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Journal of Clinical Microbiology, November 2004, p. 5058-5064, Vol. 42, No. 11
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.11.5058-5064.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Characterization of Ancestral Mycobacterium tuberculosis by Multiple Genetic Markers and Proposal of Genotyping Strategy

Department of Infectious Diseases, Tan Tock Seng Hospital,¹ Division of Medical Sciences, National Cancer Centre, Singapore,² Diagnostic Laboratory for Infectious Diseases and Perinatal Screening, National Institute for Public Health and the Environment, Bilthoven, The Netherlands,³ Laboratoire des Mécanismes Moléculaires de la Pathogenèse Bactérienne, Institut Pasteur de Lille, Lille, France⁴

Received 24 March 2004/ Returned for modification 28 June 2004/ Accepted 5 July 2004

Sixty-eight ancestral Mycobacterium tuberculosis isolates were previously identified by using the tuberculosis-specific deletion 1 (TbD1) PCR and mycobacterial interspersed-repetitive-unit-variable-number tandem repeat (MIRU-VNTR) typing (Y. J. Sun, R. Bellamy, A. S. G. Lee, S. T. Ng, S. Ravindran, S.-Y. Wong, C. Locht, P. Supply, and N. I. Paton, J. Clin. Microbiol. 42:1986-1993, 2004). These TbD1⁺ ancestral isolates were further characterized and typed in this study by IS6110 restriction fragment length polymorphism (RFLP) typing, VNTR typing using exact tandem repeats (VNTR-ETR), and spoligotyping of the direct-repeat region. To our knowledge, this is the first characterization of this genogroup by multiple genetic markers based on a fairly large sample size. In this genogroup, all spoligotypes were characterized by the absence of spacers 29 to 32 and 34. In addition, VNTR-ETR typing could add further resolution to the clustered isolates identified by MIRU-VNTR, and the combination of MIRU-VNTR and VNTR-ETR, called MIRU-ETR, showed the highest discriminatory power for these strains compared to IS6110 RFLP typing and spoligotyping alone. However, MIRU-ETR appeared to still cluster some probably epidemiologically unrelated strains, as judged by IS6110 RFLP divergence. Therefore, a typing strategy based on stepwise combination of MIRU-ETR and IS6110 RFLP is proposed to achieve maximal discrimination for unrelated TbD1⁺ strains. This typing strategy may be useful in areas where TbD1⁺ ancestral strains are prevalent.

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