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.

Quantitative Real-Time Polymerase Chain Reaction for Evaluating DNAemia due to Cytomegalovirus, Epstein-Barr Virus, and BK Virus in Solid-Organ Transplant Recipients

“…EBV is associated with posttransplantation lymphoproliferative disease (PTLD); this complication most commonly occurs in organ transplant recipients who are seronegative for the virus before transplantation [7,[25][26][27][28]. Accordingly, the highest incidence of PTLD occurs in children.…”

“…BKV and EBV are not detected in cell cultures by routine diagnostic virology methods. Detection of CMV in blood specimens can be qualitatively recognized rapidly (16 h after inoculation) in shell vial cultures, but quantitation of the CMV load with this technology is cumbersome and impractical.The first laboratory method for quantitation of CMV load was the antigenemia test [4] jective interpretation of the test results [5][6][7]. Importantly, both shell vial cell cultures (qualitative) and antigenemia tests (quantitative) have been shown in comparative studies to be less sensitive than PCR.…”

“…Importantly, both shell vial cell cultures (qualitative) and antigenemia tests (quantitative) have been shown in comparative studies to be less sensitive than PCR. In addition, DNAemia was detected in an earlier stage of nucleic acid amplification [6][7][8][9][10].As an alternative to cell culture and the antigenemia test, implementation of molecular techniques for the rapid and sensitive detection of nucleic acid targets has yielded new levels of capabilities for laboratory diagnosis of many microbial infections [11]. Implementation of these tests has been facilitated by the availability of real-time PCR instrumentation with automation of nucleic acid-target amplification and amplicondetection steps in a "closed system" (i.e., reaction tubes never opened during or after amplification).…”

Detection of Cytomegalovirus (CMV) DNA in EDTA Whole-Blood Samples: Evaluation of the Quantitative artus CMV LightCycler PCR Kit in Conjunction with Automated Sample Preparation

“…The level of CMV DNA has been found to be an important prognostic marker for the ongoing disease (1,2,4). Today, laboratories use different sample materials for the detection and quantitation of CMV DNA.…”

Detección precoz de infección por citomegalovirus en pacientes sometidos a trasplante alogéneico de precursores hematopoyéticos por reacción de polimerasa en cadena cuantitativa en tiempo real

“…Inmunocomprometidos RT-qPCR para monitoreo de infección por CMV han mostrado que existe una correlación significativa entre ambos ensayos 4,[6][7][8][9] . Un estudio de Sia y cols.…”