Application of DNA fingerprinting has disclosed that the current worldwide tuberculosis epidemic is for a significant part determined by the worldwide spread of the Beijing family of M. tuberculosis
). Therefore, knowledge on the applicability of the recently described and increasingly used VNTR typing method to characterize and differentiate this group of strains is of the utmost importance. So far, limited information was available about the utility of VNTR typing to differentiate Beijing family isolates.
In this study, 69 M. tuberculosis
isolates from Hong Kong, 74% of which were members of the Beijing family, were analyzed by VNTR typing by using 14 loci (including ETRs, MIRUs, and QUBs), IS6110
RFLP typing, and spoligotyping. Overall, the correlation between the three sets of genetic markers was high. All strains that were defined as belonging to the Beijing genotype on the basis of spoligotyping (14
) showed the Beijing-characteristic multicopy IS6110
RFLP patterns (>60.2% similarity) described by van Soolingen et al. (39
) and related VNTR patterns (>55.5% similarity). Therefore, the three methods scored equally in recognizing Beijing strains. However, spoligotyping yielded the most unambiguous identification of Beijing genotypes in the fastest and easiest way. As expected, the ability to differentiate between the 51 Beijing isolates varied between the three typing techniques. IS6110
RFLP (48 types; HGI, 0.997) was slightly superior to VNTR (39 types; HGI, 0.982), and both were highly superior to spoligotyping (5 types; HGI, 0.253).
It should be noted that the relative discriminatory powers of VNTR and RFLP typing may vary, depending on the strain collection. In this study, six of the seven VNTR-defined clusters were subdivided by RFLP, and two (including one cluster of isolates containing one copy of IS6110
) of the three RFLP-defined clusters were split up by VNTR typing. The epidemiological significance of these differences is unknown because data on epidemiological linkage between patients was not available. As previously observed, at least some of the patterns differing by one to three IS6110
bands, but clustered by VNTR, may nevertheless correspond to epidemiologically related patients (11
). However, in this study, three VNTR clusters showed M. tuberculosis
Beijing isolates with IS6110
RFLP patterns that appeared too divergent to correspond to recent epidemiological links. This suggests that VNTR alone may overestimate recent transmission of M. tuberculosis
Beijing strains. Further studies with detailed patient information are needed to investigate the relation between clustering of VNTR patterns and recent transmission of tuberculosis in more detail. Nevertheless, because of the advantages of VNTR typing over RFLP, VNTR typing can be used as a first screening method followed by additional typing in case Beijing strains with identical VNTR patterns are found.
The allelic diversity of the VNTR loci differed significantly per locus. Among the loci investigated in this study, MIRU10, QUB11a, and QUB11b were the most discriminative among Beijing strains and ETR-B, ETR-D, and MIRU16 were the least discriminative for this group of strains. The most common VNTR patterns for the Beijing strains in this study were 8684 for the QUB loci; 33733 for MIRU loci 10, 16, 26, 39, and 40; and 42435 for ETR loci A to E. This ETR profile is also the most common among members of the W family, which accounted for large outbreaks of (MDR) tuberculosis in the United States (2
) and among strains in Russia (7
) and in various other countries (15
). For all but two of the VNTR loci, the allelic diversity per locus was lower among the Beijing strains, consistent with their close genetic relationship.
Recently, Spurgiesz et al. (30
) have subjected 34 M. tuberculosis
Beijing family strains representing 14 different IS6110
RFLP types to VNTR typing with nine novel loci with very short repeats, and they were able to discriminate 7 VNTR types among these. It would be interesting to investigate whether some of these novel loci would increase the combined discriminatory power of the VNTR set used in this study or those used in previous studies. Le Fleche et al. (17
) evaluated eight other loci and tested these on 90 M. tuberculosis
complex strains, including 5 Beijing isolates. However, these loci are not likely to contribute significantly to the discriminatory power of the combined VNTR loci, as these investigators found only little polymorphism in both Beijing and non-Beijing strains. Because different investigators have used different VNTR loci, the interlaboratory comparison of VNTR results is hampered. Ideally, VNTR typing should therefore be standardized internationally. In a standardized procedure, we envisage that a limited number of loci would be amplified as a first screening, followed by a secondary set of VNTR loci in case of clustered isolates or isolates belonging to certain genotypes.
Besides the utility of VNTR typing to discriminate Beijing strains, the utility of spoligotyping with five recently disclosed additional spacers in the direct repeat region (37
) was also investigated. Among the 49 Beijing isolates that exhibited the nine-spacer spoligotype in traditional spoligotyping, three types were discriminated by using the novel spacers. Combining the data from this study and that of Van Der Zanden et al. (35
), who also subjected Beijing strains to spoligotyping with novel spacers, at least seven spoligotypes can be found among Beijing lineage strains that show the nine-spacer spoligotype in traditional spoligotyping. Thus, these findings indicate that these new spacers will be useful in studying the phylogeny of Beijing strains with greater resolution (35
The percentage of Beijing strains that showed a spoligotype pattern consisting of less than nine spacers in traditional spoligotyping was comparable to that of another study conducted in Hong Kong (5
) (4 and 5%, respectively). In our study, the IS6110
RFLP patterns of the two strains containing fewer than nine spacers in their spoligo pattern contained a high number of bands and matched those of the Beijing family, the modern lineage of the Beijing phylogenetic tree (14
), suggesting that the corresponding deletions among the last nine spacers in the DR region of these strains were relatively recent events.
Especially for Beijing family strains, quick and easy simultaneous genotype recognition and epidemiologic typing is of the utmost importance, as these strains are often epidemic and MDR (3
). Our results indicate that VNTR typing can be used for epidemiological studies of Beijing strains. However, IS6110
RFLP should be used to subtype VNTR-defined clusters.