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Discriminatory Power and Reproducibility of Novel DNA Typing Methods for Mycobacterium tuberculosis Complex Strains

Mycobacteria Reference Unit, Diagnostic Laboratory for Infectious Diseases and Perinatal Screening, National Institute of Public Health and the Environment, Bilthoven, The Netherlands,¹ Central Public Health Laboratory, London, United Kingdom,² Institute of Biotechnology of the National Institute of Agricultural Technology, Castelar, Argentina,³ Institut Pasteur, Paris, France,⁴ Robert Koch Institute, Berlin, Germany,⁵ National Center of Infectious Diseases and Parasitic Diseases, Sofia, Bulgaria,⁶ Department of Agriculture and Rural Development, Belfast, Northern Ireland,⁷ INSERM U629, Institut Pasteur de Lille, Lille, France,⁸ Medical Microbiology and Infectious Diseases, Gelre Hospitals, Apeldoorn, The Netherlands⁹

Received 2 February 2005/ Returned for modification 18 April 2005/ Accepted 23 June 2005

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.

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