The current study was aimed to evaluate the performance of direct 2,3,5-triphenyl tetrazolium chloride assay and direct microscopic observation drug susceptibility assay with indirect Löwenstein-Jensen proportion method directly on Ziehl-Neelsen smear positive sputum specimens. MethodsDirect acid fast bacilli smear positive sputum specimens (n=264) were subjected to isoniazid and rifampicin drug susceptibility testing by direct 2,3,5-triphenyl tetrazolium chloride assay, direct microscopic observation drug susceptibility assay, and the performances were compared with indirect Löwenstein-Jensen proportion method. ResultsThe direct 2,3,5-triphenyl tetrazolium chloride assay demonstrated an overall sensitivity, specificity, positive predictive value, and negative predictive value of 99.2%, 82.4%, 99.2%, and 88.5%, respectively, for the detection of isoniazid and rifampicin resistant Mycobacterium tuberculosis isolates when compared to indirect Löwenstein-Jensen proportion method. Likewise, the overall sensitivity, specificity, positive predictive value and negative predictive value of direct microscopic observation drug susceptibility assay were 98.8%, 82.4%, 99.2%, and 78.2%, respectively. ConclusionThe direct 2,3,5-triphenyl tetrazolium chloride assay was found to be an economical alternative method for the rapid and accurate detection of isoniazid and rifampicin resistance from direct acid fast bacilli smear positive sputum specimens.