Viral Immunology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health (NIH), Bethesda, MD 20892, USA.
Department of Laboratory Medicine, National Institutes of Health (NIH), Bethesda, MD 20892, USA.
Viruses. 2022 Jun 8;14(6):1246. doi: 10.3390/v14061246.
Background: Lytic infection of oligodendrocytes by the human JC polyomavirus (JCPyV) results in the demyelinating disease called progressive multifocal leukoencephalopathy (PML). The detection of viral DNA in the cerebrospinal fluid (CSF) by PCR is an important diagnostic tool and, in conjunction with defined radiological and clinical features, can provide diagnosis of definite PML, avoiding the need for brain biopsy. The main aim of this study is to compare the droplet digital PCR (ddPCR) assay with the gold standard quantitative PCR (qPCR) for the quantification of JC viral loads in clinical samples. Methods: A total of 62 CSF samples from 31 patients with PML were analyzed to compare the qPCR gold standard technique with ddPCR to detect conserved viral DNA sequences in the JCPyV genome. As part of the validation process, ddPCR results were compared to qPCR data obtained in 42 different laboratories around the world. In addition, the characterization of a novel triplex ddPCR to detect viral DNA sequence from both prototype and archetype variants and a cellular housekeeping reference gene is described. Triplex ddPCR was used to analyze the serum from six PML patients and from three additional cohorts, including 20 healthy controls (HC), 20 patients with multiple sclerosis (MS) who had never been treated with natalizumab (no-NTZ-treated), and 14 patients with MS who were being treated with natalizumab (NTZ-treated); three from this last group seroconverted during the course of treatment with natalizumab. Results: JCPyV DNA was detected only by ddPCR for 5 of the 62 CSF samples (8%), while remaining undetected by qPCR. For nine CSF samples (15%), JCPyV DNA was at the lower limit of quantification for qPCR, set at <250 copies/mL, and therefore no relative quantitation could be determined. By contrast, exact copies of JCPyV for each of these samples were quantified by ddPCR. No differences were observed between qPCR and ddPCR when five standardized plasma samples were analyzed for JCPyV in 42 laboratories in the United States and Europe. JCPyV-DNA was undetected in all the sera from HC and MS cohorts tested by triplex ddPCR, while serum samples from six patients with PML tested positive for JCPyV. Conclusion: This study shows strong correlation between ddPCR and qPCR with increased sensitivity of the ddPCR assay. Further work will be needed to determine whether multiplex ddPCR can be useful to determine PML risk in natalizumab-treated MS patients.
人类 JC 多瘤病毒（JCPyV）对少突胶质细胞的裂解性感染导致脱髓鞘疾病，称为进行性多灶性脑白质病（PML）。通过 PCR 检测脑脊液（CSF）中的病毒 DNA 是一种重要的诊断工具，结合明确的影像学和临床特征，可以提供明确的 PML 诊断，避免了脑活检的需要。本研究的主要目的是比较液滴数字 PCR（ddPCR）检测与定量 PCR（qPCR），以定量检测临床样本中的 JC 病毒载量。
对 31 例 PML 患者的 62 份 CSF 样本进行分析，以比较 qPCR 金标准技术与 ddPCR 检测 JCPyV 基因组中保守病毒 DNA 序列。作为验证过程的一部分，将 ddPCR 结果与来自全球 42 个不同实验室的 qPCR 数据进行比较。此外，还描述了一种新型三重 ddPCR 检测方法，用于检测原型和原始变异的病毒 DNA 序列和细胞管家基因参考序列。使用三重 ddPCR 分析了 6 例 PML 患者和另外 3 个队列的血清，包括 20 例健康对照（HC）、20 例从未接受过那他珠单抗治疗的多发性硬化症（MS）患者（未接受那他珠单抗治疗，no-NTZ-treated）和 14 例正在接受那他珠单抗治疗的 MS 患者（接受那他珠单抗治疗，NTZ-treated）；其中 3 例在那他珠单抗治疗过程中血清转化。
62 份 CSF 样本中，只有 5 份（8%）通过 ddPCR 检测到 JCPyV DNA，而其余样本通过 qPCR 未检测到。对于 9 份 CSF 样本（15%），qPCR 检测到的 JCPyV DNA 处于<250 拷贝/ml 的定量下限，因此无法确定相对定量。相比之下，通过 ddPCR 对每份样本进行了精确的 JCPyV 定量。在 42 个来自美国和欧洲的实验室中分析了 5 个标准化血浆样本中的 JCPyV，qPCR 和 ddPCR 之间没有差异。三重 ddPCR 检测的所有 HC 和 MS 队列的血清样本均未检测到 JCPyV-DNA，而 6 例 PML 患者的血清样本均检测到 JCPyV 阳性。
本研究表明，ddPCR 与 qPCR 具有高度相关性，ddPCR 检测的灵敏度有所提高。还需要进一步的研究来确定多重 ddPCR 是否可用于确定接受那他珠单抗治疗的 MS 患者的 PML 风险。
