Mycobacterial Interspersed Repetitive Unit Typing ofMycobacterium tuberculosisCompared to IS6110-Based Restriction Fragment Length Polymorphism Analysis for Investigation of Apparently Clustered Cases of Tuberculosis

Abstract

An evaluation of the utility of IS6110-based restriction fragment length polymorphism (RFLP) typing compared to a combination of variable number tandem repeat (VNTR) typing and mycobacterial interspersed repetitive unit (MIRU) typing was undertaken. A total of 53 patient isolates of Mycobacterium tuberculosis from four presumed episodes of cross-infection were examined. Genomic DNA was extracted from the isolates by a cetyl trimethylammonium bromide method. The number of copies of tandem repeats of the five loci ETR A to ETR E and 12 MIRU loci was determined by PCR amplification and agarose gel electrophoresis of the amplicons. VNTR typing identified the major clusters of strains in the three investigations in which they occurred (each representing a different evolutionary clade: 32333, 42235, and 32433). The majority of unrelated isolates (by epidemiology and RFLP typing) were also identified by VNTR typing. The concordance between the RFLP and MIRU typing was complete, with the exception of two isolates with RFLP patterns that differed by one band each from the rest of the major epidemiologically linked groups of isolates in investigation A. All of these isolates had identical MIRU and VNTR types. A further pair of isolates differed in the number of tandem repeat copies at two MIRU alleles but had identical RFLP patterns. The speed of the combined VNTR and MIRU typing approach enabled results for some of the investigations to be supplied in "real time," influencing choices in contact tracing. The ease of comparison of results of MIRU and VNTR typing, which are recorded as single multidigit numbers, was also found to greatly facilitate investigation management and the communication of results to health care professionals.It is widely accepted that tuberculosis is one of the most important threats to human health on a global scale. Infection with Mycobacterium tuberculosis complex causes the greatest number of deaths by a single infectious agent, and it has been estimated that approximately one-third of the world population has latent infections (9). While conventional contact tracing and treatment of contacts remain important components of control, the ability to ascribe a subtype to infecting strains is a powerful adjunct in recognizing unsuspected sources and chains of transmission. There is therefore an undoubted need for a reproducible, discriminatory, epidemiologically relevant, easy, and portable typing method. The relative merits and drawbacks of the range of molecular typing methods have recently been reviewed (6,32), and the problems with the most established typing method, IS6110-based restriction fragment length polymorphism (RFLP), have been discussed. Although this method has been widely used and is referred to as a "gold standard" (18), it is labor-intensive, requires culture, and cannot type strains with low copy numbers (Ն5) of IS6110). Possibly the biggest drawback to the method is that the data are generated as an analogue band pattern. This presents difficulties for reproducible analysis of b...

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

“…We found that spoligotyping missed two IS6110-identical pairs and that MIRU-VNTR typing failed to detect about half of the IS6110-identical matches. These results concur with observations about spoligotyping (6, 7) but appear to contrast with earlier reports about MIRU-VNTR, which suggested that patterns are generally identical within epidemiologically linked clusters (8). On further examination, we noted that for the discordant pairs, most IS6110-defined clusters had only one MIRU-VNTR locus difference between them, and spoligotypes were either identical or differed by one spacer.…”

“…The major advantage of rapid typing of isolates is the capacity to immediately provide a strain designation, which in turn may facilitate prompt public health intervention. While the technical feasibility of these methods has been well demonstrated (12,26), their public health utility remains to be determined.To date, studies of small outbreaks have shown that results from spoligotyping and MIRU-VNTR typing are often identical among isolates clustered by IS6110-based RFLP (6,7,8,11,15,16,18,23). However, the capacity of these modalities to provide unique patterns among unrelated isolates (i.e., their specificities) in a population-based study has not yet been evaluated.…”

“…To date, studies of small outbreaks have shown that results from spoligotyping and MIRU-VNTR typing are often identical among isolates clustered by IS6110-based RFLP (6,7,8,11,15,16,18,23). However, the capacity of these modalities to provide unique patterns among unrelated isolates (i.e., their specificities) in a population-based study has not yet been evaluated.…”

Use of an Automated Multiple-Locus, Variable-Number Tandem Repeat-Based Method for Rapid and High-Throughput Genotyping of Staphylococcus aureus Isolates

“…This property is likely due to the selection of short-term and long-term markers among the genes targeted in the multiplex reaction. These targets conferred a high resolution power, as reported for other bacterial species (14,29,39); reliability for the analysis of strains from outbreaks (14); and also reliability for examination of the phylogeny of geographically and temporally separated strains (35). Our results and those obtained by others suggest that the evolutionary frequency of VNTR-containing genes is also adapted to the study of transmission events.…”

Use of Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing To Examine Genetic Diversity ofMycobacterium tuberculosisin Singapore

“…An analysis of different groups of epidemiologically linked M. tuberculosis isolates has indicated that the stability of the MIRU-VNTR loci is adequate for the tracking of outbreak episodes, identification of laboratory cross-contamination, and discrimination between relapses and reinfections (12,18,23).…”