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 ~  Abstract
 ~  Introduction
 ~  Materials and Me...
 ~  Results
 ~  Discussion
 ~  References
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BRIEF COMMUNICATION
Year : 2010  |  Volume : 28  |  Issue : 4  |  Page : 363-365
 

Evaluation of nitrate reductase assay for direct detection of drug resistance in Mycobacterium tuberculosis: Rapid and inexpensive method for low-resource settings


Department of Microbiology, UCMS & GTB Hospital, Delhi - 110 095, India

Date of Submission21-Dec-2009
Date of Acceptance01-Jul-2010
Date of Web Publication20-Oct-2010

Correspondence Address:
M Gupta
Department of Microbiology, UCMS & GTB Hospital, Delhi - 110 095
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.71816

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 ~ Abstract 

The aim of this study was to evaluate a nitrate reductase assay (NRA) for the direct detection of multidrug resistance (MDR) in Mycobacterium tuberculosis from 100 smear-positive sputum samples. The NRA results were compared with the reference proportion method for 100 sputum specimens for which comparable results were available. NRA results were obtained at day 7 for 61 specimens, results for 26 specimens were obtained at day 10, and the results for 13 specimens were obtained at day 14. Thus, 87% of NRA results were obtained in 10 days. NRA is a rapid, accurate, and cost-effective method for the detection of MDR in M. tuberculosis isolates as compared to the proportion method, which is time consuming. Therefore, NRA constitutes a useful tool for detection of tuberculosis drug resistance in low-resource countries with limited laboratory facilities due to its low-cost, ease of performance and lack of requirement of sophisticated equipment.


Keywords: Drug-susceptibility testing, M. tuberculosis, nitrate reductase assay


How to cite this article:
Gupta M, Shamma, Singh N P, Kaur I R. Evaluation of nitrate reductase assay for direct detection of drug resistance in Mycobacterium tuberculosis: Rapid and inexpensive method for low-resource settings. Indian J Med Microbiol 2010;28:363-5

How to cite this URL:
Gupta M, Shamma, Singh N P, Kaur I R. Evaluation of nitrate reductase assay for direct detection of drug resistance in Mycobacterium tuberculosis: Rapid and inexpensive method for low-resource settings. Indian J Med Microbiol [serial online] 2010 [cited 2019 Sep 23];28:363-5. Available from: http://www.ijmm.org/text.asp?2010/28/4/363/71816



 ~ Introduction Top


Multidrug-resistant tuberculosis is an increasing public-health concern in many parts of the world. The spread of multidrug-resistant (MDR) strains of Mycobacterium tuberculosis has become a major concern, [1] since these bacteria often cause incurable disease, even when expensive second- and third-line drugs are available. Current methods for drug-susceptibility testing (DST) of M. tuberculosis are either slow or costly. As the prevalence of MDR strains increases, the need for fast, reliable, and inexpensive methods that can also be applied in low-resources' settings is required. The purpose of this study was to evaluate a rapid colorimetric nitrate reductase assay (NRA) for direct DST of M. tuberculosis from sputum samples and was compared with conventional proportion method as it is the reference method followed for drug susceptibility-testing under guidelines of Revised National TB Control Programme (RNTCP). An NRA is based on the ability of M. tuberculosis to reduce nitrate to nitrite, which is revealed as a color change of the culture media.


 ~ Materials and Methods Top


From Jan to June 2008, 100 sputum specimens, which were smear microscopy positive [2] submitted to Mycobacteriology laboratory of the Department of Microbiology, were included in the study. The susceptible reference strain M. tuberculosis H37Rv (ATCC 25618) was used. The specimens were processed by modified Petroff digestion-decontamination method, [3] and thereafter concentrated by centrifugation at 3200Χ g for 20 minutes. The supernatant was discarded. The small portion of the sediment was used for smear preparation and stained with Ziehl-Neelsen technique. The sputum samples that were smear positive, were included in the study. The small portion of the remaining sediment was resuspended in 3 ml of sterile distilled water. This was used to inoculate the culture medium used for culturing M. tuberculosis and for direct NRA.

Direct nitrate reductase assay drug-susceptibility testing

Nitrate reductase assay was performed as described by Musa et al. [4] with slight modification. The standard Lφwenstein-Jensen (LJ) medium, with 1000 μg of potassium nitrate (KNO3 )/ml and with or without antimicrobial agents were used. The following critical concentrations were used: 0.2 μg/ml for Isoniazid (INH), 40 μg/ml for Rifampicin (RIF), 4.0 μg/ml for Streptomycin (STR), and 3.0 μg/ml for Ethambutol (EMB). Prior to NRA testing, part of the suspension (1 : 10) was diluted 1 : 10 in sterile distilled water. For each strain, 0.2 ml of the undiluted suspension was inoculated into the tubes containing the LJ medium with KNO 3 and the antibiotics, and 0.2 ml of the 1 : 10 dilution was inoculated into three drug-free tubes containing LJ medium with KNO 3 incorporated. The latter tubes served as growth controls. The tubes were incubated at 37C, and 0.5 ml of freshly prepared reagent mixture (one part concentrated hydrochloric acid, two parts 0.2% sulfanilamide and two parts 0.1% n-1 naphthyl-ethylene diamine dihydrochloride) was added to one drug-free control tube after seven days. If any color change (strong or weak pink) seen, the corresponding antibiotic-containing tubes were also tested and the susceptibility results were read. If no color change was seen in the growth control tube this tube was discarded and other two control tubes and the antibiotic tubes were re-incubated. The procedure was then repeated at day 10 using the second growth control and, if needed, at day 14, using the last growth control tube. The results were classified as negative (no color change) or positive (pink to violet). An isolate was considered resistant to a certain drug if there was a color change in the antibiotic tube in question greater than or equal to that in the 1 : 10-diluted growth control on the same day.

Indirect proportion method

An indirect proportion technique was carried out on LJ medium according to the laboratory's standard procedure. [5] The medium was prepared with or without antimicrobial agents incorporated. Critical concentrations of anti-tuberculosis drugs were the same as were used for NRA. For each strain, 0.2 ml of the undiluted suspension was inoculated into two tubes of LJ medium without antibiotics. Then, 0.2 ml of the undiluted suspension was inoculated into the tubes containing LJ medium with antibiotics. The tubes were incubated at 37C. Final susceptibility results were reported after 42 days following the laboratory's standard procedure.

Statistical analysis

The results of the NRA and proportion method were analyzed. Sensitivity and specificity was calculated using standard formula. Kappa values (κ) were determined to know the agreement between the two methods.


 ~ Results Top


A total of 100 smear-positive sputum samples were included in the present study. Testing of 100 M. tuberculosis strains for their susceptibilities to INH, RIF, STR, and EMB gave an overall agreement of 94% between the NRA and proportion method (384 of 400 susceptibility tests) [Table 1]. The sensitivity of the NRA compared to that of the proportion method was found to be 93% (200 of 216 tests), and the specificity was found to be 94% (174 of 184 tests). Full concordant was found in results of RIF. A good correlation was also found for INH, while some problems were encountered for STR resistance and EMB resistance [Table 1]. The NRA was easy to read. Results were available in seven days for 61% of the strains, in ten days for 87% of the strains, and in 14 days for 100% of the strains. The fully susceptible strains were significantly more likely to be ready in seven days than strains with any resistance. This clearly shows that the median day to positivity is seven days and the interquartile range is 7-10 days by direct NRA. In comparison to conventional proportion method, only 30% of the positive results were available by four weeks.
Table 1: Susceptibility results, sensitivity and specificity of the NRA method compared to those of the proportion method for M. tuberculosis

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 ~ Discussion Top


Complete agreement for the results of the direct NRA and proportion methods was seen for RIF. This is essential, as RIF, together with INH, is the most valuable anti-tuberculous drug. In addition, RIF resistance is mostly combined with INH resistance [4] (in our study 52%) and might therefore be used as a marker of MDR if resources are inadequate. The direct NRA was comparable to the indirect proportion method regarding susceptibility testing of INH (sensitivity in detecting resistance - 98%; specificity - 97%) However, the sensitivities to STR and EMB were 96 and 74% respectively, whereas the specificity was 83 and 98% respectively, for both these drugs. This discordance might be overcome by adjusting the critical drug concentrations used in the NRA test, although it is well known that STR and EMB are difficult drugs to test even by conventional method. These results were almost comparable to the study done by Syre et al. [6] (the sensitivity and specificity 100 and 95%, respectively, for INH and 94 and 100%, respectively, for RIF), and Lemus et al. [7] (sensitivity of the NRA was 91.7, 96.5, 88.0, and 93.9% for INH, STR, EMB, and RMP, respectively, and the specificity was higher than 99.1% for all of the drugs). However, in both the studies, the sensitivity and specificity of NRA was compared using Bactec 460 TB system as a gold standard. In previous studies from India by Mishra et al. [2] and Sethi et al. [8] excellent agreement was observed for RIF and INH by NRA and proportion method. The results of our study thus highlights that NRA can be used directly on the sputum specimen for the early detection of MDR. In our study, we have compared direct NRA with conventional proportion method and have found excellent agreement. However, further studies are required for comparing direct NRA with direct proportion method in our setup. The spread of MDR has become a major concern, so there is a need for requirement of rapid and reliable method that can be applied in low-resource setting. Since in NRA method, color change is considered as the sign of positive, the results are available earlier than the conventional method. The time needed to complete the NRA DST in most cases was 14 days. With the indirect proportion method, which was used as the reference method, it took 42 days to get the final results, even though preliminary results could be obtained earlier for some resistant strains. In previously published NRA studies [4],[8] , the results were available after 7 to 14 days, and when Syre et al. [6] applied the NRA test with liquid medium, most results were available after five days.

Recent methods for DST are either costly, requiring heavy investment in equipments as are automatic culture systems or slow as methods based on culture on solid media. Other low-cost methods have been proposed, such as the MTT or resazurin assays. However, they make use of liquid medium in a Microplate format that makes the techniques complicated. Instead, the NRA utilizes standard solid LJ medium, although with KNO 3 incorporated and it could therefore be easily adopted in any culture laboratory.

In conclusion, the NRA constitutes a useful tool for detection of tuberculosis drug resistance in low-resource countries with limited laboratory facilities due to its low-cost, ease of performance, and lack of requirement of sophisticated equipment.

 
 ~ References Top

1.World Health Organization. 2004. The W.H.O./IUATLD global project on anti-tuberculosis drug resistance surveillance. Anti-tuberculosis drug resistance in the world, report no. 3: WHO/HTM/TB/2005.349.WHO, Geneva, Switzerland.  Back to cited text no. 1      
2.Mishra B, Muralidharan S, Srinivasa H. Direct drug susceptibility testing of Mycobacterium tuberculosis to primary anti-tubercular drugs by nitrate reductase assay. Indian J Pathol Microbiol 2009;52:343-4.  Back to cited text no. 2  [PUBMED]  Medknow Journal  
3.Training Manual for Mycobacterium tuberculosis. Available from: http://www.tbcindia.org/pdfs/DST-DRStraining manual RNTCP.pdf [last accessed on 2009 Oct 1].   Back to cited text no. 3      
4.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. 4  [PUBMED]  [FULLTEXT]  
5.Canetti G, Fox W, Khomenko A, Mahler HT, Menon NK, Mitchison DA, et al. Advances in techniques of testing mycobacterial drug sensitivity and the use of sensitivity tests in tuberculosis control programmes. Bull World Health Organ 1969;41:21-43.   Back to cited text no. 5  [PUBMED]  [FULLTEXT]  
6.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. 6  [PUBMED]  [FULLTEXT]  
7.Lemus D, Montoro E, Martin EA, 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. 7      
8.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  [PUBMED]  [FULLTEXT]  



 
 
    Tables

  [Table 1]

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