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Volume 15, Number 10–October 2009
Research
Discriminatory Ability of Hypervariable Variable Number Tandem Repeat Loci in Population-based Analysis of Mycobacterium tuberculosis Strains, London, UK
Preya Velji,1 Vladyslav Nikolayevskyy,1 Timothy Brown, and Francis Drobniewski
Author affiliations: Barts and The London School of Medicine, Queen Mary, University of London, London, UK (P. Velji, V. Nikolayevskyy); and Health Protection Agency National Mycobacterium Reference Laboratory, London (T. Brown, F. Drobniewski)

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Abstract
To address conflicting results about the stability of variable number tandem repeat (VNTR) loci and their value in prospective molecular epidemiology of Mycobacterium tuberculosis, we conducted a large prospective population-based analysis of all M. tuberculosis strains in a metropolitan setting. Optimal and reproducible conditions for reliable PCR and fragment analysis, comprising enzymes, denaturing conditions, and capillary temperature, were identified for a panel of hypervariable loci, including 3232, 2163a, 1982, and 4052. A total of 2,261 individual M. tuberculosis isolates and 265 sets of serial isolates were analyzed by using a standardized 15-loci VNTR panel, then an optimized hypervariable loci panel. The discriminative ability of loci varied substantially; locus VNTR 3232 varied the most, with 19 allelic variants and Hunter-Gaston index value of 0.909 unNN. Hypervariable loci should be included in standardized panels because they can provide consistent comparable results at multiple settings, provided the proposed conditions are adhered to.

Globally, tuberculosis (TB) accounts for almost 2 million deaths each year (1). Although TB notification rates in the United Kingdom (13.8/100,000 in 2007) remain low, rates differ substantially by region: London (43.2/100,000) accounts for ≈40% of all TB cases registered in the United Kingdom, and ≈75% of TB patients in London were born abroad (2). Rates of drug resistance also are higher in London than in the rest of the United Kingdom: 8.6% of isolates are isoniazid resistant, and 1.2% are multidrug resistant (UK Health Protection Agency; www.hpa.org.uk).

In settings where incidence of TB is low or moderate, molecular genotyping is used to investigate suspected TB outbreaks, laboratory cross-contamination, and reactivation and (at a population level) to identify clustered cases that are not apparently linked; for the latter purpose, the highest possible level of discrimination is required (3). For these purposes, insertion sequence (IS) 6110 restriction fragment length polymorphism (RFLP) analysis—often supplemented with spoligotyping and, more recently, with variable number tandem repeat (VNTR) typing—is used routinely.

The highest levels of epidemiologic discrimination of strains of the Mycobacterium tuberculosis complex (MTBC) can be achieved by using multilocus VNTR typing, but these results depend on the number and loci used, particularly for homogenous strain groups such as the Beijing family (3–5). This approach overcomes technical difficulties associated with IS6110-RFLP and is amenable to automation that results in a high throughput (6–10). A standardized panel of 15 + 9 VNTR loci (24 loci) has been proposed (7,11), but it is unclear whether sufficient discrimination would be seen when the panel is used in populations with a substantial prevalence of homogenous MTBC families (4,5,12). In addition, the discriminative power of VNTR loci may vary markedly among genetic families (7,13). Recent studies evaluating the discriminative power of VNTR typing have produced conflicting results that were generated by using convenience samples (small populations with low diversity or populations confined to a single geographic setting). These studies highlighted a need for larger population-based studies to identify discriminative VNTR loci and ascertain their applicability for various genetic groups.

Concerns about the stability and reproducibility of particularly useful hypervariable loci, such as 3232, 2163a, 3336, and 1982 (3–5,14), have been raised (7,15). As a result, they have been excluded from the proposed international panels for VNTR typing. For these reasons, we conducted a study to examine the stability of hypervariable loci and the parameters associated with reproducibility, to select loci suitable for prospective molecular epidemiologic studies, and to evaluate the discriminatory power of these loci at a population level in a metropolitan setting.

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