The mycobacterium growth indicator tube (MGIT960) automated liquid medium testing method is becoming the international gold standard for second-line drug susceptibility testing of multidrug-and extensively drug-resistant Mycobacterium tuberculosis complex isolates. We performed a comparative study of the current gold standard in the Netherlands, the Middlebrook 7H10 agar dilution method, the MGIT960 system, and the GenoType MTBDRsl genotypic method for rapid screening of aminoglycoside and fluoroquinolone resistance. We selected 28 clinical multidrug-and extensively drug-resistant M. tuberculosis complex strains and M. tuberculosis H37Rv. We included amikacin, capreomycin, moxifloxacin, prothionamide, clofazimine, linezolid, and rifabutin in the phenotypic test panels. For prothionamide and moxifloxacin, the various proposed breakpoint concentrations were tested by using the MGIT960 method. The MGIT960 method yielded results 10 days faster than the agar dilution method. For amikacin, capreomycin, linezolid, and rifabutin, results obtained by all methods were fully concordant. Applying a breakpoint of 0.5 g/ml for moxifloxacin led to results concordant with those of both the agar dilution method and the genotypic method. For prothionamide, concordance was noted only at the lowest and highest MICs. The phenotypic methods yielded largely identical results, except for those for prothionamide. Our study supports the following breakpoints for the MGIT960 method: 1 g/ml for amikacin, linezolid, and clofazimine, 0.5 g/ml for moxifloxacin and rifabutin, and 2.5 g/ml for capreomycin. No breakpoint was previously proposed for clofazimine. For prothionamide, a division into susceptible, intermediate, and resistant seems warranted, although the boundaries require additional study. The genotypic assay proved a reliable and rapid method for predicting aminoglycoside and fluoroquinolone resistance.The emergence of multidrug-resistant tuberculosis (MDR-TB) in the 1990s, and more recently, extensively drug resistant tuberculosis (XDR-TB), has revealed the need for new drugs and alternative, second-line treatment regimens. Many of these second-line drugs are either old drugs that had not been frequently used because of side effects or unproven efficacy or newer drugs intended primarily for treatment of other infections (3). Their use necessitated an evaluation of drug susceptibility testing (DST) and a determination of the critical concentrations of these alternative drugs.A variety of techniques are now available for second-line DST, of which the mycobacterium growth indicator tube automated liquid culture system (MGIT960) is probably the most used and best validated at this moment (13). The latest addition to second-line DST are genotypic methods, which detect mutations in the gyrA gene of Mycobacterium tuberculosis that are associated with fluoroquinolone resistance and mutations in the rrs operon that are associated with resistance to capreomycin and the aminoglycosides (2-5).Despite the arrival of these novel tools, many unce...