Bacteriology

Methodology: Raw beef samples were aseptically collected from 200 cattle slaughtered for consumption. Total plate count, presence of foodborne pathogens such as Listeria monocytogenes, Escherichia coli, E. coli 0157:H7, Staphylococcus aureus, Salmonella and Shigella species were determined after culture and incubation on standard microbiological media. Both liver and kidney samples were also collected from each of the 200 carcasses and tested for antibiotic residues using Original Research Article

Tuberculosis (TB) continues to be an important public health problem worldwide. Currently there are 286 TB cases per every 100,000 people in Ghana. This figure is three times higher than the TB burden estimated by the World Health Organization. The numerical contribution of bovine TB to the general TB burden is unknown. Herdsmen, livestock workers, and veterinarians and the general public are at high risk of contracting bovine TB. Zoonotic TB caused by Mycobacterium bovis is present in animals in most developing countries including Ghana. Unfortunately, activities to check and control cattle with TB infection from entering the food chain are often inadequate or unavailable. This study therefore aims to determine the prevalence of TB infection in a cohort of cattle that enters the food chain in Accra, Ghana. A cross sectional study involving five major abattoirs was conducted in the Greater Accra region between the periods of September 2012-June 2013. After routine inspection of live cattle by veterinary officials, 10 mL of blood was drawn from 94 cattle before slaughter and tested for TB infection using BOVIGAM. Six (6.4%) of the 94 cattle screened, were positive for TB infection. All except one abattoir had at least one cattle testing positive. Although the study recorded a lower prevalence of 6.4%, all animals tested were deemed fit for slaughter by veterinary officials and was to enter the food chain. The low sensitivity of routine abattoir inspection for infected organs and negative results of post-mortem examination reinforces the need for more sensitive screening tool such as BOVIGAM.

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. 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 rpoß 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. Among 2,551 isolates, 2...

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 ¼ 0.0000, RIF: 6.2%; p ¼ 0.0103, MDR: 4.6%; p ¼ 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 ¼ 0.0000) whereas the opposite was true for inhApro mutations; MAF (48.3%) compared to MTBSS (26.7%) (p ¼ 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.

For cultivation of Mycobacterium ulcerans from clinical specimens, we optimized the release of bacteria from swabs, as well as decontamination and cultivation on supplemented medium. Nevertheless, the proportions of positive cultures, 41.7% (5/12) for fine-needle-aspiration (FNA) samples and 43.8% (49/112) for swab samples, were lower than those we have previously observed for excised tissue specimens.

Buruli ulcer (BU), caused by Mycobacterium ulcerans, is the third most common mycobacterial infection in immunocompetent humans besides tuberculosis and leprosy. We have compared by ex vivo enzyme-linked immunospot analysis interferon-␥ (IFN-␥) responses in peripheral blood mononuclear cells (PBMC) from BU patients, household contacts, and individuals living in an adjacent M. ulcerans nonendemic region. PBMC were stimulated with purified protein derivative (PPD) and nonmycobacterial antigens such as reconstituted influenza virus particles and isopentenyl-pyrophosphate. With all three antigens, the number of IFN-␥ spot-forming units was reduced significantly in BU patients compared with the controls from a nonendemic area. This demonstrates for the first time that M. ulcerans infection-associated systemic reduction in IFN-␥ responses is not confined to stimulation with live or dead mycobacteria and their products but extends to other antigens. Interleukin (IL)-12 secretion by PPD-stimulated PBMC was not reduced in BU patients, indicating that reduction in IFN-␥ responses was not caused by diminished IL-12 production. Several months after surgical excision of BU lesions, IFN-␥ responses of BU patients against all antigens used for stimulation recovered significantly, indicating that the measured systemic immunosuppression was not the consequence of a genetic defect in T cell function predisposing for BU but is rather related to the presence of M. ulcerans bacteria.

Objective: The aim of this study was to use spoligotyping and large sequence polymorphism (LSP) to study the population structure of M. tuberculosis complex (MTBC) isolates. Methods: MTBC isolates were identified using standard biochemical procedures, IS6110 PCR, and large sequence polymorphisms. Isolates were further typed using spoligotyping, and the phenotypic drug susceptibility patterns were determined by the proportion method. Result: One hundred and sixty-two isolates were characterised by LSP typing. Of these, 130 (80.25%) were identified as Mycobacterium tuberculosis sensu stricto (MTBss), with the Cameroon sub-lineage being dominant (N = 59/130, 45.38%). Thirty-two (19.75%) isolates were classified as Mycobacterium africanum type 1, and of these 26 (81.25%) were identified as West-Africa I, and 6 (18.75%) as West-Africa II. Spoligotyping sub-lineages identified among the MTBss included Haarlem (N = 15, 11.53%), Ghana (N = 22, 16.92%), Beijing (4, 3.08%), EAI (4, 3.08%), Uganda I (4, 3.08%), LAM (2, 1.54%), X (N = 1, 0.77%) and S (2, 1.54%). Nine isolates had SIT numbers with no identified sub-lineages while 17 had no SIT numbers. MTBss isolates were more likely to be resistant to streptomycin (p,0.008) and to any drug resistance (p,0.03) when compared to M. africanum. Conclusion: This study demonstrated that overall 36.4% of TB in SouthWestern Ghana is caused by the Cameroon sublineage of MTBC and 20% by M. africanum type 1, including both the West-Africa 1 and West-Africa 2 lineages. The diversity of MTBC in Ghana should be considered when evaluating new TB vaccines.

Background: Different combinations of variable number of tandem repeat (VNTR) loci have been proposed for genotyping Mycobacterium tuberculosis complex (MTBC). Existing VNTR schemes show different discriminatory capacity among the six human MTBC lineages. Here, we evaluated the discriminatory power of a ''customized MIRU12'' loci format proposed previously by Comas et al. based on the standard 24 loci defined by Supply et al. for VNTR-typing of MTBC in Ghana. Method: One hundred and fifty-eight MTBC isolates classified into Lineage 4 and Lineage 5 were used to compare a customized lineage-specific panel of 12 MIRU-VNTR loci (''customized MIRU-120) to the standard MIRU-15 genotyping scheme. The resolution power of each typing method was determined based on the Hunter-Gaston-Discriminatory Index (HGDI). A minimal set of customized MIRU-VNTR loci for typing Lineages 4 (Euro-American) and 5 (M. africanum West African 1) strains from Ghana was defined based on the cumulative HGDI. Results and Conclusion: Among the 106 Lineage 4 strains, the customized MIRU-12 identified a total of 104 distinct genotypes consisting of 2 clusters of 2 isolates each (clustering rate 1.8%), and 102 unique strains while standard MIRU-15 yielded a total of 105 different genotypes, including 1 cluster of 2 isolates (clustering rate: 0.9%) and 104 singletons. Among, 52 Lineage 5 isolates, customized MIRU-12 genotyping defined 51 patterns with 1 cluster of 2 isolates (clustering rate: 0.9%) and 50 unique strains whereas MIRU-15 classified all 52 strains as unique. Cumulative HGDI values for customized MIRU-12 for Lineages 4 and 5 were 0.98 respectively whilst that of standard MIRU-15 was 0.99. A union of loci from the customised MIRU-12 and standard MIRU-15 revealed a set of customized eight highly discriminatory loci: 4052, 2163B, 40, 4165, 2165, 10,16 and 26 with a cumulative HGDI of 0.99 for genotyping Lineage 4 and 5 strains from 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.

Background-Rapid but simple diagnostic tool for detecting drug resistant (DR) tuberculosis (TB) has been acknowledged as important for effective management and control of DR-TB. Our objective was to establish a molecular line-probe assay (GenoType® MTBDRplus) for detecting DR-TB in Ghana. Method-We first screened 113 Mycobacterium tuberculosis isolates by indirect proportion method and MTBDRplus. For isolates found resistant either by phenotypic DST and/or MTBDRplus, the rpoB, and katG genes as well as the promoter regions of oxyR-ahpC and inhA were sequenced to identify mutations. We then analyzed an additional 412 isolates by MTBDRplus only. Results-Forty-three (8.2%) and 8 (1.5%) isolates were resistant to isoniazid (INH) and rifampicin (RIF), respectively, and 8 (1.5%) were multidrug-resistant. Among these resistant isolates, mutations in codon 450 of rpoB and codon 315 of katG conferring resistance to RIF and INH, respectively, dominated. We found two RIF resistant isolates with S450L substitution each harboring an additional mutation at S388L and Q409R, respectively. Using the phenotypic testing as gold standard, the MTBDRplus assays showed a sensitivity/specificity for the detection of RIF and INH resistance and MDR of 100%/100%, 83.3%/100% and 100%/100%, respectively. Conclusion-The high sensitivity makes MTBDRplus a valuable addition to the conventional TB diagnostic algorithm in Ghana.

Generalist and specialist species differ in the breadth of their ecological niches. Little is known about the niche width of obligate human pathogens. Here we analyzed a global collection of Mycobacterium tuberculosis lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses showed that, whereas the majority of human T cell epitopes were conserved in all sublineages, the proportion of variable epitopes was higher in generalists. Our data further support a European origin for the most common generalist sublineage. Hence, the global success of lineage 4 reflects distinct strategies adopted by different sublineages and the influence of human migration.

Mycobacterium africanum (Maf) causes a substantial proportion of human tuberculosis in some countries of West Africa, but little is known on this pathogen. We compared the genomes of 253 Maf clinical isolates from Ghana, including N = 175 Lineage 5 (L5) and N = 78 Lineage 6 (L6). We found that the genomic diversity of L6 was higher than in L5 despite the smaller sample size. Regulatory proteins appeared to evolve neutrally in L5 but under purifying selection in L6. Even though over 90% of the human T cell epitopes were conserved in both lineages, L6 showed a higher ratio of non-synonymous to synonymous single nucleotide variation in these epitopes overall compared to L5. Of the 10% human T cell epitopes that were variable, most carried mutations that were lineage-specific. Our findings indicate that Maf L5 and L6 differ in some of their population genomic characteristics, possibly reflecting different selection pressures linked to distinct ecological niches.

Background Bovine tuberculosis (bTB) caused by Mycobacterium bovis is a re-emerging problem in both livestock and humans. The association of some M. bovis strains with hyper-virulence, MDR-TB and disseminated disease makes it imperative to understand the biology of the pathogen. Methods Mycobacterium bovis (15) among 1755 M. tuberculosis complex (MTBC) isolated between 2012 and 2014 were characterized and analyzed for associated patient demography and other risk factors. Five of the M. bovis were whole-genome sequenced and comparatively analyzed against a global collection of published M. bovis genomes. Results Mycobacterium bovis was isolated from 3/560(0.5%) females and 12/1195(1.0%) males with pulmonary TB. The average age of M. bovis infected cases was 46.8 years (7-72years). TB patients from the Northern region of Ghana (1.9%;4/212) had a higher rate of infection with M. bovis (OR=2.7,p=0.0968) compared to those from the Greater Accra region (0.7%;11/1543). Among TB patients wi...