|
| | ORIGINAL ARTICLE | | | | | Year : 2010 | Volume : 28 | Issue : 3 | Page : 211-216 | | | Detection of Mycobacterium tuberculosis resistance mutations to rifampin and isoniazid by real-time PCR
A Hristea1, D Otelea1, S Paraschiv1, A Macri2, C Baicus3, O Moldovan2, M Tinischi1, V Arama1, A Streinu-Cercel1
1 'Prof. Dr. Matei Bals' National Institute for Infectious Diseases, Str. Calistrat Grozovici, Nr. 1, Sector 2, 021105 Bucharest, Romania
2 'Marius Nasta' National Institute of Pneumology, Sos. Viilor Nr 90, Sector 5, 050512 Bucharest, Romania
3 Department of Internal Medicine, Colentina Clinical Hospital, Sos. Stefan Cel Mare, 19-21, Sector 2, 020125 Bucharest, Romania
| Date of Submission | 28-Jul-2009 | | Date of Acceptance | 05-Apr-2010 | | Date of Web Publication | 17-Jul-2010 | Correspondence Address:
A Hristea
'Prof. Dr. Matei Bals' National Institute for Infectious Diseases, Str. Calistrat Grozovici, Nr. 1, Sector 2, 021105 Bucharest
Romania
  | 2 |
DOI: 10.4103/0255-0857.66474 PMID: 20644308 Objective: The objective of our study was to evaluate the use of a real-time polymerase chain reaction (PCR)-based technique for the prediction of phenotypic resistance of Mycobacterium tuberculosis. Materials and Methods: We tested 67 M tuberculosis strains (26 drug resistant and 41 drug susceptible) using a method recommended for the LightCycler platform. The susceptibility testing was performed by the absolute concentration method. For rifampin resistance, two regions of the rpoB gene were targeted, while for identification of isoniazid resistance, we searched for mutations in katG and inhA genes. Results: The sensitivity and specificity of this method for rapid detection of mutations for isoniazid resistance were 96% (95% CI: 88% to 100%) and 95% (95% CI: 89% to 100%), respectively. For detection of rifampin resistance, the sensitivity and specificity were 92% (95% CI: 81% to 100%) and 74% (95% CI: 61% to 87%), respectively. The main isoniazid resistance mechanism identified in our isolates is related to changes in the katG gene that encodes catalase. We found that for rifampin resistance the concordance between the predicted and observed phenotype was less than satisfactory. Conclusions: Using this method, the best accuracy for genotyping compared with phenotypic resistance testing was obtained for detecting isoniazid resistance mutations. Although real-time PCR assay may be a valuable diagnostic tool, it is not yet completely satisfactory for detection of drug resistance mutations in M tuberculosis.
Keywords: Mycobacterium tuberculosis, real-time PCR, resistance
How to cite this article:
Hristea A, Otelea D, Paraschiv S, Macri A, Baicus C, Moldovan O, Tinischi M, Arama V, Streinu-Cercel A. Detection of Mycobacterium tuberculosis resistance mutations to rifampin and isoniazid by real-time PCR. Indian J Med Microbiol 2010;28:211-6 |
How to cite this URL:
Hristea A, Otelea D, Paraschiv S, Macri A, Baicus C, Moldovan O, Tinischi M, Arama V, Streinu-Cercel A. Detection of Mycobacterium tuberculosis resistance mutations to rifampin and isoniazid by real-time PCR. Indian J Med Microbiol [serial online] 2010 [cited 2014 Mar 7];28:211-6. Available from: http://www.ijmm.org/text.asp?2010/28/3/211/66474 |
| ~ Introduction | |  |
Tuberculosis (TB) remains a major global health problem despite the availability of effective antituberculosis therapy for over 50 years. The World Health Organization (WHO) estimates that approximately one-third of the global community is infected with Mycobacterium tuberculosis. According to WHO data, with regard to infection rates, Romania is among the top five countries from the European region, with high notification rates both for new and relapse cases of TB; more than 25000 new and relapse cases are recorded every year. [1],[2]
Since the early 1990s, an alarming trend and a growing source of public health concern has been the emergence of resistance to multiple drugs. Multidrug resistance (MDR) is defined as resistance to at least isoniazid (INH) and rifampicin (RMP). Although it remains unclear whether the drug-resistant strains are less transmissible than the susceptible strains, [3] infection-control precautions need to be maintained, since patients with drug-resistant TB are likely to remain infectious for long periods. Thus the public health consequences of drug-resistant tuberculosis might be more serious than those of drug-susceptible disease.
The prevalence of M tuberculosis (MTB) drug resistance in Romania was recently evaluated by a national survey performed between 2003-2004. This showed that 3.6% of the strains isolated from newly diagnosed patients and 8.6% from relapse cases were resistant to one antituberculosis drug (INH). [2] Moreover, the results of this study indicate that MDR was observed in 2.9% of the MTB strains from newly diagnosed patients and in 11% of those isolated from relapse cases. Taking into account that in Romania more than 25000 TB cases (new cases and relapses) are reported each year, we can estimate that more than 1100 patients are infected with MDR-TB strains.
The need to limit the transmission of drug-resistant strains and to reduce the time between diagnosis and effective therapy requires rapid identification of resistance. Classical phenotypic determination of resistance may take up to 10 weeks after referral of a sample to the laboratory. Nucleic acid amplification assays can greatly shorten the detection time. Due to this major advantage, in the last few years, a lot of effort has been invested in designing performance protocols for genotyping MTB strains. Real-time PCR came to be the main approach because of its special features: high sensitivity and specificity as well as speed, with no need for any post-PCR sample manipulation. The results from fundamental research (such as the sequencing of the complete MTB genome) were used to design specific primers and probes that would allow the identification of gene mutations associated with drug resistance in MTB.
It is known that RMP interferes with RNA synthesis by binding to bacterial RNA polymerase. Resistance to RMP is conferred by mutations resulting in at least eight amino acids substitutions in the rpoB subunit of RNA polymerase. Mutations in a limited region of rpoB have been found in >95% of RMP-resistant clinical isolates of MTB and has been shown to result in high-level resistance (MIC >32 μg/mL) to RMP and cross-resistance to all rifamycins.[4]
INH acts by inhibiting an oxygen-sensitive pathway in the mycolic acid biosynthesis of the cell wall. At least four genes have been described to be involved in resistance to isoniazid: the katG gene, which encodes a catalase; the inhA gene, whose product is a target for INH; and the oxyR gene and the neighboring aphC gene, as well as their intergenic region. [5] Several real-time PCR-based methods targeting these specific genomic regions have been described. [6],[7],[8],[9],[10],[11],[12],[13] The purpose of the present study was to evaluate the LightCycler instrument in the detection of these mutations associated with resistant MTB strains isolated from Romanian patients.
| ~ Materials and Methods | | |
Strains and resistance testing
Forty-one susceptible and twenty-six resistant clinical isolates of MTB (23 resistant to both INH and RMP, 1 mono-RMP resistant, and 2 resistant to INH only) from 62 different patients were studied. The susceptibility testing was performed by the absolute concentration method (Meissner). [14] This method is based on the comparison between the growth of mycobacteria on drug-free medium with that of growth on drug-containing media (antituberculosis drugs incorporated in the medium at different concentrations) 21 days after inoculation with a standardized inoculum. Two critical concentrations were used for every tested drug: 0.2 μg/mL and 1 μg/mL for INH and 20 μg/mL and 40 μg/mL for RMP. According to this method, resistance to a drug is defined by the growth of more than 20 colonies on drug-containing media (INH 1 μg/mL, RMP 40 μg/mL).
Extraction of mycobacterial DNA
We extracted MTB DNA by the thermal lysis procedure in the presence of Chelex 100 (Amersham Pharmacia Biotech, Uppsala, Sweden). Briefly, we obtained one loopful of bacteria scraped from Lφwenstein-Jensen solid medium and suspended it in 100 μL sterile water; the same volume of Chelex 10% suspension was added and the mixture was incubated for 45 minutes at 45°C and 5 minutes at 100°C. The samples were centrifuged at 12000 g for 5 minutes and the supernatant was used in the subsequent steps of the experiment.
Real-time PCR using the Lightycler
The MTB drug-resistance genotyping was performed by adapting a previously described protocol. [6] The method published by Torres et al. was designed as a single-tube method capable of detecting RMP and INH resistance mutations; one set of primers and two fluorescently labeled hybridization probes were used for each targeted region. One set of primers and two sets of probes (rpoB1 and rpoB2) that targeted the rpoB gene were used for detection of RMP resistance and one set of primers and probes each for the katG and inhA genes in order to test for INH resistance. All primers and probes were synthesized by TIB Molbiol (DNA Synthesis Service; Roche Diagnostics, Berlin, Germany). The real-time PCR was followed by melting curve analysis, both performed on the LightCycler instrument (Roche Diagnostics, Mannheim, Germany). We used the same PCR conditions (components concentration, cycling, and melting programs) as previously described, but we added 10 more cycles of amplification to the 35 recommended. [7] We included into each experimental run one negative control (the DNA template was replaced with PCR-grade water) and one positive control (the DNA template was isolated from M tuberculosis H37Rv, a strain susceptible to both INH and RMP).
DNA sequencing
Direct PCR sequencing was performed with the commercial BigDye terminator DNA sequencing kit (Applied Biosystems, CA, USA), according to the manufacturer's recommendations. Briefly, the extracted DNA was amplified with the same primers used in the real-time PCR. The thermal cycling was performed on a GeneAmp System 9700 (Applied Biosystems, CA, USA) thermal cycler. The resulting PCR product was purified using MicroCon YM-100 concentrators (spin columns) and sequenced bidirectionally using the BigDye terminator chemistry . The capillary electrophoresis was performed on an ABI Prism 3100-Avant genetic analyzer (Applied Biosystems, CA, USA) and the raw analysis of the sequences was made by Sequencing Analysis Software, version 3.7. Finally, the sequences were aligned with BioEdit software (version 5.0.6) ( www.mbio.ncsu.edu/BioEdit/bioedit.html ) in order to generate an assembled full range sequence, which was then compared with the MTB H37Rv sequence (GenBank Accession No. NC000962).
Statistical analysis
In order to determine the cutoff of T m changes to predict mutations associated with resistance, we generated ROC curves. The area under the ROC curves was then determined, and the cutoff points were identified to maximize test sensitivity (and thus decrease the false negative rate). To further enhance sensitivity we also assessed in parallel the tests detecting the presence of either rpoB1 or rpoB2 for RMP resistance and the presence of either katG or inhA gene for INH resistance. SPSS 10.0 software (SPSS, Inc., Chicago, IL, USA) was used for the database construction and ROC curves, and CAT maker 1.1 (Centre for Evidence-Based Medicine, Oxford, GB, 2004) to calculate the attributes of the diagnostic tests studied.
| ~ Results | | |
During the real-time PCR experiments, the amplification of the DNA template was monitored by continuously measuring the fluorescence level. For samples as well as for the positive control, the fluorescent signals started to rise at a number of cycles, ranging between 20-35. The T m s for the probes annealed to the PCR product were generated by running the melting analysis program (ramping from 50°C to 85°C with 0.1°C per second) and calculated using the LightCycler software. While running different sets of samples along with the positive control, we observed that the melting temperature (T m ) for the MTB H37Rv was variable, ranging between 70.08°C and 71.26°C. Therefore, for each experimental run we analyzed the changes in T m for the PCR products derived from our collection of resistant and susceptible MTB clinical isolates as compared with the T m of the H37Rv tested in the same run rather than using directly the observed T m s.
We also noticed that the T m values for the H37Rv strain as well as for other wild-type (wt) field strains tested were lower than expected (70.08°C for H37 as compared with 72.8°C in the original communication). For the resistant strains, in one case (sample 2312), we obtained a lower T m value than the expected T m; the δ TM was however consistent with AGC > ACC mutation in position 315. In [Figure 1] we have represented the melting profiles for the positive control (H37Rv) and the other seven samples when we analyzed the katG PCR products. It can be seen that sample 2312 has a melting profile different from that of H37Rv; this is in agreement with the phenotypic results, which scored this sample as resistant to INH. The other isolates had the same melting profile as the positive control and were also found to be susceptible to INH by the phenotypic analysis.
The T m changes for the products derived from our collection of resistant and susceptible MTB clinical isolates as compared with T m of the susceptible strain varied widely: 0.00-2.29 (rpoB1), 0.00-4.63 (rpoB2), 0.01-3.32 (inhA), and 0.01-5.86 (katG).
In order to determine the cutoff of T m changes predictable for mutations associated with resistance we used the ROC curves. With regard to RMP resistance, the area under the ROC curves (with the 95% confidence intervals) for rpoB1 and rpoB2 were 0.750 (0.621 to 0.878) and 0.711 (0.584 to 0.839), respectively; for INH resistance, the areas for katG and inhA were 0.935 (0.862 to 1.008) and 0.666 (0.530 to 0.802), respectively. The cutoff points of T m changes predictable for mutations associated with resistance for rpoB1, rpoB2, inhA, and katG were, respectively, 0.90, 0.95, 1.30, and 1.10.
For INH resistance, the genotyping results correctly matched classical resistance testing in 24 (96%) of 25 isolates. There were two isolates reported as genotypically resistant and phenotypically susceptible [Table 1]. We found that 20 strains had mutations in the katG gene, while only one had mutations in the inhA gene; four strains had mutations in both genes. We can conclude that the main INH resistance mechanism identified in the MTB isolates from Romanian is related to changes in the gene that encodes catalase.
For RMP our results only partly matched those generated by the conventional testing. There were 11 isolates reported as genotypically resistant while their phenotype was susceptible; however, only two isolates among 24 phenotypically RMP-resistant strains generated a susceptible hybridization pattern [Table 2].
The sensitivity and specificity of the rapid detection of mutations for INH (presence of either inhA or katG) were 96% (95% CI: 88 to 100) and 95% (95% CI: 89 to 100), respectively, with a positive likelihood ratio (LR+) of 20 and a negative likelihood ratio (LR−) of 0.04. For RMP (presence of rpoB1 or rpoB2), the sensitivity and specificity were 92% (95% CI: 81 to 100%) and 74% (95% CI: 61 to 87%), respectively, with LR+ of 3.58 and LR− of 0.10.
Sequencing was performed in order to obtain more information about nucleotide changes in the examined genes so that differences between the results generated with the two resistance techniques could be explained. Therefore, five MTB strains were partially sequenced in the inhA and katG regions and the other six strains in the rpoB gene. Unfortunately, not all the samples with discordant results between genotyping and phenotyping were available for the sequencing experiment. For comparison, we included also samples found sensitive or resistant to INH and RMP by both tests. Nucleotide sequence analysis (three genes targeted) of the strains with discordant results revealed a number of differences from the sequence of H37Rv [Figure 2]. When translated into amino acids, no peptide sequence changes were observed.
| ~ Discussion | | |
There are a variety of methods to determine the susceptibility of MTB to antituberculosis drugs, but none of them is perfect. [15]
The objective of our study was to evaluate to what extent differences in sequence between circulating strains might hamper the use of real-time PCR-based techniques for the prediction of phenotypic resistance of MTB strains. We used a technique recommended for the LightCycler platform to analyze 67 sequences from 26 drug-resistant and 41 drug-susceptible MTB strains.
Our results suggest that this platform can be used, but there are some limitations. The least important is related to reproducibility. While testing the resistance to INH and RMP with real-time PCR we found that the T m for the sensitive as well as for the resistant strains varied from one experiment to another due to factors that could not be identified. However, the differences between the T m s of the resistant and sensitive strains were consistently observed and could be reliably associated with predicted resistance.
Although genotypic assays are very useful for the rapid detection of drug resistance, there are some limitations. First, not all MTB-DR isolates have mutations in the so-called hot spots of the genes associated with resistance. For instance, about 20%-30% of the INH-resistant strains do not have mutations in katG, inhA, kasA, or aphC genes. [4] For that reason, it is very difficult to design a test that could identify all the possible mutations that confer resistance to anti-MTB drugs. This was the case with the MTB isolates from Romanian patients. Here, only two of the main genes involved in conferring resistance to INH were targeted by PCR. We found that for the Romanian strains, targeting katG was adequate to detect INH resistance. In the analysis of data from other studies, geographical differences in the frequencies of specific mutations are also apparent: the katG gene was mutated at codon 315 in 64% of INH-resistant strains from South Africa and central and western Africa but in only 26% of Singaporean isolates. [16],[17] Furthermore, even the commercial tests for genotyping MTB drug resistance have been reported to have some limitations. A recent study evaluated the results of the two commercially available line probe assays and showed that while the accuracy for RMP resistance was very good, the sensitivity for INH was variable. [17]
We found that for RMP resistance, the concordance between the predicted and observed phenotype was less than satisfactory. This is not entirely unexpected, because a single mutation, although implicated in resistance, might not be enough to generate a resistant phenotype. Two explanations can account for these observations. The most important is the presence of mutations within the rpoB locus that are not associated with resistance but nevertheless influence the annealing properties of the probes. This is most likely why a significant number of strains were classified as resistant to RMP by genetic analysis and sensitive by phenotypic testing.
Furthermore, when performed sequencing, we observed changes at the nucleotide level that did not affect the amino acid sequence, but could alter the sensitivity of the genotypic test: an extra one or two mismatches could influence the T m value, which could affect the interpretation of the sensitivity based on hybridization. This was observed for rpoB, with a high number of false positive results (26%). A much smaller number of false negative results has been observed in strains tested for resistance to INH (4%) and RMP (8%); in this case no mutations were found in the target sequence of the tested genes. The molecular determinants of the resistant phenotype are expected to be found elsewhere.
On the other hand, it should be kept in mind that isolates that are susceptible according to molecular assays that target specific mutations may contain other unknown mechanisms of resistance, and these mechanisms will be missed by these techniques.
A much smaller number of strains were reported sensitive by the hybridization analysis and resistant by the phenotypic analysis. The explanation for this is that a small albeit significant number of strains have determinants of resistance outside the area targeted by the assays we used. A similar phenomenon has been reported by others. [18],[19],[20] Another possibility is that changes have occurred in genes whose products participate in antibiotic permeation or metabolism. [18]
In addition, the results of the absolute concentration method used in the phenotypic test are less reliable compared with the proportion method (the most preferred choice). Errors in the susceptibility testing may be related to any of the following: cultures older than 21-30 days, incorrect size of inoculum, incorrect dilution, or errors in incorporation of antibiotics in culture media. [14] This technique should be further evaluated since the circulating strains in different geographical regions might behave differently when genotypically tested.
This real-time PCR assay could be useful when investigation of drug-resistant TB is mandatory, for example, in cases with a history of one or more previous treatment(s) with several failing, discontinued regimen, or in the situation of exposure to a known source of drug-resistant TB. Although real-time PCR assays may be a valuable diagnostic tool, they are not yet completely satisfactory for MTB drug-resistance detection. Phenotype-based assays will continue to have a place in the clinical mycobacteriology laboratory.
| ~ Conclusion | | |
Thus, based on our experience, the real-time PCR assay could be used in clinical practice, albiet with caution, in cases with risk factors for resistance. The results can be used for guiding the initiation of therapy, but the treatment should be adjusted correspondingly as soon as the phenotypic testing results are available. The best accuracy for genotyping compared with phenotypic resistance testing was obtained for detecting INH resistance mutations targeting the katG gene.
| ~ References | | |
| 1. | Aziz MA, Laszlo A, Raviglione M, Rieder H, Espinal M, Wright A. Guidelines for surveillance of drug resistance in tuberculosis. 2nd ed. World Health Organization 2003. Available from: http://whqlibdoc.who.int/publications/2003/9241546336.pdf. [Accessed on 2009 July 26].  | | 2. | Stoicescu IP, Homorodean D, Chiotan D, Moldovan O, Diculencu D, Popa C, et al. Romanian anti-TB drugs resistance surveillance 2003-2004. Pneumologia 2008;57:131-7. [PUBMED] | | 3. | Soolingen D van, Kremer K, Hermans PWM. Molecular epidemiology: Breakthrough achievements and future prospects. Tuberculosis 2007. In: Palomino JC, Leao SC, Ritacco V, editors. From basic science to patient care. 2007. p. 315-40. Available from: http://www.tuberculosistextbook.com . [Accessed on 2009 July 27]. | | 4. | Cho SN, Brennan PJ. Tuberculosis: Diagnostics. Tuberculosis (Edinb) 2007;87:S14-7. [PUBMED] [FULLTEXT] | | 5. | Ramaswamy SV, Reich R, Dou SJ, Jasperse L, Pan X, Wanger A, et al. Single nucleotide polymorphisms in genes associated with isoniazid resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 2003;47:1241-50. [PUBMED] [FULLTEXT] | | 6. | Marin M, Garcia de Viedma D, Ruiz-Serrano MJ, Bouza E. Rapid direct detection of multiple rifampin and isoniazid resistance mutations in Mycobacterium tuberculosis in respiratory samples by real-time PCR. Antimicrob Agents Chemother 2004;48:4293-300. | | 7. | Torres MJ, Criado A, Palomares JC, Aznar J. Use of real-time PCR and fluorimetry for rapid detection of rifampin and isoniazid resistance-associated mutations in Mycobacterium tuberculosis. J Clin Microbiol 2000;38:3194-9. [PUBMED] [FULLTEXT] | | 8. | Garcia de Viedma D, del Sol Diaz Infantes M, Lasala F, Chaves F, Alcala L, Bouza E. New real-time PCR able to detect in a single tube multiple rifampin resistance mutations and high-level isoniazid resistance mutations in Mycobacterium tuberculosis. J Clin Microbiol 2002;40:988-95. | | 9. | Kocagoz T, Saribas Z, Alp A. Rapid determination of rifampin resistance in clinical isolates of Mycobacterium tuberculosis by real-time PCR. J Clin Microbiol 2005;43:6015-9. [PUBMED] [FULLTEXT] | | 10. | Espasa M, Gonzalez-Martin J, Alcaide F, Aragon LM, Lonca J, Manterola JM, et al. Direct detection in clinical samples of multiple gene mutations causing resistance of Mycobacterium tuberculosis to isoanizid and rifampicin using fluorogenic probes. J Antimicrob Chemother 2005;55:860-5. | | 11. | Wada T, Maeda S, Tamaru A, Imai S, Hase A, Kobayashi K. Dual-probe assay for rapid detection of drug-resistant Mycobacterium tuberculosis by real-time PCR. J Clin Microbiol 2004;42:5277-85. [PUBMED] [FULLTEXT] | | 12. | Ruiz M, Torres MJ, Llanos AC, Arroyo A, Palomares JC, Aznar J. Direct detection of rifampin- and isoanizid-resistant Mycobacterium tuberculosis in auramine-rhodamine-positive sputum specimens by real-time PCR. J Clin Microbiol 2004;42:1585-9. [PUBMED] [FULLTEXT] | | 13. | Hillemann D, Weizenegger M, Kubica T, Richter E, Niemann S. Use of the genotype MTBDR assay for rapid detection of rifampin and isoanizid resistance in Mycobacterium tuberculosis complex isolates. J Clin Microbiol 2005;43:3699-703. [PUBMED] [FULLTEXT] | | 14. | Kim SJ. Drug-susceptibility testing in tuberculosis: Methods and reliability of results. Eur Respir J 2005;25:564-9. [PUBMED] [FULLTEXT] | | 15. | Haas WH, Schilke K, Brand J, Amthor B, Weyer K, Fourie PB, et al. Molecular analysis of katG gene mutations in strains of Mycobacterium tuberculosis complex from Africa. Antimicrob Agents Chemother 1997;41:1601-3. [PUBMED] [FULLTEXT] | | 16. | Lee AS, Lim IH, Tang LL, Telenti A, Wong SY. Contribution of kasA analysis to detection of isoniazid-resistant Mycobacterium tuberculosis in Singapore. Antimicrob Agents Chemother 1999;43:2087-9. [PUBMED] [FULLTEXT] | | 17. | Ling DI, Zwerling AA, Pai M. GenoType MTBDR assays for diagnosis of multidrug-resistant tuberculosis: A meta-analysis. Eur Respir J 2008;32:1165-74. [PUBMED] [FULLTEXT] | | 18. | Kapur V, Li LL, Iordanescu S, Hamrick MR, Wanger A, Kreiswirth BN, et al. Characterization by automated DNA sequencing of mutations in the gene (rpoB) encoding the RNA polymerase beta subunit in rifampin-resistant Mycobacterium tuberculosis strains from New York City and Texas. J Clin Microbiol 1994;32:1095-8. [PUBMED] [FULLTEXT] | | 19. | Kim BJ, Kim SY, Park BH, Lyu MA, Park IK, Bai GH, et al. Mutations in the rpoB gene of Mycobacterium tuberculosis that interfere with PCR-single-strand conformation polymorphism analysis for rifampin susceptibility testing. J Clin Microbiol 1997;35:492-4. [PUBMED] [FULLTEXT] | | 20. | Telenti A, Imboden P, Marchesi F, Lowrie D, Cole S, Colston MJ, et al. Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis. Lancet 1993;341:647-50. [PUBMED] [FULLTEXT] |
[Figure 1], [Figure 2]
[Table 1], [Table 2]
| This article has been cited by | | 1 | Isoniazid MIC and KatG gene mutations among Mycobacterium tuberculosis isolates in northwest of Iran | | | Moaddab, S.R., Farajnia, S., Kardan, D., Zamanlou, S., Alikhani, M.Y. | | Iranian Journal of Basic Medical Sciences. 2011; 14(6): 540-545 | | [Pubmed] | | | 2 | Rapid molecular detection of tuberculosis [6] | | | Mohapatra, P.R. | | New England Journal of Medicine. 2011; 364(2): 184 | | [Pubmed] | | | 3 | Detection of mutations associated with multidrug-resistant Mycobacterium tuberculosis clinical isolates | | | Laila Nimri,Hala Samara,Raymond Batchoun | | FEMS Immunology & Medical Microbiology. 2011; 62(3): 321 | | [Pubmed] | | | 4 | Rapid Molecular Detection of Tuberculosis | | | | | New England Journal of Medicine. 2011; 364(2): 182 | | [Pubmed] | | | 5 | Detection of mutations associated with multidrug-resistant Mycobacterium tuberculosis clinical isolates | | | Nimri, L., Samara, H., Batchoun, R. | | FEMS Immunology and Medical Microbiology. 2011; 62(3): 321-327 | | [Pubmed] | |
| | | |
