Sensitivities and Specificities of Spoligotyping and Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing Methods for Studying Molecular Epidemiology of Tuberculosis

Abstract

The development of PCR-based genotyping modalities (spoligotyping and mycobacterial interspersed repetitive unit-variable-number tandem repeat [MIRU-VNTR] typing) offers promise for real-time molecular epidemiological studies of tuberculosis (TB). However, the utility of these methods depends on their capacity to appropriately classify isolates. To determine the operating parameters of spoligotyping and MIRU-VNTR typing, we have compared results generated by these newer tests to the standard typing method, IS6110 restriction fragment length polymorphism, in analyses restricted to high-copy-number IS6110 isolates. Sensitivities of the newer tests were estimated as the percentages of isolates with identical IS6110 fingerprints that had identical spoligotypes and MIRU-VNTR types. The specificities of these tests were estimated as the percentages of isolates with unique IS6110 fingerprints that had unique spoligotypes and MIRU-VNTR types. The sensitivity of MIRU-VNTR typing was 52% (95% confidence interval [CI], 31 to 72%), and the sensitivity of spoligotyping was 83% (95% CI, 63 to 95%). The specificity of MIRU-VNTR typing was 56% (95% CI, 51 to 62%), and the specificity of spoligotyping was 40% (95% CI, 35 to 46%). The proportion of isolates estimated to be due to recent transmission was 4% by identical IS6110 patterns, 19% by near-identical IS6110 patterns, 33% by MIRU-VNTR typing, and 53% by spoligotyping. The low calculated specificities of spoligotyping and MIRU-VNTR typing led to misclassification of cases, inflated estimates of TB transmission, and low positive predictive values, suggesting that these techniques have unsuitable operating parameters for population-based molecular epidemiology studies.Tuberculosis (TB) molecular epidemiology exploits selected bacterial DNA targets to serve as markers for Mycobacterium tuberculosis strains. The most common method of DNA fingerprinting used is IS6110-based restriction fragment length polymorphism (RFLP). In a number of studies over the past decade, this modality has been validated for tracking TB transmission through two sets of observations. First, isolates from epidemiologically linked patients generally share identical or similar patterns (2, 3, 5). Second, matched RFLP patterns, when occurring among patients without known epidemiological links, are generally observed within groups with clear risk factors for TB transmission (1, 10, 13, 24).An important practical limitation of IS6110 RFLP is that results are usually obtained weeks to months after the initial diagnosis of TB. This limitation stems from the need to grow large numbers of bacteria to extract DNA of sufficient quantity and quality for RFLP analysis. Therefore, while useful for documenting transmission events, IS6110 RFLP often provides data once outbreaks are well established. In contrast, a number of PCR-based typing modalities have been recently developed, including spoligotyping and mycobacterial interspersed repetitive unit-variable-number tandem repeat analysis (MIRU-VNTR) typing, which offer...

Genetic profiling of Mycobacterium tuberculosis in Tunisia: predominance and evidence for the establishment of a few genotypes

“…Thus it is very likely that the high level of transmission of this genotype may have contributed to its predominance. Although most of the clustered cases were epidemiologically supported, one should not dismiss the possibility that some patients may not be part of a recent transmission chain, since, as previously shown, the Spol-MIRU12 combination used in this study could lead to misclassification (Scott et al, 2005). By contrast, the ST42 genotype displayed low clustering, was more widespread than ST50, and was found to be predominant in the sample from 1990, accounting for 35.4 % (11 out of 31) of TB cases.…”

Multidrug-Resistant Tuberculosis Outbreak among US-bound Hmong Refugees, Thailand, 2005

“…Second, 20 (87.0%) of 23 MDR TB isolates were strains that matched by 3 different molecular subtyping methods. It is possible that 2 additional isolates (22 total) were part of the outbreak as well because they matched the outbreak strain according to spoligotyping and RFLP results and differed by only 1 locus according to MIRU; therefore, this difference may represent a change in genotype in the same clone over time (26). Third, elevated rates of tuberculin reactivity in household contacts of smear-positive patients suggest substantial recent transmission, not simply endemic disease.…”

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

“…Consistent with a generally lower rate of change of these 12-locus patterns than that of IS6110-RFLP (15,18,25), secondary IS6110 fingerprinting of the isolates clustered by this combined strategy is nevertheless necessary to reach the maximal resolution needed in some situations (4,18). However, another investigation suggested that this MIRU-VNTR typing combined with spoligotyping was inadequate for population-based studies because it provided a too-low resolution power and, more surprisingly, because MIRU-VNTR genotypes were apparently too frequently unstable among presumably related isolates (19). However, these parameters were determined by taking clustering defined by IS6110-RFLP as a sole reference, in the absence of any contact tracing data.…”

“…However, these parameters were determined by taking clustering defined by IS6110-RFLP as a sole reference, in the absence of any contact tracing data. Since it is known that IS6110-RFLP does not per se unambiguously define all related or unrelated isolates (see, for example, references 29, 32, and 34), unequivocal interpretation of all of the divergences between the typing methods in that study (19) was not possible. In a recent study based on an worldwide collection of tubercle bacillus isolates (24), we defined an optimized set of 24 MIRU-VNTR loci, including a subset of 15 discriminatory loci proposed to be used as a first-line typing method.…”