Monitoring low cytomegalovirus viremia in transplanted patients by a real-time PCR on plasma

Until recently, human cytomegalovirus (hCMV) infection and anti-CMV treatment in transplanted patients have been monitored essentially by pp65 antigenemia, which is time-consuming and requires experienced operators. For the last two years, pp65 antigenemia levels have tended to be lower than previously in our laboratory, which could be due to better monitoring of CMV-related risk. Results obtained by real-time PCR with a LightCycler instrument or by pp65 antigen assay were compared on 145 serial samples from bone marrow or kidney transplant recipients under the usual conditions of our laboratory. CMV DNA was extracted from plasma and quantified by using primers and probes directed to HXFL4 gene. The plasma CMV DNA load was measured by using a standard curve constructed with a commercially available quantified CMV DNA suspension. Among the 145 samples, 139 showed a pp65 antigen which was negative or lower than 20 positively stained cells per 200,000 leukocytes. In the patients with positive pp65 antigenemia, the corresponding values of CMV DNA copy number/ml were significantly higher than those observed in patients without antigenemia (P < 0.001). CMV DNA was detected from 4 up to 52 days before pp65 antigen. Elsewhere, between two dates at which pp65 antigen was positive, intermediate PCR results could be positive while the pp65 antigen was negative. This real-time quantitative PCR assay is a rapid technique adapted to monitor plasma CMV DNA in transplant setting, even for low viremia.