Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis

Abstract

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.

FIG. 1.

Discrimination by MIRU-VNTR typing alone or in combination with spoligotyping in a collection of isolates from cosmopolitan origins. The number of types distinguished among 494 isolates of the M. tuberculosis complex (A) and the corresponding clustering rates (B) obtained by different combinations of markers are shown on the y axes. Because of their particular population structure characterized by homogeneous clonal groups with horizontal gene transfers among them (15), M. prototuberculosis isolates were not considered for this analysis. Diamonds and squares correspond to values obtained using MIRU-VNTR (MV) alone and in combination with spoligotyping (spol), respectively. The composition of the different MIRU-VNTR sets is given in the inset table; 24, full set of 24 robust loci; 15, 15-locus discriminatory subset; Old12, original 12 MIRU-VNTR loci; 9, minimal set resulting in maximal resolution in the standardized collection of 90 isolates (see text).

FIG. 2.

Minimum spanning tree based on MIRU-VNTR relationships among tubercle bacilli. Circles correspond to the different types identified by the set of 24 loci among 494 M. tuberculosis complex isolates from cosmopolitan origins and 35 M. prototuberculosis isolates (as reference) and are proportional to the number of clustered isolates with an identical MIRU-VNTR type. The corresponding species names and spoligotype family designations (except T types) are indicated. Linkages by a single, double, or triple locus variation are boldfaced. EAI, East African-Indian; LAM, Latin American-Mediterranean; CAS, Central Asian; S, S spoligotype family; X, X spoligotype family.

FIG. 3.

Distribution of single, double, or triple-locus variations in 24 MIRU-VNTR loci among isolates from cosmopolitan origins. Events detected among 494 isolates from widespread geographic origins of the M. tuberculosis complex are shown. Because of their particular population structure, characterized by homogeneous clonal groups with horizontal gene transfers among them (15), M. prototuberculosis isolates were not considered for this analysis. eai, East African-Indian; lam, Latin American-Mediterranean; ory, M. bovis from oryxes; S, S spoligotype family; X, X spoligotype family; bcg, M. bovis BCG; h, Haarlem; mic, Microbacterium microti; sea, M. bovis from seals; cas, Central Asian; bov, classical M. bovis; T, T spoligotypes; bj, Beijing (including W); ug, Uganda; afr, M. africanum; gha, Ghana; cam, Cameroon; cap, Mycobacterium caprae.

FIG. 4.

IS6110 RFLP, spoligotype, and MIRU-VNTR patterns of the M. tuberculosis isolates from 125 patients in 42 IS6110-PGRS clusters, as assigned to four transmission groups (for explanation of transmission groups, see text). Designations of MIRU-VNTR loci are given according to the position (in kilobase pairs) on the M. tuberculosis H37Rv chromosome. Alias designations are in parentheses. Spoligo, spoligotyping. Results of IS6110-RFLP, spoligotyping, and MIRU-VNTR loci from VNTR 424 to 4156 were taken from reference ; results for VNTR 2165 (ETR A), VNTR 1955, 2163b (QUB-11b), and 4052 (QUB-26) are from this study. Differences in MIRU-VNTR patterns among IS6110-PGRS RFLP clustered isolates are boxed. For IS6110-PGRS RFLP cluster 37, the isolate of the patient from TG3 is compared with the isolate of the first patient from TG4, and they thus differ by an SLV in MIRU-VNTR locus 4052. Likewise, for IS6110-PGRS RFLP cluster 38, the isolates of the two patients of category TG3 differ by a four-locus variation (in loci 2165, 1955, 2163b, and 4052).