Department of Laboratory Medicine, Faculty of Medicine and Health, Örebro University, Örebro, Sweden.
Section of Virology, Imperial College, St Mary's Hospital, London, UK.
J Virol Methods. 2018 Oct;260:70-74. doi: 10.1016/j.jviromet.2018.07.003. Epub 2018 Jul 10.
Human T-lymphotrophic virus (HTLV) types 1 and 2 cause lifelong infection whereby most infected individuals are asymptomatic whilst a minority develop infection-related disease. These latter patients invariably have been found to have high proviral load (PVL). Therefore, infected patients are monitored by determining the proportion of lymphocytes that are infected with HTLV-1/2. An increase in PVL has been shown to represent an increasing risk of developing HTLV-associated diseases. Monitoring of PVL requires a reliable and sensitive method. In this study assays based on droplet digital PCR (ddPCR) were established and evaluated for detection and quantification of HTLV-1/2.
To develop two parallel assays to detect the tax genes and determine the PVL of HTLV-1 and -2.
Sixty-seven clinical samples from patients infected with HTLV-1 or HTLV-2 were analysed. The samples had previously been analysed with a qPCR and a comparison between ddPCR and qPCR was performed. The specificity of the assays were determined by analyzing samples from 20 healthy blood donors.
The ddPCR was a stable and sensitive method for detection and quantification of HTLV-1 and -2. When comparing the qPCR and ddPCR the correlation was high (Pearsons correlation coefficient 0.96). The variability of the ddPCR was very low with intra-assay coefficient of variation (CV) of 0.97-3.3% (HTLV-1) and 1.7-8.2% (HTLV-2) and inter-assay CV of 1.8-6.1% (HTLV-1) and 1.2-12.9% (HTLV-2).
The ddPCR reliably quantified HTLV DNA in clinical samples and could be a useful tool for monitoring of PVLs in HTLV-infected individuals.
人类 T 淋巴细胞病毒（HTLV）1 型和 2 型会导致终身感染，大多数受感染个体无症状，但少数会出现与感染相关的疾病。这些患者无一例外地具有高前病毒载量（PVL）。因此，通过确定感染 HTLV-1/2 的淋巴细胞比例来监测感染患者。已经表明，PVL 的增加代表了发展为 HTLV 相关疾病的风险增加。PVL 的监测需要一种可靠和敏感的方法。在这项研究中，建立了基于液滴数字 PCR（ddPCR）的检测方法，并对其进行了评估，以检测和定量 HTLV-1/2。
开发两种平行检测方法，以检测 tax 基因并确定 HTLV-1 和 -2 的 PVL。
分析了 67 例感染 HTLV-1 或 HTLV-2 的患者的临床样本。这些样本之前已经用 qPCR 进行了分析，并对 ddPCR 和 qPCR 进行了比较。通过分析 20 名健康献血者的样本来确定检测方法的特异性。
ddPCR 是一种稳定且敏感的 HTLV-1 和 -2 检测和定量方法。当比较 qPCR 和 ddPCR 时，相关性很高（Pearson 相关系数为 0.96）。ddPCR 的变异性非常低，内试验变异系数（CV）为 0.97-3.3%（HTLV-1）和 1.7-8.2%（HTLV-2），而间试验 CV 为 1.8-6.1%（HTLV-1）和 1.2-12.9%（HTLV-2）。
ddPCR 可靠地定量了临床样本中的 HTLV DNA，可能是监测 HTLV 感染个体 PVL 的有用工具。
