Detection of rifampin resistance by single-strand conformation polymorphism analysis of cerebrospinal fluid of patients with tuberculosis of the central nervous system.

Mutations in a 69-bp region of the rpoB gene of Mycobacterium tuberculosis are associated with rifampin resistance (Rif[r]). These have been detected with mycobacterial DNA extracted from bacterial suspensions or respiratory specimens that were acid-fast smear positive. We experimented with a strategy for the rapid detection of Rif(r) in cerebrospinal fluid (CSF) samples. The strategy involves the amplification of the 69-bp region of rpoB by means of PCR and the identification of nucleotide mutations by single-strand conformation polymorphism (SSCP) analysis of the amplification products. Sixty-five CSF specimens collected from 29 patients (19 patients were coinfected with human immunodeficiency virus) with culture or autopsy-confirmed (22 patients) or highly probable (7 patients) tuberculosis of the central nervous system (CNS-TB) were processed. Amplified products suitable for evaluation by SSCP analysis were obtained from 37 CSF specimens from 25 subjects (86.2%). PCR-SSCP of CSF correctly identified the rifampin susceptibility phenotype of isolates from all 17 patients for whom the results of susceptibility tests carried out with strains cultured from CSF or respiratory samples were available. Moreover, this assay revealed the rifampin susceptibility genotype of isolates from the eight patients (three patients with culture-confirmed CNS-TB and five patients in whom CNS-TB was highly probable) for whom no susceptibility test results were available; the PCR-SSCP data obtained for these patients were concordant with the outcome after a standard antituberculosis treatment. The evolution of a mutation in the rpoB gene was documented in a patient during the course of treatment. PCR-SSCP analysis of CSF seems to be an efficacious method of predicting Rif(r) and would reduce the time required for susceptibility testing from approximately 4 to 8 weeks to a few days.     

Rapid detection of rpoB gene mutations in rifampin-resistant Mycobacterium tuberculosis isolates in shanghai by using the amplification refractory mutation system

Rapid detection of drug resistance in Mycobacterium tuberculosis is essential for efficient treatment and control of this pathogen. The amplification refractory mutation system (ARMS) was used to detect mutations in the rifampin resistance-determining region of the rpoB gene. A total of 39 rifampin-resistant M. tuberculosis isolates in Shanghai were analyzed by this assay, resulting in 92.3% sensitivity (36 of 39) and 87.2% concordance (34 of 39) relative to DNA sequencing, by which 41 mutations of 11 different types, including 9 point mutations and 2 deletions, were identified in the rpoB gene. The most frequent mutations were those associated with codon 531 (21 of 39 [53.8%]) and codon 526 (9 of 39 [23.1%]). The results suggest that the ARMS assay is rapid and simple to implement and could be performed for detection of rifampin resistance in M. tuberculosis to complement conventional culture-based methods.     

Evaluation of the Genotype MTBDR assay for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis isolates

A novel PCR-based reverse hybridization method Genotype MTBDR assay (Hain Lifescience GmbH, Nehren, Germany) was evaluated for rapid detection of rifampin (RIF) and isoniazid (INH) resistance in Turkish Mycobacterium tuberculosis isolates. The Genotype MTBDR assay is designed to detect mutations within the 81-bp hotspot region of rpoB and mutations at katG codon 315. A total of 41 RIF-resistant M. tuberculosis isolates with rpoB mutations that were previously tested by the INNO-LiPA Rif.TB kit and also characterized by DNA sequencing were included in the study. Thirty-seven of these isolates were also resistant to INH. RIF resistance was correctly identified in 39 of 41 isolates (95.1%) with the Genotype MTBDR assay probes specific for these mutations. One isolate with a Gln-490-His mutation and another one with a CGG insertion between codons 514 and 515 were identified as RIF sensitive by the Genotype MTBDR assay. While the INNO-LiPA Rif.TB kit was able to determine the CGG insertion between codons 514 and 515, the Gln-490-His mutation outside the 81-bp hotspot region was not detected by the INNO-LiPA Rif.TB kit. These isolates had MICs of >or=32 microg/ml for RIF. The Genotype MTBDR assay also correctly identified 27 of 37 INH-resistant isolates (73%) with mutations in katG codon 315. In conclusion, the Genotype MTBDR assay may be useful for the rapid diagnosis of the most common mutations found in multidrug-resistant M. tuberculosis strains. However, the test results should always be confirmed with phenotypic methods.     

Newly developed primers for comprehensive amplification of the rpoB gene and detection of rifampin resistance in Mycobacterium tuberculosis

New rpoB gene primers for detecting Rif(r) in Mycobacterium tuberculosis complex bacteria achieved 100% specificity and 88% (fresh sputa) and 92% (ethanol-preserved sputa) diagnostic sensitivity and detected up to 4 CFU/sample. Of the 99 Rif(r) isolates examined, 97% had mutations within cluster I, 2% at codon 176, and 1% at codon 497.     

Mutations in the rpoB gene of multidrug-resistant Mycobacterium tuberculosis isolates from Brazil

Mutations in a 69-bp region of the rpoB gene associated with rifampin resistance (Rif(r)) in 100 isolates (82 Rif(r)) from three states of Brazil were studied. Twenty-one different kinds of mutations were identified in the Rif(r) isolates, and six new alleles are described.     

Direct detection of multidrug-resistant Mycobacterium tuberculosis in clinical specimens in low- and high-incidence countries by line probe assay

The INNO-LiPA Rif.TB assay is designed for the detection of rpoB gene mutations causing rifampin resistance in isolates. We applied the method directly to 60 Lithuanian and Danish clinical specimens to detect rifampin resistance rapidly. Results were obtained in 78.3% of clinical specimens, and all were concordant with those obtained by BACTEC 460. The assay could have major impact on the management of multidrug-resistant tuberculosis.     

Characterization of rpoB mutations in rifampin-resistant clinical isolates of Mycobacterium tuberculosis from Turkey by DNA sequencing and line probe assay

Mutations in an 81-bp region of the rpoB gene associated with rifampin resistance were studied in 41 rifampin-resistant clinical strains of Mycobacterium tuberculosis isolated in Turkey. Fourteen different rpoB alleles, three of which had not been reported before, were found. A reverse hybridization-based line probe assay (the Inno-LiPA Rif.TB test) for rapid detection of the mutations was evaluated with these isolates. Rifampin resistance was correctly identified in 23 of 41 isolates (56.1%) with the kit's R probes specific for these mutations. Seventeen of 41 isolates (41.5%) yielded hybridization patterns, with at least one negative signal obtained with the S probes for the wild type. One isolate was identified as rifampin sensitive by the line probe assay. The rate of concordance of the results of the line probe assay with the results of the in vitro susceptibility test was high (97.6%). These results demonstrate that the line probe assay kit may be useful for the rapid diagnosis of rifampin-resistant tuberculosis.     

Direct genotypic detection of Mycobacterium tuberculosis rifampin resistance in clinical specimens by using single-tube heminested PCR.

Recent analysis of the gene encoding the beta subunit of Mycobacterium tuberculosis RNA polymerase (rpoB) has demonstrated a small region that harbors the mutations most frequently associated with rifampin resistance. Earlier reports have described a high degree of sequence conservation of rpoB among mycobacteria other than M. tuberculosis and other GC-rich bacteria that can lead to false-positive amplification when applied directly to clinical specimens. We developed reagents for PCR amplification that are based on signature nucleotides discovered by comparative sequence analysis of the rpoB genes of organisms phylogenetically related to M. tuberculosis. The specificities of the reagents were challenged with 20 isolates of multiple-drug-resistant M. tuberculosis and more than 20 species of mycobacteria other than M. tuberculosis and other GC-rich organisms. A single-tube heminested PCR protocol was devised to obtain sensitivity equal to those of an IS6110-based PCR assay and culture in spiked sputum experiments. The assay correctly identified 21 of 24 (87.5%) culture-positive specimens, 13 of which were acid-fast smear-negative, in a panel of 51 clinical specimens. Three specimens that were false-positive initially were negative upon repeat testing when the assay was modified to eliminate the potential for aerosol carryover of the first-round amplification product during the open-tube addition of the second set of reaction reagents. This assay is the most sensitive and specific test to date for the direct detection of M. tuberculosis rpoB in clinical specimens. This rapid PCR-based assay can be used for the simultaneous identification of M. tuberculosis and its rifampin susceptibility genotype.     

Detection of Rifampin-Resistant Mycobacterium tuberculosis in Sputa by Nested PCR-Linked Single-Strand Conformation Polymorphism and DNA Sequencing

Either PCR-mediated single strand conformation polymorphism (SSCP) analysis or DNA sequencing of rpoB DNA (157 bp) can be used as a rapid screening method for the detection of mutations related to the rifampin resistance of Mycobacterium tuberculosis. However, due to the nonspecific amplification of rpoB DNA from nontuberculous mycobacteria these methods cannot be directly applied to clinical specimens such as sputa. We developed a nested PCR method that can specifically amplify the rpoB DNA of M. tuberculosis on the basis of rpoB DNA sequences of 44 mycobacteria. Nested PCR-linked SSCP analysis and the DNA sequencing method were applied directly in order to detect M. tuberculosis and determine its rifampin susceptibility in 56 sputa. The results obtained by nested PCR-SSCP and DNA sequencing were concordant with those of conventional drug susceptibility testing and DNA sequencing performed with culture isolates.     

Rapid detection of rifampin resistance in Mycobacterium tuberculosis isolates from India and Mexico by a molecular beacon assay

We assessed the performance of a rapid, single-well, real-time PCR assay for the detection of rifampin-resistant Mycobacterium tuberculosis by using clinical isolates from north India and Mexico, regions with a high incidence of tuberculosis. The assay uses five differently colored molecular beacons to determine if a short region of the M. tuberculosis rpoB gene contains mutations that predict rifampin resistance in most isolates. Until now, the assay had not been sufficiently tested on samples from countries with a high incidence of tuberculosis. In the present study, the assay detected mutations in 16 out of 16 rifampin-resistant isolates from north India (100%) and in 55 of 64 rifampin-resistant isolates from Mexico (86%) compared to results with standard susceptibility testing. The assay did not detect mutations (a finding predictive of rifampin susceptibility) in 37 out of 37 rifampin-susceptible isolates from India (100%) and 125 out of 126 rifampin-susceptible isolates from Mexico (99%). DNA sequencing revealed that none of the nine rifampin-resistant isolates from Mexico, which were misidentified as rifampin susceptible by the molecular beacon assay, contained a mutation in the region targeted by the molecular beacons. The one rifampin-susceptible isolate from Mexico that appeared to be rifampin resistant by the molecular beacon assay contained an S531W mutation, which is usually associated with rifampin resistance. Of the rifampin-resistant isolates that were correctly identified in the molecular beacon assay, one contained a novel L530A mutation and another contained a novel deletion between codons 511 and 514. Overall, the molecular beacon assay appears to have sufficient sensitivity (89%) and specificity (99%) for use in countries with a high prevalence of tuberculosis.     

Frequency of mutations in rpoB and codons 315 and 463 of katG in rifampin- and/or isoniazid-resistant Mycobacterium tuberculosis isolates from northeast Mexico

A total of 48 isoniazid (INH)- and rifampin (RIF)-resistant Mycobacterium tuberculosis isolates, 19 INH-resistant isolates, and 9 RIF-resistant isolates were randomly selected and tested for detecting mutations at codons 315 and 463 of katG by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and/or for detecting mutations at a 69-bp region of the rpoB gene by the INNO-LiPA Rif TB assay. Of the 67 INH-resistant isolates tested, 36 (53.7%) showed the mutation at codon 315 of katG; however, none of them showed the mutation at codon 463. The majority of the RIF-resistant samples analyzed (49 of 57, 86.0%) reacted positive with one of the four R-type probes. The R5-pattern (S531L mutation) was the most frequently observed (31 of 57, 54.4%), followed by R4a-pattern (H526Y mutation) 13 isolates (22.8%), R4b-pattern (H526D mutation) 4 isolates (7.0%), and R2-pattern (D516V mutation) 1 isolate (1.8%). Overall, there was agreement between the line probe kit and phenotypic RIF-susceptibility test for 56 (98.2%) of 57 RIF-resistant isolates tested. These results show that the mutation analysis at codon 315 of katG could be used as a screening assay prior to standard susceptibility testing, whereas mutations in the rpoB gene could be used successfully as genetic markers to rapidly detect RIF-resistant M. tuberculosis clinical isolates from northeast Mexico.     

Frequency of rpoB mutations inside and outside the cluster I region in rifampin-resistant clinical Mycobacterium tuberculosis isolates

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.     

rpoB genotypes of Mycobacterium tuberculosis Beijing family isolates from East Asian countries

The 81-bp region of the rpoB gene in 66 Rif(r) Mycobacterium tuberculosis isolates from China, Japan, Korea, and Taiwan was analyzed. Twelve single-nucleotide substitutions in the rpoB gene were detected. The most prevalent mutations were at Ser-531 (52%), Asp-516 (17%), and His-526 (11%). Mutations were not found in seven (11%) of the isolates. Higher mutation rates in 50 Beijing family isolates were found than in other isolates for mutations at Asp-516 (18 and 12.5%, respectively) and His-526 (12 and 6.3%, respectively). The different rates of mutation may reflect the choice of rifamycin analogs.     

Rapid detection of isoniazid, rifampin, and ofloxacin resistance in Mycobacterium tuberculosis clinical isolates using high-resolution melting analysis

A high-resolution melting analysis (HRMA) assay was developed to detect isoniazid, rifampin, and ofloxacin resistance in Mycobacterium tuberculosis by targeting resistance-associated mutations in the katG, mabA-inhA promoter, rpoB, and gyrA genes. A set of 28 (17 drug-resistant and 11 fully susceptible) clinical M. tuberculosis isolates was selected for development and evaluation of HRMA. PCR amplicons from the katG, mabA-inhA promoter, rpoB, and gyrA genes of all 28 isolates were sequenced. HRMA results matched well with 18 mutations, identified by sequencing, in 17 drug-resistant isolates and the absence of mutations in 11 susceptible isolates. Among 87 additional isolates with known resistance phenotypes, HRMA identified katG and/or mabA-inhA promoter mutations in 66 of 69 (95.7%) isoniazid-resistant isolates, rpoB mutations in 51 of 54 (94.4%) rifampin-resistant isolates, and gyrA mutations in all of 41 (100%) ofloxacin-resistant isolates. All mutations within the HRMA primer target regions were detected as variant HRMA profiles. The corresponding specificities were 97.8%, 100%, and 98.6%, respectively. Most false-positive results were due to synonymous mutations, which did not affect susceptibility. HRMA is a rapid, sensitive method for detection of drug resistance in M. tuberculosis which could be used routinely for screening isolates in countries with a high prevalence of tuberculosis and drug resistance or in individual isolates when drug resistance is suspected.     

Evaluation of a line probe assay kit for characterization of rpoB mutations in rifampin-resistant Mycobacterium tuberculosis isolates from New York City.

A commercial line probe assay kit (Inno-LiPA Rif.TB) for rapid identification of mutations in the rpoB gene associated with rifampin resistance in Mycobacterium tuberculosis was evaluated with a collection of 51 rifampin-resistant strains. Nine distinct rpoB mutations were identified. Concordances with automated sequence results for five wild-type kit probes and four probes for specific mutations were 94.1 and 100%, respectively. Overall concordance of the line probe assay kit with phenotypic rifampin susceptibility testing results was 90.2%.     

Detection of Rifampin Resistance in Mycobacterium tuberculosis in a Single Tube with Molecular Beacons

Current clinical assays for determining antibiotic susceptibility in Mycobacterium tuberculosis require many weeks to complete due to the slow growth of the bacilli. Here we demonstrate an extremely sensitive single-tube PCR assay that takes less than 3 h and reliably identifies rifampin-resistant M. tuberculosis in DNA extracted directly from sputum. Ninety-five percent of mutations associated with rifampin resistance occur in an 81-bp core region of the bacterial RNA polymerase gene, rpoB. All mutations that occur within this region result in rifampin resistance. The assay uses novel nucleic acid hybridization probes called molecular beacons. Five different probes are used in the same reaction, each perfectly complementary to a different target sequence within the rpoB gene of rifampin-susceptible bacilli and each labeled with a differently colored fluorophore. Together, their target sequences encompass the entire core region. The generation of all five fluorescent colors during PCR amplification indicates that rifampin-susceptible M. tuberculosis is present. The presence of any mutation in the core region prevents the binding of one of the molecular beacons, resulting in the absence of one of the five fluorescent colors. When 148 M. tuberculosis clinical isolates of known susceptibility to rifampin were tested, mutations associated with rifampin resistance were detected in 63 of the 65 rifampin-resistant isolates, and no mutations were found in any of the 83 rifampin-susceptible isolates. When DNA extracted directly from the sputum of 11 patients infected with rifampin-resistant tuberculosis was tested, mutations were detected in all of the samples. The use of this rapid assay should enable early detection and treatment of drug-resistant tuberculosis in clinical settings.     

Genetic bases of the rifampin resistance phenotype in Brucella spp

Rifampin is one of the most potent and broad-spectrum antibiotics against bacterial pathogens. Its bactericidal activity is due to its ability to bind to the beta subunit of the DNA-dependent RNA polymerase encoded by the rpoB gene. Mutations of the rpoB gene have been characterized in rifampin-resistant (Rif(r)) strains of Escherichia coli and Mycobacterium tuberculosis. The genetic bases of Rif(r) in Brucella spp. are still unknown. In the present study, the nucleotide sequences of the rpoB gene of the Rif(r) vaccine strain Brucella abortus RB51 and of 20 Rif(r) clones derived in our laboratory from two Brucella melitensis isolates were determined. These sequences were then compared to those of the respective rifampin-susceptible (Rif(s)) parental strains and to the published B. melitensis strain 16M. All Rif(r) strains carried one or more missense mutations mapping in two regions of the rpoB gene. These two "hot" regions were investigated in eight additional Rif(r) Brucella laboratory mutants and in 20 reference Rif(s) Brucella strains. rpoB mutations were found in all Rif(r) mutants. In contrast, no missense mutations were found in any analyzed Rif(s) strains. Our results represent the first from a study of the molecular characterization of rpoB mutations in resistant Brucella strains and provide an additional proof of the association of specific rpoB mutations with the development of the Rif(r) phenotype in prokaryotes. In addition, because of the relationship between Rif(r) and the attenuation of virulence in Brucella spp., studies of virulence in these mutants may provide useful information about the genetic basis of pathogenesis in Brucella.     

Rapid genotypic detection of rifampin- and isoniazid-resistant Mycobacterium tuberculosis directly in clinical specimens

A multiplex PCR DNA strip assay (Genotype MTBDR) designed to detect rifampin (rpoB) and high-level isoniazid (katG) resistance mutations in Mycobacterium tuberculosis isolates was optimized for clinical specimens. Successful genotypic results were achieved with 36 of 38 (95%) smear-positive respiratory specimens, allowing rapid therapeutic adjustments in transmittable drug-resistant tuberculosis.     

Molecular characterization of rifampin-resistant isolates of Mycobacterium tuberculosis from Hungary by DNA sequencing and the line probe assay

Two regions of rpoB associated with rifampin resistance were sequenced in 29 rifampin-resistant (determined by the proportion method) isolates of Mycobacterium tuberculosis obtained from patients from three counties in Hungary. Of the 29 resistant strains, 27 had a mutation in either the 81-bp region (26 strains) or the N-terminal region (1 strain), while the other 2 strains had no mutations in either region. The locations and frequencies of the mutations differed from those previously reported. The most common mutation in this study, D516V, was found in 38% of the Hungarian strains, a frequency 2 to 10 times higher than that found in studies from other countries. These same 29 isolates were also evaluated with the Inno-LiPA Rif. TB test (LiPA), a reverse hybridization assay for the rapid detection of rifampin resistance. Although LiPA detected the presence of an rpoB mutation in 26 of the resistant isolates, the type of mutation could not be determined in 4 isolates because the mutations present were not among those included on the LiPA strip. In addition, a silent mutation in one of the rifampin-susceptible control strains was interpreted as rifampin resistant by LiPA. These findings demonstrate the importance of validating this rapid molecular test by comparison with DNA sequence results in each geographic location before incorporating the test into routine diagnostic work.