Comparison of Methods Based on Different Molecular Epidemiological Markers for Typing of Mycobacterium tuberculosis Complex Strains: Interlaboratory Study of Discriminatory Power and Reproducibility

Abstract

In this study, the currently known typing methods forMycobacterium tuberculosis isolates were evaluated with regard to reproducibility, discrimination, and specificity. Therefore, 90 M. tuberculosis complex strains, originating from 38 countries, were tested in five restriction fragment length polymorphism (RFLP) typing methods and in seven PCR-based assays. In all methods, one or more repetitive DNA elements were targeted. The strain typing and the DNA fingerprint analysis were performed in the laboratory most experienced in the respective method. To examine intralaboratory reproducibility, blinded duplicate samples were included. The specificities of the various methods were tested by inclusion of 10 non-M. tuberculosis complex strains. All five RFLP typing methods were highly reproducible. The reliability of the PCR-based methods was highest for the mixed-linker PCR, followed by variable numbers of tandem repeat (VNTR) typing and spoligotyping. In contrast, the double repetitive element PCR (DRE-PCR), IS6110 inverse PCR, IS6110 ampliprinting, and arbitrarily primed PCR (APPCR) typing were found to be poorly reproducible. The 90 strains were best discriminated by IS6110 RFLP typing, yielding 84 different banding patterns, followed by mixed-linker PCR (81 patterns), APPCR (71 patterns), RFLP using the polymorphic GC-rich sequence as a probe (70 patterns), DRE-PCR (63 patterns), spoligotyping (61 patterns), and VNTR typing (56 patterns). We conclude that for epidemiological investigations, strain differentiation by IS6110 RFLP or mixed-linker PCR are the methods of choice. A strong association was found between the results of different genetic markers, indicating a clonal population structure of M. tuberculosis strains. Several separate genotype families within the M. tuberculosis complex could be recognized on the basis of the genetic markers used.

Identifying locations of recent TB transmission in rural Uganda: a multidisciplinary approach

“…Misclassification of cases as unique that are truly genotype-clustered is more likely to occur with a short duration of enrolment, such as our 1-year study period [34]. Misclassification of genotype-clustered cases that are truly unique also occurs more frequently with spoligotyping compared to more discriminating genotyping methods, such as whole-genome sequencing or restriction fragment polymorphism (RFLP) analysis [35]. However, misclassification by spoligotyping is less likely with the TB lineages found in this community, compared to other lineages, such as the Beijing strain [36].…”

Optimization of variable number tandem repeat typing set for differentiating Mycobacterium tuberculosis strains in the Beijing family

“…In this study, when 355 drug resistant Mycobacterium tuberculosis isolates (68.5% Beijing) were analysed, a combination of 12 VNTR loci, i.e. the same number of loci used as MIRU typing in various studies (Kremer et al, 1999;Supply et al, 2000;Supply et al, 2001), namely MIRU 10, 26, 39, 40;QUB 11a, 11b, 15, 18, 26, 3232, 3336 and ETR A, produced a comparable level of resolution (HGI = 0.9975) as IS6110 RFLP (HGI = 0.9979). Apart from the Beijing genotype, this 12-loci VNTR set also appeared to possess good differentiation power for non-Beijing M. tuberculosis (HGI = 0.9982) when compared with the 12 MIRU typing (HGI 0.9928) (data not shown).…”

Tracing isolates of bacterial species by multilocus variable number of tandem repeat analysis (MLVA)

“…The species is relatively young and specific methods for typing have been developed over the years. These include IS6110 hybridization tests and spoligotyping (Van Embden et al, 1993;Kremer et al, 1999). However, the advent of MLVA systems has led to a number of important initiatives and various detailed studies were undertaken (Frothingham & Meeker-O'Connell, 1998;Supply et al, 2000Supply et al, , 2001Mazars et al, 2001;Cowan et al, 2002;Le Fleche et al, 2002;Skuce et al, 2002;Spurgiesz et al, 2003).…”