A smooth tubercle bacillus from Ethiopia phylogenetically close to the Mycobacterium tuberculosis complex

The Mycobacterium tuberculosis complex (MTBC) includes several human- and animal-adapted pathogens. It is thought to have originated in East Africa from a recombinogenic Mycobacterium canettii-like ancestral pool. Here, we describe the discovery of a clinical tuberculosis strain isolated in Ethiopia that shares archetypal phenotypic and genomic features of M. canettii strains, but represents a phylogenetic branch much closer to the MTBC clade than to the M. canettii strains. Analysis of genomic traces of horizontal gene transfer in this isolate and previously identified M. canettii strains indicates a persistent albeit decreased recombinogenic lifestyle near the emergence of the MTBC. Our findings support that the MTBC emergence from its putative free-living M. canettii-like progenitor is evolutionarily very recent, and suggest the existence of a continuum of further extant derivatives from ancestral stages, close to the root of the MTBC, along the Great Rift Valley

External quality assessment of AFB smear microscopy performances and its associated factors in selected private health facilities in Addis Ababa, Ethiopia

Tuberculosis (TB) is still a public health problem in sub Saharan African countries. In resource-limited settings, TB diagnosis relies on sputum smear microscopy, with low and variable sensitivities, especially in paucibacillary pediatric and HIV-associated TB patients. Tuberculosis microscopy centers have several weaknesses like overworking, insufficiently trained personnel, inconsistent reagent supplies, and poorly maintained equipments; thus, there is a critical need for investments in laboratory infrastructure, capacity building, and quality assurance schemes. The performance of TB microscopy centers in the private health facilities in Addis Ababa is not known so far. The main objective of the study was to assess laboratory performance of acid fast bacilli (AFB) smear microscopy and its associated factors in selected private health facilities in Addis Ababa, Ethiopia. A cross-sectional study was conducted in 33 selected private health facilities of Addis Ababa, Ethiopia comprising 7 hospitals, 2 NGO health centers, 23 higher clinics and 1 diagnostic laboratory that provide AFB smear microscopy services. The study was conducted from January to April 2014. A total of 283 stained sputum smears were randomly collected from participant laboratories for blinded rechecking, 320 panel slides were sent to 32 microscopy centers to evaluate their performance on AFB reading, staining and reporting. Checklists were used to assess quality issues of laboratories. Data were captured, cleaned, and analyzed using SPSS version 16.0; χ2 tests, kappa statistics were used for comparison purpose. P value < 0.05 considered statistically significant. Among the 32 participant laboratories, 2-scored 100%, 15 scored 80-95% & the remaining 15 scored 50-75% for overall proficiency test performance. There were 10 (3.15%) major errors and 121 (37.8%) minor errors. The sensitivity, specificity, PPV and NPV of panel reading by microscopy centers were 89%, 96%, 96%, and 90% respectively. Out of 283 randomly selected slides for blind rechecking, 11 (3.9%) slides interpreted falsely for AFB, with overall agreement of 97.5%, sensitivity of 88.4% and specificity of 99.3%. In terms of slide quality assessment, 71.6% of AFB slides were graded as good for evenness, cleanness, thickness, size, staining and labeling. The performance score for AFB slide evenness was 56.9% (161 slides) and for labeling quality was 90.8% (257 slides); having significant difference in slide quality (p value < 0.05). On-site evaluation indicated problems in terms of infrastructure, standard operating procedure, reagent quality; equipment maintenance, data management and training issues. Most of the health facilities had poor maintenance scheme for microscope (53.5%) and poor inventory management (25.0%) system. Microscopy centers that scored a proficiency of 75.5%; which is below the acceptable minimum score of 80% and an overall error rate of 3.9% for blinded rechecking needs attention. Moreover, there are gaps identified through on site assessment including poor SOP, reagent quality, equipment maintenance, data management & lack of updated training on AFB microscopy techniques, requiring a concerted effort to alleviate the bottle neck problems and strengthening the public private partnership to control TB.The Pan African Medical Journal 2016;2

Nontuberculosis mycobacteria are the major causes of tuberculosis like lesions in cattle slaughtered at Bahir Dar Abattoir, northwestern Ethiopia

Abstract Background The main cause of bovine tuberculosis (bTB) is believed to be Mycobacterium bovis (M. bovis). Nontuberculosis mycobacteria (NTM) are neglected but opportunistic pathogens and obstacles for bTB diagnosis. This study aimed to isolate and characterize the mycobacteria organisms involved in causing TB-like lesions in cattle in northwestern Ethiopia. Results A total of 2846 carcasses of cattle were inspected for TB lesions. Ninety six tissues (including lymph nodes such as submandibular, retropharyngeal, tonsilar, mediatinal, bronchial and mesenteric, and organs such as lung, liver and kidney) with suspicious TB lesion(s) were collected and cultured on Lowenstein-Jensen medium. Twenty one showed culture growth, of which only 17 were identified containing acid fast bacilli (AFB) by Ziehl–Neelsen staining. Among the 17 AFB isolates 15 generated a polymerase chain reaction product of 1030 bp by gel electrophoresis based on the 16S ribosomal RNA gene amplification. No M. tuberculosis complex species were isolated. Further characterization by Genotype Mycobacterium CM assay showed 6 isolates identified as M. peregrinum. Eight isolates represented by mixed species, which includes M. fortuitum-peregrinum (3 isolates), M. gordonae-peregrinum (3 isolates) and M. fortuitum-gordonae-peregrinum (2 isolates). One NTM could not be interpreted. Conclusion A significant number of NTM species were isolated from TB-like lesions of grazing cattle slaughtered at Bahir Dar Abattoir. Such finding could suggest the role of NTM in causing lesions in cattle. Further investigations are recommended on the pathogenesis of the reported NTM species in cattle, and if they have public health significance

Performance of Mycobacterium Growth Indicator Tube BACTEC 960 with Lowenstein–Jensen method for diagnosis of Mycobacterium tuberculosis at Ethiopian National Tuberculosis Reference Laboratory, Addis Ababa, Ethiopia

Abstract Background Bacteriological confirmed active case detection remains the corner stone for diagnosing tuberculosis. Non-radiometric liquid culture system Mycobacterium Growth Indicator Tube with automated interface had been recommended by expert groups in addition to conventional solid culture media such as Lowenstein–Jensen. However in high burden resource limited countries advanced non-radiometric based tuberculosis diagnostic methods such as MGIT 960 is limited. Therefore we have evaluated the performance of MGIT 960 system compared to LJ for recovery of Mycobacterium complex (MTBC) from clinical specimens. Methods A cross sectional study was conducted from a total of 908 samples between January 1st, 2013 to December 31st, 2014. Clinical specimens were processed following standard procedures and the final suspension was inoculated to MGIT tubes and LJ slant. Identification and confirmation of MTBC was done by ZN staining and SD Bioline test. Data was analyzed by SPSS version 20. The sensitivity, specificity, recovery rate and the average turnaround time to recover the organism was computed. Results From a total of 908 clinical specimens processed using both LJ and BACTEC MGIT liquid culture methods the recovery rate for LJ and MGIT, for smear positive samples was 66.7% (74/111) and 87.4% (97/ 111) respectively while for smear negative samples was 13.4% (108/797) and 17.4% (139/797) for LJ and MGIT methods respectively. The overall recovery rate for MGIT is significantly higher than LJ methods [26% (236/908; vs. 20%, 182/908, P = 0.002)]. The average turnaround time for smear positive samples was 16 and 31 days for MGIT and LJ respectively. Turnaround time for smear negative samples was 20 and 36 days for MGIT and LJ respectively. The overall agreement between MGIT and LJ was fairly good with Kappa value of 0.59 (P < 0.001). In the present study the contamination rate for MGIT is higher than the LJ methods, 15 and 9.3% respectively. Conclusions The BACTEC MGIT liquid culture system has better MTBC recovery rate with shorter turnaround time for both smear positive and negative clinical specimens compared to Conventional LJ method. However, efforts should be made in order to reduce the high contamination rate in BACTEC MGIT system and to lesser extent to LJ methods

Molecular characterization and drug resistance patterns of Mycobacterium tuberculosis complex in extrapulmonary tuberculosis patients in Addis Ababa, Ethiopia.

BackgroundMolecular characterization of Mycobacterium tuberculosis (MTB) is important to understand the pathogenesis, diagnosis, treatment, and prevention of tuberculosis (TB). However, there is limited information on molecular characteristics and drug-resistant patterns of MTB in patients with extra-pulmonary tuberculosis (EPTB) in Ethiopia. Thus, this study aimed to determine the molecular characteristics and drug resistance patterns of MTB in patients with EPTB in Addis Ababa, Ethiopia.MethodsThis study was conducted on frozen stored isolates of EPTB survey conducted in Addis Ababa, Ethiopia. A drug susceptibility test was performed using BACTEC-MGIT 960. Species and strain identification were performed using the Geno-Type MTBC and spoligotyping technique, respectively. Data were entered into the MIRU-VNTRplus database to assess the spoligotype patterns of MTB. Analysis was performed using SPSS version 23, and participants' characteristics were presented by numbers and proportions.ResultsOf 151 MTB isolates, 29 (19.2%) were resistant to at least one drug. The highest proportion of isolates was resistant to Isoniazid (14.6%) and Pyrazinamide (14.6%). Nine percent of isolates had multidrug-resistant TB (MDR-TB), and 21.4% of them had pre-extensively drug-resistant TB (pre-XDR-TB). Among the 151 MTB isolates characterized by spoligotyping, 142 (94.6%) had known patterns, while 9 (6.0%) isolates were not matched with the MIRU-VNTRplus spoligotype database. Of the isolates which had known patterns, 2% was M.bovis while 98% M. tuberculosis. Forty-one different spoligotype patterns were identified. The most frequently identified SpolDB4 (SIT) wereSIT149 (21.2%), SIT53 (14.6%) and SIT26 (9.6%). The predominant genotypes identified were T (53.6%), Central Asia Strain (19.2%) and Haarlem (9.9%).ConclusionThe present study showed a high proportion of MDR-TB and pre-XDR-TB among EPTB patients. The strains were mostly grouped into SIT149, SIT53, and SIT26. The T family lineage was the most prevalent genotype. MDR-TB and pre-XDR-TB prevention is required to combat these strains in EPTB. A large scale study is required to describe the molecular characteristics and drug resistance patterns of MTB isolates in EPTB patients

Optimization of the Simple One-Step Stool Processing Method to Diagnose Tuberculosis: Evaluation of Robustness and Stool Transport Conditions for Global Implementation

ABSTRACT Stool is recommended as an alternative specimen for the diagnosis of tuberculosis (TB) in young children, as they cannot easily produce sputum. The Simple One-Step (SOS) stool processing method is a new and simple stool processing method for the detection of Mycobacterium tuberculosis (MTB) using Xpert MTB/RIF Ultra (Xpert-Ultra). We determined the robustness of the SOS stool processing method and stool specimen transport conditions in participants with confirmed TB. We processed stool using the standard protocol after simulated “transport,” varying time, and temperature, and experimented with slightly modified processing steps. We included 2,963 Xpert-Ultra test results from 132 stool specimens of 47 TB participants, including 11 children aged 0.8 g of stool. We found that almost all steps in the current SOS stool processing method provide optimal Xpert-Ultra results but recommend an adjustment to use a wider range of stool amounts (0.3 to 0.8 g) than advised previously (0.8 g). With this adaptation, stool-based diagnosis of TB using the SOS stool processing method can be scaled-up. IMPORTANCE The manuscript will support the global implementation and scale-up of the SOS stool method in routine settings. It also provides important insights on the optimal stool transport conditions and robustness of the SOS method, which can be used for bacteriological diagnosis of TB in children at the lowest levels of the healthcare system, avoiding lengthy healthcare-seeking pathways and additional costs

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