Comparison of 2 highly automated nucleic acid extraction systems for quantitation of human cytomegalovirus in whole blood

Mengelle, Catherine; Mansuy, Jean‐Michel; Silva, Isabelle Da; Davrinche, C.; Izopet, Jacques

doi:10.1016/j.diagmicrobio.2010.08.011

Cited by 19 publications

(14 citation statements)

References 17 publications

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“…However, results of measurements of CMV DNA loads performed with commercially available assays might differ significantly, particularly according to the extraction method used, which is a source of variability with whole blood (WB) (9)(10)(11)). An interlaboratory comparison of CMV viral load assays in 33 different laboratories in the United States, Canada, and Europe has shown significant variability, with Ͼ40% of results having a delta log above 0.5, which may have an impact on patient care and limit interinstitutional comparisons (12).…”

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confidence: 99%

Fully Automated Quantification of Cytomegalovirus (CMV) in Whole Blood with the New Sensitive Abbott RealTi m e CMV Assay in the Era of the CMV International Standard

et al. 2013

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f Fully standardized reproducible and sensitive quantification assays for cytomegalovirus (CMV) are needed to better define thresholds for antiviral therapy initiation and interruption. We evaluated the newly released Abbott RealTime CMV assay for CMV quantification in whole blood (WB) that includes automated extraction and amplification (m2000 RealTime system). Sensitivity, accuracy, linearity, and intra-and interassay variability were validated in a WB matrix using Quality Control for Molecular Diagnostics (QCMD) panels and the WHO international standard (IS). The intra-and interassay coefficients of variation were 1.37% and 2.09% at 5 log 10 copies/ml and 2.41% and 3.80% at 3 log 10 copies/ml, respectively. According to expected values for the QCMD and Abbott RealTime CMV methods, the lower limits of quantification were 104 and <50 copies/ml, respectively. The conversion factor between international units and copies (2.18), determined from serial dilutions of the WHO IS in WB, was significantly different from the factor provided by the manufacturer (1.56) (P ‫؍‬ 0.001). Results from 302 clinical samples were compared with those from the Qiagen artus CMV assay on the same m2000 RealTime system. The two assays provided highly concordant results (concordance correlation coefficient, 0.92), but the Abbott RealTime CMV assay detected and quantified, respectively, 20.6% and 47.8% more samples than the Qiagen/artus CMV assay. The sensitivity and reproducibility of the results, along with the automation, fulfilled the quality requirements for implementation of the Abbott RealTime CMV assay in clinical settings. Our results highlight the need for careful validation of conversion factors provided by the manufacturers for the WHO IS in WB to allow future comparison of results obtained with different assays.

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Fully Automated Quantification of Cytomegalovirus (CMV) in Whole Blood with the New Sensitive Abbott RealTi m e CMV Assay in the Era of the CMV International Standard

et al. 2013

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“…Likewise, there are scarce data published on the performance of automated extraction systems for CMV plasma DNA quantitation (5,11,(14)(15)(16)20). The above information could, to some extent, allow the direct comparison and interpretation of CMV DNA loads obtained using different methods.…”

Section: Vol 49 2011 Qrt-pcr Automated Extraction Method and CMV mentioning

confidence: 99%

“…Whole blood specimens yield higher CMV DNA loads than plasma samples and permit the earlier detection of active CMV infection in this clinical setting (23). In a prior study (14), differences in the performance of several automated extraction methods for CMV DNA quantitation in whole blood specimens using "in-house" QRT-PCR assays were reported. Overall, the magnitude of these differences was slightly higher than those observed in the current study for plasma specimens.…”

Section: Vol 49 2011 Qrt-pcr Automated Extraction Method and CMV mentioning

confidence: 99%

“…Automated nucleic acid extraction systems are less time-consuming, less prone to analytical errors, and, overall, more efficient than manual methods. A few studies have directly compared the extraction efficiency of different automated systems for the quantitation of CMV DNA in plasma by 14,16). In this study, we evaluated the performance characteristics of three automated DNA extraction platforms coupled with three different QRT-PCR assays for CMV DNA quantitation in plasma obtained from Allo-SCT recipients.…”

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Comparative Evaluation of Three Automated Systems for DNA Extraction in Conjunction with Three Commercially Available Real-Time PCR Assays for Quantitation of Plasma Cytomegalovirus DNAemia in Allogeneic Stem Cell Transplant Recipients

et al. 2011

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Limited data are available on the performance of different automated extraction platforms and commercially available quantitative real-time PCR (QRT-PCR) methods for the quantitation of cytomegalovirus (CMV) DNA in plasma. We compared the performance characteristics of the Abbott mSample preparation system DNA kit on the m24 SP instrument (Abbott), the High Pure viral nucleic acid kit on the COBAS AmpliPrep system (Roche), and the EZ1 Virus 2.0 kit on the BioRobot EZ1 extraction platform (Qiagen) coupled with the Abbott CMV PCR kit, the LightCycler CMV Quant kit (Roche), and the Q-CMV complete kit (Nanogen), for both plasma specimens from allogeneic stem cell transplant (Allo-SCT) recipients (n ‫؍‬ 42) and the OptiQuant CMV DNA panel (AcroMetrix). The EZ1 system displayed the highest extraction efficiency over a wide range of CMV plasma DNA loads, followed by the m24 and the AmpliPrep methods. The Nanogen PCR assay yielded higher mean CMV plasma DNA values than the Abbott and the Roche PCR assays, regardless of the platform used for DNA extraction. Overall, the effects of the extraction method and the QRT-PCR used on CMV plasma DNA load measurements were less pronounced for specimens with high CMV DNA content (>10,000 copies/ ml). The performance characteristics of the extraction methods and QRT-PCR assays evaluated herein for clinical samples were extensible at cell-based standards from AcroMetrix. In conclusion, different automated systems are not equally efficient for CMV DNA extraction from plasma specimens, and the plasma CMV DNA loads measured by commercially available QRT-PCRs can differ significantly. The above findings should be taken into consideration for the establishment of cutoff values for the initiation or cessation of preemptive antiviral therapies and for the interpretation of data from clinical studies in the Allo-SCT setting.Quantitative real-time PCR (QRT-PCR) assays are being increasingly used for the surveillance of active cytomegalovirus (CMV) infection in allogeneic stem cell transplant (Allo-SCT) recipients (23). Several CMV QRT-PCR assays targeting different CMV genes and using different chemistries are commercially available (23). The analytical performance and clinical usefulness of these assays have been assessed, mostly in comparison with the pp65 antigenemia test or quantitative endpoint PCR assays (1, 2, 6, 9, 10-13, 15, 17, 19, 21, 22, 24-26). Limited data are available on how these QRT-PCR tests compare to each other for the quantitation of CMV plasma DNAemia (5,11,12,18,24). This information may allow, at least to some extent, direct comparisons of CMV DNA loads measured at different centers.Nucleic acid extraction is a critical step in QRT-PCR testing, and it has been shown to be a major source of assay variability in viral DNA quantitation (7). Automated nucleic acid extraction systems are less time-consuming, less prone to analytical errors, and, overall, more efficient than manual methods. A few studies have directly compared the extraction efficiency of different automat...

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“…While recently reports have shown improvement in the sensibility obtained by the automated extraction instruments in comparison with the manual extraction kits (Gartner et al, 2004;Mengelle et al, 2011), and several studies performed on different herpesviruses have shown increased sensibility when automated extraction was performed compared to manual extraction kits (Nicholson et al;Griffiths et al;, our laboratory recently demonstrated that the DNA extraction method from Affigene was more efficient than the automated system from Abbott providing a more accurate estimation of CMV DNA load (Gracia-Ahufinger et al, 2010). Our data proving that the manual DNA extraction method from Affigene resulted in a more efficient DNA extraction in comparison with that of an automated procedure from Abbott were somewhat surprising and are in contrast to previously published studies showing just the opposite (Kalpoe et al, 2004;Limaye et al, 2001).…”

Section: Dna Extraction Methodsmentioning

confidence: 99%

Detection of CMV Infection in Allogeneic SCT Recipients: The Multiple Assays

Lobo¹,

BenMarzouk-Hidalgo²,

Pérez‐Romero³

2012

Advances in Hematopoietic Stem Cell Research

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Comparison of 2 highly automated nucleic acid extraction systems for quantitation of human cytomegalovirus in whole blood

Cited by 19 publications

References 17 publications

Fully Automated Quantification of Cytomegalovirus (CMV) in Whole Blood with the New Sensitive Abbott RealTi m e CMV Assay in the Era of the CMV International Standard

Fully Automated Quantification of Cytomegalovirus (CMV) in Whole Blood with the New Sensitive Abbott RealTi m e CMV Assay in the Era of the CMV International Standard

Comparative Evaluation of Three Automated Systems for DNA Extraction in Conjunction with Three Commercially Available Real-Time PCR Assays for Quantitation of Plasma Cytomegalovirus DNAemia in Allogeneic Stem Cell Transplant Recipients

Detection of CMV Infection in Allogeneic SCT Recipients: The Multiple Assays

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