Evaluation and strategy for use of MIRU-VNTRplus, a multifunctional database for online analysis of genotyping data and phylogenetic identification of Mycobacterium tuberculosis complex isolates

Abstract

Because of its portable data, discriminatory power, and recently proposed standardization, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing has become a major method for the epidemiological tracking of Mycobacterium tuberculosis complex (MTBC) clones. However, no public MIRU-VNTR database based on well-characterized reference strains has been available hitherto for easy strain identification. Therefore, a collection of 186 reference strains representing the primary MTBC lineages was used to build a database, which is freely accessible at http://www.MIRU-VNTRplus.org. The geographical origin and the drug susceptibility profile of each strain were stored together with comprehensive genetic lineage information, including the 24-locus MIRU-VNTR profile, the spoligotyping pattern, the single-nucleotide- and large-sequence-polymorphism profiles, and the IS6110 restriction fragment length polymorphism fingerprint. Thanks to flexible import functions, a single or multiple user strains can be analyzed, e.g., for lineage identification with or without the use of reference strains, by best-match or tree-based analyses with single or combined marker data sets. The results can easily be exported. In the present study, we evaluated the database consistency and various analysis parameters both by testing the reference collection against itself and by using an external population-based data set comprising 629 different strains. Under the optimal conditions found, lineage predictions based on typing by 24-locus MIRU-VNTR analysis optionally combined with spoligotyping were verified in >99% of the cases. On the basis of this evaluation, a user strategy was defined, which consisted of best-match analysis followed, if necessary, by tree-based analysis. The MIRU-VNTRplus database is a powerful tool for high-resolution clonal identification and has little equivalent in terms of functionalities among the bacterial genotyping databases available so far.

FIG. 1.

Typical output of a best-match analysis. Visualization of the MIRU-VNTR copy numbers and spoligotype patterns of a test isolate and its best matches among the reference strain collection is presented along with information on the best-match reference strains, including the strain lineage, country of isolation, MIRU-VNTR type, SNP and RD types, and SpolDB4 shared type and lineage classification. Differences in genotype profiles between the test strain and strains in the reference database are highlighted in red for better recognition.

FIG. 2.

Phylogenetic distribution of isolates with unassigned or T spoligotypes. A radial tree was constructed by using the 24-locus MIRU-VNTR typing data for the reference strains in the MIRU-VNTRplus database and 187 genotypes with unassigned or T spoligotypes from the Brussels population-based collection by using the neighbor-joining algorithm and categorical distance coefficient. The positions of Brussels genotypes with unassigned or T spoligotypes are indicated by yellow circles. Lineages containing exclusively T-spoligotype strains or lineages corresponding to well-characterized, classical clades of the Euro-American superlineage (such as the Haarlem lineage) comprising T-spoligotype strains are denoted T-specific and T-aspecific, respectively. EAI, East African-Indian; West Afri 2, West African 2 (M. africanum 2); West Afri 1, West African 1 (M. africanum 1); Tur, Turkish; BJ, Beijing; CAS, Central Asian; LAM, Latin American-Mediterranean.

FIG. 3.

Strategy for use of phylogenetic identification. If no match is detected after an initial best-match analysis with a stringent distance cutoff of 0.17, tree-based analysis is preferred upon best-match analysis with a relaxed distance cutoff of 0.3 as a second step, because the former method minimizes the fine-tuned mismatching that can occur as an exception among strains belonging to the Euro-American superlineage (see text). NJ, neighbor-joining.