Comparative Study of IS 6110 Restriction Fragment Length Polymorphism and Variable-Number Tandem-Repeat Typing of Mycobacterium tuberculosis Isolates in the Netherlands, Based on a 5-Year Nationwide Survey

Abstract

mIn order to switch from IS6110 and polymorphic GC-rich repetitive sequence (PGRS) restriction fragment length polymorphism (RFLP) to 24-locus variable-number tandem-repeat (VNTR) typing of Mycobacterium tuberculosis complex isolates in the national tuberculosis control program in The Netherlands, a detailed evaluation on discriminatory power and agreement with findings in a cluster investigation was performed on 3,975 tuberculosis cases during the period of 2004 to 2008. The level of discrimination of the two typing methods did not differ substantially: RFLP typing yielded 2,733 distinct patterns compared to 2,607 in VNTR typing. The global concordance, defined as isolates labeled unique or identically distributed in clusters by both methods, amounted to 78.5% (n ‫؍‬ 3,123). Of the remaining 855 cases, 12% (n ‫؍‬ 479) of the cases were clustered only by VNTR, 7.7% (n ‫؍‬ 305) only by RFLP typing, and 1.8% (n ‫؍‬ 71) revealed different cluster compositions in the two approaches. A cluster investigation was performed for 87% (n ‫؍‬ 1,462) of the cases clustered by RFLP. For the 740 cases with confirmed or presumed epidemiological links, 92% were concordant with VNTR typing. In contrast, only 64% of the 722 cases without an epidemiological link but clustered by RFLP typing were also clustered by VNTR typing. We conclude that VNTR typing has a discriminatory power equal to IS6110 RFLP typing but is in better agreement with findings in a cluster investigation performed on an RFLPclustering-based cluster investigation. Both aspects make VNTR typing a suitable method for tuberculosis surveillance systems. DNA fingerprinting of Mycobacterium tuberculosis isolates has been applied for the investigation of epidemiological links between tuberculosis (TB) cases in several countries since DNA typing techniques were developed in the early 1990s (1-4). In The Netherlands, IS6110 restriction fragment length polymorphism (RFLP) typing was used nationwide from 1993 until the end of 2008. Although RFLP typing revolutionized studies on the transmission of M. tuberculosis, the method remained technically demanding and time consuming. The analysis of the complex IS6110 RFLP banding pattern requires a sophisticated computer application and a high degree of expertise (5). Moreover, in the Netherlands, it took on average 44 days to the deliver the RFLP typing result to the Municipal Health Services, after the isolate had reached the laboratory (D. van Soolingen, personal communication). This major drawback significantly limited the usefulness of DNA fingerprinting in routine examination of transmission, as it in fact only offered retrospective confirmation of suspected epidemiological links. In 2006, 24-locus variable-number tandemrepeat (VNTR) typing was favorably evaluated and proposed as the new gold standard for typing of M. tuberculosis (6). In order to switch from RFLP to VNTR typing in 2008 in the Netherlands, it was considered necessary to establish a retrospective VNTR pattern database of possible sources of infection for a pr...

Diversity and Evolution of Mycobacterium tuberculosis: Moving to Whole-Genome-Based Approaches

“…As only scarce single-locus variations were observed among such clonal isolates, a cluster suggestive of clonal transmission is defined based on strict conservation of the 24 alleles among clinical isolates. Such cluster analysis based on 24 loci, optionally combined with spoligotyping, was shown to provide a close to similar predictive value as IS6110 fingerprinting for tracing TB transmission in several Western Europe countries or settings (e.g., Oelemann et al 2007;Allix-Beguec et al 2008a;Bidovec-Stojkovic et al 2011;Roetzer et al 2011;de Beer et al 2013) (Fig. 3).…”

Optimization of Standard In-House 24-Locus Variable-Number Tandem-Repeat Typing for Mycobacterium tuberculosis and Its Direct Application to Clinical Material

“…Compared to the level of discrimination of the formerly used restriction fragment length polymorphism (RFLP) typing method, that of 24-locus VNTR typing has proven to be sufficient to trace the transmission of tuberculosis (TB) in low-burden settings (1,2). The advantages of VNTR typing over RFLP typing include simplified comparison of the results and applicability to small amounts of DNA, by which the turnaround time decreased from an average of 44 days to 15 days at our laboratory.…”

Transmission and Progression to Disease of Mycobacterium tuberculosis Phylogenetic Lineages in The Netherlands

“…In Western countries, studies on transmission are more feasible, as all cases undergo extended diagnostic algorithms and all clinical and demographic data are recorded. Current molecular typing methods, such as variable number of tandem repeat (VNTR) typing and restriction fragment length polymorphism (RFLP) typing, allow identification of clusters of Mycobacterium tuberculosis isolates with identical genotypes that, in population-based studies, reveal recent transmission (2,3). Spoligotyping and VNTR typing can identify the genotype family of the isolate, revealing bacterial variation via the identification of phylogenetic lineages (4,5).…”