Semiquantitative PCR analysis of Epstein-Barr virus DNA in clinical samples of patients with EBV-associated diseases

The laboratory diagnosis of primary and reactivated Epstein-Barr virus (EBV ) infection is based on serologic methods in immunocompetent patients. Howe ver, in immunocompromised patients, serologic data are difficult to interpr et and do not often correlate with clinical data. In order to find a useful and practical marker for diagnosis of EBV-related diseases, a polymerase c hain reaction (PCR) assay was established for semiquantitative detection of EBV sequences. The method was based on a nested PCR, using primers of the virus capsid antigen p23 region and an endpoint dilution. This method was c arried out on 68 plasma samples, 68 samples of peripheral blood mononuclear cells and 5 cerebrospinal fluid samples of 39 patients with various diseas es to evaluate the EBV-genome copy number. Samples from patients suffering from infectious mononucleosis served as positive controls for active EBV in fection. In 5 patients with infectious mononucleosis, high copy numbers of EBV genomes in peripheral blood mononuclear cells were detected within a ra nge of 1,000-40,000 copies in 105 peripheral blood mononuclear cells. In co ntrast, samples from 19 latently infected persons either showed low copy nu mbers (10-100 in 10(5) peripheral blood mononuclear cells) or were EBV PCR negative. Comparable results were observed in seven renal transplant patien ts without any symptoms. The practical value of the semiquantitative detect ion of EBV DNA was demonstrated in three bone marrow transplant recipients. Two developed a lymphoproliferative disease associated with extremely high amounts of EBV DNA in plasma (16,000 and 50,000 copies/ml, respectively) a nd peripheral blood mononuclear cells (100,000 and 6.5 million copies in 10 (5) peripheral blood mononuclear cells, respectively). The high EBV load in plasma and peripheral blood mononuclear cells was reduced dramatically aft er successful anti-viral therapy in one case. The third bone marrow transpl ant recipient developed an EBV-induced transverse myelitis with an increase d number of EBV-genome copies in peripheral blood mononuclear cells and EBV -positive cerebrospinal fluid samples. After combined antiviral and immune therapy, the EBV-genome copy numbers decreased and the patient recovered co mpletely. These data demonstrate a good correlation between semiquantitativ e detection of EBV genomes and clinical findings. The method is recommended for the diagnosis of EBV-associated diseases in patients after transplanta tion, as well as for monitoring the response to therapy. (C) 2001 Wiley-Lis s, Inc.