Comparison of Diagnostic Performance of Three Real-Time PCR Kits for Detecting<i>Mycobacterium</i>Species

Cho, Sun Young; Kim, Min Jin; Suh, Jeong; Lee, Hee Joo

doi:10.3349/ymj.2011.52.2.301

Cited by 24 publications

(23 citation statements)

References 14 publications

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“…In general, the results found in this study are in line with those of recently published studies on the performance of the Cobas TaqMan assay (20,21,22,23,24,25,26). However, those studies were restricted mostly to respiratory samples, discrepant test results were not analyzed in detail, and sequence analysis of the amplicon was not used for validation of the test results.…”

Section: Discussionsupporting

confidence: 89%

Evaluation of Cobas TaqMan MTB for Direct Detection of the Mycobacterium tuberculosis Complex in Comparison with Cobas Amplicor MTB

et al. 2013

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The Roche Cobas Amplicor MTB assay, recently replaced by the Roche Cobas TaqMan MTB assay, was one of the first commercially available assays for detection of the Mycobacterium tuberculosis complex based on nucleic acid amplification. We reported previously on the limited specificity of the Cobas Amplicor MTB assay, in particular for positive samples with an optical density at 660 nm (OD 660 ) of <2.0. Using a selected set of respiratory samples, which were scored as false positive by the Cobas Amplicor test, we demonstrate here that the specificity of the Cobas TaqMan assay is significantly improved. In addition, our study of a set of 133 clinical samples revealed that the Cobas TaqMan MTB assay showed significantly less PCR inhibition than the Cobas Amplicor test. An overall concordance of 98.2% was observed between the two assays. In a subsequent prospective study, we evaluated the performance of the Roche Cobas TaqMan MTB assay on 1,143 clinical specimens, including respiratory (n ‫؍‬ 838) and nonrespiratory (n ‫؍‬ 305) specimens. Using culture as the gold standard, we found a sensitivity of 88.4% and a specificity of 98.8% for the 838 respiratory specimens, compared to a sensitivity of 63.6% and a specificity of 94.6% for the 305 nonrespiratory specimens. We conclude that the Cobas TaqMan MTB assay is a significantly improved tool for the direct detection of M. tuberculosis DNA in clinical specimens. The Cobas Amplicor test is based on amplification of a 584-bp 5= part of the 16S rRNA gene (1, 2) using biotinylated primers, a capture probe, and photometric staining for quantification (3, 4). The Cobas Amplicor test was extensively evaluated for various clinical specimens and demonstrated high sensitivity and specificity, in particular for smear-positive samples (5). We reported previously on a substantial rate of false-positive samples, in particular when the Cobas Amplicor MTB test showed results with optical density at 660 nm (OD 660 ) values of Ͼ0.35 and Ͻ2.0 (6). The observed false-positive results were demonstrated to be due to cross-reactivity of the capture probe with closely related species, such as nontuberculous mycobacterial species and Corynebacterium spp. (6).Recently, Roche Diagnostics (Rotkreuz, Switzerland) replaced the Cobas Amplicor MTB test with the Cobas TaqMan MTB test. The Cobas TaqMan MTB test is a real-time PCR assay that amplifies part of the 16S rRNA gene with the use of a TaqMan probe for the detection of Mycobacterium tuberculosis complex DNA in clinical specimens (7). Here, we evaluated the Cobas TaqMan MTB assay and compared its performance with that of the Cobas Amplicor MTB assay. In a prospective study, we analyzed the performance of the Cobas TaqMan MTB assay for routine mycobacteriology laboratory use during a 6-month period in which 1,143 specimens were submitted for MTB PCR testing. MATERIALS AND METHODSPatient population. The Institute of Medical Microbiology (IMM) serves the 850-bed tertiary University Hospital of Zurich and smaller surrounding hospitals. The patients ...

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Section: Discussionsupporting

confidence: 89%

Evaluation of Cobas TaqMan MTB for Direct Detection of the Mycobacterium tuberculosis Complex in Comparison with Cobas Amplicor MTB

et al. 2013

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“…However, these assays have not been compared in a systematic manner. The clinical sensitivity of a part of the assays has been reported in only a few studies, using culture positivity and/or clinical diagnosis as the "gold standard" (2)(3)(4)(5)(6).…”

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Comparison of 14 Molecular Assays for Detection of Mycobacterium tuberculosis Complex in Bronchoalveolar Lavage Fluid

Akkerman

Werf

Boer³

et al. 2013

J Clin Microbiol

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g We compared 14 molecular assays for their ability to detect the Mycobacterium tuberculosis complex in bronchoalveolar lavage fluid samples. Three approaches were followed. First, by using DNA from Mycobacterium bovis BCG, we determined the detection limits of the assays using routine molecular methods. Second, in order to determine the analytical sensitivities of the assays, we added one of four M. tuberculosis isolates with various numbers of the insertion sequence IS6110 to N-acetyl-L-cysteine (NALC)-NaOH-treated bronchoalveolar lavage fluid samples in dilutions of 1:10 to 1:10,000,000. Third, intertest variabilities were measured and defined by the standard deviations for the quantitation cycle (Cq) values of three positive test results per dilution per assay. The 14 assays tested had similar analytical sensitivities, except for GeneXpert, which had an analytical sensitivity that was 10-to 100-fold lower than that of the other assays. The MP MTB/NTM test and the in-house TaqMan-10 revealed the best performances for the detection limit and had the highest analytical sensitivities. Most of the tests performed well regarding detection limit and analytical sensitivity for the detection of the M. tuberculosis complex in serial dilutions, and the differences were small. The MP MTB/NTM and the in-house TaqMan-10 assays revealed the best, and GeneXpert the worst, overall performances. I n 2011, 6.2 million patients were diagnosed with tuberculosis (TB) worldwide and reported this to the national tuberculosis control programs (NTP) (1). Control of this high-burden disease heavily depends on improved rapid diagnosis and optimal treatment. Real-time (RT)-PCR is the standard DNA amplification technique currently used in TB laboratories. In a short time span, a substantial number of commercial nucleic acid amplification tests for Mycobacterium tuberculosis complex (MTC) infections have become available. However, these assays have not been compared in a systematic manner. The clinical sensitivity of a part of the assays has been reported in only a few studies, using culture positivity and/or clinical diagnosis as the "gold standard" (2-6).The insertion sequence IS6110 has long been appreciated as a target in the molecular detection of the M. tuberculosis complex in clinical material, and it is the most abundant IS element in the genome of M. tuberculosis. However, among clinical isolates worldwide, the number of IS6110 copies in the genome of M. tuberculosis varies from 0 to 25, potentially influencing the detection limits of related assays (7-10).In this study, we compared the analytical performances of 14 assays for the molecular detection of MTC in three different approaches. We hypothesized that not all assays would have equally good analytical sensitivities and that the analytical sensitivity of assays targeting IS6110 would depend on the number of target copies in the genomes of M. tuberculosis strains studied. We therefore compared IS6110-targeting tests with those not targeting this element or that explored unkno...

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“…The identification of mycobacteria responsible for diseases has important ramifications for infection control and selection of antimicrobial therapy. Identification, however, is hampered by the slow growth of most mycobacteria, which may take as long as 2 months using traditional culture methods (6, 7).Molecular methods represent a reliable and rapid alternative for laboratory diagnostics of mycobacteria in clinical samples (1,3,4). Several PCR assays have already been described for the detection of mycobacteria; however, some of them are conventional PCR requiring post-PCR processing, others use melting curve analysis and need additional interpretation, and some have not been used directly from clinical samples (8-11).…”

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confidence: 99%

“…Tuberculosis is still a major global public health problem and one of the leading infectious causes of death, especially in developing countries (1,3,4). In contrast, the prevalence of nontuberculous mycobacterial infection has been increasing in developed countries.…”

mentioning

confidence: 99%

“…Molecular methods represent a reliable and rapid alternative for laboratory diagnostics of mycobacteria in clinical samples (1,3,4). Several PCR assays have already been described for the detection of mycobacteria; however, some of them are conventional PCR requiring post-PCR processing, others use melting curve analysis and need additional interpretation, and some have not been used directly from clinical samples (8-11).…”

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Validation of a Multiplex Real-Time PCR Assay for Detection of Mycobacterium spp., Mycobacterium tuberculosis Complex, and Mycobacterium avium Complex Directly from Clinical Samples by Use of the BD Max Open System

et al. 2016

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A multiplex real-time PCR was validated on the BD Max open system to detect different Mycobacterium tuberculosis complex, Mycobacterium avium complex, and Mycobacterium spp. directly from clinical samples. The PCR results were compared to those with traditional cultures. The multiplex PCR assay was found to be a specific and sensitive method for the rapid detection of mycobacteria directly from clinical specimens. Mycobacterium tuberculosis complex (MTC) and Mycobacterium avium complex (MAC) are the most common slowgrowing mycobacteria isolated from respiratory infections worldwide (1, 2). Tuberculosis is still a major global public health problem and one of the leading infectious causes of death, especially in developing countries (1,3,4). In contrast, the prevalence of nontuberculous mycobacterial infection has been increasing in developed countries. MAC isolates represent the organisms most frequently associated with nontuberculous mycobacterial lung diseases in most of the world (1, 2, 5). The identification of mycobacteria responsible for diseases has important ramifications for infection control and selection of antimicrobial therapy. Identification, however, is hampered by the slow growth of most mycobacteria, which may take as long as 2 months using traditional culture methods (6, 7).Molecular methods represent a reliable and rapid alternative for laboratory diagnostics of mycobacteria in clinical samples (1,3,4). Several PCR assays have already been described for the detection of mycobacteria; however, some of them are conventional PCR requiring post-PCR processing, others use melting curve analysis and need additional interpretation, and some have not been used directly from clinical samples (8-11). The Cepheid Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA) is an FDAcleared assay that provides direct detection of MTC and rifampin (RIF) resistance in clinical samples, and it displays good sensitivity relative to that of culture (12). However, it does not detect MAC or of other Mycobacterium species. The BD Max system (BD Diagnostics, Sparks, MD) is an open fully integrated automated molecular platform that combines specimen processing and real-time PCR. In addition to a number of FDA-cleared assays, the BD Max also offers generic extraction kits and PCR reagents to be used on an open platform that allows users to create their own assay using their own set of primers and probes (13,14). The aim of this study was to validate a multiplex PCR test to detect Mycobacterium spp. (pan-Mycobacterium [PAN]), MTC, and MAC directly from clinical respiratory samples using a user-developed protocol (UDP) on the BD Max open-mode system. This test is for primary diagnosis only and was not designed to detect RIF resistance. The PAN target usually encompasses broad-based characterizations of gene content in a given group of organisms. The pan-Mycobacterium primers used in this study were based on the amplification of the 16S rRNA gene from Mycobacterium species.A total of 120 frozen clinical specimens previously identified...

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Comparison of Diagnostic Performance of Three Real-Time PCR Kits for DetectingMycobacteriumSpecies

Cited by 24 publications

References 14 publications

Evaluation of Cobas TaqMan MTB for Direct Detection of the Mycobacterium tuberculosis Complex in Comparison with Cobas Amplicor MTB

Evaluation of Cobas TaqMan MTB for Direct Detection of the Mycobacterium tuberculosis Complex in Comparison with Cobas Amplicor MTB

Comparison of 14 Molecular Assays for Detection of Mycobacterium tuberculosis Complex in Bronchoalveolar Lavage Fluid

Validation of a Multiplex Real-Time PCR Assay for Detection of Mycobacterium spp., Mycobacterium tuberculosis Complex, and Mycobacterium avium Complex Directly from Clinical Samples by Use of the BD Max Open System

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