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Journal of Clinical Microbiology, January 2001, p. 107-110, Vol. 39, No. 1
Institut fuer Medizinische Mikrobiologie und
Hygiene, Universität Regensburg,
Regensburg,1 and Forschungszentrum
Borstel, National Reference Center for Mycobacteria,
Borstel,2 Germany
Received 28 August 2000/Returned for modification 25 September
2000/Accepted 31 October 2000
The prevalence of recently described mutation V176F,
located in the beginning of the rpoB gene and associated
with rifampin resistance and the wild-type cluster I sequence, was
determined by analyzing the distribution of rpoB mutations
among 80 rifampin (RIF)-resistant Mycobacterium
tuberculosis strains isolated in Germany during 1997. The most
frequent rpoB mutations were changes in codon 456 (52 isolates, 65%), followed by changes in codon 441 (13 isolates, 16%)
and codon 451 (11 isolates, 14%). The V176F mutation was detected in
one isolate of the study population and in 5 of 18 RIF-resistant
strains with no cluster I mutation from six previously published
studies. In three isolates, a mixture of resistant and susceptible
subpopulations (heteroresistance) prohibited the detection of
rpoB mutations in the initial analysis; however, in these
isolates, cluster I mutations could be verified after a passage on
RIF-containing medium. IS6110 DNA fingerprinting of 76 strains revealed eight clusters comprising 27 strains with identical
restriction fragment length polymorphism patterns that mainly also show
identical rpoB mutations and identical or similar drug
resistance patterns. In conclusion, our results indicate that the V176F
mutation should be included in molecular tests for prediction of RIF
resistance in M. tuberculosis. We further demonstrated that
heteroresistance caused by a mixture of mycobacterial subpopulations
with different susceptibilities to RIF may influence the sensitivity of
molecular tests for detection of resistance.
One of the most alarming trends
concerning tuberculosis (TB) is the emergence of drug-resistant
Mycobacterium tuberculosis strains, which has become a
worldwide health care problem (13). Drug-resistant TB is
increasing in many parts of the world, and high rates of drug-resistant
and multidrug-resistant TB-causing (MDR-TB) isolates (resistant to at
least isoniazid [INH] and rifampin [RIF]) have been reported in
several countries (16). So far in Germany, the incidence
of TB, and also of drug resistance, is low compared to international
data (16), but this situation may change because of
importation of drug-resistant strains from countries with high rates of
drug resistance, e.g., the former Soviet Union (11).
Early detection of drug resistance in clinical M. tuberculosis isolates is crucial for appropriate treatment to
prevent the development of further resistance and the spread of
resistant strains. Compared to conventional methods using solid media,
the introduction of manual and automated methods for susceptibility testing in liquid media has resulted in a reduction of turnaround times
for susceptibility results from 4 to 6 weeks to 3 to 15 days (6,
17). More recently, the identification of resistance mutations,
e.g., the genetic basis for RIF resistance, enables the development of
molecular tests which may allow the detection of resistant strains
within 1 day (18).
RIF is one of the most potent antituberculous drugs. More than 90% of
RIF-resistant TB-causing isolates are also resistant to INH, and RIF
resistance is therefore a valuable surrogate marker for MDR-TB
(15), which is a tremendous obstacle for TB therapy. In
contrast to INH resistance, resistance to RIF is associated mainly with
single point mutations in a small 81-bp hot-spot region (codons 432 to
458, cluster I) of the RNA polymerase gene (rpoB) (18) that can easily be amplified by PCR. Thus, several
rapid molecular assays for prediction of RIF resistance targeting these mutations have been established or are under investigation
(12). However, the detection of RIF resistance by these
methods fails to match with a resistant phenotype in about 5%
of all cases (15). Recently, we have reported a
mutation in the beginning of the rpoB gene conferring
resistance to RIF on Helicobacter pylori (V149F) and
M. tuberculosis (V176F) (5). The amino acid
exchange is located amino terminal to cluster I in M. tuberculosis and may account for false-negative test results
obtained by molecular methods that only detect mutations within
the cluster I region. Furthermore, we were able to show that, as
described for Escherichia coli, mutations in clusters
II and III located carboxy terminal to cluster I can reduce
the susceptibility of H. pylori to rifamycins (4).
To determine the frequency of the V176F mutation, we analyzed the
prevalence of mutations inside and outside of the cluster I region of
the rpoB gene in a collection of RIF-resistant clinical M. tuberculosis isolates during a 1 year (1997) survey at
the German National Reference Center for Mycobacteria (NRC). Moreover, 18 samples of RIF-resistant M. tuberculosis strains, 16 DNA
samples, and 2 culture samples described in six previous reports
(2, 3, 7, 10, 21, 22) to have no cluster I mutation were obtained and analyzed.
(Parts of the results presented here were presented as a poster at the
100th General Meeting of the American Society for Microbiology in Los
Angeles, Calif. [section U-2]).
Strains analyzed.
In total, 93 RIF-resistant M. tuberculosis isolates from 80 patients obtained during 1997 by the
NRC were analyzed in this study. Moreover, 18 DNA samples, including 2 culture samples, of RIF-resistant strains with no known cluster I
mutation provided by six other centers were investigated (Table
1).
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.1.107-110.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Frequency of rpoB Mutations Inside and
Outside the Cluster I Region in Rifampin-Resistant
Clinical Mycobacterium tuberculosis Isolates
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
TABLE 1.
rpoB codon exchanges in DNA samples from
RIF-resistant clinical isolates without cluster I mutations
described in recent studies
PCR amplification conditions. For analysis of the presence of the V176F mutation, a 365-bp fragment of the rpoB gene was amplified using primers TB-176-F (5'-CTTCTCCGGGTCGATGTCGTTG-3') and TB-176-R (5'-CGCGCTTGTCGACGTCAAACTC-3') as recently described (5). Five microliters of a 200-fold dilution (in double-distilled water) of total bacterial DNA (see DNA genotyping techniques) was used per PCR mixture.
Amplification of a 749-bp rpoB DNA fragment comprising the complete region from cluster I to cluster III was carried out with primers TBB-1 (5' ATCACACCGCAGACGTTG-3') and TBB-2 (5'TGCATCACAGTGATGTAGTCG-3'). The 50-µl reaction mixture contained 5 µl of 1:200-diluted total DNA, 2.5 µl of dimethyl sulfoxide, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 µM each deoxynucleoside triphosphate, 30 pmol of each primer, and one U of Ampli Taq DNA polymerase (Perkin-Elmer, Foster City, Calif.). PCR amplifications were performed using the following protocol: initial denaturation at 94°C for 5 min; 40 cycles of denaturation at 94°C for 45 s, annealing at 64°C for 1 min, and extension at 72°C for 2 min; and a final extension at 72°C for 7 min.DNA sequencing analysis. Direct sequencing of the rpoB PCR fragments was performed by cycle sequencing using the BigDye Ready Reaction Terminator Cycle Sequencing Kit (Perkin-Elmer) and the ABI Prism 377 DNA sequencer (Perkin-Elmer) as instructed by the manufacturer. The PCR primers were also used as sequencing primers. The DNASIS program (version 2.1; Hitachi, San Bruno, Calif.) was used for DNA sequence comparisons. DNA sequences were compared with the most recent version of the GenBank NR data bank using the BLASTN algorithm (1).
The sequence of DNA sample 84 (Table 1), provided by Yang et al. (21) and showing mutation V176F, is in the GenBank database under accession number AF177294.DNA genotyping techniques. Extraction of genomic DNA from mycobacterial strains and DNA fingerprinting using IS6110 as a probe were performed in accordance with a standardized protocol described elsewhere (11, 19). The IS6110 fingerprint patterns of mycobacterial strains were analyzed using the Gelcompare software (Windows 95, version 4.2; Applied Maths, Kortrijk, Belgium) as described previously (11, 19).
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Patient demographics. Of the 80 patients studied, data on age and sex were available for 76 patients, of whom 53 (70%) were males. The male-to-female ratio was 2.3:1 and comparable to the ratio described for the total number of patients from whom M. tuberculosis strains were isolated in Germany in 1997 (16). Most (75%) of the patients were between 20 and 50 years old. Indications of foreign birth were seen in up to 58% of the patients, strongly suggesting that MDR-TB, to a great extent, is a problem of certain risk groups in Germany so far.
First-line drug resistance patterns. The susceptibilities to the first-line drugs INH, RIF, ethambutol (EMB), and pyrazinamide (PZA) of the clinical M. tuberculosis isolates obtained from the NRC were determined by conventional methods. Of the 80 strains tested (1 isolate per patient) only 7 showed resistance to only RIF, while 73 isolates (91%) were at least resistant to INH and RIF. Of these MDR-TB isolates, a majority of 60 strains (82%) showed further resistance to at least one other first-line drug and 21 (28%) were resistant to RIF, INH, PZA, and EMB. Since 91% of all RIF-resistant strains also were resistant to INH, our data confirm the use of RIF resistance as a surrogate marker for the rapid detection of MDR-TB in clinical M. tuberculosis isolates.
Distribution and frequency of rpoB mutations.
The
rpoB genes of the 80 RIF-resistant M. tuberculosis strains were analyzed by direct sequencing of two
rpoB DNA fragments either containing codon 176 or
encompassing the whole region from cluster I to cluster III
(4). The distribution and frequency of the rpoB
mutations are shown in Fig. 1 using two
classifications, (i) the codon numbering system used by Telenti et al.
(18) deriving from homologous mutations in E. coli and (ii) the codon designations deriving from the M. tuberculosis genome. Only the latter is used in the text. In the
initial analyses, a cluster I mutation could be detected in 76 of the
80 RIF-resistant strains. In accordance with previous data, mutation of
codon 456 was most frequent (52 isolates, 65%) among the strains
analyzed. However, in contrast to other studies, e.g., in the United
States (8) and Australia (22), the rate of
mutation in codon 451 was low (11 isolates, 14%) and nearly identical
to the rate of mutation in codon 441 (13 isolates, 16%). Double
mutations were detected in 17 isolates (22%), of which 11 isolates
possessed a second mutation outside the cluster I region (Fig. 1).
Although the combination of two single point mutations has been
described previously for RIF-resistant M. tuberculosis
strains (8, 20), the high percentage of double mutations
found among the strains isolated in Germany differed clearly from the
lower prevalence of double mutations in other studies. It might be of
interest that several amino acid exchanges which occurred in
combination with a second mutation in clusters I and II or between
clusters II and III have been described to be very rare and/or to
induce a lower level of resistance (15). Mutation S456A
(S531A in the E. coli numbering system) might represent a
newly described allele.
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Prevalence of mutation V176F. In one of four isolates showing no mutation in the cluster I to cluster III region in the initial analysis, the V176F mutation could be found in the beginning of the rpoB gene. This mutation was also found in 5 out of the 18 samples with no mutation in cluster I, II, or III, respectively, obtained from six other studies comprising M. tuberculosis cultures, of strains from Asia, Africa, the United States, and Australia (Table 1). For two M. tuberculosis cultures, of which one had the V176F mutation and the other had a cluster II (I497F) mutation (kindly provided by D. Caugant; Table 1), MIC testing was performed. Mutation V176F in the first strain was associated with a RIF MIC of 256 µg/ml and a rifabutin MIC of 16 µg/ml. The second strain showed a cluster II mutation (I497V) so far observed only in one isolate from Australia (22); the RIF and rifabutin MICs were 64 and 2 µg/ml, respectively. These data confirm our previous observation that the V176F mutation in the rpoB gene is associated with high-level resistance to RIF in clinical M. tuberculosis isolates and may be responsible for more than 1% of resistant isolates.
Heteroresistance. No rpoB mutation was detected by PCR and sequencing in 3 of the 80 isolates. However, re-examination using bacterial cultures grown on RIF-containing medium led to the identification of cluster I mutations in all three cases. Hence, a mixture of wild-type and resistant subpopulations in the initial culture (heteroresistance) presumably prohibited the detection of resistance mutations by means of PCR in the initial analyses of bacterial cultures grown on medium without RIF. In five other isolates, mixed bacterial populations showing the wild-type sequence and a typical point mutation could be detected in the initial sequence analysis. These results indicate that heteroresistance seems to occur in a considerable number of clinical M. tuberculosis isolates (8 out of 80 in our study) and may influence the sensitivity of molecular tests for the prediction of drug resistance if the percentage of the resistant subpopulation is comparably low. Methods which are able to detect small resistant subpopulations in a clinical sample, e.g., by specific hybridization, might be more sensitive in these cases.
IS6110 restriction fragment length polymorphism
analysis.
To analyze the genetic relationship of the clinical
M. tuberculosis strains obtained from the NRC, DNA
fingerprinting using IS6110 as a probe was performed for 76 isolates (11). Forty-nine strains showed unique
IS6110 patterns, confirming independent acquisition of
resistance. However, 27 strains (35%) clustered in eight groups
comprising two to seven isolates with identical restriction fragment
length polymorphism patterns (Table 2). Transmission of the same RIF-resistant M. tuberculosis
strain among the patients of one cluster could further be confirmed by the fact that in most clusters all of the isolates showed the same
rpoB mutations and identical or very similar drug resistance patterns (Table 2). In only two clusters did isolates display different
rpoB mutations, indicating independent acquisition of RIF
resistance. Thus, recent transmission of resistant M. tuberculosis strains may have occurred in Germany. The results
presented here indicate that at least the rpoB mutations
found in the clustered strains seem not to affect the virulence of
M. tuberculosis isolates in a way that transmission is not
possible. Frequent transmission of resistant M. tuberculosis
strains might also explain the disequilibrium in the distribution of
rpoB mutations observed in RIF-resistant M. tuberculosis isolates from different countries (8, 14, 22).
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Conclusion. In the M. tuberculosis isolates evaluated in this study, RIF resistance was associated with the V176F mutation of the rpoB gene when cluster I to III mutations and heteroresistance were excluded. In the remaining 12 DNA samples from the literature with unexplained resistance, additional independent resistance mechanisms, heteroresistance, or even rpoB mutations outside the regions investigated cannot be ruled out. Mutation V176F seems to confer high-level resistance in clinical M. tuberculosis isolates and may account for more than 1% of all RIF-resistant strains. Appropriate molecular tests should be able to detect such mutations for early and reliable prediction of RIF susceptibility in clinical M. tuberculosis samples or isolates. The presence of a mixture of susceptible and resistant subpopulations in mycobacterial cultures isolated from clinical specimens, so-called heteroresistance, seems to represent an important obstacle to molecular drug resistance testing and also to successful therapy.
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ACKNOWLEDGMENTS |
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We thank I. Radzio, F. Schaefer, B. Schlüter, and A. Zyzik, Borstel, Germany, and R. Birngruber, Regensburg, Germany, for excellent technical assistance and C. Abe, S. Kohno, D. Williams, D. Caugant, P. Kiepiela, and L. Yuen for providing DNA samples from recent surveys.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, Herrman-Herder-Str-11, D-79104 Freiburg, Germany. Phone: 49 761 2036546. Fax: 49 761 2036562. E-mail: Markus-Heep{at}web.de.
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Journal of Clinical Microbiology, January 2001, p. 107-110, Vol. 39, No. 1
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.1.107-110.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Murphy, C. K., Mullin, S., Osburne, M. S., van Duzer, J., Siedlecki, J., Yu, X., Kerstein, K., Cynamon, M., Rothstein, D. M.
(2006). In Vitro Activity of Novel Rifamycins against Rifamycin-Resistant Staphylococcus aureus. Antimicrob. Agents Chemother.
50: 827-834
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Viveiros, M., Leandro, C., Rodrigues, L., Almeida, J., Bettencourt, R., Couto, I., Carrilho, L., Diogo, J., Fonseca, A., Lito, L., Lopes, J., Pacheco, T., Pessanha, M., Quirim, J., Sancho, L., Salfinger, M., Amaral, L.
(2005). Direct Application of the INNO-LiPA Rif.TB Line-Probe Assay for Rapid Identification of Mycobacterium tuberculosis Complex Strains and Detection of Rifampin Resistance in 360 Smear-Positive Respiratory Specimens from an Area of High Incidence of Multidrug-Resistant Tuberculosis. J. Clin. Microbiol.
43: 4880-4884
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Hillemann, D., Weizenegger, M., Kubica, T., Richter, E., Niemann, S.
(2005). Use of the Genotype MTBDR Assay for Rapid Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Complex Isolates. J. Clin. Microbiol.
43: 3699-3703
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McCammon, M. T., Gillette, J. S., Thomas, D. P., Ramaswamy, S. V., Graviss, E. A., Kreiswirth, B. N., Vijg, J., Quitugua, T. N.
(2005). Detection of rpoB Mutations Associated with Rifampin Resistance in Mycobacterium tuberculosis Using Denaturing Gradient Gel Electrophoresis. Antimicrob. Agents Chemother.
49: 2200-2209
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O'Sullivan, D. M., McHugh, T. D., Gillespie, S. H.
(2005). Analysis of rpoB and pncA mutations in the published literature: an insight into the role of oxidative stress in Mycobacterium tuberculosis evolution?. J Antimicrob Chemother
55: 674-679
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Zenkin, N., Kulbachinskiy, A., Bass, I., Nikiforov, V.
(2005). Different Rifampin Sensitivities of Escherichia coli and Mycobacterium tuberculosis RNA Polymerases Are Not Explained by the Difference in the {beta}-Subunit Rifampin Regions I and II. Antimicrob. Agents Chemother.
49: 1587-1590
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Hillemann, D., Kubica, T., Rusch-Gerdes, S., Niemann, S.
(2005). Disequilibrium in Distribution of Resistance Mutations among Mycobacterium tuberculosis Beijing and Non-Beijing Strains Isolated from Patients in Germany. Antimicrob. Agents Chemother.
49: 1229-1231
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Yam, W. C., Tam, C. M., Leung, C. C., Tong, H. L., Chan, K. H., Leung, E. T. Y., Wong, K. C., Yew, W. W., Seto, W. H., Yuen, K. Y., Ho, P. L.
(2004). Direct Detection of Rifampin-Resistant Mycobacterium tuberculosis in Respiratory Specimens by PCR-DNA Sequencing. J. Clin. Microbiol.
42: 4438-4443
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Deng, J.-Y., Zhang, X.-E., Lu, H.-B., Liu, Q., Zhang, Z.-P., Zhou, Y.-F., Xie, W.-H., Fu, Z.-J.
(2004). Multiplex Detection of Mutations in Clinical Isolates of Rifampin-Resistant Mycobacterium tuberculosis by Short Oligonucleotide Ligation Assay on DNA Chips. J. Clin. Microbiol.
42: 4850-4852
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Sajduda, A., Brzostek, A., Poplawska, M., Augustynowicz-Kopec, E., Zwolska, Z., Niemann, S., Dziadek, J., Hillemann, D.
(2004). Molecular Characterization of Rifampin- and Isoniazid-Resistant Mycobacterium tuberculosis Strains Isolated in Poland. J. Clin. Microbiol.
42: 2425-2431
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Enne, V. I., Delsol, A. A., Roe, J. M., Bennett, P. M.
(2004). Rifampicin resistance and its fitness cost in Enterococcus faecium. J Antimicrob Chemother
53: 203-207
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Mokrousov, I., Otten, T., Vyshnevskiy, B., Narvskaya, O.
(2003). Allele-Specific rpoB PCR Assays for Detection of Rifampin-Resistant Mycobacterium tuberculosis in Sputum Smears. Antimicrob. Agents Chemother.
47: 2231-2235
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Dreses-Werringloer, U., Padubrin, I., Kohler, L., Hudson, A. P.
(2003). Detection of Nucleotide Variability in rpoB in both Rifampin-Sensitive and Rifampin-Resistant Strains of Chlamydia trachomatis. Antimicrob. Agents Chemother.
47: 2316-2318
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Yue, J., Shi, W., Xie, J., Li, Y., Zeng, E., Wang, H.
(2003). Mutations in the rpoB Gene of Multidrug-Resistant Mycobacterium tuberculosis Isolates from China. J. Clin. Microbiol.
41: 2209-2212
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Martin-Galiano, A. J., de la Campa, A. G.
(2003). High-Efficiency Generation of Antibiotic-Resistant Strains of Streptococcus pneumoniae by PCR and Transformation. Antimicrob. Agents Chemother.
47: 1257-1261
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Hwang, H.-Y., Chang, C.-Y., Chang, L.-L., Chang, S.-F., Chang, Y.-H., Chen, Y.-J.
(2003). Characterization of rifampicin-resistant Mycobacterium tuberculosis in Taiwan. J Med Microbiol
52: 239-245
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Cavusoglu, C., Hilmioglu, S., Guneri, S., Bilgic, A.
(2002). Characterization of rpoB Mutations in Rifampin-Resistant Clinical Isolates of Mycobacterium tuberculosis from Turkey by DNA Sequencing and Line Probe Assay. J. Clin. Microbiol.
40: 4435-4438
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Garcia de Viedma, D., del Sol Diaz Infantes, M., Lasala, F., Chaves, F., Alcala, L., Bouza, E.
(2002). 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.
40: 988-995
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Vogler, A. J., Busch, J. D., Percy-Fine, S., Tipton-Hunton, C., Smith, K. L., Keim, P.
(2002). Molecular Analysis of Rifampin Resistance in Bacillus anthracis and Bacillus cereus. Antimicrob. Agents Chemother.
46: 511-513
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Bartfai, Z., Somoskovi, A., Kodmon, C., Szabo, N., Puskas, E., Kosztolanyi, L., Farago, E., Mester, J., Parsons, L. M., Salfinger, M.
(2001). Molecular Characterization of Rifampin-Resistant Isolates of Mycobacterium tuberculosis from Hungary by DNA Sequencing and the Line Probe Assay. J. Clin. Microbiol.
39: 3736-3739
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