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

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 teste...

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Bibliographic Details
Published in:Journal of antimicrobial chemotherapy 2008-01, Vol.61 (1), p.123-127
Main Authors: Martin, Anandi, Cubillos-Ruiz, Andrés, Von Groll, Andrea, Del Portillo, Patricia, Portaels, Françoise, Palomino, Juan Carlos
Format: Article
Language:English
Subjects:
Antibiotics. Antiinfectious agents. Antiparasitic agents
Antitubercular Agents - pharmacology
Bacteria
Bacterial diseases
Base Sequence
Biological and medical sciences
Deoxyribonucleic acid
diagnostic
DNA
DNA, Bacterial - genetics
Drug resistance
Drug Resistance, Multiple, Bacterial - drug effects
Drug Resistance, Multiple, Bacterial - genetics
drug susceptibility testing
Genes, Bacterial
Human bacterial diseases
Humans
Infectious diseases
Medical sciences
Microbial Sensitivity Tests - methods
Microbiology
Molecular Sequence Data
Mutation
Mycobacterium tuberculosis
Mycobacterium tuberculosis - drug effects
Mycobacterium tuberculosis - enzymology
Niacinamide - pharmacology
Nitrate Reductase - metabolism
Pharmacology. Drug treatments
pncA
Pyrazinamide - pharmacology
Time Factors
Tuberculosis
Tuberculosis and atypical mycobacterial infections
Tuberculosis, Multidrug-Resistant - drug therapy
Tuberculosis, Multidrug-Resistant - microbiology
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Summary: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.
ISSN:0305-7453
1460-2091
DOI:10.1093/jac/dkm418