COMPARISON OF TWO REAL TIME PCR METHODS AND A CONVENTIONAL PCR TEST FOR THE QUANITIFICATION OF CYTOMEGALOVIRUS (CMV) IN PLASMA

Quantification of CMV DNA in blood has become the standard of care for diagnosis and for monitoring therapeutic efficacy in immunocompromised patients. Commercial quantification reagents are available in “research use only” (RUO) and analyte-specific reagent (ASR) formats. In this study we describe performance characteristics of an in-house developed, quantitative real time CMV assay and a commercial ASR real time CMV test (artus CMV, Qiagen, Hamburg, Germany). Linear range, limit of detection (LOD), precision and correlation of quantification were determined. Correlation studies comparing these two real time methods with COBAS Amplicor CMV Monitor test (CAM, Roche Diagnostics, Indianapolis, IN) were performed. All samples tested by the real time methods were extracted on the BioRobot M48. Results were linear from 2.0 to 7.6 log10 copies/ml and from 2.4 to 8.0 log10 copies/ml for the in-house and artus CMV methods. The LOD was 2.0 log10 copies/ml for both methods. The in-house real time PCR assay results for intra-run and inter-run variability (2.0/5.0 log10 copies/ml) were 4.2/2.1 and 11.8/1.8. For artus CMV ASR, the intra-run and inter-run variability (2.7/3.7/5.7 log10 copies/ml) was 8.5/1.5/0.8 and 7.3/2.2/0.9. Clinical samples tested by CAM with CMV levels between 2.8 -6.8 log10 copies/ml were quantified using the real time PCR methods. The mean difference in quantification across the range of specimens tested was 0.05 log10 copies/ml for both the in-house real time PCR method and the artus CMV real time ASR. Characteristics of the regression lines included slope=0.98, y intercept= 0.04 for the in-house method and slope=0.98, y intercept=0.05 for the artus CMV ASR. Agreement was better between artus CMV ASR and CAM (R2=0.91) than the in-house real time PCR and CAM (R2=0.85). The real time methods with automated extraction have advantages over CAM including broader linear range thereby eliminating the need for repeat testing on diluted samples.