Comparison of LightCycler-Based PCR, COBAS Amplicor CMV Monitor, and pp65 Antigenemia Assays for Quantitative Measurement of Cytomegalovirus Viral Load in Peripheral Blood Specimens from Patients after Solid Organ Transplantation

Abstract

In order to evaluate the LightCycler-based PCR (LC-PCR) as a diagnostic assay technique, a classical pp65antigenemia assay and the commercially available COBAS Amplicor CMV Monitor (CACM) assay were compared to the LC-PCR assay for the detection and quantitation of cytomegalovirus (CMV) load in 404 parallel specimens of peripheral blood from 66 patients after solid organ transplantation. A good correlation existed among these three assays (r Х 0.6, P < 0.0001). The LC-PCR assay was the most sensitive (54% of specimens positive) compared to the CACM (48.6%) and the pp65 antigenemia (26%) assays. The LC-PCR assay detected all samples found positive by using both the CMV pp65 antigenemia assay and the CACM assay. The LC-PCR also had the widest dynamic range (from 250 to 10 7 DNA copies/ml of plasma). No cross-reactions were found among CMV and Epstein-Barr virus, varicella-zoster virus, or herpes simplex virus in the LC-PCR by using amplification with specifically designed primer pairs. Precision, expressed as the coefficient of variation, was <3% with standard DNA from cell cultures and between 6.55 and 14.1% with clinical specimens in repeat LC-PCR runs. One run of the LC-PCR took half of the time required for the semiautomated CACM procedure. Because of its sensitivity, specificity, cost-effectiveness, and simplicity, the LC-PCR assay could replace the pp65 antigenemia and the CACM assays as the preferred technique for the surveillance, diagnosis, and monitoring of response of CMV diseases in high-risk populations.

Comparison of a Duplex Quantitative Real-Time PCR Assay and the COBAS Amplicor CMV Monitor Test for Detection of Cytomegalovirus

“…This superior sensitivity for qPCR facilitates the management of CMV-infected patients and enables faster preemptive antiviral therapy. Our results are in agreement with two recent reports (14,15) but contrast with a previous evaluation of the quantitative COBAS test in which it had a similar sensitivity to a real-time PCR assay with fluorescence resonance energy transfer probes (17). An alternative explanation for the discrepancy between the methods would be mismatching nu- cleotides in the primer and/or probe regions used in the COBAS assay.…”

“…Another explanation could be a different accuracy in definitions of the number of genomes in the two standards. Notably, the phenomenon of higher values by the qPCR assay than by the COBAS system has even been reported in previous evaluations (4,14).…”

CMV Infection in CMV-Seropositive Kidney Transplant Recipients

“…A recent multicenter comparison of viral load assays demonstrated up to a 1000 folds variation among them. Standardization may be achieved in the future with quantitative viral load assays (Pang et al, 2003).…”

A Commutable Cytomegalovirus Calibrator Is Required to Improve the Agreement of Viral Load Values between Laboratories

“…Real-time PCR was performed with a laboratorydeveloped real-time PCR test that amplifies and detects the UL55 (glycoprotein B) gene of CMV. Thermal cycling and real-time detection were carried out with the Roche LightCycler instrument (16,17 ). The assay was calibrated with DNA from CMV strain AD169 spiked into plasma.…”