Molecular typing based on 12 loci containing variable numbers of tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTRs) has been adopted in combination with spoligotyping as the basis for large-scale, high-throughput genotyping of Mycobacterium tuberculosis. However, even the combination of these two methods is still less discriminatory than IS6110 fingerprinting. Here, we define an optimized set of MIRU-VNTR loci with a significantly higher discriminatory power. The resolution and the stability/robustness of 29 loci were analyzed, using a total of 824 tubercle bacillus isolates, including representatives of the main lineages identified worldwide so far. Five loci were excluded for lack of robustness and/or stability in serial isolates or isolates from epidemiologically linked patients. The use of the 24 remaining loci increased the number of types by 40%-and by 23% in combination with spoligotyping-among isolates from cosmopolitan origins, compared to those obtained with the original set of 12 loci. Consequently, the clustering rate was decreased by fourfold-by threefold in combination with spoligotyping-under the same conditions. A discriminatory subset of 15 loci with the highest evolutionary rates was then defined that concentrated 96% of the total resolution obtained with the full 24-locus set. Its predictive value for evaluating M. tuberculosis transmission was found to be equal to that of IS6110 restriction fragment length polymorphism typing, as shown in a companion population-based study. This 15-locus system is therefore proposed as the new standard for routine epidemiological discrimination of M. tuberculosis isolates and the 24-locus system as a high-resolution tool for phylogenetic studies.The genotyping of Mycobacterium tuberculosis isolates contributes to tuberculosis (TB) control by, e.g., indicating possible epidemiological links between TB patients, detecting (un)suspected outbreaks and laboratory cross-contamination, and distinguishing exogenous reinfection from endogenous reactivation in relapse cases. For these purposes, IS6110 restriction fragment length polymorphism (RFLP) typing (48) has been used as the gold standard method for more than a decade. However, this method is labor-intensive, requires weeks for culturing the isolates and subsequent DNA purification, and suffers from problems of interpretability and portability of the complex banding patterns. In addition, it provides insufficient discrimination among isolates with low (Ͻ6) IS6110 copy numbers, a problem that is only partly overcome by using PCR-based spoligotyping as a secondary method (6).Genotyping based on variable numbers of tandem repeats (VNTRs) of different classes of interspersed genetic elements named mycobacterial interspersed repetitive units (MIRUs) (12,25,32,36,40,43,44) is increasingly used to solve these problems. This method relies on PCR amplification of multiple loci using primers specific for the flanking regions of each repeat locus and on the determination of the sizes of the amplicons...

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