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Indian Journal of Medical Microbiology, Vol. 30, No. 1, January-March, 2012, pp. 81-84 Brief Communication Comparison of the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide tube method with the conventional method and real-time polymerase chain reaction for the detection of rifampicin resistance in Mycobacterium tuberculosis U Raut1, S Rantai2, P Narang3, DS Chauhan4, M Chahar4, R Narang3, DK Mendiratta3 1 Departments of Microbiology, Jawaharlal Nehru Medical College, Datta Meghe Institute of Medical Sciences, Sawangi (M), Wardha, Maharashtra, India Correspondence Address: Date of Submission: 14-Jun-2011 Code Number: mb12015 PMID: 22361766 DOI: 10.4103/0255-0857.93047 Abstract Colorimetric methods are cheap, reproducible, and rapid methods of detecting drug resistance in Mycobacterium tuberculosis. The MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) method is one such technique that has been established in our laboratory to detect rifampicin resistance. The present study compared the results of the MTT method with those of the proportion method and real-time polymerase chain reaction (RTPCR) in order to establish sensitivity and specificity of MTT. The mutations for rifampicin resistance occur in rpoB gene, and the commonest reported are in codons 526 and 531. Therefore, RTPCR was targeted at these two codons. The concordance of MTT with the proportion method and RTPCR was 94 and 72.77%, respectively, and that of RTPCR with the proportion method was 77.77%. While the study confirmed that the MTT method is a good method for detecting rifampicin resistance, it also brought out the fact that RTPCR when targeted for limited mutations is not a good tool. Either the genotypic method used should target the total 81-bp rpoB genome or methods such as DNA sequencing should be used. For resource-constraint laboratories, the MTT method can be considered as a better choice. Keywords: MTT, Mycobacterium tuberculosis, real-time polymerase chain reaction, rpoB gene Introduction Control of tuberculosis is threatened by the widespread emergence of multidrug resistance (MDR) in Mycobacterium tuberculosis (MTB). The situation is further compounded by the emergence of XDR-TB (extensively drug-resistant TB) and very recently of extremely drug resistant (XXDR) strains. [1] Hence, an early detection of drug resistance in MTB constitutes the priority of TB control programs as it allows the initiation of appropriate treatment in patients and also surveillance of drug resistance. 'Rapid detection of drug resistance using genotypic methods have the advantage of shorter turnaround time, possibility of direct application on clinical samples, lower biohazard risk and feasibility of automation'. [2] Most of the time these methods are directed toward the detection of rifampicin resistance since it is considered a good surrogate marker for MDR-TB especially in settings with the high prevalence of MDR-TB. However, not all the molecular mechanisms of drug resistance are known. Phenotypic methods, on the other hand, are in general simpler to perform and easier to implement on a routine basis in clinical microbiology laboratories. [2] Recently, some workers have used colorimetric methods, which are relatively rapid and fairly accurate. [3],[4],[5] One such method using the MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) dye was standardized in our department and showed encouraging results. [6] The present study is an extension and has been taken up to assess the efficiency of the MTT method for the detection of rifampicin resistance in comparison with the molecular method using mutation for rpoB gene at the commonest site, i.e., at codons His526 and Ser531. The proportion method on LJ medium was taken as the gold standard. Materials and Methods Fifty clinical isolates of MTB were randomly selected from the stock strains and revived by subculturing on two Lowenstein-Jensen (LJ) slopes. The isolates were identified and reconfirmed as M. tuberculosis by using standard biochemical reactions. [7] They were then subjected to susceptibility testing for rifampicin on LJ medium by using the proportion method [8] taken as the gold standard and by using the MTT tube method as described in the previous study. [6] Briefly, for the MTT method, 0.5 ml of inoculum (10 7 cfu/ml) prepared in Middlebrook's 7H9 broth supplemented with oleic acid, albumin, dextrose, and catalase was added to 0.5 ml of rifampicin (1 μg/ml) solution. This was incubated at 37°C for 4 days along with drug-free controls. The MTT assay was then performed to find out change in color from yellow to purple, indicating growth in the tubes. The optical density for each tube was also calculated by a colorimeter at 570 nm. A strain with relative optical density unit of ≤0.2 was considered as sensitive and that with >0.2 was considered as resistant. A H 37 Rv strain (ATCC 27394) was also included as an experimental control. Furthermore, 20 isolates were picked randomly from 50 of the aforementioned strains and were looked for rifampicin susceptibility by detecting mutations at rpoB gene through real-time polymerase chain reaction (RTPCR) at the National JALMA Institute of Leprosy and Other Mycobacterial Diseases, Agra, Uttar Pradesh, India. DNA isolation was done after a fresh subculture on LJ medium by using the method described by van soolingen et al.[9] DNA extracts were then amplified by RTPCR as described by Torres et al[10] using TR8 (5'-GTGCACGTCGCGGACC-TCCA) and TR9 (5'-TCGCCGCGATCAAGGAGT) primers and detected by fluorescence resonance energy transfer probes comprising rpoB sensor (5'- 640c-ACCCACAAGCGCCGACTGCTGG-3'phosd) and rpoB anchor (5'-TTCATGGACCAGAACAACCCGCTGT-CGGT-3'Fe). Both primers (TR8 and TR9) and probes (rpoB anchor and rpoB sensor) used were designed and synthesized by TIB MOLBIOL (DNA Synthesis Service, Roche Diagnostics, Berlin, Germany). The melting temperature (Tm ) for each isolate was obtained and compared with that of H 37 Rv. Any deviation in Tm of ≥(2 ± 0.5) from the Tm of H 37 Rv was considered to have mutations at either 526 or 531 codon. Results The results obtained by using the MTT method were read visually and colorimetrically, and they matched perfectly with each other. When the results of the MTT method were compared with those of the proportion method, a concordance of 94% was seen between them, with three discordant results [Table - 1]. Thus, the agreement between the two tests was found to be very good (kappa coefficient = 0.868; 95% CI = 0.724-1.013). The sensitivity and specificity of the MTT method was calculated as 94.1 and 93.2%, respectively. Further testing for the presence of mutations at codons 531 and 526 in rpoB gene was done only on 20 isolates. These included the three discordant isolates found by using the MTT and proportion methods and 17 randomly selected isolates irrespective of their susceptibility. Of these, 15 showed no mutation at codon 531 or 526, 2 (Lab No. 1128 and 1011) could not be amplified, and 3 isolates (Lab No. 1319, 1424, and 1361) were found to have a deviation in Tm of ≥(2 ± 0.5) from the Tm of H 37 Rv, indicating the presence of mutation at codon 531(TCG→TTG) [Table - 2]. Comparison of results obtained by all three methods for 20 isolates [Table - 2] showed that excluding 2 isolates that could not be amplified by RTPCR, concordance between the proportion method and RTPCR was 77.77% (kappa coefficient = 0.478; 95% CI = 0.094-0.863) while that of the MTT method and RTPCR was 72.22% (kappa coefficient = 0.400; 95% CI = 0.042-0.758). Thus, by considering the proportion method as the gold standard, the sensitivity and specificity of RTPCR for the detection of mutations at codons 531 and 526 were found to be 42.85 and 100%, respectively. Discussion Molecular methods have shown a great potential for detecting drug-resistant strains. However, they await complete acceptance, and there is a need to standardize them by taking the conventional method as a gold standard. Moreover, these methods are highly technical and expensive and require great expertise. Colorimetric methods give rapid results in lesser cost. In the present study, concordance between the MTT method and the proportion method was 94% and that between RTPCR and the MTT method was 72.22% and between RTPCR and the proportion method was 77.77%, which is rather poor. The RTPCR detected all rifampicin-sensitive strains as nonmutants, giving a specificity of 100%. However, it could detect only three of the seven resistant strains with mutations at codon 531. None showed mutation at codon 526. More than 95% of rifampicin-resistant strains are associated with mutations at 81-bp rpoB region, with more than 70% of the mutations being present at the level of codon Ser531 and His526. Torres et al. had designed the primers and probes directed to amplify only the fragment spanning codon 526 and 531 of rpoB gene with good results. [10] On an earlier occasion, National JALMA Institute used same probes and primers and had reported 100% concordance with their resistant strains tested by sequencing. [11] The same primers and probes were used in the present study, which showed a concordance of 77.77% with the proportion method. Codon 516 has also been suggested as an important site for mutation, and probes should be designed to detect this in the system. Fan et al. reported a concordance of only 87.2% between the phenotypic method and DNA sequencing even after detecting mutations at codons 531, 526, and 516. [12] The reason for the discrepancy reported by them was probably the presence of mutations beyond the 81-bp region of rpoB gene or the involvement of at least one additional mechanism of resistance. Although the number tested was small, it did indicate that RTPCR used in the study was not adequate to detect all the resistant strains and the molecular type of resistant strains in our area are different from those found at Agra. Also, as described by Lee et al., different kinds of point mutations within a single codon can also be present. [13] Hence, there is an obvious need to do sequencing of three mutants detected in the study in order to determine the exact site of mutation and to find out any other mechanism of resistance existing in them. Also, sequencing is required to confirm the results of three discordant isolates (Lab No. 352, 901, and 1562) and those found resistant by both the proportion method and the MTT method as well. The RTPCR otherwise is considered a good technique because it gives rapid amplification and test can be completed within 2 hours including the time for DNA extraction. It also correctly detected all strains sensitive by the conventional proportion method. If molecular methods are used, they should incorporate primers and probes that target mutation sites common for the given area as the strains may vary from one geographical area to another. Alternatively, the system should use primers to detect total codons in 81-bp genome or methods such as DNA sequencing should be looked for. The study recommends that as molecular methods and DNA sequencing are very expensive and require special technical expertise and laboratories, resource-constrained laboratories can utilize the MTT tube method for performing drug susceptibility test on isolates. Acknowledgements We thank Dr. V.M. Katoch, the then Director of the National JALMA Institute of Leprosy and Other Mycobacterial Diseases, Agra, Uttar Pradesh, India, for his valuable guidance and support in carrying out molecular part of the study at his esteemed institute. References
Copyright 2012 - Indian Journal of Medical Microbiology The following images related to this document are available:Photo images[mb12015t1.jpg] [mb12015t2.jpg] |
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