['Conventional culture-based drug susceptibility testing (DST) for the second-line antituberculosis drugs is slow, leading to diagnostic delay with associated exacerbation of transmission, amplification of resistance, and increased mortality.', 'To assess the diagnostic performance of the GenoType MTBDRsl line probe assay (LPA) for the rapid detection of mutations conferring resistance to ofloxacin (OFX), amikacin (AMK), and ethambutol and to determine the impact of implementation on the turnaround time in a high-throughput diagnostic laboratory.', 'Six hundred and fifty-seven direct patient acid-fast bacilli smear-positive specimens resistant to isoniazid, rifampin, or both according to the GenoType MTBDRplus assay were consecutively tested, using the GenoType MTBDRsl LPA. The diagnostic performance was assessed relative to the "gold standard" culture-based method, and the laboratory turnaround times for both methods were determined.', 'A total of 516 of 657 patient specimens had valid results for both tests. The sensitivity for detecting OFX, AMK, and extensive drug resistance, using the GenoType MTBDRsl LPA, was 90.7% (95% confidence interval [CI], 80.1-96.0%), 100% (95% CI, 91.8-100%), and 92.3% (95% CI, 75.9-97.9%), respectively, and the specificity for detection was 98.1% (95% CI, 96.3-99.0%), 99.4% (95% CI, 98.2-99.8%), and 99.6% (95% CI, 98.5-99.9%), respectively. Implementation of this test significantly reduced the turnaround time by 93.3% (P < 0.001), calculated from the date that the specimen was received at the laboratory to reporting second-line results. In addition, a significant increase in diagnostic yield of 20.1% and 19.3% (P < 0.001) for OFX and AMK resistance, respectively, was obtained for isolates that were either contaminated or had lost viability.'] Does genotype MTBDRsl line probe assay shorten time to diagnosis of extensively drug-resistant tuberculosis in a high-throughput diagnostic laboratory?