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

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Abstract
The laboratory diagnosis of primary and reactivated Epstein‐Barr virus (EBV) infection is based on serologic methods in immunocompetent patients. However, in immunocompromised patients, serologic data are difficult to interpret 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 chain 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 carried out on 68 plasma samples, 68 samples of peripheral blood mononuclear cells and 5 cerebrospinal fluid samples of 39 patients with various diseases to evaluate the EBV‐genome copy number. Samples from patients suffering from infectious mononucleosis served as positive controls for active EBV infection. In 5 patients with infectious mononucleosis, high copy numbers of EBV genomes in peripheral blood mononuclear cells were detected within a range of 1,000–40,000 copies in 105 peripheral blood mononuclear cells. In contrast, samples from 19 latently infected persons either showed low copy numbers (10–100 in 105 peripheral blood mononuclear cells) or were EBV PCR negative. Comparable results were observed in seven renal transplant patients without any symptoms. The practical value of the semiquantitative detection 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) and peripheral blood mononuclear cells (100,000 and 6.5 million copies in 105 peripheral blood mononuclear cells, respectively). The high EBV load in plasma and peripheral blood mononuclear cells was reduced dramatically after successful antiviral therapy in one case. The third bone marrow transplant recipient developed an EBV‐induced transverse myelitis with an increased 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 completely. These data demonstrate a good correlation between semiquantitative detection of EBV genomes and clinical findings. The method is recommended for the diagnosis of EBV‐associated diseases in patients after transplantation, as well as for monitoring the response to therapy. J. Med. Virol. 65:348–357, 2001.