Three-year population-based evaluation of standardized mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing of Mycobacterium tuberculosis

Abstract

Standardized mycobacterial interspersed repetitive-unit-variable-number tandem repeat (MIRU-VNTR) typing based on 15 and 24 loci recently has been proposed for Mycobacterium tuberculosis genotyping. So far, this optimized system has been assessed in a single, 1-year population-based study performed in Germany (M. C. Oelemann, R. Diel, V. Vatin, W. Haas, S. Rusch-Gerdes, C. Locht, S. Niemann, and P. Supply, J. Clin. Microbiol. 45:691-697, 2007). Here, we evaluated these optimized formats in a much larger population-based study conducted during 39 months in the Brussels capital region of Belgium. Isolates from 807 patients were genotyped. The resolution power, cluster, and lineage identification by the standardized MIRU-VNTR sets were compared to those obtained using standardized IS6110-restriction fragment length polymorphism (RFLP), spoligotyping, and a previous 12-MIRU-VNTR-locus set. On a subset representing 77% of the cases during a 16-month period, a high concordance was observed between unique isolates or strain clusters as defined by standardized MIRU-VNTR and IS6110-RFLP (i.e., more than five IS6110 bands). When extended to the entire population-based collection, the discriminatory subset of 15 loci decreased the strain-clustering rate by almost twofold compared to that of the old 12-locus set. The addition of the nine ancillary MIRU-VNTR loci and/or spoligotyping only slightly further decreased this strain-clustering rate. Familial, social, and/or geographic proximity links were found in 48% of the clusters identified, and well-known risk factors for tuberculosis transmission were identified. Finally, an excellent correspondence was determined between our MIRU-VNTR-spoligotyping strain identifications and external reference strain lineages included in the MIRU-VNTRplus database and identified by, e.g., large sequence polymorphisms. Our results reinforce the proposal of standardized MIRU-VNTR typing as a new reference genotyping method for the epidemiological and phylogenetic screening of M. tuberculosis strains.

FIG. 1.

Concordance observed between MIRU-VNTR groupings and spoligotype assignations among the 446 isolates from well-defined lineages. MIRU-VNTR groupings are visualized by a dendrogram calculated using a neighbor-joining algorithm and rooted using an “M. prototuberculosis” C/D genotype (20). Asterisks indicate examples in which the MIRU-VNTR groupings confirmed the genetic classifications that initially were presumed on the basis of spoligotypes sharing only partial spoligoprototype signatures.

FIG. 2.

Distribution of major M. tuberculosis lineages according to patients' regions of origin. Asterisks indicate statistically significant associations (P < 0.001).

FIG. 3.

Distribution of branches of the Euro-American M. tuberculosis superlineage according to patients' regions of origin. Asterisks indicate statistically significant associations, and respective P values are indicated.