Discriminatory Power and Reproducibility of Novel DNA Typing Methods forMycobacterium tuberculosisComplex Strains

Abstract

In recent years various novel DNA typing methods have been developed which are faster and easier to perform than the current internationally standardized IS6110 restriction fragment length polymorphism typing method. However, there has been no overview of the utility of these novel typing methods, and it is largely unknown how they compare to previously published methods. In this study, the discriminative power and reproducibility of nine recently described PCR-based typing methods for Mycobacterium tuberculosis were investigated using the strain collection of the interlaboratory study of Kremer et al. (J. Clin. Microbiol. 37:2607-2618, 1999). This strain collection contains 90 M. tuberculosis complex and 10 non-M. tuberculosis complex mycobacterial strains, as well as 31 duplicated DNA samples to assess reproducibility. The highest reproducibility was found with variable numbers of tandem repeat typing using mycobacterial interspersed repetitive units (MIRU VNTR) and fast ligation-mediated PCR (FLiP), followed by second-generation spoligotyping, ligation-mediated PCR (LM-PCR), VNTR typing using five repeat loci identified at the Queens University of Belfast (QUB VNTR), and the Amadio speciation PCR. Poor reproducibility was associated with fluorescent amplified fragment length polymorphism typing, which was performed in three different laboratories. The methods were ordered from highest discrimination to lowest by the Hunter-Gaston discriminative index as follows: QUB VNTR typing, MIRU VNTR typing, FLiP, LM-PCR, and spoligotyping. We conclude that both VNTR typing methods and FLiP typing are rapid, highly reliable, and discriminative epidemiological typing methods for M. tuberculosis and that VNTR typing is the epidemiological typing method of choice for the near future.

Discordance between Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat Typing and IS 6110 Restriction Fragment Length Polymorphism Genotyping for Analysis of Mycobacterium tuberculosis Beijing Strains in a Setting of High Incidence of Tuberculosis

“…The inclusion of the three hypervariable loci improved the discriminatory power of the MIRU-VNTR genotyping method in this Beijing lineage, thereby supporting a previous suggestion for their inclusion (10). However, the use of these loci needs further evaluation in other evolutionary lineages, as difficulties associated with amplification reproducibility have been reported (15,21,36).…”

“…The inclusion of the three hypervariable loci improved the discriminatory power of the MIRU-VNTR genotyping method in this Beijing lineage, thereby supporting a previous suggestion for their inclusion (10). However, the use of these loci needs further evaluation in other evolutionary lineages, as difficulties associated with amplification reproducibility have been reported (15,21,36).We conclude that the PCR-based MIRU-VNTR genotyping method could be applied as an epidemiological tool to measure the performance of a TB control program over time in a defined geographical setting. However, the observed concordance in the estimate of recent and ongoing transmission when using the IS6110 RFLP or MIRU-VNTR genotyping methods was only coincidental.…”

“…In their study they showed that VNTR loci 3820, 3232, and 4120 were highly polymorphic in Beijing genotype strains and thus proposed the use of these loci to enhance the discriminatory power of the proposed 15-MIRU-VNTR genotyping method. However, other studies have excluded these loci due to difficulties associated with the reproducibility of PCR amplification (15,21,36).Subsequently, a study in Hong Kong, which also examined strains of the Beijing genotype, showed that a different combination of 12 VNTR and QUB (Queen's University of Belfast) loci gave a Hunter-Gaston discriminatory index value which was almost equal to that obtained in IS6110 RFLP genotyping (12, 13). However, this was refuted by a more recent study from China which suggested that MIRU-VNTR genotyping may overestimate transmission in isolates with the Beijing genotype (11).…”

“…In their study they showed that VNTR loci 3820, 3232, and 4120 were highly polymorphic in Beijing genotype strains and thus proposed the use of these loci to enhance the discriminatory power of the proposed 15-MIRU-VNTR genotyping method. However, other studies have excluded these loci due to difficulties associated with the reproducibility of PCR amplification (15,21,36).…”

Molecular Epidemiology of Multidrug-ResistantMycobacterium bovisIsolates with the Same Spoligotyping Profile as Isolates from Animals

“…PCR-based characterization techniques such as spoligotyping (15) and mycobacterial interspersed repetitive unit (MIRU) variable-number tandem repeat (VNTR) typing (9,31) have been adopted in most laboratories for M. bovis typing because they are faster, simpler, and cost-effective, with the added advantage that results are qualitative (present or absent or in the form of a number). The degree of differentiation and reproducibility is good, but spoligotyping has a lower discriminatory power than MIRU-VNTR (1,16).…”