Nitrate reductase assay for the rapid detection of pyrazinamide resistance in Mycobacterium tuberculosis using nicotinamide.
OBJECTIVE
The purpose of this study was to develop the nitrate reductase assay (NRA) for the rapid detection of pyrazinamide resistance in Mycobacterium tuberculosis using nicotinamide resistance as a marker of pyrazinamide resistance in Löwenstein-Jensen (LJ) medium at neutral pH.


METHODS
We tested 68 M. tuberculosis isolates using nicotinamide at three different concentrations (1000, 500 and 250 mg/L) by the NRA in LJ medium and compared the results with those obtained with the BACTEC 460-TB or the BACTEC MGIT 960 as reference standard methods. Mutations in the pncA gene were detected by DNA sequencing of the pyrazinamide-resistant isolates.


RESULTS
Out of 34 M. tuberculosis pyrazinamide-resistant isolates, 31 were found to be resistant to 1000 and 500 mg/L nicotinamide giving sensitivity and specificity of 91% and 94%, respectively. At 250 mg/L nicotinamide, the sensitivity and specificity decreased to 91% and 71%, respectively. Results were obtained in an average of 10 days. Based on these results, a tentative breakpoint concentration of 500 mg/L nicotinamide was defined. DNA sequencing of the pncA gene detected mutations in 26 out of 34 M. tuberculosis isolates resistant to pyrazinamide.


CONCLUSIONS
The NRA using nicotinamide to detect resistance to pyrazinamide in LJ medium is a rapid and accurate method that could be useful in limited-resource countries where the BACTEC 460-TB or the BACTEC MGIT 960 system is not available.
Martin
Anandi
A
Mycobacteriology Unit, Institute of Tropical Medicine, Nationalestraat 155, Antwerp 2000, Belgium. amartin@itg.be
Cubillos-Ruiz
Andrés
A
Von Groll
Andrea
A
Del Portillo
Patricia
P
Portaels
Françoise
F
Palomino
Juan Carlos
JC
D000995:Antitubercular Agents / Q000494:pharmacology*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D024901:Drug Resistance, Multiple, Bacterial / Q000187:drug effects* / Q000235:genetics; D005798:Genes, Bacterial; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology; D009536:Niacinamide / Q000494:pharmacology*; D050901:Nitrate Reductase / Q000378:metabolism*; D011718:Pyrazinamide / Q000494:pharmacology*; D013997:Time Factors; D018088:Tuberculosis, Multidrug-Resistant / Q000188:drug therapy / Q000382:microbiology
D000995:Antitubercular Agents; D004269:DNA, Bacterial; D009536:Niacinamide; D011718:Pyrazinamide; D050901:Nitrate Reductase