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ORIGINAL ARTICLE Table of Contents   
Year : 2009  |  Volume : 52  |  Issue : 3  |  Page : 343-344
Direct drug susceptibility testing of Mycobacterium tuberculosis to primary anti-tubercular drugs by nitrate reductase assay


Department of Microbiology, St John's Medical College, Bangalore - 560 034, India

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Date of Web Publication12-Aug-2009
 

   Abstract 

Objectives: Traditional drug susceptibility testing for Mycobacterium tuberculosis takes weeks and/or expensive. In this study, we evaluated nitrate reductase assay for drug susceptibility testing which is faster than the visual detection of colonies. Materials and Methods: 32 clinical specimens (direct microscopy positive for AFB with 1+, 2+ or 3+ grading) were decontaminated and the sediment was inoculated onto the L-J medium with INH or Rifampicin incorporated with Potassium nitrate and the same medium without antibiotics at 1;10 dilution as control. After 2 weeks, the control was first tested for color change with addition of nitrate reductase reagents. If found positive, the media with antibiotics were tested and compared. Futher incubation was done if the control was found to be negative. The results obtained was compared with standard direct proportion method for drug susceptibility testing. Results: Resistance of isolates as shown by both methods for INH and Rifampicin was 37.5% and 31.3% respectively. The results showed that NRA and proportion method do not differ significantly ( P < 0.05 for both drugs). Thus an excellent agreement between the results of NRA and proportion method was found for two primary anti-tubercular drugs, 87.5% for INH and 97% for Rifampicin. Conclusion: Nitrate reductase assay is a rapid and inexpensive method for susceptibility testing of M. tuberculosis for primary anti-tubercular drugs and could be an alternative to existing methods, particularly in resource poor settings.

Keywords: Mycobacterium tuberculosis , nitrate reductase assay, proportion method

How to cite this article:
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

How to cite this URL:
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 [serial online] 2009 [cited 2019 Dec 11];52:343-4. Available from: http://www.ijpmonline.org/text.asp?2009/52/3/343/54989



   Introduction Top


Tuberculosis is a major public health problem worldwide. Emergence of multidrug-resistant (MDR) tuberculosis is now a major obstacle to the effective treatment of this condition. Hence, early reporting of the sensitivity for M. tuberculosis has become imperative, so that appropriate drugs can be chosen for effective cure. However, conventional culture and sensitivity takes weeks to yield results. Automated culture though takes only 10-14 days, is too expensive. There is an urgent need for an alternative sensitivity testing method for anti-tubercular drugs which is rapid, simple and affordable. Several investigators have suggested nitrate reductase assay (NRA) as an alternative method which is simple and suitable for resource-poor countries. [1],[2],[3],[4],[5],[6] However, there is a need for comparing this new method with the existing standard method . Hence, a study was conducted to evaluate the role of NRA in comparison to conventional proportion method (PM) of sensitivity testing for primary anti-tubercular drugs. Sensitivity was detected for Isoniazid (INH) and Rifampicin.


   Materials and Methods Top


The study was conducted for a period of seven months from January to July 2007. A total of 32 sputum samples were processed (one sample per patient). Direct susceptibility testing was done for two primary anti-tubercular drugs (INH and Rifampicin) by NRA [1],[2],[3],[4],[5] and conventional PM. [5],[7],[8]

Smear-positive sputum samples (Positive for AFB with 1+, 2+ or 3+ grade) were inoculated directly for NRA and PM after decontamination and concentration by modified Petroff's method. [9] The sediment (3 ml) was divided into two halves. One half of the sediment was used for NRA and the other half for the PM. For NRA, the first half of the sediment was suspended in 3 ml of sterile distilled water and 200 l was inoculated onto Lowenstein-Jenson (L-J) medium with 1,000 g/ml of potassium nitrate with antibiotics (0.2g/ml for INH and 40 g/ml for Rifampicin separately). The control for this test was an L-J slant with 1,000 g/ml of potassium nitrate without any antibiotics. A total of three of these media were inoculated at 1:10 dilution of the above mentioned sediment. All tubes were incubated at 37 C. NRA was based on ability of Mycobacterium tuberculosis to reduce nitrate to nitrite, which was revealed by color change in the medium after addition of Griess reagent. [4] The results were classified as negative (sensitive) if no color change occurred in the antibiotic containing media and positive (resistant) if there was pink to red color development. Color intensity was compared with that of the control. After 14 days of incubation, Griess reagent was added to one of the control tubes. If found positive (pink to red color), it was then tested for the antibiotic-containing tubes [Figure 1]. Any change of color (more than the control tube) was taken as resistance. If the control tube did not show any color change after addition of Griess reagent, other tubes were further incubated and the procedure was repeated at 21 days and 28 days. The results of the NRA were compared with the standard PM.

The PM was performed using L-J medium according to the standard procedure with the recommended critical concentration of INH and Rifampicin. [2],[4],[8] H 37 RV served as a standard control for both the tests.


   Results Top


A total of 32 M. tuberculosis isolates were subjected to both NRA and PM and compared [Table 1]. The results showed that NRA and PM do not differ significantly ( P < 0.05) for both drugs. Thus an excellent agreement between the results of NRA and PM was found for two primary anti- tubercular drugs; 87.5% for INH and 97% for Rifampicin. In our study, results were available in 14 days for nine strains (28.1%), in 21 days for 21 strains (65.6%) and in 28 days for the remaining two strains (6.3%). In 30 (94.1%) cases results were available within three weeks. When compared to the PM, the results of NRA were available one to two weeks ahead of PM.


   Discussion Top


For proper and effective management of tuberculosis, particularly pulmonary tuberculosis, drug sensitivity testing is a must. However, several reasons such as lack of technical expertise, cost, time factor make the standard method out of reach for many clinicians managing tuberculosis. So much so many cases are over-treated without any benefit and many a time with unwarranted side-effects.

In the present study, NRA was explored as an alternative sensitivity testing method to anti-tubercular drugs and compared with the existing sensitivity testing method. The results showed excellent agreement between the two methods; 87.5% for INH and 97% for Rifampicin. With reference to NRA, Sethi et al. , [2] have shown 100% agreement for Rifampicin and 99% for INH. Musa et al. , [4] have shown 93% and 100% agreement for INH and Rifampicin respectively.

As the NRA was dependent on color change, results could be read well ahead of the conventional PM (two to three weeks for NRA/ four to six weeks for PM). Similar findings were also observed by Angeby et al. , [1] Sethi et al. , [2] Lemus et al. , [5] and Affolabi et al . [6] This time gain is an additional advantage and will be useful for early institution of appropriate therapy. Possibly, a liquid medium-based NRA could reduce this time further if applied on smear microscopy positive samples. An additional advantage of NRA was an ease of reading the result without the help of any sophisticated instrument.

More than 99% of M. tuberculosis strains possess nitrate reductase enzyme and hence are capable of reducing nitrate, making NRA an excellent alternative method for anti-tubercular drug susceptibility testing. Some other species like M. kansasii, M. smegmatis also possess this enzyme. [2] However, these strains are not frequently encountered and can be identified by the morphology and various biochemical tests.

To conclude, the NRA is rapid, inexpensive, and easy to perform. It does not require costly equipment and expensive substrates or reagents. Nevertheless, more studies are required to further assess the accuracy and reproducibility before this method can be considered for clinical use. We believe that NRA can become an important tool for rapid detection of MDR tuberculosis.

 
   References Top

1.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. 1  [PUBMED]  [FULLTEXT]
2.Sethi S, Sharma S,Sharma SK, Meharwal SK, Jindal SK, Sharma M. Drug susceptibility of Mycobacterium tuberculosis to primary anti-tubercular drugs by nitrate reductase assay. Indian J Med Res 2002;120:468-71.  Back to cited text no. 2    
3.Coban AY, Birinci A, Ekinci B, Durupinar B. Drug susceptibility testing of Mycobacterium tuberculosis with nitrate reductase assay. Int J Antimicrob Agents 2004;24:304-6.  Back to cited text no. 3  [PUBMED]  [FULLTEXT]
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.Lemus D, Montoro E, Echemendia M, Martin A, Portaels F, Palomino JC. Nitrate reduction assay for detection of drug resistance in Mycobacterium tuberculosis : Simple and in expensive method for low-resource laboratories. J Med Microbiol 2006;55:861-3.  Back to cited text no. 5    
6.Affolabi D, Odoun M, Martin A, Palamino JC, Anagonou S, Portaels F. Evaluation of direct detection of Mycobacterium tuberculosis Rifampicin resistance by nitrate reductase assay applied to sputum samples in Cotonou, Benin. J Clin Microbiol 2007;45:2123-5.  Back to cited text no. 6    
7.Canetti G, Fox W, Khomenko AL, 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 programs. Bull World Health Organ 1969;41:21-43.  Back to cited text no. 7    
8.Kent PT, Kubica GP. Public Health Mycobacteriology: A guide for the level III laboratory. U.S. Department of Health and laboratory services, Centre for disease control and prevention, Atlanta,Ga.  Back to cited text no. 8    
9.Laidlaw M, Mycobacterium: Tubercle bacilli. In: Collee JG, Fraser AG, Marmion BP, editors. Mackie and McCartney Practical Medical Microbiology. 13 th ed. New York: Charchill Livingstone; 1989. p. 412-3.  Back to cited text no. 9    

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Correspondence Address:
Baijayanti Mishra
Department of Microbiology, St John's Medical College, Bangalore - 560034
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.54989

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