Nitrate reductase assay for detection of drug resistance in Mycobacterium tuberculosis: simple and inexpensive method for low-resource laboratories

Lemus, Dihadenys; Montoro, Ernesto; Echemendía, Miguel; Martin, Anandi; Portaels, Françoise; Palomino, Juan Carlos

doi:10.1099/jmm.0.46540-0

Cited by 44 publications

(28 citation statements)

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“…The current findings with these two drugs agreed with previous and recent data for nitrate reductase assay (5,21,22), demonstrating insufficient concordance of the assay with the normally recommended standards to recommend them for routine use. Nonetheless, Rosales et al (23) have recently evaluated the nitrate reductase assay for the rapid detection of resistance to secondline drugs such as ofloxacin and kanamycin.…”

Section: Discussionsupporting

confidence: 47%

Utilidad del ensayo de nitrato reductasa en la detección de Mycobacterium tuberculosis multirresistente en caso de recursos limitados

López

Álvarez

2011

biomedica

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Introduction. The performance of a drug susceptibility test may change when moving from the research stage to implementation on a population level in actual public health practice. Objective. The performance of a rapid drug susceptibility test was described for detecting multidrugresistant Mycobacterium tuberculosis when implemented in the routine workflow of a low-resource reference laboratory. Materials and methods. A prospective study was done comparing the performance of the nitrate reductase assay with the conventional proportion method for rifampicin and isoniazid on 364 isolates were obtained from multidrug-resistant tuberculosis risk patients referred from diffrent Colombian laboratories.Results. When compared with the proportion method, the nitrate reductase assay sensitivity was 86.8% and 84.9% for rifampicin and isoniazid, respectively, whereas nitrate reductase assay specificity was 100% for isoniazid and rifampicin. Nitrate reductase assay sensitivity was significantly higher when the age of isolate was less than 70 days. A sensitivity of 94.4% dropped to 78.1% for rifampicin resistance for fresh and old isolates, respectively (Fisher exact test, p=0.05). For isoniazid resistance using fresh and old isolates, 94.7% vs.74.3% sensitivities, were achieved (chi square test, p=0.03). The proportion of nitrate reductase assay ambiguous results was significantly higher in multidrug-resistant than in nonmultidrug-resistant isolates (17.6% vs. 4.0%, chi square test, p<0.005). Conclusions.The nitrate reductase assay demonstrated provided reliable results for antibiotic resistance. However, using old cultures leds to a higher proportion of false sensitive results; furthermore, the nitrate reductase assay capability to detect multidrug-resistant tuberculosis decreased due to a higher proportion of non-interpretable results.

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Section: Discussionsupporting

confidence: 47%

Utilidad del ensayo de nitrato reductasa en la detección de Mycobacterium tuberculosis multirresistente en caso de recursos limitados

López

Álvarez

2011

biomedica

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“…NRA was successfully tested in previous studies (1,(17)(18)(19) and has the advantage of being performed on classical LJ medium that does not need to be acidified. The MGMT assay should be easy to implement in clinical TB laboratories, and its microtube format may pose fewer biosafety risks than colorimetric microplate methods (4,16,26).…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Colorimetric Methods Using Nicotinamide for Rapid Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis

et al. 2010

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The direct detection of pyrazinamide resistance in Mycobacterium tuberculosis is sufficiently difficult that many laboratories do not attempt it. Most pyrazinamide resistance is caused by mutations that inactivate the pyrazinamidase enzyme needed to convert the prodrug pyrazinamide to its active form. We evaluated two newer and simpler methods to assess pyrazinamidase activity, the nitrate reductase and malachite green microtube assays, using nicotinamide in place of pyrazinamide. A total of 102 strains were tested by these methods and the results compared with those obtained by the classic Wayne assay. Mutations in the pncA gene were identified by sequencing the pncA genes from all isolates in which pyrazinamide resistance was detected by any of the three methods. Both the nitrate reductase and malachite green microtube assays showed sensitivities of 93.75% and specificities of 97.67%. Mutations in the pncA gene were found in 14 of 16 strains that were pyrazinamide resistant and in 1 of 4 strains that were sensitive by the Wayne assay. Both of these simple methods, used with nicotinamide, are promising and inexpensive alternatives for the rapid detection of pyrazinamide resistance in limited-resource countries.

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“…This test is based on the ability of Mycobacterium tuberculosis to reduce nitrate to nitrite, which is revealed as a color change in the culture medium, using the Griess method (10). The indirect (using isolates) NRA yields results in less than 14 days but requires an initial 3 to 4 weeks for cultivation of the isolate (7,13,14).…”

mentioning

confidence: 99%

“…Recently, alternative rapid methods have been developed (13). Among them, the nitrate reductase assay (NRA) on Löwenstein-Jensen (LJ) medium is simple to perform and has been successfully implemented in low-income countries (7,13,14). This test is based on the ability of Mycobacterium tuberculosis to reduce nitrate to nitrite, which is revealed as a color change in the culture medium, using the Griess method (10).…”

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Evaluation of Direct Detection of Mycobacterium tuberculosis Rifampin Resistance by a Nitrate Reductase Assay Applied to Sputum Samples in Cotonou, Benin

et al. 2007

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The aim of this study was to evaluate a nitrate reductase assay (NRA) performed on smear-positive sputa for the direct detection of rifampin resistance in Mycobacterium tuberculosis. A total of 213 smear-positive sputa with a positivity score of 1؉ or more (>1 acid-fast bacillus per field by fluorescence microscopy) were used in the study. The samples were decontaminated using the modified Petroff method, and portions of the resulting suspension were used to perform the NRA. The NRA results were compared with the reference indirect proportion method for 177 specimens for which comparable results were available. NRA results were obtained at day 10 for 15 specimens (9%), results for 88 specimens (50%) were obtained at day 14, results for 66 specimens (37%) were obtained at day 18, and results for the remaining 8 specimens (4%) were obtained at day 28. Thus, 96% of NRA results were obtained in 18 days. Of the 177 specimens, there was only one discrepancy (susceptible according to the NRA and resistant according to the indirect proportion method). NRA is simple to perform and provides a rapid, accurate, and cost-effective means for the detection of rifampin resistance in M. tuberculosis isolates.Tuberculosis (TB) remains a major public health problem worldwide. In recent years, the incidence of TB has been rising. There is also an emergence of multidrug-resistant (MDR) tuberculosis (defined as resistance to at least rifampin [RMP] and isoniazid) that is worsening the impact of this disease (1,4,21).Previous studies suggest that RMP resistance could be a surrogate marker for multidrug resistance, especially in settings with a high prevalence of drug resistance (8, 18). Therefore, the detection of resistance to this major anti-TB drug is essential for the optimal control of TB.Conventional tests for the detection of drug resistance require several weeks to yield results (5). Recently, alternative rapid methods have been developed (13). Among them, the nitrate reductase assay (NRA) on Löwenstein-Jensen (LJ) medium is simple to perform and has been successfully implemented in low-income countries (7,13,14). This test is based on the ability of Mycobacterium tuberculosis to reduce nitrate to nitrite, which is revealed as a color change in the culture medium, using the Griess method (10). The indirect (using isolates) NRA yields results in less than 14 days but requires an initial 3 to 4 weeks for cultivation of the isolate (7,13,14).So far, only a few studies have evaluated the NRA applied directly to sputum samples. The results of these studies (which were done in high-incidence settings) were concordant with results obtained by the reference method (15, 17). However, to our knowledge, no direct NRA study has been done in a setting with low resistance prevalence or in Africa, where there is potentially a high frequency of nitrate reductase-negative M. tuberculosis complex strains (11,12).The objective of this study was to evaluate NRA applied directly to smear-positive sputa in the West African country of Benin in orde...

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Nitrate reductase assay for detection of drug resistance in Mycobacterium tuberculosis: simple and inexpensive method for low-resource laboratories

Cited by 44 publications

References 11 publications

Utilidad del ensayo de nitrato reductasa en la detección de Mycobacterium tuberculosis multirresistente en caso de recursos limitados

Utilidad del ensayo de nitrato reductasa en la detección de Mycobacterium tuberculosis multirresistente en caso de recursos limitados

Evaluation of Colorimetric Methods Using Nicotinamide for Rapid Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis

Evaluation of Direct Detection of Mycobacterium tuberculosis Rifampin Resistance by a Nitrate Reductase Assay Applied to Sputum Samples in Cotonou, Benin

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