Quantitation of cytomegalovirus DNA by real-time polymerase chain reaction in peripheral blood specimens of patients with solid organ transplants: comparison with end-point PCR and pp65 antigen test.J Med Virol. 2006 Jul; 78(7):915-22.JM
The polymerase chain reaction (PCR) for cytomegalovirus (CMV) DNA quantitation provides sensitive and specific data for detecting CMV as well as monitoring the infection and determining the appropriate antiviral strategy. A recently introduced real-time PCR assay for CMV DNA quantitation was applied on 158 peripheral blood leukocytes (PBLs) from 32 liver-transplanted patients with CMV asymptomatic infection and correlated with a commercial quantitative end-point PCR (COBAS AMPLICOR CMV Monitor) and CMV pp65 antigenemia. A good correlation was found between real-time PCR and pp65 antigen test (r2 = 0.691) and the two PCR assays (r2 = 0.761). Real-time PCR data were higher in pre-emptive treated patients (>20 pp65 + positive cells, median CMV DNA value: 3.8 log(10) copies/500,000 PBLs) than in not-treated ones (2.9 logs). According to pp65 levels of 0, 1-10, 11-20, 21-50, 51-100, and >100 positive cells/200,000 PBLs, median CMV DNA by real-time PCR was 2.6, 3.0, 3.6, 4.0, 4.2, and 4.8 logs, respectively, (CMV DNA levels by COBAS AMPLICOR: 2.8, 2.9, 3.8, 3.7, 3.9, and 4.0 logs). For samples with >20 pp65 + cells, real-time PCR gave significantly higher values than in groups with <20 pp65 + cells, whereas the COBAS AMPLICOR results showed a slower progression rate. Dilutions of CMV AD169 strain were used to probe real-time PCR reproducibility (between and intra-assay variability <2%) and sensitivity (100% detection rate at 10 copies/reaction, 28.5% with end-point PCR). In conclusion, real-time PCR significantly improves the study of CMV DNA dynamics due to a more reliable quantitation of CMV DNA for moderate and high DNA level compared to end-point PCR with better sensitivity and specificity. Real-time PCR provides more precise information for evaluating infection progress and assessing antiviral response, simplifying and accelerating the process of producing a reliable quantitation of CMV DNA for clinical purposes.