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JAC Advance Access published online on November 1, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm418
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Nitrate reductase assay for the rapid detection of pyrazinamide resistance in Mycobacterium tuberculosis using nicotinamide

Anandi Martin1,2,*, Andrés Cubillos-Ruiz3, Andrea Von Groll4, Patricia Del Portillo3, Françoise Portaels1 and Juan Carlos Palomino1

1 Mycobacteriology Unit, Institute of Tropical Medicine, Nationalestraat 155, Antwerp 2000, Belgium 2 Médecins Sans Frontières, rue Saint Sabin, 8, 75011 Paris, France 3 Corpogen, Carrera 5, 66a-34, Bogota DC, Colombia 4 Fundacion Universidade Federal do Rio Grande, Rua General Osorio, 96200-400 Rio Grande, Brazil

Received 6 August 2007; returned 30 August 2007; revised 5 October 2007; accepted 8 October 2007

* Corresponding author. Tel: +32-3-2476334; Fax: +32-3-2476333; E-mail: amartin{at}itg.be

Objectives: 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.

Key Words: drug susceptibility testing , tuberculosis , pncA , diagnostic


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P. Jureen, J. Werngren, J.-C. Toro, and S. Hoffner
Pyrazinamide Resistance and pncA Gene Mutations in Mycobacterium tuberculosis
Antimicrob. Agents Chemother., May 1, 2008; 52(5): 1852 - 1854.
[Abstract] [Full Text] [PDF]


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