Comparison of phenotypic and genotypic methods for pyrazinamide susceptibility testing with Mycobacterium tuberculosis

Mycobacterium tuberculosis converts pyrazinamide to its active form by usin g 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 Mycobact erium 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 enzym e was sought by using the Wayne assay, which was positive in ail of the sen sitive isolates and four of the resistant isolates. The pncA gene was ampli fied by PCR, and mutations were sought by single-strand conformation polymo rphism (SSCP) analysis, We identified four isolates which were phenotypical ly resistant to pyrazinamide, but which had active pyrazinamide enzyme on t he 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 an y mutations. This implies that an alternative mechanism of resistance exist s in these strains. We conclude that genotypic assessment of pyrazinamide r esistance is unreliable, because it depends on the identification of a sing le resistance mechanism. Phenotypic methods such as the BACTEC 460TB techni que remain the best methods for pyrazinamide susceptibility testing.