Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan.
Department of Genetics, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan; Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Stockholm, Sweden.
J Mol Diagn. 2023 Jun;25(6):403-409. doi: 10.1016/j.jmoldx.2023.03.004. Epub 2023 Mar 23.
Human adenovirus (AdV) reactivation after hematopoietic stem cell transplantation (HSCT) is associated with life-threatening clinical manifestations. Although real-tme quantitative PCR (qPCR) has been widely used to measure AdV loads, it has not been standardized for AdV. Droplet digital PCR (ddPCR) is a novel pathogen detection technology that enables the absolute quantification of viral loads. ddPCR would enable a more accurate AdV DNA detection compared with qPCR. In this study, ddPCR was developed for AdV DNA and its performance characteristics compared with those of qPCR. AdV DNAemia incidence during the first 12 weeks after allogenic HSCT was then retrospectively examined by qPCR and ddPCR in 97 HSCT procedures using the preserved 545 DNA samples. ddPCR exhibited better reproducibility and sensitivity, as well as equivalent quantifiability, compared with qPCR. AdV DNA among HSCT patients was detected in 11 (2.0%) and 49 (9.0%) of 545 samples by qPCR and ddPCR, respectively. AdV DNA levels >1000 copies/mL were observed in five cases by qPCR and/or ddPCR. However, two patients developed fulminant hepatitis and died; other patients remained asymptomatic with subsequently undetectable AdV DNA. In conclusion, ddPCR was more sensitive and reproducible in detecting AdV DNA among pediatric HSCT recipients than qPCR. ddPCR offers the potential to provide a more accurate DNAemia detection, determine cutoff values for treatment initiation, and enable antiviral efficacy assessment.
人腺病毒（AdV）在造血干细胞移植（HSCT）后再激活与危及生命的临床表现有关。虽然实时定量 PCR（qPCR）已广泛用于测量 AdV 负荷，但尚未对 AdV 进行标准化。数字液滴 PCR（ddPCR）是一种新型的病原体检测技术，可实现病毒载量的绝对定量。与 qPCR 相比，ddPCR 可实现更准确的 AdV DNA 检测。在这项研究中，开发了用于 AdV DNA 的 ddPCR，并比较了其与 qPCR 的性能特征。在 97 例接受同种异体 HSCT 的患者中，使用保存的 545 个 DNA 样本，通过 qPCR 和 ddPCR 回顾性检查了移植后 12 周内的前 AdV DNA 血症发生率。与 qPCR 相比，ddPCR 表现出更好的重现性和灵敏度，以及等效的定量能力。qPCR 和 ddPCR 分别在 545 个样本中的 11（2.0%）和 49（9.0%）个样本中检测到 HSCT 患者的 AdV DNA。qPCR 和/或 ddPCR 观察到 5 例 AdV DNA 水平>1000 拷贝/ml。然而，两名患者发生暴发性肝炎并死亡；其他患者无症状，随后 AdV DNA 检测不到。总之，ddPCR 比 qPCR 更灵敏、更能重现地检测儿科 HSCT 受者中的 AdV DNA。ddPCR 有可能提供更准确的 DNA 血症检测、确定开始治疗的截止值，并能够评估抗病毒疗效。
