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

Abstract

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...

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

“…Results showed a good correlation of the results but higher sensitivity for the RT-PCR assay (Gimeno et al, 2008). More recently the Abbott CMV RT-PCR assay was also evaluated in SCT recipients compared with other two commercial tests (Roche and Nanogen) (Bravo et al, 2011). The results found variations in the performance of the tests which limited to establish a common cutoff between different assays.…”

“…However, there are not many studies based on the application of these commercial assays in SCT recipients (Bravo et al;Gimeno et al, 2008;Gouarin et al, 2007;Gracia-Ahufinger et al, 2010;Hanson et al, 2007). As it will be described below, these studies evaluated the suitability of the commercial assays for the surveillance of active CMV infection in these patients and compared the performance of the different tests.…”

“…It consists of cultured CMV that has been diluted in defibrinated, delipidized normal human plasma at different concentrations. The panel can be used with any test procedure designed for measuring CMV DNA in human serum or plasma and it has been widely used as a standard in several studies (Bravo et al;Forman et al, 2011;Hanson et al, 2007;Raggam et al, 2010) to compare techniques with the same laboratory and inter-laboratory.…”

Preemptive antiviral therapy for CMV infection in allogeneic stem cell transplant recipients guided by the viral doubling time in the blood

“…This is because most commercially available QRT-PCR assays show an exquisite linearity above their limit of quantification, with slope coefficients that vary minimally and approach R2 values of 1. 8,9,12 Despite the advent of international standards for CMV DNA quantification, such as the one made available by the World Health Organization, 13 this can hardly be achieved by using preestablished CMV DNA cutoff values, given the suboptimal across-center precision and reproducibility of both in-house or commercially available QRT-PCR. 14 In summary, in this proof-of-principle study, we showed that the CMV dt in the blood compartment might be a valuable parameter for guiding preemptive antiviral therapy for both initial and recurrent episodes of active CMV infection developing in a subset of Allo-SCT recipients, particularly in those at highest risk of CMV disease.…”

“…CMV DNA load monitoring in plasma was performed as previously indicated, 3,5,7 using the new RealTime CMV PCR (Abbott Molecular, Des Plaines, IL, USA), whose limit of detection and quantification is 20 copies/mL (95% confidence interval). 8,9 Antiviral therapy with valganciclovir either at conventional doses (900 mg/12 h) or at reduced doses (450 mg/12 h) in patients meeting one or more of the following criteria: o 50 kg body weight, reduction of the glomerular filtrate ⩽ 60 mL/min per 1.73 m 2 , o1000 neutrophils/μL or o 50 000 platelets/μL, was initiated when the CMV dt was ⩽ 2.0 days, or alternatively when the plasma CMV DNA load reached levels of 41000 copies/mL, whichever occurred first. The CMV dts were calculated using the CMV DNA load values measured in the first two positive QRT-PCR results (Table 2).…”