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.
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