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Journal of Clinical Microbiology, September 2009, p. 2937-2943, Vol. 47, No. 9
0095-1137/09/$08.00+0     doi:10.1128/JCM.01594-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Sputum PCR-Single-Strand Conformational Polymorphism Test for Same-Day Detection of Pyrazinamide Resistance in Tuberculosis Patients{triangledown}

Patricia Sheen,1 Melissa Méndez,1 Robert H. Gilman,1,2 Lizeth Peña,1 Luz Caviedes,1 Mirko J. Zimic,3 Ying Zhang,4 David A. J. Moore,1,2,5 and Carlton A. Evans1,2,5*

Laboratory of Infectious Diseases Research, Department of Microbiology, Faculty of Sciences, Universidad Peruana Cayetano Heredia, Lima, Peru,1 Department of International Health, Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland,2 Laboratory of Bioinformatics, Department of Biochemistry and Molecular Biology, Faculty of Sciences, Universidad Peruana Cayetano Heredia, Lima, Peru,3 Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland,4 Wellcome Trust Centre for Clinical Tropical Medicine, Department of Infectious Diseases & Immunity, Imperial College London Hammersmith Hospital Campus, London, United Kingdom5

Received 16 August 2008/ Returned for modification 27 October 2008/ Accepted 8 June 2009

Pyrazinamide is a first-line drug for treating tuberculosis, but pyrazinamide resistance testing is usually too slow to guide initial therapy, so some patients receive inappropriate therapy. We therefore aimed to optimize and evaluate a rapid molecular test for tuberculosis drug resistance to pyrazinamide. Tuberculosis PCR-single-strand conformational polymorphism (PCR-SSCP) was optimized to test for mutations causing pyrazinamide resistance directly from sputum samples and Mycobacterium tuberculosis isolates. The reliability of PCR-SSCP tests for sputum samples (n = 65) and Mycobacterium tuberculosis isolates (n = 185) from 147 patients was compared with four tests for pyrazinamide resistance: Bactec-460 automated culture, the Wayne biochemical test, DNA sequencing for pncA mutations, and traditional microbiological broth culture. PCR-SSCP provided interpretable results for 96% (46/48) of microscopy-positive sputum samples, 76% (13/17) of microscopy-negative sputum samples, and 100% of Mycobacterium tuberculosis isolates. There was 100% agreement between PCR-SSCP results from sputum samples and Mycobacterium tuberculosis isolates and 100% concordance between 50 blinded PCR-SSCP rereadings by three observers. PCR-SSCP agreement with the four other tests for pyrazinamide resistance varied from 89 to 97%. This was similar to how frequently the four other tests for pyrazinamide resistance agreed with each other: 90 to 94% for Bactec-460, 90 to 95% for Wayne, 92 to 95% for sequencing, and 91 to 95% for broth culture. PCR-SSCP took less than 24 hours and cost approximately $3 to $6, in contrast with the other assays, which took 3 to 14 weeks and cost $7 to $47. In conclusion, PCR-SSCP is a relatively reliable, rapid, and inexpensive test for pyrazinamide resistance that indicates which patients should receive pyrazinamide from the start of therapy, potentially preventing months of inappropriate treatment.

* Corresponding author. Mailing address: Laboratorio de Investigación y Desarrollo, Depto. Microbiologia, Universidad Peruana Cayetano Heredia Facultad de Ciencia, Av. Honorio Delgado #430, Urb. Ingeniería, San Martin de Porres, Lima, Peru. Phone: 51 1 382 0929. Fax: 51 1 616 5501. E-mail: CarltonEvans{at}yahoo.com

{triangledown} Published ahead of print on 17 June 2009.

Journal of Clinical Microbiology, September 2009, p. 2937-2943, Vol. 47, No. 9
0095-1137/09/$08.00+0     doi:10.1128/JCM.01594-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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