Molecular typing of Mycobacterium tuberculosis complex isolated from pulmonary tuberculosis patients in central Ethiopia.

BACKGROUND: Identification of the types of strains of Mycobacterium tuberculosis (M. tuberculosis) complex causing tuberculosis (TB) could contribute to TB control program of specific geographic region as well as it could add knowledge onto the existing literature on TB worldwide. The objective of the present study was to identify the species and strains of M. tuberculosis complex causing pulmonary tuberculosis in central Ethiopia. METHODS: A health institution- based cross-sectional study was conducted on 338 smear positive TB cases visiting three hospitals between October 2012 and September 2013. Morning and spot sputum samples were collected before the starting of treatment regimens. Thus, a total of 338 pooled sputum samples collected from these cases. Samples were cultured on Löwenstein Jensen media and the isolates were identified by the region of difference (RD) 9 based polymerase chain reaction (PCR) and spoligotyping. RESULT: Of the total isolates 98.6% of the isolates were identified to be M. tuberculosis while the remaining 1.4% were identified as M. africanum. Further, typing of M. tuberculosis using spoligotyping lead to the identification of 90 different strains of M. tuberculosis. Of these strains, 32 were clustered consisting of more than one isolate while the remaining 58 strains were unique consisting of single isolate. Thus, 79.3% (223/281) of the isolates were found in the clustered while only 20.6% (58/281) of the strains were unique. Forty-five of the spolgotyping patterns were registeredin the SITVIT2 or SpolDB4 database in while the remaining 45 were notfound in the database and hence were orphan strains. The dominant strains were SIT53, SIT149, and SIT54, consisting of 43, 37 and 34 isolates, respectively. Classification of the spoligotype patterns using TB-insight RUN TB-Lineage showed that 86.8, 6.4, 5, 1.4% ofthe isolatesbelonged to the Euro-American lineage, East-African-Indian, Indo-oceanic and M. africanum, respectively. CONCLUSION: The identification of clustered and new strains using spolygotyping in present study does not give conclusive finding as spoligotyping has low discriminatory power. Thus, further identification of these isolates using mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VENTR) and or whole genome sequencing (WGS) recommended.

In vitro anti-mycobacterial activity of selected medicinal plants against Mycobacterium tuberculosis and Mycobacterium bovis strains.

BACKGROUND: Tuberculosis (TB) is a global burden with one -third of the world's population infected with the pathogen Mycobacterium tuberculosis complex and annually 1.4 million deaths occur due to the disease. This high incidence of infection and the increased rate of multi-drug resistant and extensively-drug resistant strains of the organism further complicated the problem of TB control and have called for an urgent need to develop new anti-TB drugs from plants. In this study, the in vitro activity of root of Calpurnia aurea, seeds of Ocimum basilicum, leaves of Artemisia abyssinica, Croton macrostachyus, and Eucalyptus camaldulensis were evaluated against M. tuberculosis and M. bovis strains. METHODS: Five Ethiopian medicinal plants, root of Calpurnia aurea, seeds of Ocimum basilicum, leaves of Artemisia abyssinica, Croton macrostachyus, and Eucalyptus camaldulensis used locally for the management of TB. They were investigated for in vitro antimycobacterial activity against M. tuberculosis and M. bovis strains. 80% methanolic extracts of the plant materials were obtained by maceration. The antimycobacterial activity was determined using 96 wells of microplate with the help of visual Resazurin Microtiter Assay. RESULTS: The crude 80% methanolic extracts of the root of C. aurea, seeds of O. basilicum, and leaves of A. abyssinica, C. macrostachyus, and E. camaldulensis had anti-mycobacterial activity with minimum inhibitory concentration (MIC) ranging from 6.25-100 µg/mL. The MIC of 80% methanol extracts in the order mentioned above ranged 25-100 µg/ml and 12.5-75 µg/mL, 25-100 µg/mL and 25-50 µg/mL, 6.25-50 µg/mL and 12.5-50 µg/mL, 12.5-100 µg/mL and 18.25-50 µg/mL and 6.25-50 µg/mL and 12.5-50 µg/mL, respectively for M. tuberculosis and M. bovis strains. CONCLUSIONS: The results support the local use of these plants in the treatment of TB and it is suggested that these plants may have therapeutic value in the treatment of TB. However, further investigations are needed on isolating chemical constituents responsible for eliciting the observed activity in these plants.

Field evaluation of specific mycobacterial protein-based skin test for the differentiation of Mycobacterium bovis-infected and Bacillus Calmette Guerin-vaccinated crossbred cattle in Ethiopia.

Bovine tuberculosis (bTB) challenges intensive dairy production in Ethiopia and implementation of the test and slaughter control strategy is not economically acceptable in the country. Vaccination of cattle with Bacillus Calmette-Guerin (BCG) could be an important adjunct to control, which would require a diagnostic test to differentiate Mycobacterium bovis (M. bovis)-infected and BCG-vaccinated animals (DIVA role). This study describes an evaluation of a DIVA skin test (DST) that is based on a cocktail (DSTc) or fusion (DSTf) of specific (ESAT-6, CFP-10 and Rv3615c) M. bovis proteins in Zebu-Holstein-Friesians crossbred cattle in Ethiopia. The study animals used were 74 calves (35 BCG vaccinated and 39 unvaccinated) aged less than 3 weeks at the start of experiment and 68 naturally infected 'TB reactor' cows. Six weeks after vaccination, the 74 calves were tested with the DSTc and the single intradermal cervical comparative tuberculin (SICCT) test. The TB reactor cows were tested with the DSTc and the SICCT test. Reactions to the DSTc were not observed in BCG-vaccinated and unvaccinated calves, while SICCT test reactions were detected in vaccinated calves. DSTc reactions were detected in 95.6% of the TB reactor cows and single intradermal tuberculin positive reactions were found in 98.2% (95% confidence interval, CI, 92.1-100%). The sensitivity of the DSTc was 95.6% (95% CI, 87.6-99.1%), and significantly (p < .001) higher than the sensitivity (75%, 95% CI, 63.0-84.7%) of the SICCT test at 4 mm cut-off. DSTf and DSTc reactions were correlated (r = 0.75; 95% CI = 0.53-0.88). In conclusion, the DSTc could differentiate M. bovis-infected from BCG-vaccinated cattle in Ethiopia. DST had higher sensitivity than the SICCT test. Hence, the DSTc could be used as a diagnostic tool for bTB if BCG vaccination is implemented for the control of bTB in Ethiopia and other countries.

Spoligotype analysis of Mycobacterium bovis isolates from cattle and assessment of zoonotic TB transmission among individuals working in bovine TB-infected dairy farms in Ethiopia.

Bovine tuberculosis (bTB) is a disease with impact on dairy productivity, as well as having the potential for zoonotic transmission. Understanding the genetic diversity of the disease agent Mycobacterium bovis is important for identifying its routes of transmission. Here we investigated the level of genetic diversity of M. bovis isolates and assessed the zoonotic potential in risk groups of people working in bTB-infected dairy farms in central Ethiopia. M. bovis was isolated and spoligotyped from tissue lesions collected from slaughtered cattle as well as from raw milk collected from bTB positive cows in dairy farms from six urban areas of central Ethiopia. From consented dairy farm workers, knowledge and practices related to zoonotic TB transmission, together with demographic and clinical information, was collected through interviews. Sputum or Fine Needle Aspirate (FNA) samples were collected from suspected TB cases. Spoligotyping of 55 M. bovis isolates that originated either from cattle tissues with tuberculous lesion or from raw milk revealed seven spoligotype patterns where SB1176 was the most prevalent type (47.3%). Most isolates (89.1%) were of the M. bovis African 2 clonal complex. All sputum and FNA samples from 41 dairy farm workers with symptoms of TB were culture negative for any mycobacteria. Among the 41 TB suspected farm workers, 61% did not know about bTB in cattle and its zoonotic potential, and over two-third of these workers practiced raw milk consumption. Our spoligotype analysis suggests a wider transmission of a single spoligotype in the study area. The results reported here may be useful in guiding future work to identify the source and direction of bTB transmission and hence design of a control strategy. Isolation of M. bovis from milk, knowledge gap on zoonotic TB and practice of consumption of raw milk in the study population showed potential risk for zoonotic transmission.

Pathology of Bovine Tuberculosis in Three Breeds of Dairy Cattle and Spoligotyping of the Causative Mycobacteria in Ethiopia.

Different breeds of cattle were observed to have a variable degree of susceptibility to bovine tuberculosis (bTB). The screening of bTB was conducted on 720 dairy cattle consisting of three breeds using the single intradermal cervical comparative tuberculin (SICCT) test. Besides this, 43 SICCT test-positive cattle were used to compare the severity of the pathology of bTB among the three breeds and to identify the causative mycobacteria using spoligotyping. The overall SICCT test positivity was 17.92% (129/720) by pooling all animals in the three farms. There was a significant difference in SICCT test positivity among the three breeds (χ2 = 71.06; p < 0.001); the highest (25.34%) was recorded in the crossbreed followed by the Boran breed (10.08%), while the least (3.14%) was recorded in the Jersey breed. On other hand, the highest median pathology score (10.0, interquartile range, IQR = 6.0-17.0) was recorded in Boran followed by cross (5.0, IQR = 3.5-7.5), while the least (3.0, IQR = 2.25-3.0) was recorded in Jersey. Thus, the difference in the median pathology scores was significant [Kruskal Wallis χ ( 2 ) 2 = 18.78, p < 0.001] among the three breeds. Furthermore, multivariate analysis using ordinal logistic regression by considering age, sex, breed, reproductive status, and location of the farms also showed a significant [ χ ( 2 ) 2 = 11.97, p < 0.01] difference in pathology scores among the three breeds of cattle. Even at a single-herd level at Holeta, the difference in severity of pathology between the Boran and crossbreeds was significant (U = 33.5; p < 0.01). Culture positivity was 39% in 108 suspicious tissues. Fourteen Mycobacterium bovis (M. bovis) and two Mycobacterium tuberculosis (M. tuberculosis) were isolated from the lesions. All the 14 M. bovis isolates belonged to SB0912, while the two M. tuberculosis belonged to SIT54. In conclusion, although the frequency of the SICCT test positivity was high in the crossbreed, a more severe pathology was observed on the Boran (zebu) breed. In addition M. tuberculosis was isolated from TB lesions of dairy cattle, demonstrating the role of M. tuberculosis in causing TB in cattle.

Protein and Microbial Biomarkers in Sputum Discern Acute and Latent Tuberculosis in Investigation of Pastoral Ethiopian Cohort.

Differential diagnosis of tuberculosis (TB) and latent TB infection (LTBI) remains a public health priority in high TB burden countries. Pulmonary TB is diagnosed by sputum smear microscopy, chest X-rays, and PCR tests for distinct Mycobacterium tuberculosis (Mtb) genes. Clinical tests to diagnose LTBI rely on immune cell stimulation in blood plasma with TB-specific antigens followed by measurements of interferon-γ concentrations. The latter is an important cytokine for cellular immune responses against Mtb in infected lung tissues. Sputum smear microscopy and chest X-rays are not sufficiently sensitive while both PCR and interferon-γ release assays are expensive. Alternative biomarkers for the development of diagnostic tests to discern TB disease states are desirable. This study's objective was to discover sputum diagnostic biomarker candidates from the analysis of samples from 161 human subjects including TB patients, individuals with LTBI, negative community controls (NCC) from the province South Omo, a pastoral region in Ethiopia. We analyzed 16S rRNA gene-based bacterial taxonomies and proteomic profiles. The sputum microbiota did not reveal statistically significant differences in α-diversity comparing the cohorts. The genus Mycobacterium, representing Mtb, was only identified for the TB group which also featured reduced abundance of the genus Rothia in comparison with the LTBI and NCC groups. Rothia is a respiratory tract commensal and may be sensitive to the inflammatory milieu generated by infection with Mtb. Proteomic data supported innate immune responses against the pathogen in subjects with pulmonary TB. Ferritin, an iron storage protein released by damaged host cells, was markedly increased in abundance in TB sputum compared to the LTBI and NCC groups, along with the α-1-acid glycoproteins ORM1 and ORM2. These proteins are acute phase reactants and inhibit excessive neutrophil activation. Proteomic data highlight the effector roles of neutrophils in the anti-Mtb response which was not observed for LTBI cases. Less abundant in the sputum of the LTBI group, compared to the NCC group, were two immunomodulatory proteins, mitochondrial TSPO and the extracellular ribonuclease T2. If validated, these proteins are of interest as new biomarkers for diagnosis of LTBI.

Evaluation of the Efficacy of BCG in Protecting Against Contact Challenge With Bovine Tuberculosis in Holstein-Friesian and Zebu Crossbred Calves in Ethiopia.

Bovine tuberculosis (bTB) is prevalent in intensive dairy farms in Ethiopia. Vaccination could be an alternative control approach given the socio-economic challenges of a test-and-slaughter control strategy. The efficacy of the BCG was evaluated on 40 Holstein-Friesian (HF) and zebu crossbred calves recruited from single intradermal cervical comparative tuberculin (SICCT) test negative herds and randomly allocated into two groups. Twenty-two calves were vaccinated within 2 weeks of age, and 18 were kept as a control. Six weeks post-vaccination, the two groups were exposed and kept mixed with known SICCT test positive cows for 1 year. Immune responses were monitored by interferon gamma (IFN-γ) release assay (IGRA), SICCT test, and antibody assay. Vaccinated calves developed strong responses to the SICCT test at the sixth week post-vaccination, but did not respond to ESAT-6/CFP-10 peptide antigen-based IGRA. During the exposure, IFN-γ response to the specific peptide cocktail [F (2.44, 92.67) = 26.96; p < 0.001] and skin reaction to the specific proteins cocktail [F (1.7, 64.3); p < 0.001] increased progressively in both groups while their antibody responses were low. The prevalence of bTB was 88.9% (95% CI: 65.3-98.6) and 63.6% (95% CI: 40.7-83.8) in the control and vaccinated calves, respectively, based on Mycobacterium bovis isolation, giving a direct protective efficacy estimate of 28.4% (95% CI: -2.7 to 50.1). The proportion of vaccinated calves with lesion was 7.0% (34/484) against 11.4% (45/396) in control calves, representing a 38% (95% CI: 5.8-59.4) reduction of lesion prevalence. Besides, the severity of pathology was significantly lower (Mann-Whitney U-test, p < 0.05) in vaccinated (median score = 2.0, IQR = 0-4.75) than in control (median score = 5, IQR = 3.0-6.25) calves. Moreover, survival from M. bovis infection in vaccinated calves was significantly (log-rank test: χ2 = 6.749, p < 0.01) higher than that of the control calves. In conclusion, the efficacy of BCG was low, but the reduced frequency and severity of lesion in vaccinated calves could suggest its potential role in containing onward transmission.

Utility of urine as a clinical specimen for the diagnosis of pulmonary tuberculosis in people living with HIV in Addis Ababa, Ethiopia.

BACKGROUND: Tuberculosis is a common cause of mortality and morbidity among people living with HIV/AIDS. Despite the increased prognosis of tuberculosis among HIV infected patients, diagnosis of pulmonary tuberculosis (PTB) smear microscopy has a low sensitivity due to low bacterial load in a sputum specimen of HIV patients. Having alternative specimens for increasing detection of Mycobacterium tuberculosis (Mtb) is very important. OBJECTIVE: The aim of this study was to evaluate the efficacy of urine as clinical specimen for the diagnosis of pulmonary tuberculosis in people living with HIV. METHOD: A total of 117 HIV-seropositive individuals from three public health facilities in Addis Ababa, Ethiopia were enrolled consecutively from December 2013 to July 2014. A total of 117 paired morning sputum and urine samples were simultaneously collected from anti-retroviral therapy (ART) naïve PTB suspected individuals living with HIV. Both sputum and urine samples were processed for culture using Lowenstein-Jensen medium, and the left was subjected to PCR using RD9 primers. Chi-square test and kappa value were used to compare different methods used. RESULT: Out of 117 suspected PTB HIV-infected people, sputum culture alone detected more mycobacterial isolates 33 (28.2%) than the urine specimen alone 17 (14.5%). Of the 33 patients positive for sputum culture, 13 patients were observed as a urine culture positive. Of the 84 individuals negative for mycobacterial by sputum culture, four (4.8%) were urine culture positive and thus, the sensitivity, and the agreement between urine culture as compare to sputum culture were 39.4% and 0.49, respectively. On the other hand, the sensitivity of RD9-based PCR directly on urine was 72.7% by considering sputum culture as a reference standard. Moreover, RD9-based PCR directly on sputum detected 9 (7.7%) individuals who were sputum culture negative for M. Tuberculosis. The detection rate of M. tuberculosis from urine in patients those who couldn't produce sputum were 9(34.6%). CONCLUSION: PCR and culture examination of urine samples also can improve the detection rate of M. tuberculosis in PTB suspected HIV positive individuals.

Spoligotype-based population structure of Mycobacterium tuberculosis in the Jimma Zone, southwest Ethiopia.

BACKGROUND: To understand the population dynamics and propose more effective preventive strategies, defining the population structure of the circulating Mycobacterium tuberculosis strains is important. METHODS: A total of 177 M. tuberculosis complex isolates from pulmonary tuberculosis (TB) cases in southwest Ethiopia were genotyped by spoligotyping. Of the strains included in this study, 126 were pan-susceptible strains while the remaining 51 isolates were resistant to one or more first-line anti-TB drugs. The genotyping results were compared to the international spoligotyping (SITVIT) database of the Pasteur Institute of Guadeloupe and the newly revised publicly available international multi-marker database (SITVITWEB/SPOLDB4). An online tool Run TB-Lineage was also used to predict the major lineages using a conformal Bayesian network analysis. RESULTS: The spoligotyping of the 177 isolates resulted in 69 different spoligotype patterns of which 127 (71.8%) were clustered into 19 spoligoclusters (with clustering rate of 61.02%). Each cluster contains 2-29 isolates. Of the isolates with corresponding SIT in SITVIT/SDB4, the predominant strains identified were SIT 37 of the T3 subfamily with 29 isolates followed by SIT 53 of the T1 subfamily with 20 isolates. SIT 777 of the H4 subfamily and SIT 25 of the CAS1_DELHI subfamily each consisting of six isolates were identified. Eighty spoligotype patterns were orphan as they were not recorded in the SITVIT2/SPDB4 database. Further classification of the isolates on the basis of major lineages showed that 82.5% and 14.1% of the isolates belonged to Euro-American and East African Indian lineages, respectively, while 2.8% of the isolates belonged to Mycobacterium africanum and 0.6% to Indo-Oceanic. CONCLUSION: The ill-defined T and H clades were predominant around Jimma. The substantial number of orphans recorded in the study area warrants for additional studies with genotyping methods with better resolution and covering whole areas of southwest Ethiopia.

Genetic diversity and drug susceptibility profiles of Mycobacterium tuberculosis obtained from Saint Peter's TB specialized Hospital, Ethiopia.

BACKGROUND: Tuberculosis (TB) is one of the major public health problems in Ethiopia. Data on genetic diversity and resistance profile of circulating TB strains is critical for informing the national TB control program. METHODS: A cross-sectional study was conducted on 213 smear positive pulmonary TB patients between 2015 and 2016. Sputum samples were cultured on LJ media following the Petroff's method. Region of difference-9 (RD9)-deletion typing and spoligo-typing were performed for molecular analysis of M. tuberculosis at species and strain levels, respectively. Drug sensitivity and mutation patterns of the isolates were assessed by the conventional indirect proportion method and molecular line probe assays (LPAs), respectively. Data were analyzed using statistical package for social sciences (SPSS) software version 20. RESULTS: Spoligo-typing of 150 M. tuberculosis isolates led to 57 different patterns of which 25 were new strains. The majority (71.6%) of the isolates were grouped in to 17 clusters consisting 2 to 24 isolates. The majority of the strains belonged to Euro-American lineage and the predominant spoligotypes were SIT 37 and SIT 149. MDR-TB was detected in 5.2% and 20.3% of new and retreatment cases, respectively. Two MDR-TB isolates exhibited additional resistance to one of the second line anti-TB drugs. Common gene mutations including S531L, S315T1 and M306V were detected in RIF, INH and EMB resistant strains, respectively. CONCLUSIONS: The identification of several new strains, higher proportion of MDR-TB and higher clustering rate in this study, warrants the need for re-enforcement of the national TB control program. The detection of common gene mutations in the majority drug resistant strains might suggest the feasibility of LPAs for rapid screening of drug resistant M. tuberculosis strains in Ethiopia.

Genotyping of mycobacterium tuberculosis isolated from pulmonary tuberculosis patients among people living with HIV in Addis Ababa: Cross-sectional study.

BACKGROUND: Tuberculosis is a serious infection that is common in people living with HIV and increases the mortality and morbidity from the diseases. The study of genetic diversity among strains of M. tuberculosis has a great impact in studying pathogenicity and transmissibility, design for vaccines production, identification of nominee genes for drug targets, and improving molecular diagnostic techniques. The aim of this study was to characterize Mycobacterium tuberculosis (Mtb) isolated from suspected pulmonary tuberculosis among people living with HIV. METHOD: A total of 143 sputum samples was collected and transported to Akililu Lemma TB laboratory. The collected samples were processed for culture using Lowenstein-Jensen medium. For 45 culture positive isolates, genotyping of mycobacterial DNA was performed by spoligotyping and isolates were assigned to families using the SpolDB4 and the model-based program 'SPOTCLUST'. Categorical data were analyzed by Chi-square test. RESULT: A high level of diversity was found among the 45 isolates. Twenty six different Spoligo patterns were obtained. The T (46.7%), Family33 (44.4%) and Central Asian (CAS): (4.4%) families were the dominant isolates comprising 91.5% of the total strains. Of 44% of the Euro-American, 6/20(30%) and 9/20(45%), identified were lineage belonged to Spoligo-International-Type (SIT336) and SIT149. Of the total strains, 12 (22%) were unique and have not been described in SpolDB4 to date. CONCLUSION: We found the high diversity of Mtb in pulmonary tuberculosis patients in this setting. T3_ETH family identified as the numerous M.tuberculosis strains circulating in the community.

Detection of Mycobacterium tuberculosis from the stool of HIV sero-positive individuals suspected of pulmonary tuberculosis.

BACKGROUND: The impact of tuberculosis (TB) is exacerbated in Africa because of the human immunodeficiency virus (HIV) pandemic. Pulmonary tuberculosis (PTB) diagnosis is difficult in HIV-infected patients and negative sputum results are more common which leads to diagnostic delay and increases morbidity and mortality. Extra-pulmonary samples such as stool may be easier to obtain and our approach may therefore significantly improve PTB detection in people living with HIV. OBJECTIVE: To detect Mycobacterium tuberculosis from the stool of HIV sero-positive individuals suspected of pulmonary TB. METHOD: A total of 117 HIV-infected individuals from three public health facilities in Addis Ababa, Ethiopia were enrolled consecutively in the study. Paired morning sputum and stool samples were simultaneously collected from anti-retroviral therapy (ART) naïve individuals living with HIV and suspected for PTB. The diagnostic accuracy of the smear microscopy, culture and region of difference (RD)9-based polymerase chain reaction (PCR) in stool was compared with the accuracy of sputum testing. Chi-square test and kappa value were used to compare different method used. RESULTS: Sputum culture positivity for mycobacteria was confirmed in 33(28.2%) of the study subjects. Of 33 individuals positive for sputa culture, 10 individuals were observed to be stools culture positive. Of the 84 individuals negative for mycobacteria by sputum culture, three (3.6%) were stool culture positive and thus, the sensitivity and agreement between stool culture as compare to sputum culture were 30.3% and 0.33, respectively. Of 117 individuals, 11(9.4%) were sputum smear positive and of 11 sputum smear positive three were also stool smear positive. While of the 106 sputum smear negative individuals', only one was stool smear positive resulting in 12.1% sensitivity and 0.18 agreements against sputum culture. On the other hand, the sensitivity of RD9-based PCR directly on stool was 69.7% by considering sputum culture as a reference standard. Moreover, RD9-based PCR directly on sputum detected 7(6.0%) individuals who were sputum culture negative for M. tuberculosis. CONCLUSION: M. tuberculosis was detected in stool of individuals living with HIV who were negative for sputum smear microscopy and culture. Hence, examination of stool samples alongside with sputum samples increases the detection of PTB in individuals living with HIV.

Evaluation of the GenoType MTBDRplus assay for detection of rifampicin- and isoniazid-resistant Mycobacterium tuberculosis isolates in central Ethiopia.

OBJECTIVE/BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) is growing globally and becoming a major challenge for national TB control programs. Therefore, rapid identification of MDR strains of Mycobacterium tuberculosis and monitoring their transmission could contribute significantly to the control of TB. The GenoType MTBDRplus assay has been recommended by the World Health Organization to identify rifampicin (RIF)- and isoniazid (INH)-resistant M. tuberculosis isolates. This study was carried out to evaluate the performance of the GenoType MTBDRplus assay for the detection of RIF- and INH-resistant M. tuberculosis isolates in central Ethiopia. METHODS: A total of 279 M. tuberculosis strains isolated from active TB cases in central Ethiopia were evaluated for their drug sensitivity by the conventional drug-susceptibility test (DST) and compared with data derived from the GenoType MTBDRplus assay. The DST served as the gold standard for evaluating the GenoType MTBDRplus assay. RESULTS: The sensitivity and specificity of the GenoType MTBDRplus assay for the detection of RIF-resistant M. tuberculosis isolates were 80.0% and 99.6%, respectively. Its sensitivity and specificity for the detection of INH-resistant M. tuberculosis isolates were 82.7% and 99.6%, respectively, whereas they were 75.0% and 100%, respectively, for the detection of MDR M. tuberculosis strains. The concordances of the GenoType MTBDRplus assay and the conventional DST for the detection of RIF and INH susceptibility were 80% (8/10) and 86.2% (25/29), respectively. Furthermore, the concordance of the two tests for the detection of MDR M. tuberculosis strains was 75%. Specific mutations were detected in 55.6% (5/9) of the RIF-resistant isolates, with the highest mutation rate (33.3%) for the rpoB gene (Codon S531L). For INH-resistant isolates, the highest mutation rate (88.8%) related to a katG mutation (Codon S315T1). CONCLUSION: The findings of this study revealed that the GenoType MTBDRplus assay has high sensitivity and specificity for the detection of RIF and INH resistance. These preliminary data support the notion that the assay should be considered as an alternative to the DST for the characterization of MDR in M. tuberculosis isolates and the control of TB.

Genetic Diversity of Mycobacterium tuberculosis Complex Isolated from Tuberculosis Patients in Bahir Dar City and Its Surroundings, Northwest Ethiopia.

The knowledge of the diversity of strains of Mycobacterium tuberculosis complex (MTBC) species in a specific geographical region can contribute to the control of tuberculosis (TB). This study was conducted to identify the MTBC isolates to the species and spoligotype international type (SIT) level by spoligotyping. A total of 168 MTBC isolates were recovered from TB patients, spoligotyped, and their patterns were compared with those of the strains registered in the SITVIT2 database. Of 168 isolates spoligotyped, 89 patterns were identified. Ninety-eight isolates were clustered into 19 strain groups with clustering percentage of 58.3%. Forty-four strains matched the preexisting SITs in the SITVIT2 database. The dominant strains were SIT289, SIT134, and SIT3411, comprising 16.7% (28/168), 7.14% (12/168), and 4.76% (8/168) of the isolates, respectively. Euro-American (51.2%), East-African-Indian (34.5%), and M. africanum (9.52%) were the major lineages identified. Two strains of M. bovis were isolated from TB lymphadenitis cases. The high percentage of clustered strains of M. tuberculosis could suggest that a small number of lineages of M. tuberculosis are causing the disease in the area while isolation of M. bovis could suggest its zoonotic potential. Additionally, identification of M. africanum requires further confirmation by tools with a better discriminatory power.