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Journal of Clinical Microbiology, December 2006, p. 4498-4510, Vol. 44, No. 12
0095-1137/06/$08.00+0     doi:10.1128/JCM.01392-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Proposal for Standardization of Optimized Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing of Mycobacterium tuberculosis{triangledown} ,{dagger}

Philip Supply,1,2* Caroline Allix,3 Sarah Lesjean,1,2 Mara Cardoso-Oelemann,1,2,{ddagger} Sabine Rüsch-Gerdes,4 Eve Willery,1,2 Evgueni Savine,1,2 Petra de Haas,5 Henk van Deutekom,6 Solvig Roring,7 Pablo Bifani,1,2,3,8 Natalia Kurepina,8 Barry Kreiswirth,8 Christophe Sola,9 Nalin Rastogi,9 Vincent Vatin,2,10 Maria Cristina Gutierrez,11 Maryse Fauville,3 Stefan Niemann,4 Robin Skuce,7 Kristin Kremer,4 Camille Locht,1,2 and Dick van Soolingen4

INSERM, U629,1 Institut Pasteur de Lille, Lille, France,2 Institut Pasteur de Bruxelles, Laboratoire Tuberculose et Mycobactéries, Brussels, Belgium,3 Forschungszentrum Borstel, National Reference Center for Mycobacteria, Borstel, Germany,4 Diagnostic Laboratory for Infectious Diseases and Perinatal Screening, National Institute for Public Health and the Environment, 3720 Bilthoven,5 Department of Tuberculosis Control, Municipal Health Service, Amsterdam, The Netherlands,6 Veterinary Sciences Division, Department of Agriculture and Rural Development, Belfast, Northern Ireland,7 TB Center, Public Health Research Institute, Newark, New Jersey,8 Unité de la Tuberculose et des Mycobactéries, Institut Pasteur de Guadeloupe, Guadeloupe,9 CNRS, UMR 8090, Lille, France,10 Laboratoire de Référence des Mycobactéries, Institut Pasteur, Paris, France,11

Received 6 July 2006/ Returned for modification 18 August 2006/ Accepted 12 September 2006

Molecular typing based on 12 loci containing variable numbers of tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTRs) has been adopted in combination with spoligotyping as the basis for large-scale, high-throughput genotyping of Mycobacterium tuberculosis. However, even the combination of these two methods is still less discriminatory than IS6110 fingerprinting. Here, we define an optimized set of MIRU-VNTR loci with a significantly higher discriminatory power. The resolution and the stability/robustness of 29 loci were analyzed, using a total of 824 tubercle bacillus isolates, including representatives of the main lineages identified worldwide so far. Five loci were excluded for lack of robustness and/or stability in serial isolates or isolates from epidemiologically linked patients. The use of the 24 remaining loci increased the number of types by 40%—and by 23% in combination with spoligotyping—among isolates from cosmopolitan origins, compared to those obtained with the original set of 12 loci. Consequently, the clustering rate was decreased by fourfold—by threefold in combination with spoligotyping—under the same conditions. A discriminatory subset of 15 loci with the highest evolutionary rates was then defined that concentrated 96% of the total resolution obtained with the full 24-locus set. Its predictive value for evaluating M. tuberculosis transmission was found to be equal to that of IS6110 restriction fragment length polymorphism typing, as shown in a companion population-based study. This 15-locus system is therefore proposed as the new standard for routine epidemiological discrimination of M. tuberculosis isolates and the 24-locus system as a high-resolution tool for phylogenetic studies.


* Corresponding author. Mailing address: Laboratoire de Mécanismes Moléculaires de la Pathogenèse Bactérienne, INSERM U629, Institut Pasteur de Lille, 1, rue du Prof. Calmette, F-59019 Lille Cedex, France. Phone: (33) 320.87.11.54. Fax: (33) 320.87.11.58. E-mail: Philip.Supply{at}pasteur-lille.fr.

{triangledown} Published ahead of print on 27 September 2006.

{dagger} Supplemental material for this article may be found at http://jcm.asm.org/.

{ddagger} Present affiliation: Laboratório de Biologia Molecular Aplicada a Micobactérias, Departamento de Micobacterioses, Instituto Oswaldo Cruz-Fiocruz, Rio de Janeiro, RJ, Brazil.


Journal of Clinical Microbiology, December 2006, p. 4498-4510, Vol. 44, No. 12
0095-1137/06/$08.00+0     doi:10.1128/JCM.01392-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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