title: 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.
abstract: 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.
authors.affiliation: Microbiology Laboratory, Molinette Hospital, Turin, Italy.
authors.forename: Tiziano
authors.initials: T
authors.lastname: Allice
authors.forename: Marco
authors.initials: M
authors.lastname: Enrietto
authors.forename: Fabrizia
authors.initials: F
authors.lastname: Pittaluga
authors.forename: Silvia
authors.initials: S
authors.lastname: Varetto
authors.forename: Alessandro
authors.initials: A
authors.lastname: Franchello
authors.forename: Giovanna
authors.initials: G
authors.lastname: Marchiaro
authors.forename: Valeria
authors.initials: V
authors.lastname: Ghisetti
mesh_terms: D000956:Antigens, Viral / Q000097:blood; D003587:Cytomegalovirus / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000175:diagnosis / Q000209:etiology / Q000821:virology; D004279:DNA, Viral / Q000097:blood* / Q000235:genetics*; D006801:Humans; D007118:Immunoassay / Q000379:methods / Q000706:statistics & numerical data; D007962:Leukocytes / Q000821:virology; D016031:Liver Transplantation / Q000009:adverse effects; D016377:Organ Transplantation / Q000009:adverse effects; D010750:Phosphoproteins / Q000097:blood / Q000276:immunology; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D014763:Viral Matrix Proteins / Q000097:blood / Q000276:immunology; D014773:Virology / Q000379:methods / Q000706:statistics & numerical data
chemical_list: D000956:Antigens, Viral; D004279:DNA, Viral; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa