Rapid identification of mycobacteria by the gen-probe accuprobesystem

Lumb, Richard; Lanser, J.B.K.; Lim, Irene

doi:10.3109/00313029309066597

Cited by 28 publications

(11 citation statements)

References 16 publications

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“…Since PCR detection commonly suffers from severe contamination problems, the SAT-TB assay should reduce the risk of laboratory contamination and false-positive results (1). The SAT-TB assay uses the same principles of isothermal RNA amplification as the MTD assay (12,18,21,25,27,28,35,39). NAA based on isothermal amplification of RNA has been used to detect several pathogens, including M. tuberculosis, human immunodeficiency virus type 1, Chlamydia trachomatis, and Neisseria gonorrhoeae (10,16,24,45).…”

Section: Discussionmentioning

confidence: 99%

Novel Real-Time Simultaneous Amplification and Testing Method To Accurately and Rapidly Detect Mycobacterium tuberculosis Complex

Cui

Wang²,

Fang

et al. 2012

J Clin Microbiol

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The aim of this study was to establish and evaluate a simultaneous amplification and testing method for detection of the Mycobacterium tuberculosis complex (SAT-TB assay) in clinical specimens by using isothermal RNA amplification and real-time fluorescence detection. In the SAT-TB assay, a 170-bp M. tuberculosis 16S rRNA fragment is reverse transcribed to DNA by use of Moloney murine leukemia virus (M-MLV) reverse transcriptase, using specific primers incorporating the T7 promoter sequence, and undergoes successive cycles of amplification using T7 RNA polymerase. Using a real-time PCR instrument, hybridization of an internal 6-carboxyfluorescein-4-[4-(dimethylamino)phenylazo] benzoic acid N-succinimidyl ester (FAM-DABCYL)-labeled fluorescent probe can be used to detect RNA amplification. The SAT-TB assay takes less than 1.5 h to perform, and the sensitivity of the assay for detection of M. tuberculosis H37Rv is 100 CFU/ml. The TB probe has no cross-reactivity with nontuberculous mycobacteria or other common respiratory tract pathogens. For 253 pulmonary tuberculosis (PTB) specimens and 134 non-TB specimens, the SAT-TB results correlated with 95.6% (370/387 specimens) of the Bactec MGIT 960 culture assay results. The sensitivity, specificity, and positive and negative predictive values of the SAT-TB test for the diagnosis of PTB were 67.6%, 100%, 100%, and 62.0%, respectively, compared to 61.7%, 100%, 100%, and 58.0% for Bactec MGIT 960 culture. For PTB diagnosis, the sensitivities of the SAT-TB and Bactec MGIT 960 culture methods were 97.6% and 95.9%, respectively, for smear-positive specimens and 39.2% and 30.2%, respectively, for smear-negative specimens. In conclusion, the SAT-TB assay is a novel, simple test with a high specificity which may enhance the detection rate of TB. It is therefore a promising tool for rapid diagnosis of M. tuberculosis infection in clinical microbiology laboratories.T he rapid and accurate diagnosis of pulmonary tuberculosis (PTB) plays a critical role in its successful management, as it is essential to treat patients with the appropriate therapy as soon as possible to minimize the risk of transmission. A number of tests based on nucleic acid amplification (NAA) have been developed to identify Mycobacterium tuberculosis in clinical specimens rapidly and directly (8,34,35,44). Among the approved commercial NAA methods, the amplified M. tuberculosis direct test (MTD test; Gen-Probe) detects M. tuberculosis RNA by using a specific isothermal transcription-mediated amplification method (21). The MTD test has been shown to be a sensitive, specific, and rapid method for use with clinical samples (9, 17, 18); however, it requires the use of expensive specialized detection equipment, which prevents the assay from being applied widely in clinical laboratories. The aim of this study was to report and evaluate a simultaneous amplification and testing method for detection of the M. tuberculosis complex (SAT-TB assay) for use with clinical sputum specimens, based on real-time fluorescence dete...

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Section: Discussionmentioning

confidence: 99%

Novel Real-Time Simultaneous Amplification and Testing Method To Accurately and Rapidly Detect Mycobacterium tuberculosis Complex

Cui

Wang²,

Fang

et al. 2012

J Clin Microbiol

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“…Unfortunately, many of these methods are not only time-consuming but they have poor accuracy. As a result, these have been augmented by molecular-based methods in the United States, which include commercially available DNA probes (7)(8)(9) and DNA sequencing of a variety of target genes (e.g., 16S rRNA genes and rpoB) (10)(11)(12). While accurate, these methods are expensive to perform and may not necessarily be cost-effective in populations of low mycobacterial prevalence (13).…”

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confidence: 99%

Comparison of the Bruker Biotyper and Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Systems for Identification of Mycobacteria Using Simplified Protein Extraction Protocols

2014

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Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been described as a fast and inexpensive method for the identification of mycobacteria. Although mycobacteria require extraction prior to MALDI-TOF MS analysis, previously published protocols have been relatively complex, involving significant hands-on time and materials not often found in the clinical laboratory. In this study, we tested two simplified protein extraction protocols developed at the University of Washington (UW) and by bioMérieux (BMX) for use with two different mass spectrometry platforms (the Bruker MALDI Biotyper and the bioMérieux Vitek MS, respectively). Both extraction protocols included vortexing with silica beads in the presence of ethanol. The commercial Bruker database was also augmented with an in-house database composed of 123 clinical Mycobacterium strains. A total of 198 clinical strains, representing 18 Mycobacterium species, were correctly identified to the species level 94.9% of the time when extracted using the UW protocol and compared to the augmented database. The BMX protocol and Vitek MS system resulted in correct species-level identifications for 94.4% of these strains. In contrast, only 79.3% of the strains were identified to the species level by the nonaugmented Bruker database, although the use of a lower identification score threshold (>1.7) increased the identification rate to 93.9%, with two misidentifications that were unlikely to be clinically relevant. The two simplified protein extraction protocols described in this study are easy to use for identifying commonly encountered Mycobacterium species.

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“…Traditional identification of mycobacterial species has mainly relied on phenotypic and biochemical traits. More recent methods involve the chromatographic analysis of mycolic acid profiles or the use of DNA probes which target the most common Mycobacterium species (7)(8)(9)(10). To date, the gold standard for species identification remains DNA sequencing of 16S rRNA, rpoB, and/or hsp65 genes (11,12).…”

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confidence: 99%

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Combined Species Identification and Drug Sensitivity Testing in Mycobacteria

et al. 2017

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Species identification and drug susceptibility testing (DST) of mycobacteria are important yet complex processes traditionally reserved for reference laboratories. Recent technical improvements in matrix-assisted laser desorption ionizationtime of flight mass spectrometry (MALDI-TOF MS) has started to facilitate routine mycobacterial identifications in clinical laboratories. In this paper, we investigate the possibility of performing phenotypic MALDI-based DST in mycobacteriology using the recently described MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA). We randomly selected 72 clinical Mycobacterium tuberculosis and nontuberculous mycobacterial (NTM) strains, subjected them to MBT-ASTRA methodology, and compared its results to current gold-standard methods. Drug susceptibility was tested for rifampin, isoniazid, linezolid, and ethambutol (M. tuberculosis, n ϭ 39), and clarithromycin and rifabutin (NTM, n ϭ 33). Combined species identification was performed using the Biotyper Mycobacteria Library 4.0. Mycobacterium-specific MBT-ASTRA parameters were derived (calculation window, m/z 5,000 to 13,000, area under the curve [AUC] of Ͼ0.015, relative growth [RG] of Ͻ0.5; see the text for details). Using these settings, MBT-ASTRA analyses returned 175/177 M. tuberculosis and 65/66 NTM drug resistance profiles which corresponded to standard testing results. Turnaround times were not significantly different in M. tuberculosis testing, but the MBT-ASTRA method delivered on average a week faster than routine DST in NTM. Databases searches returned 90.4% correct species-level identifications, which increased to 98.6% when score thresholds were lowered to 1.65. In conclusion, the MBT-ASTRA technology holds promise to facilitate and fasten mycobacterial DST and to combine it directly with high-confidence species-level identifications. Given the ease of interpretation, its application in NTM typing might be the first in finding its way to current diagnostic workflows. However, further validations and automation are required before routine implementation can be envisioned.KEYWORDS drug susceptibility testing, MALDI-TOF, MBT-ASTRA, mycobacteria T he genus Mycobacterium comprises at least 175 recognized species (List of Prokaryotic Names with Standing in Nomenclature [LPSN] database) with a wide spectrum of pathogenicity. Mycobacterium tuberculosis, the leading cause of tuberculosis (TB), is among the greatest public health threats in low-income countries. According to the latest WHO report, TB ranks now alongside HIV as a primary cause of death

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Rapid identification of mycobacteria by the gen-probe accuprobesystem

Cited by 28 publications

References 16 publications

Novel Real-Time Simultaneous Amplification and Testing Method To Accurately and Rapidly Detect Mycobacterium tuberculosis Complex

Novel Real-Time Simultaneous Amplification and Testing Method To Accurately and Rapidly Detect Mycobacterium tuberculosis Complex

Comparison of the Bruker Biotyper and Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Systems for Identification of Mycobacteria Using Simplified Protein Extraction Protocols

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Combined Species Identification and Drug Sensitivity Testing in Mycobacteria

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