Biphasic Medium Using Nicotinamide for Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis

Reliable drug susceptibility testing of pyrazinamide (PZA) is technically difficult, since PZA activity is pH sensitive. The aim of this study was to evaluate a biphasic medium assay (BMA) for the reliable detection of PZA resistance in Mycobacterium tuberculosis (MTB) using nicotinamide (NIC) as a surrogate for PZA and identifying the appropriate cut-off value for the assay. The PZA susceptibility of 122 multidrug-resistant tuberculosis (MDR-TB) isolates and 39 drug-susceptible tuberculosis (DS-TB) isolates was examined using the BMA with NIC at four different concentrations (250, 500, 1000, and 2000 mg/L) and comparing the results with results from the BACTEC MGIT 960 reference method. Out of 122 MDR-TB isolates, 40 were identified as resistant by the BACTEC MGIT 960 system, of which 92.5% contained mutations within their pncA gene plus promoter region. A minimum inhibitory concentration of NIC ≥ 1000 mg/L was used as the cut-off concentration to define resistance in correlation with the MGIT 960 outcomes. NIC-BMA had a sensitivity of 90.91%, a specificity of 100%, and an accuracy of 97.52% compared with the MGIT 960 method. NIC-BMA is a promising assay to screen PZA resistance in microbiological laboratories without automation or advanced molecular instruments.