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Journal of Clinical Microbiology, August 1999, p. 2607-2618, Vol. 37, No. 8
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Comparison of Methods Based on Different Molecular Epidemiological Markers for Typing of Mycobacterium tuberculosis Complex Strains: Interlaboratory Study of Discriminatory Power and Reproducibility

K. Kremer,1,* D. van Soolingen,1 R. Frothingham,2 W. H. Haas,3 P. W. M. Hermans,4 C. Martín,5 P. Palittapongarnpim,6 B. B. Plikaytis,7 L. W. Riley,8 M. A. Yakrus,9 J. M. Musser,10 and J. D. A. van Embden11

Diagnostic Laboratory for Infectious Diseases and Perinatal Screening1 and Research Laboratory for Infectious Diseases,11 National Institute of Public Health and the Environment, 3720 BA Bilthoven, and Laboratory of Pediatrics, Erasmus University Rotterdam, 3000 DR Rotterdam,4 The Netherlands; Molecular Mycobacteriology Laboratory, University of Heidelberg, 69120 Heidelberg, Germany3; Durham VA Medical Center, Durham, North Carolina 277052; National Center for Infectious Diseases7 and Diagnostics and Molecular Epidemiology Section,9 Centers for Disease Control and Prevention, Atlanta, Georgia 30333; School of Public Health, University of California, Berkeley, Berkeley, California 947208; Institute for the Study of Human Bacterial Pathogenesis, Department of Pathology, Baylor College of Medicine, Houston, Texas 7703010; Department of Microbiology, University of Zaragoza, 50009 Zaragoza, Spain5; and Department of Microbiology, Mahidol University, Bangkok 10400, Thailand6

Received 29 January 1999/Returned for modification 1 April 1999/Accepted 13 May 1999

In this study, the currently known typing methods for Mycobacterium tuberculosis isolates were evaluated with regard to reproducibility, discrimination, and specificity. Therefore, 90 M. tuberculosis complex strains, originating from 38 countries, were tested in five restriction fragment length polymorphism (RFLP) typing methods and in seven PCR-based assays. In all methods, one or more repetitive DNA elements were targeted. The strain typing and the DNA fingerprint analysis were performed in the laboratory most experienced in the respective method. To examine intralaboratory reproducibility, blinded duplicate samples were included. The specificities of the various methods were tested by inclusion of 10 non-M. tuberculosis complex strains. All five RFLP typing methods were highly reproducible. The reliability of the PCR-based methods was highest for the mixed-linker PCR, followed by variable numbers of tandem repeat (VNTR) typing and spoligotyping. In contrast, the double repetitive element PCR (DRE-PCR), IS6110 inverse PCR, IS6110 ampliprinting, and arbitrarily primed PCR (APPCR) typing were found to be poorly reproducible. The 90 strains were best discriminated by IS6110 RFLP typing, yielding 84 different banding patterns, followed by mixed-linker PCR (81 patterns), APPCR (71 patterns), RFLP using the polymorphic GC-rich sequence as a probe (70 patterns), DRE-PCR (63 patterns), spoligotyping (61 patterns), and VNTR typing (56 patterns). We conclude that for epidemiological investigations, strain differentiation by IS6110 RFLP or mixed-linker PCR are the methods of choice. A strong association was found between the results of different genetic markers, indicating a clonal population structure of M. tuberculosis strains. Several separate genotype families within the M. tuberculosis complex could be recognized on the basis of the genetic markers used.

* Corresponding author. Mailing address: National Institute of Public Health and the Environment (RIVM), Laboratory for Infectious Diseases and Perinatal Screening, Mycobacteria Department (pb22), P.O. Box 1, 3720 BA Bilthoven, The Netherlands. Phone: 31-30-2742261. Fax: 31-30-2744418. E-mail: kristin.kremer{at}rivm.nl.

Journal of Clinical Microbiology, August 1999, p. 2607-2618, Vol. 37, No. 8
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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