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JAC Advance Access originally published online on June 2, 2006
Journal of Antimicrobial Chemotherapy 2006 58(2):327-331; doi:10.1093/jac/dkl231
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© The Author 2006. 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

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

Anandi Martin1,*, Howard Takiff2, Peter Vandamme3, Jean Swings4, Juan Carlos Palomino1 and Françoise Portaels1

1 Mycobacteriology Unit, Institute of Tropical Medicine Nationalestraat 155, Antwerpen, B-2000 Belgium 2 Instituto Venezolano de Investigaciones Científicas Caracas, Venezuela 3 Laboratorium voor Microbiologie, University Gent Belgium 4 Laboratorium voor Microbiologie and BCCM/LMG Culture Collection, University Gent Belgium

Received 3 March 2006; returned 29 March 2006; revised 31 March 2006; accepted 9 May 2006

*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 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.

Keywords: drug resistance , first-line drug , resazurin , susceptibility testing


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