Assessment of an Optimized Mycobacterial Interspersed Repetitive- Unit-Variable-Number Tandem-Repeat Typing System Combined with Spoligotyping for Population-Based Molecular Epidemiology Studies of Tuberculosis

Abstract

An optimized set of 24 mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) loci, including a discriminatory subset of 15 loci, has recently been defined for the typing of Mycobacterium tuberculosis. Here, we evaluated the performances of this MIRU-VNTR typing system in combination with spoligotyping for the detection of transmission chains in a population-based study comprising 91% of culture-confirmed tuberculosis patients reported in 2003 in Hamburg, Germany. Of the 154 isolates investigated, more than 90% had high IS6110 copy numbers (>6). IS6110 restriction fragment length polymorphism (RFLP) typing resulted in 13 clusters, 5 of which had a confirmed epidemiological link. All five, as well as six of the eight IS6110 clusters with no identified epidemiological link, were perfectly matched by MIRU-VNTR typing with the 24 loci. Two IS6110 clusters were split by differences into 6 to 12 MIRU-VNTR loci, clearly supporting the absence of a link, as judged by contact tracing data. In contrast, only one MIRU-VNTR cluster, grouping what were probably epidemiologically unlinked isolates, was split by IS6110 RFLP. However, these isolates were also distinguished by spoligotyping. Both the optimized 24-locus and 15-locus sets thus showed a comparable to slightly better predictive value, especially when combined with spoligotyping, than the current gold standard IS6110 RFLP for the study of tuberculosis transmission in Hamburg. Because the epidemiological characteristics of this setting are similar to those of many developed countries, these results support the wide applicability of this real-time genotyping approach for population-based studies of M. tuberculosis transmission.Population-based molecular epidemiological studies of tuberculosis (TB) aim to identify pathways and predictors of ongoing TB transmission by determining the proportion of patients with identical isolates (clustering rate), as defined by molecular typing at regional or national scales. IS6110 restriction fragment length polymorphism (RFLP) typing of Mycobacterium tuberculosis is the gold standard method for this purpose (30). Consistently, M. tuberculosis outbreak isolates often show identical IS6110 RFLP patterns, and clustering by identical patterns is associated with well-identified risk factors for transmission, whereas unrelated patients often have isolates with different IS6110 fingerprints (see, for example, references 6, 21, 31, and 33).However, IS6110 fingerprinting is laborious and requires weeks of M. tuberculosis culturing, which limits the possibilities to use typing prospectively for more efficient TB control. Furthermore, comparison of the fingerprints from large data sets requires highly standardized experimental and computerized procedures (30). These problems complicate the determination of clustering rates in population-based studies and the exchange of data.Typing based on PCR amplification of multiple loci containing mycobacterial interspersed repetitive-unit-variable-number tandem repeats (MIRU-...

Evaluation of Mycobacterium tuberculosis Typing Methods in a 4-Year Study in Schleswig-Holstein, Northern Germany

“…Not sur- prisingly, the IS6110 low-copy-number M. bovis cluster was clearly split by MIRU-VNTR typing (4,11,13).…”

“…In comparative populationbased evaluations, we and others found a similar if not slightly better performance of 15-or 24-loci MIRU-VNTR typing compared to IS6110 RFLP typing for discrimination of clinical isolates and detection of transmission chains in TB patient populations (13). Few subsequent studies compared MIRU-VNTR typing with the "gold standard" IS6110 RFLP typing, because the latter is rather laborious and more difficult to perform (6,7,15).…”

“…Furthermore, all isolates were analyzed by the spoligotyping technique described by Kamerbeek et al (10). For MIRU-VNTR genotyping, 24 loci were amplified by PCR analysis as described previously (13,16). Briefly, analyses were performed by using multiplex PCRs, the ROX-labeled MapMarker 1,000-bp-size standard (BioVentures, Inc., Murfreesboro, VT), and the ABI 3130xl sequencer with 16 capillaries (Applied Biosystems, Foster City, CA).…”

“…Taken on their own, all methods have their particular disadvantages: IS6110 fingerprinting is laborious, time-consuming, and requires highly standardizes procedures; spoligotyping has the lowest discrimination power to differentiate strains; and MIRU-VNTR remains a rather new procedure. Pilot population-based studies have shown that MIRU-VNTR's discriminatory power and predictive value for tracing TB transmission was close to that of IS6110 RFLP in some populations (1,13); however, this needs to be further evaluated in different settings. In any case, the combination of spoligotyping and MIRU-VNTR is likely to be the new gold standard method for large population-based studies due to their rapid and simple applicability (1,13).…”

Contribution of spoligotyping to the characterization of the population structure of Mycobacterium tuberculosis isolates in Portugal

“…Spoligotyping is highly reproducible and useful to broadly study population structure and attempt to identify main lineages, however it must be combined with other typing methods to be useful for identification of transmission chains and discriminate isolates up to the clonal level (Cowan et al, 2005;Oelemann et al, 2007).…”