Comparative evaluation of drug susceptibility testing of Mycobacterium tuberculosis by Nitrate reductase assay on direct sputum samples and Conventional proportion method

Background and Objectives: The routinely used methods for anti-tubercular drug susceptibility testing are either costly or slow. As the prevalence of multidrug-resistant strains is increasing, the need for fast, reliable, and inexpensive methods that can be applied in settings with limited resources is essential. Methods: It was a study of 100 sputum samples from smear positive patients at RNTCP centre. The samples were subjected to anti-tubercular drug susceptibility by Nitrate reductase assay on direct sputum samples and the routine indirect Conventional proportion method for two primary anti-tubercular drugs, i.e., Isoniazid, and Rifampicin. Results: Out of 100 samples, 94 were sensitive to Isoniazid and Rifampicin by both Conventional Proportion Method, and Nitrate Reductase Assay. Four isolates were detected as MDR-TB strains (resistant to both Isoniazid and Rifampicin) and two were resistant only to Isoniazid by both the methods. Conclusion: Drug susceptibility detected by Nitrate Reductase Assay has excellent agreement with the gold standard Conventional proportion method for Mycobacterium tuberculosis in our study. Hence in countries like India, where there is burden of tuberculosis cases and especially of drug resistant cases, NRA is a very valuable tool in the detection, treatment and follow up of tuberculosis cases for drug resistance.


Introduction
Tuberculosis (TB) has been one of the fatal human infections with many serious consequences, affecting humans since antiquity. The situation has been worsened due to the emergence of multi drug resistant tuberculosis (MDR-TB), extensively drug resistant tuberculosis (XDR-TB) and HIV-TB co-infection [1]. The rapid identification of multi drug resistant isolates is essential for the effective treatment of patients. Conventional drug susceptibility test results become available only after 4 to 6 weeks after isolation by culture. Culture itself takes additional 4 to 8 weeks. Liquid culture methods like BACTEC MGIT 960 take much lesser time but the initial infrastructure requirement and the cost per test are a bit high to afford in many laboratories across India. An efficient and rapid drug susceptibility testing becomes necessary in such a situation.
The nitrate reductase assay (NRA), is a simple and less expensive technique based on the characteristic ability of M. tuberculosis to reduce nitrate to nitrite. Hence, in this study we tried to study the efficacy of Nitrate reductase assay as an alternative to the Conventional proportion method (CPM) for anti-tubercular drug susceptibility testing.

Research Article
Tropical Journal of Pathology & Microbiology Available online at: www.pathologyreview.in 220 | P a g e

Materials and Methods
Sputum samples from smear positive patients visiting our RNTCP centre were collected in a sterile wide mouthed container. The collected samples were subjected to decontamination and concentration by modified Petroff's method [2]. Biosafety class II cabinet was used for procedure.
The control for this test was an L-J slant with 1,000 μg/ml of potassium nitrate without any antibiotics. A total of three of these control 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 [3]. 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 7 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 antibioticcontaining tubes. 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 14 days and 21 days. The results of the NRA were compared with the CPM. The CPM was performed using L-J medium according to the standard procedure [4] with the recommended critical concentration of INH, and RIF. H37Rv served as a standard control for both the tests.

Results
A total of 100 sputum samples with smear positivity were subjected to both direct NRA (without isolation of Mycobacterium tuberculosis) and indirect CPM (after isolation M. tuberculosis) and compared. Out of 100 samples, 94 were sensitive to both Isoniazid and Rifampicin by both Conventional Proportion Method, and Nitrate Reductase Assay (Table 1). Four isolates were detected as MDR-TB strains, i.e., resistant to both Isoniazid and Rifampicin, and two were resistant only to Isoniazid by both the methods. The results showed that NRA and CPM do not differ for both drugs. Thus an excellent agreement between the results of NRA and CPM was found for Isoniazid and Rifampicin of 100%. In our study, results were available in 7 days for 42 strains, 14 days for 39 strains, in 21 days for 19 strains. When compared to the CPM, the results of NRA were available two to three weeks ahead of CPM. Also another added advantage is that there is no need to isolate the organism by culture as the drug-susceptibility by NRA was done on direct sputum samples, thereby further reducing the time.   The same result is obtained by both the methods, i.e., conventional proportion method and nitrate reductase assay.

Discussion
NRA is simple to perform and does not require additional equipment and reagents than those used for Conventional Proportion Method. NRA is performed on classical LJ media used routinely in all TB laboratories.
Results are simple to interpret by change of color. Also, biosafety problems are limited as the test is performed in a solid medium reducing the risk of production of aerosols during work. observed by other authors [7, 8, 9, 10, 11, and 12]. The findings were analyzed and supported in the metaanalysis by Martin et al [13], in which they have mentioned the need for more such studies in countries with high prevalence of tuberculosis.
When compared to the CPM, the results of direct NRA were available two to three weeks ahead of CPM with an added advantage of no necessity to isolate the organism. Liquid culture methods like BACTEC MGIT 960 take much lesser time but the initial infrastructure requirement and the cost per test are a bit high to afford in many laboratories across India. Nitrate reductase negative strains of M. tuberculosis are rare (<1%) and would create no false results since the control would be negative and the test would therefore be invalid [7]. No such strains were encountered in our study.

Conclusion
NRA is simple to perform, and provides a rapid, accurate, and cost effective means for the detection of Isoniazid and Rifampicin resistance in M. tuberculosis. Drug susceptibility detected by direct Nitrate Reductase Assay (NRA) has excellent agreement with the gold standard Conventional proportion method for M. tuberculosis. NRA is also a phenotypic method as