title: Sputum PCR-single-strand conformational polymorphism test for same-day detection of pyrazinamide resistance in tuberculosis patients.
abstract: 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.
authors.affiliation: Laboratory of Infectious Diseases Research, Department of Microbiology, Faculty of Sciences, Universidad Peruana Cayetano Heredia, Lima, Peru.
authors.forename: Patricia
authors.initials: P
authors.lastname: Sheen
authors.forename: Melissa
authors.initials: M
authors.lastname: Méndez
authors.forename: Robert H
authors.initials: RH
authors.lastname: Gilman
authors.forename: Lizeth
authors.initials: L
authors.lastname: Peña
authors.forename: Luz
authors.initials: L
authors.lastname: Caviedes
authors.forename: Mirko J
authors.initials: MJ
authors.lastname: Zimic
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.forename: David A J
authors.initials: DA
authors.lastname: Moore
authors.forename: Carlton A
authors.initials: CA
authors.lastname: Evans
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000191:economics / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D018807:Polymorphism, Single-Stranded Conformational; D011718:Pyrazinamide / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology; D013997:Time Factors; D014376:Tuberculosis / Q000382:microbiology*
chemical_list: D000995:Antitubercular Agents; D004269:DNA, Bacterial; D011718:Pyrazinamide