A new rapid and simple colorimetric method to detect pyrazinamide resistance in Mycobacterium tuberculosis using nicotinamide.
OBJECTIVE
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.
Martin
Anandi
A
Mycobacteriology Unit, Institute of Tropical Medicine Nationalestraat 155, Antwerpen, B-2000 Belgium. amartin@itg.be
Takiff
Howard
H
Vandamme
Peter
P
Swings
Jean
J
Palomino
Juan Carlos
JC
Portaels
Françoise
F
D000581:Amidohydrolases / Q000235:genetics; D000995:Antitubercular Agents / Q000494:pharmacology*; D003124:Colorimetry / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial; D005189:False Positive Reactions; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D008826:Microbial Sensitivity Tests / Q000379:methods*; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000378:metabolism; D009536:Niacinamide / Q000378:metabolism*; D010078:Oxazines / Q000378:metabolism; D010084:Oxidation-Reduction; D011718:Pyrazinamide / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014966:Xanthenes / Q000378:metabolism
D000995:Antitubercular Agents; D004269:DNA, Bacterial; D007202:Indicators and Reagents; D010078:Oxazines; D014966:Xanthenes; C005843:resazurin; D009536:Niacinamide; D011718:Pyrazinamide; D000581:Amidohydrolases; C487496:PncA protein, Mycobacterium tuberculosis