Real-time ed end-point Polymerase Chain Reaction per la quantizzazione del DNA di Citomegalovirus: confronto tra metodi e con il test per l’antigene pp65

Quantitave Polymerase Chain Reaction (PCR) for Cytomegalovirus (CMV) DNA provides highly sensitive and specific data for detecting CMV as well as monitoring the infection and determining the appropriate antiviral strategy.To determine the clinical application of a recently introduced real-time (RT) PCR assay for CMV DNA quantitation in peripheral blood leukocytes (PBLs) and defining its correlation with the commercial quantitative end-point (EP) PCR method COBAS AMPLICOR CMV Monitor and pp65 antigen test. Sequential PBL samples (n=158) from 32 liver transplanted patients with CMV asymptomatic infection and positive for CMV DNA by EP-PCR were retrospectively analysed with RT-PCR and studied according to pp65 antigen levels. A good correlation was found between RT-PCR and pp65 antigen test (r=0.691) and between the two PCR assays (r=0.761). RT-PCR data were significantly higher in pre-emptive treated patients (those with >20 pp65+positive cells, median value: 3.8 log10 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 load by RT-PCR was 2.6, 3.0, 3.6, 4.0. 4.2 and 4.8, log10 copies/ 500,000 PBLs, respectively (EP-PCR CMV DNA levels: 2. 8, 2.9, 3.8, 3.7, 3.9 and 4.0 logs). For samples with >20 pp65+cells, that is above the level at which pre-emptive therapy was started, RT-PCR values were significantly higher than in groups with less than 20 pp65+cells, whereas EP-PCR values did not significantly differ and showed a slower progression rate. Dilutions of DNA from CMV AD169 strain were used to probe RT-PCR reproducibility (between and intra-assay variability < 2%) and sensitivity (100% detection rate at 10 copies/reaction, 28.5% with EP-PCR). A significant improvement is coming from the introduction of RT-PCR to the study of CMV DNA dynamics in differently CMV infected patients due to a more reliable quantitation of CMV DNA for moderate and high DNA level compared to EP-PCR with better sensitivity and specificity. RTPCR gives more precise informations on viral load kinetics for evaluating the infection progress and assessing antiviral response, significantly simplifying and accelerating the process of producing a reliable quantification of CMV DNA for clinical purposes

Quantitation of human cytomegalovirus DNA in peripheral blood leukocytes of heart transplant recipients: relationship with pp65 antigenernia and with antiviral therapy

ObjectiveTo retrospectively determine DNA levels in blood polymorphonuclear leukocytes (PMNLs) of 21 heart transplant patients who suffered from HCMV infection and who were monitored by the antigenemia assay (pp65 test) during follow-up, by use of a quantitative competitive polymerase chain reaction (PCR) assay for human cytomegalovirus (HCMV) DNA.MethodsQuantitation of HCMV DNA by PCR was expressed as genome equivalents (GE) per 200 000 PMNLs.ResultsTen patients experienced symptomatic HCMV infection (five primary infections and five reactivations) with mild symptoms and received ganciclovir treatment, whereas 11 asymptomatic HCMV infections were not treated. Therapy was discontinued when a 90% reduction of the pretreatment antigenic load was achieved in a symptomless patient. The mean HCMV DNA and antigenic loads were significantly higher in symptomatic than in asymptomatic patients: 4.6×105±4.7×105 GE and 1.1×104 GE (p < 0.0001) and 390±350 versus 25±12 pp65-positive PMNLs (p < 0.0001), and in primary than in secondary infections (583±403 pp65-positive PMNLs versus 85±111, p=0.002 and 5.2×105±5.2×105 GE instead of 1.5×105±3.2×105 GE, p=0.02). A single course of 14−21 days of ganciclovir caused a marked decrease of HCMV DNA and antigenemia in eight of 10 patients in whom a 90% reduction of the antigenic load correlated with a 98% DNA reduction of the pretreatment levels. In two primary infections, a 90% antigenic reduction was achieved by 21 days of ganciclovir treatment, but those data only correlated with a DNA load reduction of 28% and 60% of the pretreatment levels. Fifteen and 12 days later, respectively, the two patients relapsed and underwent a second ganciclovir course, at the end of which a 90% reduction of the antigenic load correlated with a >98% DNA drop. GCV was discontinued and the patients recovered completely. In those two patients we retrospectively found persistent high DNA levels before the second ganciclovir course, whereas the antigenic load slowly increased after an apparent reduction.Conclusions: Our data suggest that: (1) DNA levels have the same trend as the pp65 antigen test—they are significantly higher in symptomatic and in primary HCMV-infected patients than in asymptomatic patients and those with secondary infection; (2) a 90% antigenic load reduction from the pretreatment level may be a less reliable predictor of the efficacy of anti-HCMV therapy than DNA load, at least in primary infection, in which a much higher viral load and much more severe disease are present; and (3) a DNA load reduction of >98% of the pretreatment value is required for therapeutic success

Comparison of polymerase chain reaction and pp65 antigen test for early detection of human cytomegalovirus in blood leukocytes of cardiac transplant recipients

ObjectiveTo establish whether polymerase chain reaction (PCR) for cytomegalovirus deoxyribonucleic acid (DNA) can provide clinical information for the management of the infection.MethodsLeukocytes in 30 heart transplant recipients were monitored by pp65 antigen testing and PCR for 82 to 365 days after transplantation.ResultsOf the 30 patients, 26 developed cytomegalovirus infection, nine of whom were symptomatic. Altogether, 300 leukocyte samples were examined. The concordance between PCR and pp65 antigen test was 82.6%. In symptomatic patients after surgery, PCR detected cytomegalovirus infection after 38 ± 16 days and the pp65 antigen test, after 48 ± 15 days. Symptomatic infection correlated with a higher number of pp65-positive leukocytes than did asymptomatic infection: 310 ± 356 vs 24 ± 35 (p < 0.005)/200,000 examined, respectively. Clearance of virus was observed by PCR after 125 ± 73 days (range 29 to 225) in symptomatic, and after 82 ± 70 days (range 16 to 301) in asymptomatic, cases of infection.ConclusionsThe positive predictive value of PCR for symptomatic infection was 34.6%. Our findings correlate with previous reports and show that the qualitative detection of cytomegalovirus DNA is not associated with overt disease whereas quantitation of pp65-positive leukocytes closely correlate with symptom onset. Insofar as the results are not quantitative, PCR is not a marker of clinically apparent infection. Careful monitoring of cytomegalovirus infection based on quantitative pp65 antigen assay can fulfill all clinical needs for early diagnosis and proper management of the infectio