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Journal of Clinical Microbiology, August 2007, p. 2662-2668, Vol. 45, No. 8
0095-1137/07/$08.00+0     doi:10.1128/JCM.00244-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Drug Susceptibility Testing of Mycobacterium tuberculosis Complex by Use of a High-Throughput, Reproducible, Absolute Concentration Method{triangledown}

Bert van Klingeren, Mirjam Dessens-Kroon, Tridia van der Laan, Kristin Kremer, and Dick van Soolingen*

Mycobacteria Reference Laboratory, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands

Received 31 January 2007/ Returned for modification 24 April 2007/ Accepted 21 May 2007

Accurate drug susceptibility testing (DST) for Mycobacterium tuberculosis is highly important for both therapy guidance and surveillance of drug resistance. Although liquid medium DST methods are used increasingly and seem most efficient and fast, the high costs hamper widespread implementation. In addition, an inability to check the colony morphology of the growing bacteria is a disadvantage of these methods. Moreover, these methods discriminate only between susceptibility and resistance and do not determine the MIC. In this paper, we describe a low-cost, reproducible, high-throughput, proportional absolute concentration DST method. The method uses a concentration series of antituberculosis drugs, including pyrazinamide in 7H10 medium, distributed semiautomatically in 25-well plates. The performance of this 25-well DST method was evaluated by the World Health Organization and the International Union against Tuberculosis and Lung Disease in 10 rounds of proficiency testing regarding sensitivity, specificity, efficiency, reproducibility, and predictive value for resistance and susceptibility. The performance of the method for these characteristics was 100% for isoniazid and from 96 to 100% for rifampin, 91 to 100% for streptomycin, and 85 to 100% for ethambutol. The method was 100% reproducible for all four drugs. The levels of drug resistance and the MIC distributions for the first-line antituberculosis drugs were determined for all 7,956 M. tuberculosis strains isolated in The Netherlands from 1998 to 2005 and amounted to 7.5% for isoniazid, 1.4% for rifampin, 8.5% for streptomycin, and 1.0% for ethambutol. Pyrazinamide testing was successful for 7,026 (88.3%) of the isolates and showed a resistance level of 0.8%.

* Corresponding author. Mailing address: Mycobacteria Reference Laboratory (Pb 22), Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), P.O. Box 1 (LIS, pb 22), 3720 BA Bilthoven, The Netherlands. Phone: 31-30-2742363. Fax: 31-30-2744418. E-mail: d.van.soolingen{at}rivm.nl

{triangledown} Published ahead of print on 30 May 2007.

Journal of Clinical Microbiology, August 2007, p. 2662-2668, Vol. 45, No. 8
0095-1137/07/$08.00+0     doi:10.1128/JCM.00244-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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