Prevalence of non-tuberculous mycobacteria among previously treated TB patients in the Gulf of Guinea, Africa.

Objective: Differentiation between non-tuberculous mycobacteria (NTM) and Mycobacterium tuberculosis complex (MTBC) is crucial for case management with the appropriate antimycobacterials. This study was undertaken in three West and Central African countries to understand NTM associated with pulmonary tuberculosis in the sub-region. Methods: A collection of 503 isolates (158 from Cameroon, 202 from Nigeria and 143 from Ghana) obtained from solid and liquid cultures were analysed. The isolates were tested for drug susceptibility, and MTBC were confirmed using IS6110. All IS6110-negative isolates were identified by 65-kilodalton heat shock protein (hsp65) gene amplification, DNA sequencing and BLAST analysis. Results: Overall, the prevalence of NTM was 16/503 (3.2%), distributed as 2/202 (1%) in Nigeria, 2/158 (1.3%) in Cameroon and 12/143 (8.4%) in Ghana. The main NTM isolates included 5/16 (31.3%) M. fortuitum, 2/16 (12.5%) M. intracellulare and 2/16 (12.5%) M. engbaekii. Eight (57.1%) of the 14 previously treated patients harboured NTM (odds ratio 0.21, 95% confidence interval 0.06-0.77; P=0.021). Three multi-drug-resistant strains were identified: M. engbaekii, M. fortuitum and M. intracellulare. Conclusion: NTM were mainly found among individuals with unsuccessful treatment. This highlights the need for mycobacterial species differentiation using rapid molecular tools for appropriate case management, as most are resistant to routine first-line antimycobacterials.

A pilot study on the genetic diversity of Mycobacterium tuberculosis complex strains from tuberculosis patients in the Littoral region of Cameroon.

BACKGROUND: The re-emergence of tuberculosis (TB) worldwide, compounded by multi-drug resistance (MDR) of the causative agents constitutes a major challenge to the management of the disease. Rapid diagnosis and accurate strain identification are pivotal to the control of the disease. This pilot study investigated the genetic diversity of Mycobacterium tuberculosis complex (MTBC) strains from TB patients in the Littoral region of Cameroon as well as their resistance to rifampicin (RIF). PATIENTS AND METHODS: This was a cross sectional hospital-based study carried out between January and December 2017 and including 158 isolates from sputum smear positive individuals [105 (66.5%) males and 53 (33.5%) females]. Sputum samples were tested using Xpert MTB/RIF, followed by culture on Lowenstein-Jensen medium. Isolates were further subjected to molecular characterization using IS6110 typing, deletion analysis and spoligotyping. RESULTS: Thirteen (8.8%) of the 147 isolates with susceptibility results available were resistant to RIF. Drug resistance occurred in 5/50 (10%) female compared to 8/97 (8.2%) male (OR, 0.81; 0.25-2.62; p = 0.764), and there was no significant difference across the age ranges (p = 0.448). On the other hand, RIF resistance was associated (OR, 0.18, 95%CI, 0.05-0.69; p = 0.023) with previously treated patients [(4/14 (28.6%)] compared to new ones [9/133 (6.8%)]. The 150 identified lineages included among others 54 (36%) Cameroon, 18 (12%) UgandaI, 32 (21.3%) Haarlem, 17 (11.3%) Ghana, 9(6%) West African 1, 7(4.7%) Delhi/CAS, 4 (2.7%) LAM and 3 (2%) UgandaII. Of the 150 isolates, the major cluster was the Cameroon SIT 61, with 43(28.7%) isolates. Six (35.3%) of the 17 UgandaI sub-lineage were RIF resistant (OR, 9.58; 95%CI, 2.74-33.55, p = 0.001). CONCLUSION: The cosmopolitan Littoral region presents with a wide Mycobacterium tuberculosis (MTB) strains diversity and the UgandaI sub-lineage likely associated with RIF resistance. Understanding the spread of this clade through surveillance will enhance TB control in the region.

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 ß-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.

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

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.

Diagnosis of tuberculosis in ghana: the role of laboratory training.

OBJECTIVES: The laboratory is considered the cornerstone of tuberculosis (TB) control programme. International review of Ghana's programme in the late nineties identified the laboratory services as the weakest component. Sputum smear microscopy (SSM) being the main method of diagnosing pulmonary TB in Ghana, the training objectives were to: (i) strengthen the knowledge and skills of laboratory personnel on SSM (ii) impart necessary techniques in biosafety and (iii) introduce a Quality Assurance (QA) system in order to strengthen SSM services. METHODS: Personnel were selected for training during a nationwide situation analysis of SSM centres in 2000/2001. Four training sessions on SSM/QA were held between 2001/2004. RESULTS: A total of 80 personnel were trained: 10 regional TB coordinators and 70 laboratory personnel. The participants upon return to their respective regions also organized training within their districts. This approach resulted in another 100 district TB coordinators and 200 laboratory personnel being trained. Improvement in smear preparation, staining and reading ability of the participants were observed during the post-test and subsequent visit to their respective laboratories. The training has led to strengthening of TB laboratory services in the country and has contributed to increase in case detection from 10,745 in 2000 to 11,827 in 2004 and 14,022 in 2008. It was observed during the post-training follow-up and quarterly supervision visits that morale of the personnel was high. CONCLUSION: Continuous training and re-training of laboratory personnel on SSM and QA at regular intervals do play an important role for effective and efficient TB control programme.

Mycobacterial species causing pulmonary tuberculosis at the korle bu teaching hospital, accra, ghana.

SUMMARY OBJECTIVE: Characterize mycobacterial species causing pulmonary tuberculosis (PTB) at the Korle-Bu Teaching Hospital in Ghana. DESIGN: Sputum smear positive samples, two (2) from 70 patients diagnosed as having tuberculosis, after they had consented, were collected from the Korle-Bu Teaching Hospital Chest Clinic between January and July 2003. SETTING: Korle-Bu Teaching Hospital Chest Clinic, Accra. RESULTS: Sixty-four mycobacterial isolates were obtained and confirmed as members of Mycobacterium tuberculosis complex by colonial morphology and conventional biochemical assays. Forty-seven (73%) were M. tuberculosis, the human strain, 2 (3%) M. bovis, the bovine strain, 13 (20%) M. africanum I (West Africa type), and 2 (3%) M. africanum II (East Africa type). CONCLUSION: The results indicate that, there are various strains causing PTB at the Korle-Bu Teaching Hospital and of great concern is M. bovis, which mostly causes extra-PTB in humans but found to cause PTB in this study. This calls for the need to conduct a nationwide survey using both conventional and molecular techniques to characterize various mycobacterial species causing TB in Ghana. This will result in better understanding of the various strains circulating in the country and inform individual TB treatment regimen especially the inclusion or exclusion of pyrazinamide.

Situation analysis of TB microscopy centres in Ghana.

SETTING: Public health laboratories in Ghana performing tuberculosis (TB) microscopy. OBJECTIVE: To assess the situation of the laboratories in terms of staff strength, technical skills, documentation, biosafety practices, equipment, supplies and disposal systems. DESIGN: Methods used for data collection were interviews using a structured questionnaire, informal observation of laboratory registers, disposal systems and safety measures for sputum handling. RESULTS: Of 114 laboratories visited between 2000 and 2001, 102 (89.5%) were performing TB microscopy. Of the staff working in the laboratories, 9% were medical technologists, 24% laboratory technicians, 37% laboratory assistants and 30% orderlies. Average false-negative and -positive rates were respectively 13% and 14%. Although most of the centres (85.3%) were using the recommended TB laboratory register for recording, in most cases they were not filled in accurately or completely. The majority of the available microscopes had mechanical or optical faults. Availability of other materials for smear preparation and staining ranged from 44% to 82%. The main methods employed for disposal of laboratory waste were burning and burying, but conditions were poor in most of the facilities visited. CONCLUSION: Training of laboratory personnel in TB microscopy and establishment of a quality assurance system are needed in Ghana.

Improving the laboratory diagnosis of TB in Ghana: the impact of a quality assurance system.

SETTING: Greater Accra region, Ghana. OBJECTIVE: To establish a pilot quality assurance (QA) system in sputum smear microscopy and to evaluate its impact. DESIGN: Quarterly supporting visits were paid to participating laboratories between 2000 and 2002. Fifteen examined slides were selected randomly from each laboratory during the visits and blindly re-assessed. Feedback was given promptly to the various laboratories. Training and stakeholder workshops were organised whenever necessary. RESULTS: General improvements in smear preparation and staining as well as the reading ability of the laboratory personnel included in the study were observed. The average marks for specimen quality, staining ability, smear cleanness, thickness, size and evenness increased from 64%, 79%, 69%, 46%, 67% and 60% in the last quarter of 2000 to 81%, 90%, 86%, 79%, 80% and 74%, respectively, 24 months after the establishment of the QA system. Within the same period, the rate of false-positives and -negatives decreased from respectively 14.8% and 20.5% to 0%, and agreements in positivity grade increased from 74% to 95%. The performance of the participating laboratories in keeping the laboratory registers up to date also improved. CONCLUSION: The QA system needs to be extended to the rest of the country.

Microbiological quality of "khebab" consumed in the accra metropolis.

SummaryThe study was carried out in 2003 to evaluate the microbial load in "khebab", meat products from pork, and beef, which are vended in most of the streets and some public drinking places, either with alcoholic or non-alcoholic drinks.Osu (Alata), Nima-Kotobabi and Central Accra (Adabraka - very close to the main lorry station), all in the Accra Metropolis, were selected for the investigation. The main reason for the selection of these sites was based on the population density as well as patronage for the khebab. Our main interest for this investigation was to assess the microbial load in khebab as far as enteric pathogen and other pathogenic micro-organisms reported earlier in the raw meat are concerned. Thirty samples of khebab were bought from these sampling points. Results obtained from samples at Osu recorded mean total plate count (TPC) of 5.02, Accra Central samples had TPC of 4.08 and those from Nima had TPC of 4.80 log(10) colony-forming units (cfu) per gram of khebab. Samples from Accra Central recorded the highest mean coliform count (5.12) whist samples bought from Osu and Nima recorded 4.41 and 3.70 log(10) cfu/g respectively. Accra Central samples again recorded the highest faecal coliforms (4.4 log(10) cfu/g) as compared to 3.98 and 3.80 log10 cfu/g for samples bought from Osu and Nima respectively. Salmonella ssp were not isolated from the samples bought at the three sampling sites. Khebab samples from sites were contaminated with E. coli, other gram-negative bacteria and Staphylococcus species, whose virulence factor(s) are yet to be determined. The faecal coliforms enumerated could originate from either humans or the animals slaughtered for the khebab.Staphylococcus species could originate from the vendors. Vendors have to be educated on hygienic practices which could help reduce risks of food-borne infection. Skin disinfection can be done by a thorough wash. Vendors could also be educated to stop selling their products to customers once they have bouts of diarrhoea, vomiting and "fever". Washing of their hands with soap and water before serving their customers could also help reduce the risk of food-borne infection from eating their products.

Application of a simple multiplex PCR to aid in routine work of the mycobacterium reference laboratory.

A PCR specific for spacer regions 33 and 34 of the direct repeat region of the Mycobacterium tuberculosis complex was developed to complement the biochemical differentiation of M. tuberculosis, Mycobacterium bovis, M. bovis BCG, and Mycobacterium africanum subtypes I and II. In addition, this approach was incorporated into a multiplex PCR that included primers specific for IS6110 and the 65-kDa antigen gene in order to differentiate members of the M. tuberculosis complex from atypical mycobacteria.