Multicenter evaluation of the nitrate reductase assay for drug resistance detection of Mycobacterium tuberculosis

Universidad Peruana Cayetano Heredia, Λίμα, Lima, Peru
Journal of Microbiological Methods (Impact Factor: 2.03). 12/2005; 63(2):145-50. DOI: 10.1016/j.mimet.2005.03.004
Source: PubMed


The performance of the nitrate reductase assay was evaluated in a multicenter laboratory study to detect resistance of Mycobacterium tuberculosis to the first-line anti-tuberculosis drugs rifampicin, isoniazid, ethambutol and streptomycin using a set of coded isolates. Compared with the gold standard proportion method on Löwenstein-Jensen medium, the assay was highly accurate in detecting resistance to rifampicin, isoniazid and ethambutol with an accuracy of 98%, 96.6% and 97.9%, respectively. For streptomycin, discrepant results were obtained with an overall accuracy of 85.3%. The assay proved easy to be implemented in countries with limited laboratory facilities.

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    • "The GeneXpert MTB/RIF molecular assay [1], which has been endorsed by World Health Organization (WHO) [2], is sensitive, rapid and relatively easy to use. However, it remains costly in resource-limited settings [3,4]. "
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    ABSTRACT: Background The resazurin microtiter assay (classic REMA), a colorimetric liquid culture-based drug susceptibility assay for Mycobacterium tuberculosis (MTB), has been endorsed by the World Health Organization. The assay requires 8-16 days to obtain results, delaying management of drug resistant tuberculosis patients. A modified REMA which allows results in as little as 24 hours for bacterial strains, has been developed and validated using Staphylococcus aureus, but has not yet been evaluated for MTB. Therefore we assessed the performance of the modified REMA for rifampicin (RIF) and isoniazid (INH) susceptibility, using the classic REMA as the reference standard. We also compared simplicity (from the technicians¿ point of view), time taken to obtain results (rank-sum testing), specificity and Kappa statistics of the two methods.ResultsThe modified REMA, which is a one-step procedure, was found to be simpler to perform and results were obtained in a significantly shorter time (5 versus 9 days, p¿<¿0.0001) compared to the classic REMA due to addition of indicator and strain at the same time. The specificity of the modified REMA was low {46.8% (35.5% - 58.4%) for RIF and 13.9% (7.2% - 23.5%) for INH}. Kappa statistics were 16.0% for RIF and 2.0% for INH. Low specificity and kappa statistics are due to indicator reduction by the strains before complete drug activity.Conclusion Although modified REMA is faster and simpler compared to classic REMA, it is not reliable for MTB drug susceptibility testing.
    Full-text · Article · Oct 2014 · BMC Microbiology
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    • "Recently, inexpensive, rapid and reliable colourimetric methods have attracted increased interest; of these, the resazurin microplate method and the nitrate reductase assay are the most popular (Angeby et al. 2002, Palomino et al. 2002, Syre et al. 2003, Coban et al. 2004, Martin et al. 2005, Montoro et al. 2005 , Bwanga et al. 2010, Dixit et al. 2012). Crystal violet (CV) is a triphenylmethane dye that is antimicrobial and toxic to mammalian cells. "
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    ABSTRACT: The aim of this study was to investigate the performance of a new and accurate method for the detection of isoniazid (INH) and rifampicin (RIF) resistance among Mycobacterium tuberculosis isolates using a crystal violet decolourisation assay (CVDA). Fifty-five M. tuberculosis isolates obtained from culture stocks stored at -80ºC were tested. After bacterial inoculation, the samples were incubated at 37ºC for seven days and 100 µL of CV (25 mg/L stock solution) was then added to the control and sample tubes. The tubes were incubated for an additional 24-48 h. CV (blue/purple) was decolourised in the presence of bacterial growth; thus, if CV lost its colour in a sample containing a drug, the tested isolate was reported as resistant. The sensitivity, specificity, positive predictive value, negative predictive value and agreement for INH were 92.5%, 96.4%, 96.1%, 93.1% and 94.5%, respectively, and 88.8%, 100%, 100%, 94.8% and 96.3%, respectively, for RIF. The results were obtained within eight-nine days. This study shows that CVDA is an effective method to detect M. tuberculosis resistance to INH and RIF in developing countries. This method is rapid, simple and inexpensive. Nonetheless, further studies are necessary before routine laboratory implementation.
    Full-text · Article · Mar 2014 · Memórias do Instituto Oswaldo Cruz
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    • "The performance of NRA was evaluated in a multicentre study by Martin et al. to ascertain the susceptibility of MTB against first-line antitubercular drugs (29). The accuracy was greater than 97% for INH, EMB, and RIF while that for STR was inferior (85.3%). "
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    ABSTRACT: The objective of the study was to evaluate the performance of nitrate reductase assay (NRA) as a rapid, reliable and inexpensive method for drug-susceptibility testing (DST) of Mycobacterium tuberculosis against first-line antitubercular drugs, such as rifampicin (RIF), isoniazid (INH), streptomycin (STR), and ethambutol (EMB). In total, 286 isolates were subjected to test by proportion method (PM) and NRA. By comparing the results of NRA with those of the gold standard PM, sensitivities and specificities were 98.4%, 97%, 88.5%, and 94.2% and 100%, 100%, 94%, and 99% for RIF, INH, STR, and EMB respectively. The positive predictive values were 100%, 100%, 95%, and 98% for RIF, INH, STR, and EMB respectively. The negative values were 99%, 98%, 87%, and 96% for RIF, INH, STR, and EMB respectively. The median time of obtaining results was shorter using NRA (10 days) compared to PM (28 days). An excellent agreement was observed between the two phenotypic tests with the K values of 0.98, 0.97, 0.81, and 0.93 for RIF, INH, STR, and EMB respectively. The results demonstrated that NRA is suitable for the early determination of INH and RIF resistance and has the potential to be a useful tool for rapid drug-sensitivity test of M. tuberculosis in resource-constrained settings.
    Full-text · Article · Feb 2011 · Journal of Health Population and Nutrition
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