Meerbach A, Gruhn B, Egerer R, Reischl U, Zintl F, Wutzler P
Institute for Antiviral Chemotherapy, University of Jena, Jena, Germany.
J Med Virol. 2001 Oct;65(2):348-57. doi: 10.1002/jmv.2040.
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 10(5) peripheral blood mononuclear cells. In contrast, samples from 19 latently infected persons either showed low copy numbers (10-100 in 10(5) 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 10(5) 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.
在免疫功能正常的患者中，原发性和再激活的爱泼斯坦-巴尔病毒（EBV）感染的实验室诊断基于血清学方法。然而，在免疫功能低下的患者中，血清学数据难以解释，且往往与临床数据不相关。为了找到一种用于诊断EBV相关疾病的有用且实用的标志物，建立了一种聚合酶链反应（PCR）检测方法，用于半定量检测EBV序列。该方法基于巢式PCR，使用病毒衣壳抗原p23区域的引物和终点稀释法。对39例患有各种疾病的患者的68份血浆样本、68份外周血单个核细胞样本和5份脑脊液样本进行该方法检测，以评估EBV基因组拷贝数。患有传染性单核细胞增多症患者的样本用作活动性EBV感染的阳性对照。在5例传染性单核细胞增多症患者中，在外周血单个核细胞中检测到高拷贝数的EBV基因组，在10⁵个外周血单个核细胞中的拷贝数范围为1000 - 40000个。相比之下，19例潜伏感染患者的样本要么显示低拷贝数（10⁵个外周血单个核细胞中有10 - 100个），要么EBV PCR检测为阴性。在7例无任何症状的肾移植患者中也观察到了类似结果。在3例骨髓移植受者中证明了EBV DNA半定量检测的实用价值。其中2例发生了与血浆中极高量的EBV DNA（分别为16000和50000拷贝/ml）以及外周血单个核细胞中极高量的EBV DNA（10⁵个外周血单个核细胞中分别为100000和6500000拷贝）相关的淋巴增殖性疾病。在1例患者成功进行抗病毒治疗后，血浆和外周血单个核细胞中的高EBV载量显著降低。第3例骨髓移植受者发生了EBV诱导的横贯性脊髓炎，外周血单个核细胞和EBV阳性脑脊液样本中的EBV基因组拷贝数增加。在联合抗病毒和免疫治疗后，EBV基因组拷贝数下降，患者完全康复。这些数据表明EBV基因组的半定量检测与临床发现之间具有良好的相关性。该方法推荐用于移植后患者EBV相关疾病的诊断，以及监测治疗反应。
