Longitudinal monitoring of latent and active human cytomegalovirus infections in peripheral blood of heart transplant recipients by single-tube nested RT-PCR

Abstract

PCR is a sensitive diagnostic tool for the detection of human cytomegalovirus (HCMV) DNA in the peripheral blood of immunosuppressed transplant recipients. However, its specificity as a prognostic marker for clinical disease is unclassified, because infections considered to be latent may be detected by this method. In order to diagnose active viral infections, we used reverse transcription-PCR (RT-PCR) to identify HCMV mRNA in blood. We developed a single-tube nested RT-PCR with preformed PCR mixes embedded in a trehalose matrix. Blood samples of 48 heart transplant recipients were investigated for HCMV DNA. 8 patients detected to be HCMV DNA positive after transplantation were investigated in longitudinal monitoring for at least 6 months. HCMV mRNA was found in 5 patients who developed HCMV related symptoms during the period of RNA detection. There was no clear relation between the onset of DNA detection and the first demonstration of mRNA. In 2 patients HCMV DNA could be detected 74 and 81 days before the appearance of mRNA, suggesting long persistence until active infection is started. In 3 patients HCMV mRNA disappeared during or immediately after the end of ganciclovir therapy. In contrast, HCMV DNA was detectable continuously for prolonged periods after therapy, indicating that the persistance of HCMV DNA is not influenced by ganciclovir treatment. In summary, HCMV mRNA detection seems to be a reliable early marker for the differentiation of persistent and active HCMV infections in immunosuppressed patients. Our data show that viral mRNA detection is probably a better predictor of the effectiveness of antiviral therapy than viral DNA detection.