A new rapid and simple colorimetric method to detect pyrazinamide resistance in Mycobacterium tuberculosis using nicotinamide

Objectives: The purpose of this study was to develop and assess a rapid method for pyrazinamide resistance detection in Mycobacterium tuberculosis using nicotinamide in a colorimetric resazurin assay. Methods: We have tested M. tuberculosis isolates using nicotinamide in a 96-well format with the redox indicator resazurin (REMA) and compared results using the BACTEC 460-TB system with two concentrations of pyrazinamide (100 and 300 mg/L), as well as the Wayne method for detecting pyrazinamidase activity. Mutations in the pncA gene were detected by DNA sequencing of the pyrazinamide-resistant strains. Results: Out of 95 clinical isolates of M. tuberculosis tested, 25 were determined to be resistant by the Wayne, BACTEC (300 mg/L), and the REMA nicotinamide methods. Using a nicotinamide MIC > 250 mg/L as the cut-off for defining resistance, only one strain was falsely labelled as resistant. The REMA nicotinamide assay demonstrated a sensitivity of 100% and a specificity of 98%. The BACTEC (100 mg/L) falsely classified 8 strains as resistant. DNA sequencing detected mutations in 18/22 of the pncA genes from pyrazinamide-resistant strains. Conclusions: The REMA plate using nicotinamide to detect resistance to pyrazinamide is a simple and rapid method that could be useful in limited-resource countries.