Multicenter evaluation of the nitrate reductase assay for drug resistance detection of Mycobacterium tuberculosis.
ABSTRACT 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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ABSTRACT: Background:Despite recent advances, tuberculosis diagnosis remains imperfect in resource-limited setting due to complexity, cost, poor sensitivity or long time to reporting. We present a report on the use of colorimetric methods, based on the detection of mycobacterial growth using colorimetric indicators, for the detection of Mycobacterium tuberculosis from sputum specimens.Methods:We evaluated the nitrate reductase assay (NRA), a modified method (NRAp) using paranitrobenzoic acid (PNB), and resazurin tube assay using PNB (RETAp) to differentiate tuberculous and non-tuberculous mycobacteria. The performances were assessed at days 18 and 28 using mycobacterium growth indicator tube (MGIT) and Löwenstein Jensen (LJ) culture methods as the reference standards.Results:We enrolled 690 adults suspected of pulmonary tuberculosis from a regional referral hospital in Uganda between March 2010 and June 2011. At day 18 the sensitivity and specificity of NRA were 84.6% and 90.0%; 84.1% and 92.6% for NRAp; and 71.2% and 99.3% for RETAp. At day 28, the sensitivity of RETAp increased to 82.6%. Among smear-negative TB suspects, sensitivity at day 28 was 64.7% for NRA, 61.3% for NRAp and 50% for RETAp. Contamination was found in 5.4% in NRA and 6.7% in RETAp results compared to 22.1% in LJ and 20.4% in MGIT. The median times for positivity were 10, 7, and 25 days for colorimetric methods, MGIT and LJ, respectively.Conclusion:Whereas the low specificity of NRA/NRAp precludes it from being used for TB diagnosis, RETAp could provide an alternative to LJ to accelerate time to culture results in resource-poor settings.Journal of clinical microbiology 05/2013; · 4.16 Impact Factor
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ABSTRACT: To perform a multicentre study evaluating the performance of the direct nitrate reductase assay (NRA) for the detection of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis in sputum samples. The study was conducted in six laboratories performing tuberculosis diagnosis that were located in six different countries. The NRA was performed directly on sputum samples in parallel with the reference method used at each site. Detection of resistance was performed for rifampicin, isoniazid, ofloxacin and kanamycin. Excellent agreement was obtained for all drugs tested at the majority of sites. The accuracy was 93.7%-100% for rifampicin, 88.2%-100% for isoniazid, 94.6%-100% for ofloxacin and 100% for kanamycin. The majority of NRA results were available at day 21 for sites 1, 2 and 5. Site 3 had a turnaround time of 13.9 days, at site 4 it was 18.4 days and at site 6 it was 16.2 days. The contamination rate ranged between 2.5% and 12%. Rapid detection of drug resistance by the direct NRA on sputum smear-positive samples was accurate and easy to implement in clinical diagnostic laboratories, making it a good alternative for rapid screening for MDR and XDR tuberculosis.Journal of Antimicrobial Chemotherapy 09/2013; · 5.34 Impact Factor
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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.Memórias do Instituto Oswaldo Cruz 03/2014; · 1.36 Impact Factor