Rapid prediction of rifampin susceptibility of Mycobacterium tuberculosis.

Ohno, Hideaki; Koga, Hironobu; Kuroita, Toshihiro; Tomono, Kazunori; Ogawa, Kazuhiko; Yanagihara, Katsunori; Yamamoto, Yoshihiro; Miyamoto, Junko; Tashiro, Takayoshi; Kohno, Shigeru

doi:10.1164/ajrccm.155.6.9196115

Cited by 27 publications

(24 citation statements)

References 24 publications

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“…Ohno and colleagues documented MICs of Ն64 g/ml up to 512 g/ml for mutations in codon 531 and a variable level of RMP susceptibility among strains containing amino acid substitutes in either codon 516 or 526 when they used the broth dilution method (14).…”

Section: Discussionmentioning

confidence: 99%

Rifampin Resistance Missed in Automated Liquid Culture System for Mycobacterium tuberculosis Isolates with Specific rpoB Mutations

et al. 2013

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b WHO-endorsed phenotypic drug susceptibility testing (DST) methods for Mycobacterium tuberculosis are assumed to be the gold standard for identifying rifampin (RMP) resistance. However, previous results indicated that low-level, yet probably clinically relevant, RMP resistance linked to specific rpoB mutations is easily missed by some growth-based methods. We aimed to compare the level of resistance detected on Löwenstein-Jensen (LJ) medium with resistance detected by the Bactec MGIT 960 automated DST (MGIT-DST) system for various rpoB mutants. Full agreement between LJ and MGIT-DST was observed for mutations located at codons 513 (Lys or Pro) and 531 (Leu, Trp), which were always resistant by both methods. For mutations 511Pro, 516Tyr, 533Pro, 572Phe, and several 526 mutations, LJ and MGIT results were highly discordant, with MGIT-DST failing to give a result or declaring the strains susceptible. Our data show that phenotypic RMP resistance testing of M. tuberculosis is not a binary phenomenon for some rpoB mutations and that the widely used automated MGIT 960 system is prone to miss some RMP resistance-conferring mutations, while careful DST on LJ missed hardly any. Given the association of these mutations with poor clinical outcome, our findings suggest that the gold standard for rifampin resistance should be reconsidered, in order to address the present confusion caused by discrepancies between phenotypic and genotypic results. The impacts of these mutations will depend on the frequency of their occurrence, which may vary from one setting to another.

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Section: Discussionmentioning

confidence: 99%

Rifampin Resistance Missed in Automated Liquid Culture System for Mycobacterium tuberculosis Isolates with Specific rpoB Mutations

et al. 2013

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“…The same is true for RIF resistance (which results most frequently from changes in codons 526 and 531 in the rpoB gene), but different investigators have found miscellaneous additional genetic changes (in some cases, these changes are related to certain geographic settings) which lead to high degrees of variability in RIF resistance mutations (3,9,10,17). Different genotypic assays have been proposed for the detection of mutations associated with resistance to anti-TB drugs.…”

Section: Discussionmentioning

confidence: 99%

“…More than 95% of RIF-resistant (RIF r ) strains are associated with mutations within an 81-bp region of the rpoB gene (17). Between 60 and 70% of the INH-resistant (INH r ) strains encode mutations in katG, and a specific mutation (in codon 315 of the katG gene) is responsible for many of these cases of resistance (19).…”

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confidence: 99%

New Real-Time PCR Able To Detect in a Single Tube Multiple Rifampin Resistance Mutations and High-Level Isoniazid Resistance Mutations in Mycobacterium tuberculosis

et al. 2002

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The emergence of resistance to antituberculosis drugs is a relevant matter worldwide, but the retrieval of antibiograms for Mycobacterium tuberculosis is severely delayed when phenotypic methods are used. Genotypic methods allow earlier detection of resistance, although conventional approaches are cumbersome or lack sensitivity or specificity. We aimed to design a new real-time PCR method to detect rifampin (RIF)-and isoniazid (INH)-resistant M. tuberculosis strains in a single reaction tube. First, we characterized the resistant isolates in our area of Spain by DNA sequencing. Some mutation was found within the rpoB core region in all the RIF-resistant (RIF r ) strains. Forty-six percent of the INH-resistant (INH r ) strains showed a mutation in katG codon 315, and most of these were associated with high MICs. Eighteen of the RIF r , INH r , and multidrugresistant strains sequenced were tested by our real-time PCR assay; and full concordance of the results of the PCR with the sequencing data was obtained. In addition, a blind test was performed with a panel of 15 different susceptible and resistant strains from throughout Spain, and our results were also in 100% agreement with the sequencing data. Ours is the first assay based on rapid-cycle PCR able to simultaneously detect in a single reaction tube a large variety of mutations associated with RIF resistance (12 different mutations affecting 8 independent codons, including the most prevalent mutations at positions 526 and 531) and the most frequent INH resistance mutations. Our design could be a model for new, rapid genotypic methods able to simultaneously detect a wide variety of antibiotic resistance mutations.The emergence of resistance to antituberculosis (anti-TB) drugs is a relevant matter worldwide and has been reported in several studies (5,18,20). The standard treatment for TB is a multidrug regimen that is based on isoniazid (INH) and rifampin (RIF), the drugs most efficaceous against Mycobacterium tuberculosis infection. The development of resistance to these two drugs, however, means that the efficacy of standard anti-TB treatment is reduced by up to 77% (12).The detection of resistant M. tuberculosis strains is generally performed by phenotypic assays, which require the isolate to be cultured in the presence of the different drugs. This usually means unacceptable delays in the detection of resistance and makes it difficult to adhere to the recommendations of the Centers for Disease Control and Prevention for the reporting of resistance patterns within 28 days of receipt of the specimen in the laboratory (25).Methods that guarantee the early detection of resistant M. tuberculosis strains are required in order to avoid delays in the initiation of effective therapies and to prevent the transmission of multidrug-resistant (MDR) strains, which have been responsible for notorious outbreaks (1, 4, 7).The molecular basis of resistance to anti-TB drugs is becoming clearer (2, 19). More than 95% of RIF-resistant (RIF r ) strains are associated with mutation...

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“…The DNA sequencing [6,12] PCR was done by using forward primer RP5T and reverse primer RP13T to know the exact nucleotide sequence of RRDR of rpo B gene.…”

Section: Methodsmentioning

confidence: 99%

Rapid Detection of Mutation in RRDR of rpo B Gene for Rifampicin Resistance in MDR-Pulmonary Tuberculosis by DNA Sequencing

Patra

Jain

Sherwal

et al. 2010

Indian J Clin Biochem

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To detect the site of mutation in RRDR of rpo B gene for rifampicin resistance in MDR-TB by DNA sequencing. 50 MDR-TB patients were enrolled in our study after informed written consent. Mycobacterial DNA was extracted from sputum samples by Universal Sample Processing (USP) method and RRDR of rpo B gene was amplified by PCR using primers RP4T and RP8T and then sequenced by automated DNA sequencing. The nucleotide sequences of RRDR of rpo B gene were compared with the reference sequence. We observed three different types of mutation in the RRDR of rpo B gene. The frequency of mutation in codon 531 (TCG ? TTG), 526 (CAC ? TAC) and 516 (GAC ? GTC) are 60, 26.6 and 6.6% respectively. Of the total cases studied, 6.6% cases, although resistant to rifampicin, did not show any mutation in the RRDR of rpo B gene. Codon 531 (TCG ? TTG) is the most common site of mutation in RRDR of rpo B gene for rifampicin resistance in MDR-pulmonary tuberculosis followed by codon 526 (CAC ? TAC) and codon 516 (GAC ? GTC).

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Rapid prediction of rifampin susceptibility of Mycobacterium tuberculosis.

Cited by 27 publications

References 24 publications

Rifampin Resistance Missed in Automated Liquid Culture System for Mycobacterium tuberculosis Isolates with Specific rpoB Mutations

Rifampin Resistance Missed in Automated Liquid Culture System for Mycobacterium tuberculosis Isolates with Specific rpoB Mutations

New Real-Time PCR Able To Detect in a Single Tube Multiple Rifampin Resistance Mutations and High-Level Isoniazid Resistance Mutations in Mycobacterium tuberculosis

Rapid Detection of Mutation in RRDR of rpo B Gene for Rifampicin Resistance in MDR-Pulmonary Tuberculosis by DNA Sequencing

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