Optimization of quantitative detection of cytomegalovirus DNA in plasma by real-time PCR

Abstract

Previous studies have shown that detection of cytomegalovirus (CMV) DNA in plasma is less sensitive than the antigenemia assay for CMV surveillance in blood. In 1,983 blood samples, plasma PCR assays with three different primer sets (UL125 alone, UL126 alone, and UL55/UL123-exon 4) were compared to the pp65 antigenemia assay and blood cultures. Plasma PCR detected CMV more frequently in blood specimens than either the antigenemia assay or cultures, but of the three PCR assays, the double-primer assay (UL55/UL123-exon 4) performed best with regard to sensitivity, specificity, and predictive values compared to antigenemia: 122 of 151 antigenemia-positive samples were detected (sensitivity, 80.1%), and there were 122 samples that were PCR positive-antigenemia negative (specificity, 93%). Samples with discrepant results had a low viral load (median, 0.5 cells per slide; 1,150 copies per ml) and were often obtained from patients receiving antiviral therapy. CMV could be detected by other methods in 15 of 29 antigenemia positive-PCR negative samples compared to 121 of 122 PCR positive-antigenemia negative samples (P < 0.001). On a per-subject basis, 21 of 25 patients (antigenemia positive-PCR negative) and all 57 (PCR positive-antigenemia negative) could be confirmed at different time points during follow-up. The higher sensitivity of the double-primer assay resulted in earlier detection compared to antigenemia in a time-to-event analysis of 42 CMV-seropositive stem cell transplant recipients, and two of three patients with CMV disease who were antigenemia negative were detected by plasma PCR prior to the onset of disease. Interassay variability was low, and the dynamic range was >5 log(10). Automated DNA extraction resulted in high reproducibility, accurate CMV quantitation (R = 0.87, P < 0.001), improved sensitivity, and increased speed of sample processing. Thus, primer optimization and improved DNA extraction techniques resulted in a plasma-based PCR assay that is significantly more sensitive than pp65 antigenemia and blood cultures for detection of CMV in blood specimens.

FIG. 1.

Correlation of CMV quantitation by CMV pp65 antigenemia and UL55/UL123 plasma PCR among samples determined to be positive by both methods (R = 0.62, P < 0.0001) (A) and among samples found to be positive by either method (R = 0.57, P = 0.0001) (B).

FIG. 2.

Time to first detection by double-primer PCR at different levels versus pp65 antigenemia in CMV-seropositive HSCT recipients.

FIG. 3.

PCR results in three patients with CMV disease who were missed by antigenemia surveillance. The dashed arrows indicate positive PCR results prior to the onset of the disease, and solid arrows represent positive tests after the onset. The numbers indicate copy numbers per milliliter of plasma.

FIG. 4.

Manual versus automated extraction in 70 samples (UL55/UL123 assay; R = 0.87, P < 0.001).