Adane Worku

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.

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.

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

BackgroundIdentification 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.MethodsA 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.ResultOf 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.ConclusionThe 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.

Mycobacterium tuberculosis in central Ethiopia: drug sensitivity patterns and association with genotype

Drug resistance tuberculosis (TB) and the emergence of multidrug resistant (MDR) isolates are significant concerns regarding TB control programs in several countries. This study was undertaken to evaluate the drug sensitivity of Mycobacterium tuberculosis and to assess its association with strains and lineages of M. tuberculosis. A total of 279 M. tuberculosis strains isolated from Central Ethiopia were tested for their drug sensitivity patterns to first line TB drugs using the conventional proportion method on Löwenstein Jensen media. The association between drug sensitivity and strain type was assessed on 263 isolates of the 279 isolates. Of the 268 M. tuberculosis isolates obtained from new cases, 209 (78%) were susceptible to first line TB drugs, and 59 (22.2%) bacterial isolates were resistant to at least one of the first line drugs. The highest mono-resistance (7.5%) pertained to streptomycin (STM). Remarkably, seven of eleven isolates (63.6%) previous treatment for TB were resistant to at least one of the first line drugs. The prevalence of MDR-TB was 1.5% (4/268) for newly identified TB cases, all of which were members of the Euro-American Lineage. There was no statistically significant association (P > 0.05) between drug sensitivity, and either strains, sub-lineages or main lineages of M. tuberculosis. A significant proportion of M. tuberculosis was resistant to at least one first line anti-TB drug. Moreover, the frequencies of resistance to either isoniazid or rifampicin were high compared to data that were previously reported in some part of the country.

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.

Genotyping and molecular detection of multidrug-resistant Mycobacterium tuberculosis among tuberculosis lymphadenitis cases in Addis Ababa, Ethiopia

Multidrug-resistant tuberculosis (MDR-TB) has emerged as a major public health problem. Drug-resistance surveillance data show that 3.9% of new and 21% of previously treated TB cases were estimated to have had rifampicin/ multidrug-resistant tuberculosis (MDR/RR-TB) in 2015. This implies that the MDR-TB is increasing alarmingly. Hence, a better understanding of drug resistance mechanisms and genotypes associated with multidrug resistance in M. tuberculosis is crucial for improving diagnostic and therapeutic methods to treat individuals with MDR-TB. The aim of this study was to analyze molecular drug resistance mutations of MDR-TB isolates from the cases of TB-lymphadenitis in relation to its genetic lineages. A cross-sectional study was conducted on culture positive cases from July to October, 2014 in Addis Ababa, Ethiopia. Sixty isolates were included to analyze drug resistance mutated gene responsible for MDR-TB in relation to its molecular genotyping. Mycobacterial culture, GenoTypeMTBDR plus and Spoligotyping were used to undertake the study. Of 60 TBLN isolates, 8.3% were identified MDR-TB cases and one isolate was isoniazid mono-resistant. Eleven isolates in T3-ETH genetic sub lineage were sensitive to both RMP and INH, while only 2 isolates were MDR-TB. Most of the RMP- resistant isolates showed mutation in codon S531L and all isolates mutated in the katG gene conferring INH resistant strains had mutations in codon of S315T1. Screening for the rpoB and katG gene mutation of tuberculosis lymphadenitis is useful in Ethiopia for an early detection and treatment of MDR-TB. Besides, there is a drug resistance variation among different lineages of Tuberculosis lymphadenitis which has important consequences for the development of efficient control strategies.

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

To understand the population dynamics and propose more effective preventive strategies, defining the population structure of the circulating Mycobacterium tuberculosis strains is important.

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.