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Because of the considerable impact of human cytomegalovirus (HCMV) infection, sensitive, specific, and standardized methods are required for rapid and accurate evaluation of viral load in monitoring transplant recipients. The aim of the present study was to evaluate the usefulness of a multiplex polymerase chain reaction (PCR) for the coamplification of HCMV-DNA and beta-globin genomic sequence in polymorphonuclear leukocytes (PMNL). Analysis and quantification of PCR products were carried out by a DNA enzyme immunoassay (DEIA), which is based on the hybridization of amplified DNA with a single-stranded DNA probe, which coats microtitre wells. Colorimetric detection of the DNA-antibody complex was carried out and optical density (O.D.) was recorded at 450/630 nm. To quantify HCMV/DNA load, a standard curve to which samples O.D. refer was obtained by amplifying serial dilutions of recombinant PGEM-3Z plasmid DNA containing a genomic fragment of glycoprotein B. 340 PMNL specimens from 102 solid organ recipients were tested for the detection of pp65 antigen and HCMV-DNA. The results showed a good correlation between viral load and clinical symptoms of HCMV infection; high specificity and predictive values for HCMV disease were found by PCR, using a cut-off limit of 10(3) genomic copies per 2 x 10(5) PMNL. These findings indicate that the system described is an efficient and reproducible diagnostic method easy to apply for routine diagnosis and therapeutic monitoring of transplanted patients., (Copyright 2000 Wiley-Liss, Inc.)

The use of sensitive and specific methods for rapid and reliable diagnosis is required due to the considerable impact of human cytomegalovirus (HCMV) in organ transplant recipients. For this purpose the demonstration of the presence of viral antigens in peripheral blood leukocytes (PMNLs) and of viral nucleic acids in the same cells or in sera would seem to be of valid support. The present study was designed to test pp65 antigen, HCMV DNA and HCMV late mRNA in order to provide clinical information for the management of the infection. Fifty solid organ recipients were monitored for six months after transplant. The data obtained from the various tests were analysed from the first evidence of HCMV infection revealed by positive antigenaemia and/or DNA-polymerase chain reaction (PCR). In 3 asymptomatic and in 7 symptomatic patients, PCR became positive 1-2 weeks before antigenaemia but PCR did not discriminate the clinical evolution of HCMV infection. The antigenaemia test well correlated to the development of viral infection being positive in all symptomatics and in 31, 2% of asymptomatics. The antigenic load > 100/2 x 10(5) positive cells was always associated with clinical signs of illness. The detection of late mRNA was more indicative of the virus replicative status in the follow-up of patients treated with ganciclovir. In some cases there was evidence, prior to the other two tests, the block of viral replication due to the antiviral therapy and in others the onset of HCMV infection relapse.