Comparative Evaluation of Three Commercial Quantitative Cytomegalovirus Standards by Use of Digital and Real-Time PCR

Abstract

The recent development of the 1st WHO International Standard for human cytomegalovirus (CMV) and the introduction of commercially produced secondary standards have raised hopes of improved agreement among laboratories performing quantitative PCR for CMV. However, data to evaluate the trueness and uniformity of secondary standards and the consistency of results achieved when these materials are run on various assays are lacking. Three concentrations of each of the three commercially prepared secondary CMV standards were tested in quadruplicate by three real-time and two digital PCR methods. The mean results were compared in a pairwise fashion with nominal values provided by each manufacturer. The agreement of results among all methods for each sample and for like concentrations of each standard was also assessed. The relationship between the nominal values of standards and the measured values varied, depending upon the assay used and the manufacturer of the standards, with the degree of bias ranging from ؉0.6 to ؊1.0 log 10 IU/ml. The mean digital PCR result differed significantly among the secondary standards, as did the results of the real-time PCRs, particularly when plotted against nominal log 10 IU values. Commercially available quantitative secondary CMV standards produce variable results when tested by different real-time and digital PCR assays, with various magnitudes of bias compared to nominal values. These findings suggest that the use of such materials may not achieve the intended uniformity among laboratories measuring CMV viral load, as envisioned by adaptation of the WHO standard.R outine viral load measurements have become the standard of care for many patients, particularly those with severely compromised immune systems (1-5). However, despite their widespread clinical use, current testing strategies still have many limitations. Except for HIV and hepatitis B and C viruses, there has been little standardization of the testing process. Most methods are based on real-time PCR and have a high degree of result variability, particularly when testing among institutions is compared (6-9). The reasons for this variability are myriad. Real-time PCR is a dynamic process, with quantification based on normalization of the time to signal generation to a calibration curve that is in turn based on the use of calibration material with "known" values. Variations in any part of this complex procedure might theoretically affect result accuracy or precision. In fact, several factors have been shown to play a role (10); however, the most emphasis in the literature has been placed on the lack of universally accepted calibrators (11, 12). The lack of available international quantitative standards for many of the commonly tested viral analytes has led to the use of a wide variety of materials, intuitively reducing the agreement of results when common samples have been tested by different centers. It has been widely hoped that the development of such international reference material would help improve this situation. The...

Commutability of the First World Health Organization International Standard for Human Cytomegalovirus

“…Other factors may come into play as well. Commercially produced standards may not be equivalent in all aspects; they may not have equal trueness or comparability to WHO material, nor may they behave the same among assays or reagents (13).…”

“…This is a commonplace practice, but one that is fraught with challenge. Recent work has examined whether such secondary materials are actually comparable in value and provide an accurate representation of the WHO standards (13). Commutability should be assessed at all phases of the process of quantification, including calibrators, higher-order, traceable standards, and considering other aspects of reaction chemistry and performance that can affect quantitative results.…”

Calibration of BK Virus Nucleic Acid Amplification Testing to the 1st WHO International Standard for BK Virus

“…However, previous work has shown that, for a given nominal value, secondary standards do not always contain equal copy numbers of the targeted sequence (17). This applies to interstandard comparisons and may also apply within a given standard, for example, if a particular region of the genome is deleted or multiplicated.…”

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.…”