was 9.4% (282/2990). TBDM cases were significantly associated with weight loss, poor appetite, night sweat and fatigue (pConclusionOur findings confirms the importance of screening for diabetes during TB diagnosis and highlights the association between genetic diversity and diabetes. in Ghana.

Rapid but simple diagnostic tools for the detection of drug-resistant (DR) tuberculosis (TB) have been acknowledged as being important for its effective management and control.; To establish a molecular line-probe assay (GenoType(®) MTBDRplus) for detecting DR-TB in Ghana.; We first screened 113 Mycobacterium tuberculosis isolates using the indirect proportion method and MTBDRplus. The rpoB and katG genes and the promoter regions of oxyR-ahpC and inhA were sequenced to identify mutations in isolates found to be resistant on phenotypic drug susceptibility testing and/or MTBDRplus. We then analysed an additional 412 isolates using only MTBDRplus.; Respectively 43 (8.2%) and 8 (1.5%) isolates were resistant to isoniazid (INH) and rifampicin (RMP), while 8 (1.5%) were multidrug-resistant. In resistant isolates, mutations in codon 450 of rpoB and codon 315 of katG, conferring resistance to respectively RMP and INH, dominated. We found two RMP-resistant isolates with a S450L substitution, each harbouring an additional mutation at S388L and Q409R. Using phenotypic testing as gold standard, the MTBDRplus assay showed a sensitivity and specificity in the detection of RMP and INH resistance and multidrug resistance of respectively 100% and 100%, 83.3% and 100%, and 100% and 100%.; The high sensitivity of MTBDRplus makes it a valuable addition to the conventional TB diagnostic algorithm in Ghana.

The introduction of antibiotic therapy as first-line treatment of Buruli ulcer underlines the importance of laboratory confirmation of clinical diagnosis. Because smear microscopy has very limited sensitivity, the technically demanding and more expensive IS2404 diagnostic polymerase chain reaction (PCR) has become the main method for confirmation. By optimization of the release of mycobacteria from swab specimen and concentration of bacterial suspensions before smearing, we were able to improve the detection rate of acid-fast bacilli by microscopy after Ziehl-Neelsen staining. Compared with IS2404 PCR, which is the gold standard diagnostic method, the sensitivity and specificity of microscopy with 100 concentrated specimens were 58.4% and 95.7%, respectively. We subsequently evaluated a stepwise laboratory confirmation algorithm with detection of AFB as first-line method and IS2404 PCR performed only with those samples that were negative in microscopic analysis. This stepwise approach reduced unit cost by more than 50% to $5.41, and the total costs were reduced from $917 to $433

Background Buruli ulcer (BU), a necrotizing skin infection caused by Mycobacterium ulcerans is the third most important mycobacterial disease globally after tuberculosis and leprosy in immune competent individuals. This study reports on the retrospective analyses of microbiologically confirmed Buruli ulcer (BU) cases in seventy-five health facilities in Ghana. Method/Principal findings Pathological samples were collected from BU lesions and transported either through courier services or by car directly to the laboratory. Samples were processed and analysed by IS2404 PCR, culture and Ziehl-Neelsen staining for detection of acid-fast bacilli. From 2008 to 2016, we analysed by PCR, 2,287 samples of 2,203 cases from seventy-five health facilities in seven regions of Ghana (Ashanti, Brong Ahafo, Central, Eastern, Greater Accra, Northern and Volta). The mean annual positivity rate was 46.2% and ranged between 14.6% and 76.2%. The yearly positivity rates from 2008 to 2016 were 52.3%, 76.2%, 56.7%, 53.8%, 41.2%, 41.5%, 22.9%, 28.5% and 14.6% respectively. Of the 1,020 confirmed cases, the ratio of female to male was 518 and 502 respectively. Patients who were 15 years of age and below accounted for 39.8% of all cases. The median age was 20 years (IQR = 10–43). Ulcerative lesions were 69.2%, nodule (9.6%), plaque (2.9%), oedema (2.5%), osteomyelitis (1.1%), ulcer/oedema (9.5%) and ulcer/plaque (5.2%). Lesions frequently occurred on the lower limbs (57%) followed by the upper limbs (38%), the neck and head (3%) and the least found on the abdomen (2%). Conclusions/Significance Our findings show a decline in microbiological confirmed rates over the years and therefore call for intensive education on case recognition to prevent over-diagnosis as BU cases decline., Author summary Buruli ulcer (BU), a necrotizing skin disease caused by Mycobacterium ulcerans, is currently reported in 33 countries, with the greatest disease burden mostly in West African countries along the gulf of Guinea. The lack of pain associated with BU disease enhances delay in seeking medical treatment that could result to complications. The current existing control strategy is early case detection. Previously BU diagnosis was based solely on clinical evidence by a healthcare worker, however, since other skin conditions present similar clinical signs as BU there is the need for further laboratory diagnosis. We microbiological confirmed all clinically diagnosed cases by IS2404 PCR, and Ziehl-Neelsen. We found that over 50% of the clinically diagnosed cases were not BU, thereby averting any unnecessary antimycobacterial treatment with the associated side effects.

BACKGROUND: There is a perception that genomic differences in the species/lineages of the nine species making the Mycobacterium tuberculosis complex (MTBC) may affect the efficacy of distinct control tools in certain geographical areas. We therefore analyzed the prevalence and spatial distribution of MTBC species and lineages among isolates from pulmonary TB cases over an 8-year period, 2007-2014. METHODOLOGY: Mycobacterial species isolated by culture from consecutively recruited pulmonary tuberculosis patients presenting at selected district/sub-district health facilities were confirmed as MTBC by IS6110 and rpos PCR and further assigned lineages and sub lineages by spoligotyping and large sequence polymorphism PCR (RDs 4, 9, 12, 702, 711) assays. Patient characteristics, residency, and risks were obtained with a structured questionnaire. We used SaTScan and ArcMap analyses to identify significantly clustered MTBC lineages within selected districts and spatial display, respectively. RESULTS: Among 2,551 isolates, 2,019 (79.1%), 516 (20.2%) and 16 (0.6%) were identified as M. tuberculosis sensu stricto (MTBss), M. africanum (Maf), 15 M. bovis and 1 M. caprae, respectively. The proportions of MTBss and Maf were fairly constant within the study period. Maf spoligotypes were dominated by Spoligotype International Type (SIT) 331 (25.42%), SIT 326 (15.25%) and SIT 181 (14.12%). We found M. bovis to be significantly higher in Northern Ghana (1.9% of 212) than Southern Ghana (0.5% of 2339) (p = 0.020). Using the purely spatial and space-time analysis, seven significant MTBC lineage clusters (p< 0.05) were identified. Notable among the clusters were Ghana and Cameroon sub-lineages found to be associated with north and south, respectively. CONCLUSION: This study demonstrated that overall, 79.1% of TB in Ghana is caused by MTBss and 20% by M. africanum. Unlike some West African Countries, we did not observe a decline of Maf prevalence in Ghana.

Background We identified secondary infection of Buruli ulcer (BU) wounds as a cause of healing delay. In order to contribute to the improvement of wound management and reduction of healing delay, we initiated a study to gain understanding of the possible routes of infection and also characterised the resistant profiles of Gram negative bacteria isolated from the wounds of patients attending two health facilities in Ghana. Methods Staphylococcus aureus isolates were characterised by the spa gene, mecA and the Pantone Valentine Leukocidin (PVL) toxin followed by spa sequencing and whole genome sequencing of a subset of isolates. Phenotypic antibiotic susceptibility testing of Gram negative clinical isolates was performed and multidrug-resistant Pseudomonas aeruginosa identified. The Enterobacteriaceae were further investigated for ESBL and carbapenem production, and some resistance conferring genes were analysed by PCR. Results Twenty-four isolates were identified as methicillin-resistant S. aureus (MRSA), and lukFS genes encoding PVL were identified in 67 isolates. Typing and sequencing of the spa gene from 91 isolates identified 29 different spa types with t355 (ST152), t186 (ST88), and t346 dominating. While many distinct strains were isolated from both health centres, genotype clustering was identified within centres pointing to possible health care-associated transmission. Phylogenomic analysis confirmed these clusters. Among the GNB, phenotype screening showed widespread resistance to ampicillin, chloramphenicol, ticarcillin-clavulanic acid, cefuroxime and sulphamethoxazole-trimethoprim. ESBL production was confirmed in 15 isolates phenotypically while 61.5% of screen-positive isolates harboured at least one ESBL-conferring gene. Carbapenem encoding genes were detected in 41% of the isolates. Conclusions Our findings indicate that the health-care environment likely contributes to superinfection of BU wounds and calls for training in wound management and infection control techniques. The observed frequency of ESBL and carbapenem resistance indicates the need to set up surveillance networks and strictly enforce policies which guide the rational use of antibiotics.

Background: The worldwide emergence of drug resistant forms of the Mycobacterium tuberculosis complex, the causative agents of tuberculosis (TB), has the potential to make this important human infectious disease which is generally treatable, virtually incurable if not controlled. Our aim therefore was to determine the in vitro drug susceptibilities of M. tuberculosis complex isolates and to correlate this with the clinical response of patients from whom the isolates were obtained.

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, Author Summary Tuberculosis remains one of the main global public health problems. Human tuberculosis is caused by bacteria known as the Mycobacterium tuberculosis complex (MTBC). The MTBC includes a variant called Mycobacterium africanum, which causes up to half of all tuberculosis cases in West Africa. For reasons unknown, M. africanum does not occur in other parts of the world. To explore the possible reasons for this geographic restriction of M. africanum, we analysed a large collection of bacterial strains isolated from tuberculosis patients in Ghana. We genetically characterized these bacterial isolates and collected relevant socio-demographic and epidemiological data. We found tuberculosis patients infected with M. africanum were more likely to belong to the Ewe ethnic group, compared to patients carrying other MTBC bacteria. The Ewes are indigenous inhabitants of coastal regions in West Africa that have previously been shown to harbour a high prevalence of M. africanum. Our findings support the hypothesis that different variants of MTBC have adapted to different human populations, and offer a possible explanation for the geographical restriction of M. africanum to West Africa.

Mycobacterium ulcerans (M. ulcerans), the causative agent of the devastating skin disease Buruli ulcer (BU), is characterized by an extremely low level of genetic diversity. Recently, we have reported the first discrimination of closely related M. ulcerans variants in the BU endemic Densu River Valley of Ghana. In the study real-time PCR-based single nucleotide polymorphism (SNP) typing at 89 predefined loci revealed the presence of ten M. ulcerans haplotypes circulating in the BU endemic region. Here we describe the development of temperature-switch PCR (TSP) assays that allow distinguishing these haplotypes by conventional agarose gel-based analysis of the PCR products. After validation of the accuracy of typing results, the TSP assays were successfully established in a reference laboratory in Ghana. Development of the cost-effective and rapid TSP-based genetic fingerprinting method will thus allow investigating the spread of M. ulcerans clones by regular genetic monitoring in BU endemic countries., Author Summary As the third most common mycobacterial disease after tuberculosis and leprosy, Buruli ulcer constitutes a considerable health problem in many parts of the world, but in particular in West and Central Africa. Although this emerging skin disease is commonly associated with proximity to aquatic habitats, the mode of transmission remains obscure. While the clonal population structure of Mycobacterium ulcerans provides a great potential to trace transmission pathways and evolutionary relationships, micro-epidemiological studies have long been hampered by a striking lack of genetic diversity among African M. ulcerans populations. Whole genome comparison of M. ulcerans strains isolated from patients living in the BU endemic Densu River Valley of Ghana led to the identification of single nucleotide polymorphisms between these closely related strains. The subsequent development of SNP typing assays enabled a differentiation of ten M. ulcerans haplotypes in the BU endemic region. Here we selected canonical SNP markers for a spatio-temporal analysis of M. ulcerans variants in the Densu River Valley of Ghana using a temperature-switch PCR-based approach.