Department of Urology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466-8550, Japan.
J Clin Microbiol. 2010 Mar;48(3):825-30. doi: 10.1128/JCM.01699-09. Epub 2010 Jan 6.
In recent years, virus-induced nephropathy caused mainly by BK polyomavirus (BKPyV), JC polyomavirus (JCPyV), and adenovirus has emerged as a problem in renal transplant patients. In the present study, we developed a multiplex real-time PCR assay to quantify the viral load of BKPyV, JCPyV, and adenovirus simultaneously. The dynamic range covered at least 6 orders of magnitude. This system was specific and reproducible, even in the presence of large amounts of DNA of other viruses. To validate this assay, urine samples from 124 renal transplant patients and serum samples from 18 hemorrhagic cystitis patients after hematopoietic stem cell transplantation were examined. In the urine samples from renal transplant patients, BKPyV was detected in 28 patients (22.6%), JCPyV was detected in 51 patients (41.1%), and adenovirus was detected in 2 patients (1.6%). The maximum amounts of each virus detected were 2.7 x 10(9), 8.7 x 10(8), and 1.2 x 10(2) copies/ml, respectively. Decoy cells were observed in 31 patients. The quantities of both BKPyV and JCPyV DNA were greater in samples with decoy cells. Two patients whose BKPyV viral loads exceeded 10(8) copies/ml had elevated serum creatinine levels and were diagnosed with BKPyV nephropathy based on graft biopsies. In serum samples from hemorrhagic cystitis patients, BKPyV, JCPyV, and adenovirus was detected in six, two, and three patients, respectively. Strong correlations were observed between the viral DNA copy numbers determined in the multiplex assays and those determined in single assays. Since this new assay is rapid, sensitive, and specific, it can be used for quantitative analyses of viruses in urine and serum samples after transplantation.
近年来，由 BK 多瘤病毒（BKPyV）、JC 多瘤病毒（JCPyV）和腺病毒引起的病毒相关性肾病已成为肾移植患者的一个问题。在本研究中，我们开发了一种多重实时 PCR 检测方法，用于同时定量 BKPyV、JCPyV 和腺病毒的病毒载量。该系统的检测范围至少涵盖了 6 个数量级。该系统具有特异性和可重复性，即使存在大量其他病毒的 DNA 也是如此。为了验证该检测方法，我们检测了 124 例肾移植患者的尿液样本和 18 例造血干细胞移植后发生出血性膀胱炎患者的血清样本。在肾移植患者的尿液样本中，检测到 28 例（22.6%）患者存在 BKPyV、51 例（41.1%）患者存在 JCPyV，2 例（1.6%）患者存在腺病毒。每种病毒的最大检测量分别为 2.7×10(9)、8.7×10(8)和 1.2×10(2)拷贝/ml。在 31 例患者中观察到诱饵细胞。诱饵细胞存在的样本中 BKPyV 和 JCPyV DNA 数量更大。2 例 BKPyV 病毒载量超过 10(8)拷贝/ml 的患者血清肌酐水平升高，基于移植肾活检诊断为 BKPyV 相关性肾病。在出血性膀胱炎患者的血清样本中，分别在 6、2 和 3 例患者中检测到 BKPyV、JCPyV 和腺病毒。多重检测方法和单重检测方法确定的病毒 DNA 拷贝数之间存在很强的相关性。由于该新检测方法快速、敏感且特异性高，因此可用于移植后尿液和血清样本中病毒的定量分析。
