Isolation of Mycobacterium tuberculosis Strains with a Silent Mutation in
            <i>rpoB</i>
            Leading to Potential Misassignment of Resistance Category

Alonso, María; Palacios, Juan José; Herránz, Marta; Penedo, Ana; Menéndez, Ángela; Bouza, Emilio; Viedma, Darío García de

doi:10.1128/jcm.00659-11

Cited by 45 publications

(34 citation statements)

References 15 publications

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“…However, several incidents of detection of rpoB mutations not conferring RIF resistance resulted in erroneous reporting of false RIF resistance. Other investigators also encountered problems with detection of false RIF resistance with commercial probe-based assays (12)(13)(14)(15). One of these problems was rooted in a general assumption that the presence of any mutation in the core region of rpoB predicts RIF resistance.…”

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confidence: 99%

Pyrosequencing for Rapid Detection of Extensively Drug-Resistant Mycobacterium tuberculosis in Clinical Isolates and Clinical Specimens

et al. 2014

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cTreating extensively drug-resistant (XDR) tuberculosis (TB) is a serious challenge. Culture-based drug susceptibility testing (DST) may take 4 weeks or longer from specimen collection to the availability of results. We developed a pyrosequencing (PSQ) assay including eight subassays for the rapid identification of Mycobacterium tuberculosis complex (MTBC) and concurrent detection of mutations associated with resistance to drugs defining XDR TB. The entire procedure, from DNA extraction to the availability of results, was accomplished within 6 h. The assay was validated for testing clinical isolates and clinical specimens, which improves the turnaround time for molecular DST and maximizes the benefit of using molecular testing. A total of 130 clinical isolates and 129 clinical specimens were studied. The correlations between the PSQ results and the phenotypic DST results were 94.3% for isoniazid, 98.7% for rifampin, 97.6% for quinolones (ofloxacin, levofloxacin, or moxifloxacin), 99.2% for amikacin, 99.2% for capreomycin, and 96.4% for kanamycin. For testing clinical specimens, the PSQ assay yielded a 98.4% sensitivity for detecting MTBC and a 95.8% sensitivity for generating complete sequencing results from all subassays. The PSQ assay was able to rapidly and accurately detect drug resistance mutations with the sequence information provided, which allows further study of the association of drug resistance or susceptibility with each mutation and the accumulation of such knowledge for future interpretation of results. Thus, reporting of false resistance for mutations known not to confer resistance can be prevented, which is a significant benefit of the assay over existing molecular diagnostic methods endorsed by the World Health Organization. Regional increases in the prevalence of tuberculosis (TB) with drug resistance and a broad distribution of multidrug-resistant (MDR) TB and extensively drug-resistant (XDR) TB (1, 2) may reverse recent gains in global TB control (1). Molecular detection of mutations associated with drug resistance has facilitated rapid detection of drug resistance in the Mycobacterium tuberculosis complex (MTBC) (3-7), and the use of such molecular tools has become increasingly important in TB control and TB patient management (5, 7-10). Recognizing the advantages and disadvantages of various molecular methods will help in the selection of optimal methods for improved prediction of drug resistance in MTBC, which is critical in advancing molecular diagnostic approaches for this defined purpose.In 2003, the Microbial Diseases Laboratory (MDL) at the California Department of Public Health developed a real-time, probe-based assay using molecular beacons for screening for MDR TB (11). Remarkable improvements in turnaround times for predicting resistance to isoniazid (INH) and rifampin (RIF) and significant impacts on the management of MDR TB (9) were realized. However, several incidents of detection of rpoB mutations not conferring RIF resistance resulted in erroneous reporting of false RIF resi...

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Pyrosequencing for Rapid Detection of Extensively Drug-Resistant Mycobacterium tuberculosis in Clinical Isolates and Clinical Specimens

et al. 2014

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“…Given the very high level of agreement between our XDR assay and Sanger sequencing, our assay should be highly specific. The only likely cause of a false resistance call would be a heteroresistant sample (which should arguably be treated as a resistant TB case) or the rare presence of a synonymous mutation in a probe binding site, as is occasionally seen with the Xpert MTB/RIF assay and rifampin resistance detection (52). The Xpert MTB/RIF assay and GeneXpert MTB/RIF instruments are already widely used in countries with a high burden of TB (26,51), and our XDR assay can easily be added to sites that already detect TB using the GeneXpert MTB/RIF system.…”

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confidence: 99%

Detection of Isoniazid-, Fluoroquinolone-, Amikacin-, and Kanamycin-Resistant Tuberculosis in an Automated, Multiplexed 10-Color Assay Suitable for Point-of-Care Use

Chakravorty

Roh

Glass³

et al. 2017

J Clin Microbiol

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Extensively drug-resistant (XDR) tuberculosis (TB) cannot be easily or quickly diagnosed. We developed a rapid, automated assay for the detection of XDR-TB plus resistance to the drug isoniazid (INH) for point-of-care use. Using a simple filter-based cartridge with an integrated sample processing function, the assay identified a wide selection of wild-type and mutant sequences associated with XDR-TB directly from sputum. Four new large-Stokes-shift fluorophores were developed. When these four Stokes-shift fluorophores were combined with six conventional fluorophores, 10-color probe detection in a single PCR tube was enabled. A new three-phase, double-nested PCR approach allowed robust melting temperature analysis with enhanced limits of detection (LODs). Finally, newly designed sloppy molecular beacons identified many different mutations using a small number of probes. The assay correctly distinguished wild-type sequences from 32 commonly occurring mutant sequences tested in gyrA, gyrB, katG, and rrs genes and the promoters of inhA and eis genes responsible for resistance to INH, the fluoroquinolone (FQ) drugs, amikacin (AMK), and kanamycin (KAN). The LOD was 300 CFU of Mycobacterium tuberculosis in 1 ml sputum. The rate of detection of heteroresistance by the assay was equivalent to that by Sanger sequencing. In a blind study of 24 clinical sputum samples, resistance mutations were detected in all targets with 100% sensitivity, with the specificity being 93.7 to 100%. Compared to the results of phenotypic susceptibility testing, the sensitivity of the assay was 75% for FQs and 100% each for INH, AMK, and KAN and the specificity was 100% for INH and FQ and 94% for AMK and KAN. Our approach could enable testing for XDR-TB in point-of-care settings, potentially identifying highly drug-resistant TB more quickly and simply than currently available methods.KEYWORDS 10-color assay, three-phase PCR, XDR-TB, point-of-care test

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“…TB (detecting resistance to RMP only) and the GenoType MTBDRplus (detecting resistance to RMP and INH), as well as the real-time PCR-based automated Xpert MTB/RIF (Xpert) assay (detecting resistance to RMP only) (3,5). However, these tests are not specific, as silent mutations in the rpoB gene occasionally lead to the detection of false-positive RMP resistance (6)(7)(8)(9). The current WHO recommendations are to use the Xpert assay as the initial diagnostic test and start treatment for MDR-TB if an RMP resistance result is expected, or, if unexpected, to repeat Xpert assay testing on another sputum sample, particularly in settings in which the prevalence of RMP-resistant TB is Ͻ15% (10).…”

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“…The Xpert assay result represents real false-positive RMP resistance, since silent mutations do not change the properties of encoded proteins, and this reinforces the recommendations of the WHO to perform a confirmatory DST by phenotypic or other genotypic methods and to resolve any discordant RMP susceptibility results by sequencing of the rpoB gene (10). Silent mutations in the rpoB gene have been reported at codon Thr508 in Haitian isolates (9), Gln510 in New Zealand isolates (7), Leu511 and Gln513 in South Korean isolates (21), Phe514 in Spanish and American isolates (6,22), Thr525 in Chinese isolates (23), Ala532 in Indian isolates (24), and Leu533 in Indian and Belgian isolates (8,24). The growing body of literature on silent mutations within the RRDR of the rpoB gene is alarming, since the Xpert assay was designed for the rapid diagnosis of MDR-TB for effective management of such patients, but it may actually result in overdiagnosis of MDR-TB in resource-poor settings due to limited access to confirmatory DST by phenotypic methods or rpoB sequencing.…”

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Discordance between Xpert MTB/RIF Assay and Bactec MGIT 960 Culture System for Detection of Rifampin-Resistant Mycobacterium tuberculosis Isolates in a Country with a Low Tuberculosis (TB) Incidence

et al. 2015

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Among 452 samples that were positive by the Xpert MTB/RIF (Xpert) assay and MGIT 960 system (MGIT), 440 and 10 Mycobacterium tuberculosis samples were detected as rifampin susceptible and rifampin resistant, respectively. Two isolates that were rifampin susceptible by the MGIT system were rifampin resistant by the Xpert assay. rpoB sequencing identified a silent (CTG521TTG) mutation in one isolate and a missense (GAC516TAC) mutation in another. The detection of rifampin resistance is imperfect with both the Xpert assay and MGIT system. Any discordant rifampin resistance results should be confirmed by sequencing of the rpoB gene. Multidrug-resistant tuberculosis (MDR-TB) (defined as infection with a Mycobacterium tuberculosis strain resistant to at least the two most effective, rifampin and isoniazid, anti-TB drugs) is prevalent throughout the world, difficult to treat, and associated with higher rates of clinical failure and disease relapse (1, 2). The rapid and accurate laboratory diagnosis of MDR-TB is crucial for effective treatment, which will also limit the transmission of MDR-TB (2, 3). The resistance of M. tuberculosis to rifampin (RMP) in nearly 97% of isolates is due to mutations in an 81-bp rifampin resistance-determining region (RRDR) of the rpoB gene (4). Other RMP-resistant isolates contain mutations in either the N-terminal or cluster II region of the rpoB gene, or the resistance is due to other mechanisms (2, 4). Resistance to RMP is a key determinant in treatment failure and also correlates well with MDR-TB, since Ͼ85% of RMP-resistant M. tuberculosis isolates worldwide are also resistant to isoniazid (INH) (2-4). Molecular assays detect mutations in the RRDR of the rpoB gene for the rapid detection of RMP-resistant M. tuberculosis in clinical specimens and culture isolates (2, 3). The World Health Organization (WHO)-approved tests include two line probe assays, the INNO-LiPA Rif. TB (detecting resistance to RMP only) and the GenoType MTBDRplus (detecting resistance to RMP and INH), as well as the real-time PCR-based automated Xpert MTB/RIF (Xpert) assay (detecting resistance to RMP only) (3, 5). However, these tests are not specific, as silent mutations in the rpoB gene occasionally lead to the detection of false-positive RMP resistance (6-9). The current WHO recommendations are to use the Xpert assay as the initial diagnostic test and start treatment for MDR-TB if an RMP resistance result is expected, or, if unexpected, to repeat Xpert assay testing on another sputum sample, particularly in settings in which the prevalence of RMP-resistant TB is Ͻ15% (10). For those settings, treatment for MDR-TB should be initiated when the Xpert assay repeatedly detects RMP resistance. Treatment should be optimized by following susceptibility testing with other first-line and second-line drugs and confirmatory testing for RMP resistance by phenotypic or other genotypic methods; any discordant RMP susceptibility results can be resolved by sequencing of the rpoB gene (10).Phenotypic drug susceptibility testing ...

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Isolation of Mycobacterium tuberculosis Strains with a Silent Mutation in rpoB Leading to Potential Misassignment of Resistance Category

Cited by 45 publications

References 15 publications

Pyrosequencing for Rapid Detection of Extensively Drug-Resistant Mycobacterium tuberculosis in Clinical Isolates and Clinical Specimens

Pyrosequencing for Rapid Detection of Extensively Drug-Resistant Mycobacterium tuberculosis in Clinical Isolates and Clinical Specimens

Detection of Isoniazid-, Fluoroquinolone-, Amikacin-, and Kanamycin-Resistant Tuberculosis in an Automated, Multiplexed 10-Color Assay Suitable for Point-of-Care Use

Discordance between Xpert MTB/RIF Assay and Bactec MGIT 960 Culture System for Detection of Rifampin-Resistant Mycobacterium tuberculosis Isolates in a Country with a Low Tuberculosis (TB) Incidence

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