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 Table of Contents  
Year : 2019  |  Volume : 24  |  Issue : 2  |  Page : 78-81

Evaluation of nitrate reductase assay on Lowenstein–Jensen medium for drug susceptibility testing of Mycobacterium tuberculosis

1 Department of Microbiology, Universal Diagnostic Laboratory, Pusad, Yavatmal District, Wardha, Maharashtra, India
2 Department of Microbiology, Mahatma Gandhi Institute of Medical Sciences, Wardha, Maharashtra, India

Date of Web Publication17-Sep-2019

Correspondence Address:
Dr. Rahul Narang
Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jmgims.jmgims_59_16

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Introduction: Multidrug-resistant tuberculosis is of increasing public health concern and a rapid and inexpensive drug susceptibility test is needed in resource limited settings. Aim: This study aims to compare the efficacy of drug susceptibility testing (DST) of Mycobacterium tuberculosis using nitrate reductase assay (NRA) on solid Lowenstein–Jensen (LJ) medium with proportion method on LJ medium as reference standard. Materials and Methods: From November 2011 to July 2012, 100 isolates of M. tuberculosis were tested for susceptibility to first-line drugs on LJ medium by proportion method and NRA using LJ with potassium nitrate. Results: For various drugs, the sensitivity and specificity by NRA on solid medium were as follows: streptomycin 96.6% and 98.5%; isoniazid 96.87% and 97%; rifampicin 96.3% and 96%; and ethambutol 78.9% and 91.4%, respectively. The concordance for rifampicin and isoniazid between NRA method and LJ proportion method was found to be >95%. Conclusion: NRA was found to be a rapid and sensitive method for DST of M. tuberculosis.

Keywords: India, multidrug-resistant tuberculosis, nitrate reductase assay, tuberculosis

How to cite this article:
Ghodmare R, Narang R, Narang P. Evaluation of nitrate reductase assay on Lowenstein–Jensen medium for drug susceptibility testing of Mycobacterium tuberculosis. J Mahatma Gandhi Inst Med Sci 2019;24:78-81

How to cite this URL:
Ghodmare R, Narang R, Narang P. Evaluation of nitrate reductase assay on Lowenstein–Jensen medium for drug susceptibility testing of Mycobacterium tuberculosis. J Mahatma Gandhi Inst Med Sci [serial online] 2019 [cited 2022 Nov 26];24:78-81. Available from: https://www.jmgims.co.in/text.asp?2019/24/2/78/267015

  Introduction Top

Tuberculosis (TB) is a major public health problem in the developing countries. In 2016, 10.4 million people had TB and 1.3 million HIV-negative and 374,000 HIV-positive people died of TB. An estimated 600,000 people developed rifampicin-resistant TB of which 490,000 had multidrug-resistant (MDR) TB. Almost half (47%) of these cases were in India, China, and the Russian Federation.[1] Data from 52 countries in the World Health Organization's global project on TB drug resistance surveillance reported a median prevalence of 1.8% (range, 0%–18.1%) for MDR strains and 11.1% (range, 2.9%–40.8%) for strains with any drug resistance.[2] Timely detection of MDR-TB patients is therefore important to avoid the spread of MDR M. tuberculosis strains in the community.

The standard methods for drug susceptibility testing (DST) of M. tuberculosis such as absolute concentration method, resistance ratio method, and proportion method are used globally but are time-consuming as they require 4–6 weeks for the result.[3] The WHO approved commercial tests, namely BACTEC MGIT 960 automated system, HAIN Line Probe Assay and Cepheid Gene Xpert are costly, require sophisticated equipment and are technically demanding.[4] For a country like India, we require economic methods which are simple, rapid, and technically less demanding.

Colorimetric tests are such methods that are easy to perform and are cost-effective with the main advantage of giving results in 7–14 days.[5] In these tests, mycobacterial growth in media with antitubercular drugs is detected by color developing system which indicates bacterial resistance to that particular drug.

The nitrate reductase assay (NRA) has been used for DST by various authors using solid Lowenstein–Jensen medium (LJ)[6],[7],[8] and liquid Middlebrook 7H9 medium[9],[10] for indirect DST. Some articles have also mentioned the use of NRA on direct sputum samples, which is again very quick but less reliable because of more chances of contamination.[3],[11] Considering the above, this study was planned to evaluate NRA using solid LJ medium for DST of M. tuberculosis against proportion method on LJ as the reference standard.

  Materials and Methods Top

This study was done as a cross-sectional study in Microbiology department, MGIMS Sevagram from November 1, 2011, to July 31, 2012. One hundred M. tuberculosis isolates from clinical specimen possessing nitrate reductase activity were used as study samples after approval from the Institutional Ethics Committee. M. tuberculosis H37Rv ATCC (27294) was used as a control.

Proportion method

Drug susceptibility of all 100 nonrepetitive isolates to streptomycin (S), isoniazid (I), rifampicin (R), and ethambutol (E) was performed by standard method.[12]

Nitrate reductase assay for drug susceptibility

LJ medium containing potassium nitrate (KNO3) 1 mg/ml with and without antitubercular drugs were prepared. The following critical concentrations were used: streptomycin 4 μg/ml, INH 0.2 μg/ml, rifampicin 40 μg/ml, and for ethambutol 2.0 μg/ml.[6] For each isolate, inoculum of No. 1 and 1:10 dilution of No. 1 McFarland turbidity were prepared. Total seven bottles, i.e., four drug containing media and three drug-free media with KNO3 were inoculated. The drug media were inoculated with No. 1 McFarland turbidity and the control media were inoculated with 1:10 dilution of No. 1 turbidity. Bottles were incubated at 37° C and reading was taken on 7th, 10th, and 14th day by adding 0.5 ml mixture of reagents - 1 part 50% (vol/vol) concentrated hydrochloric acid, 2 parts 0.2% (wt/vol) sulfanilamide, and 2 parts 0.1% (wt/vol) n-1-naphthylethylenediamine dihydrochloride mixed shortly before use. First, the drug-free medium was tested, and if its color changed to pink, then bottles with drugs were tested. An isolate was considered resistant if, when tested on the same day, the color change (pink or deep red to violet) in the drug tube in question was greater than in the 1:10 diluted growth control. If the tubes did not show any color change and remained the same, these were further incubated for 10 days and for 14 days.

Analysis of data

The data were entered into Microsoft Excel sheet and analyzed. Sensitivity, specificity, positive predictive value, negative predictive value, and concordance were calculated taking proportion method on LJ as reference standard.

  Results Top

Among these 100 patients, 70 were male and 30 were female. Age wise majority of the patients belonged to economically productive age group of 15–54 years. There was male dominance in all the age groups. The isolates used in this study were obtained from different types of clinical specimens. The majority were from pulmonary specimens (89%). Others were extrapulmonary-pus, pleural fluid, aspirate, and urine.

One hundred isolates were tested for four drugs, streptomycin, isoniazid, rifampicin, and ethambutol using critical concentrations. It was observed that 62 isolates were sensitive to all the four drugs by both proportion method and solid NRA. MDR cases were found in 26 and 27 isolates by proportion method and solid NRA, respectively. Monoresistance was observed for all the drugs except rifampicin. Other patterns were also observed in [Table 1].
Table 1: Drug susceptibility pattern of 100 Mycobacterium tuberculosis isolates by gold standard and test method using solid nitrate reduction assay on Lowenstein–Jensen medium

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Resistance pattern of 100 isolates by gold standard proportion method was as follows: 62 isolates were sensitive to all drugs. Seven isolates showed monoresistance; five isolates showed resistance to two drugs other than MDR isolates, while 26 isolates were MDR.

Sensitivity, specificity, positive predictive values, negative predictive values and concordance are depicted in [Table 2].
Table 2: Comparison of drug susceptibility testing results obtained by solid medium using nitrate reductase assay with gold standard proportion method on Lowenstein–Jensen medium

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  Discussion Top

In the present study, for nitrate reductase method using solid medium, sensitivity and specificity of streptomycin was 96.55% and 98.59%, respectively which is similar to values of Lemus et al.[13] study (sensitivity-96.5% and specificity >99.1%), Angeby et al.[6] (sensitivity 95% and specificity 83%), Montoro et al.[7] (sensitivity 93.7% and specificity 98%), and Gupta et al.[14] (sensitivity 88.5% and specificity 94%). The concordance was 98% for streptomycin which was 100% in a study by Sethi et al.[8]

The sensitivity and specificity of isoniazid of the present study was 96.87% and 97.05%, respectively. These values are comparable to the studies of Angeby et al. (sensitivity 97% and specificity 96%),[6] Montoro et al. (sensitivity 95.6% and specificity 100%),[7] Lemus et al. (sensitivity 91.7% and specificity >99%),[13] and Gupta et al. (sensitivity 97% and specificity 100%).[14] The agreement was 97% while it was 99% in Sethi et al. and 98.5% in Gupta et al. studies.[8],[14]

The sensitivity and specificity of rifampicin (R) were 96.29% and 95.89%, respectively in the present study which is lower than Angeby et al. and Montoro et al. (sensitivity and specificity both 100%);[6],[7] however, it is comparable to the study of Lemus et al. (sensitivity 93.9% and specificity >99.1%).[13] In the present study, the concordance was 96% while in a study by Sethi et al. there was 100% agreement and of Gupta et al. it was 99.2%.[8],[14] Values of sensitivity and specificity for ethambutol (EMB) were 78.94% and 91.35%, comparable with the studies of Angeby et al. (sensitivity 75% and specificity 98%),[6] Lemus et al. (sensitivity 88% and specificity >99%).[13] In the study of Montoro et al.,[7] sensitivity and specificitywas 98.7% and 100%. In Sethi et al.[8] and Gupta et al.[14] studies agreement was 99% and in 96.6% in comparison to 89% of this study.

In this colorimetric method, time duration to obtain results was 7 days to 14 days for NRA using a solid medium with thirteen isolates positive by 7th day of incubation, 30 isolates by 10th day and remaining 57 isolates by 14th day of incubation. This period to positivity was quite early as compared to the proportion method which takes 4–6 weeks. Depending on drug resistance surveillance conducted in India, MDR among new cases is 2.84% (confidence interval [CI] 2.27%–3.5%) and in previously treated cases is 11.60% (CI 10.21%–13.15%).[15] In the present study, MDR cases were found to be higher. This can be explained by the fact that as this laboratory is located in a tertiary care hospital, most specimens submitted for DST were from patients who had received some antitubercular treatment or were special cases not responding to treatment. As history of drug intake was not taken directly from the patients in the present study, minimal history available in laboratory register could not clarify whether the patient had taken treatment for TB in any form. Hence, a division of MDR cases on the basis of new and previously treated cases was not done. Among 100 isolates, 26% were found to be MDR, while 62% isolates were sensitive to all drugs.

In the present study, the NRA method was found to be cost effective. The cost for Proportion method on LJ medium was Rs.~200 ($ 3.04) while that of NRA using LJ was Rs.~210 ($ 3.19).

The test has given concordance of >95% for three drugs, namely streptomycin, INH, and rifampicin, while it was low for ethambutol 89%. In a meta-analysis of DST for four first-line drugs, good sensitivity was observed with INH, rifampicin, and ethambutol, while it was low with streptomycin.[16] Considering this, NRA technique can be applied to DST for INH and rifampicin and with caution for streptomycin and ethambutol. The technique has strength in good concordance with a reference standard and less time to report the results. Cost wise, it is cheaper than other rapid alternatives. One limiting feature to perform DST by this method could be the absence of nitrate reductase enzyme in certain isolates of M. tuberculosis. In the present study, the nitrate test was performed first as it was an evaluation study.

  Conclusion Top

NRA for DST using solid medium is a rapid, cheap, easy to perform test and good concordance of results was found with that of reference standard proportion method.

Financial support and sponsorship

Kasturba Health Society, Sevagram

Conflicts of interest

There are no conflicts of interest.

  References Top

World Health Organization. Global Tuberculosis Report 2017. Geneva: World Health Organization; 2017.  Back to cited text no. 1
World Health Organization. The WHO/IUATLD Global Project on Anti Tuberculosis Drug Resistance Surveillance. Anti-Tuberculosis Drug Resistance in the World, Report No. 2: Prevalence and Trends. Geneva, Switzerland: World Health Organization; 2000.  Back to cited text no. 2
Shikama ML, Ferro e Silva R, Villela G, Sato DN, Martins MC, Giampaglia CM, et al. Multicentre study of nitrate reductase assay for rapid detection of rifampicin-resistant M. Tuberculosis. Int J Tuberc Lung Dis 2009;13:377-80.  Back to cited text no. 3
Martin A, Paasch F, Von Groll A, Fissette K, Almeida P, Varaine F, et al. Thin-layer agar for detection of resistance to rifampicin, ofloxacin and kanamycin in Mycobacterium tuberculosis isolates. Int J Tuberc Lung Dis 2009;13:1301-4.  Back to cited text no. 4
Martin A, Portaels F, Palomino JC. Colorimetric redox-indicator methods for the rapid detection of multidrug resistance in Mycobacterium tuberculosis: A systematic review and meta-analysis. J Antimicrob Chemother 2007;59:175-83.  Back to cited text no. 5
Angeby KA, Klintz L, Hoffner SE. Rapid and inexpensive drug susceptibility testing of Mycobacterium tuberculosis with a nitrate reductase assay. J Clin Microbiol 2002;40:553-5.  Back to cited text no. 6
Montoro E, Lemus D, Echemendia M, Martin A, Portaels F, Palomino JC. Comparative evaluation of the nitrate reduction assay, the MTT test, and the resazurin microtitre assay for drug susceptibility testing of clinical isolates of Mycobacterium tuberculosis. J Antimicrob Chemother 2005;55:500-5.  Back to cited text no. 7
Sethi S, Sharma S, Sharma SK, Meharwal SK, Jindal SK, Sharma M. Drug susceptibility of Mycobacterium tuberculosis to primary antitubercular drugs by nitrate reductase assay. Indian J Med Res 2004;120:468-71.  Back to cited text no. 8
Poojary A, Nataraj G, Kanade S, Mehta P, Baveja S. Rapid antibiotic susceptibility testing of Mycobacterium tuberculosis: Its utility in resource poor settings. Indian J Med Microbiol 2006;24:268-72.  Back to cited text no. 9
[PUBMED]  [Full text]  
Syre H, Phyu S, Sandven P, Bjorvatn B, Grewal HM. Rapid colorimetric method for testing susceptibility of Mycobacterium tuberculosis to isoniazid and rifampin in liquid cultures. J Clin Microbiol 2003;41:5173-7.  Back to cited text no. 10
Musa HR, Ambroggi M, Souto A, Angeby KA. Drug susceptibility testing of Mycobacterium tuberculosis by a nitrate reductase assay applied directly on microscopy-positive sputum samples. J Clin Microbiol 2005;43:3159-61.  Back to cited text no. 11
Manual of Standard Operating Procedures (SOPs). Culture of Mycobacterium tuberculosis and Drug Susceptibility Testing on Solid Medium. Version No. 01.01.2009. New Delhi: Revised National Tuberculosis Control Programme, Central TB Division; 2009.  Back to cited text no. 12
Lemus D, Montoro E, Echemendía M, Martin A, Portaels F, Palomino JC. Nitrate reductase assay for detection of drug resistance in Mycobacterium tuberculosis: Simple and inexpensive method for low-resource laboratories. J Med Microbiol 2006;55:861-3.  Back to cited text no. 13
Gupta A, Sen MR, Mohapatra TM, Anupurba S. Evaluation of the performance of nitrate reductase assay for rapid drug-susceptibility testing of Mycobacterium tuberculosis in North India. J Health Popul Nutr 2011;29:20-5.  Back to cited text no. 14
Report of theFirst National Antituberculosis Drug Resistance Survey India 2014-16. Ministry of Health and Family Welfare, Government of India; 2018.  Back to cited text no. 15
Coban AY, Deveci A, Sunter AT, Martin A. Nitrate reductase assay for rapid detection of isoniazid, rifampin, ethambutol, and streptomycin resistance in Mycobacterium tuberculosis: A systematic review and meta-analysis. J Clin Microbiol 2014;52:15-9.  Back to cited text no. 16


  [Table 1], [Table 2]


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