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Journal of Clinical Microbiology, October 2000, p. 3686-3688, Vol. 38, No. 10
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Comparison of Phenotypic and Genotypic Methods for Pyrazinamide Susceptibility Testing with Mycobacterium tuberculosis

A. P. Davies,1 O. J. Billington,1 T. D. McHugh,1 D. A. Mitchison,2 and S. H. Gillespie1,*

Department of Medical Microbiology, Royal Free and University College Medical School,1 and Department of Medical Microbiology, St George's Hospital Medical School,2 London, United Kingdom

Received 18 April 2000/Returned for modification 28 June 2000/Accepted 8 August 2000

Mycobacterium tuberculosis converts pyrazinamide to its active form by using the enzyme pyrazinamidase. This enzyme is coded for on the pncA gene, and mutations in the pncA gene result in absence of active enzyme, conferring resistance to the drug pyrazinamide. We investigated 27 strains of Mycobacterium tuberculosis suspected of being multidrug resistant. Each isolate was tested for susceptibility to pyrazinamide by the BACTEC 460TB method, and 19 were pyrazinamide resistant. The presence of active pyrazinamidase enzyme was sought by using the Wayne assay, which was positive in all of the sensitive isolates and four of the resistant isolates. The pncA gene was amplified by PCR, and mutations were sought by single-strand conformation polymorphism (SSCP) analysis. We identified four isolates which were phenotypically resistant to pyrazinamide, but which had active pyrazinamide enzyme on the Wayne assay and normal pncA gene SSCP. MICs measured by BACTEC 460TB and susceptibility testing at a lower pH of 5.5 confirmed genuine resistance. The pncA gene was sequenced in these four isolates and found not to have any mutations. This implies that an alternative mechanism of resistance exists in these strains. We conclude that genotypic assessment of pyrazinamide resistance is unreliable, because it depends on the identification of a single resistance mechanism. Phenotypic methods such as the BACTEC 460TB technique remain the best methods for pyrazinamide susceptibility testing.

* Corresponding author. Mailing address: Department of Medical Microbiology, Royal Free and University College Medical School, Royal Free Campus, Rowland Hill St., London NW3 2PF, United Kingdom. Phone: 0171 794 500, ext. 3539. Fax: 0171 794 0433. E-mail: stepheng{at}rfc.ucl.ac.uk.

Journal of Clinical Microbiology, October 2000, p. 3686-3688, Vol. 38, No. 10
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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