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Background: Emergence of multidrug- and extensively drug-resistant tuberculosis (M/XDR-TB) is a major hurdle for TB control programs especially in developing countries like China. Resistance to fluoroquinolones is high among MDR-TB patients. Early diagnosis of MDR/pre-XDR-TB is essential for lowering transmission of drug-resistant TB and adjusting the treatment regimen.Methods: Smear-positive sputum specimens (n = 186) were collected from Wuhan Institute for Tuberculosis Control. The DNA was extracted from the specimens and run through a Sanger sequencing assay to detect mutations associated with MDR/pre-XDR-TB including the rpoB core region for rifampicin (RIF) resistance; katG and inhA promoter for isoniazid (INH) resistance; and gyrA for fluoroquinolone (FQ) resistance. Sequencing data were compared to phenotypic Lowenstein-Jensen (L-J) proportion method drug susceptibility testing (DST) results for performance analysis.Results: By comparing the mutation data with phenotypic results, the detection rates of MDR-TB and pre-XDR-TB were 84.31% (43/51) and 83.33% (20/24), respectively. The sequencing assay illustrated good sensitivity for the detection of resistance to RIF (96.92%), INH (86.89%), FQ (77.50%). The specificities of the assay were 98.35% for RIF, 99.20% for INH, and 97.26% for FQ.Conclusions: The sequencing assay is an efficient, accurate method for detection of MDR-TB and pre-XDR-TB from clinical smear-positive sputum specimens, should be considered as a supplemental method for obtaining early DST results before the availability of phenotypic DST results. This could be of benefit to early diagnosis, adjusting the treatment regimen and controlling transmission of drug-resistant TB. [ABSTRACT FROM AUTHOR]