Beijing Institute of Hepatology, Beijing Youan Hospital, Capital Medical University, Beijing, China.
Clin Transl Gastroenterol. 2022 Jul 1;13(7):e00509. doi: 10.14309/ctg.0000000000000509. Epub 2022 Jun 8.
Hepatitis delta virus (HDV) far exceeds our expected level. There remains a lack of reliable quantitative assays for HDV RNA detection. We sought to develop a new method based on digital droplet polymerase chain reaction (ddPCR) for HDV quantitative detection.
With plasmid (pMD19T) containing HDV full genome, we determined the method for ddPCR-based HDV RNA quantification. To compare various assays for HDV detection, 30 cases diagnosed with hepatitis D and 14 controls were examined using enzyme-linked immunosorbent assay, reverse-transcriptase PCR (RT-PCR), and ddPCR. A total of 728 hepatitis B virus-related patients, including 182 patients with chronic hepatitis B, 182 with liver cirrhosis, 182 with hepatocellular carcinoma, and 182 with liver failure, were screened for HDV infection.
The detection limit of ddPCR for HDV is significantly low, with lower limit of detection and lower limit of quantitation of 0.29 IU/mL (95% confidence interval: 1.93 × 10-3-1.22 IU/mL) and 8.76 IU/mL (95% confidence interval: 1.83-1.03 × 106 IU/mL), respectively. Among the 44 samples, the enzyme-linked immunosorbent assay detected 30 cases positive, ddPCR reported 24 samples, and RT-PCR reported 10 samples positive for HDV RNA. Moreover, the positive rates of anti-HDV were 1.1%, 3.3%, 2.7%, and 7.1% in patients with chronic hepatitis B, liver cirrhosis, hepatocellular carcinoma, and liver failure, respectively; the detection rates of RT-PCR in HDV RNA were 0%, 16.67%, 15.4%, and 20%, respectively. However, the detection rates of ddPCR were 0%, 33.33%, 30.77%, and 60%, respectively.
We establish a high sensitivity and specificity quantitative HDV RNA detection method based on ddPCR. Hepatitis B virus-related end-stage liver diseases, especially liver failure, are associated with a remarkably high rate of HDV infection.
乙型肝炎 delta 病毒（HDV）的感染远超我们的预期。目前仍缺乏可靠的 HDV RNA 定量检测方法。本研究旨在建立一种基于数字微滴聚合酶链反应（ddPCR）的新型 HDV 定量检测方法。
以包含 HDV 全基因组的质粒（pMD19T）为模板，确定了 ddPCR 检测 HDV RNA 的方法。为了比较各种 HDV 检测方法，我们检测了 30 例确诊的丁型肝炎患者和 14 例对照者，使用酶联免疫吸附试验、逆转录 PCR（RT-PCR）和 ddPCR。共筛查了 728 例乙型肝炎病毒相关患者，包括 182 例慢性乙型肝炎患者、182 例肝硬化患者、182 例肝细胞癌患者和 182 例肝功能衰竭患者，以了解其 HDV 感染情况。
ddPCR 检测 HDV 的检测下限显著降低，检测下限和定量下限分别为 0.29 IU/mL（95%置信区间：1.93×10-3-1.22 IU/mL）和 8.76 IU/mL（95%置信区间：1.83-1.03×106 IU/mL）。在 44 个样本中，酶联免疫吸附试验检测到 30 个阳性样本，ddPCR 报告 24 个样本，RT-PCR 报告 10 个样本 HDV RNA 阳性。此外，慢性乙型肝炎、肝硬化、肝细胞癌和肝功能衰竭患者的抗-HDV 阳性率分别为 1.1%、3.3%、2.7%和 7.1%；HDV RNA 的 RT-PCR 检测率分别为 0%、16.67%、15.4%和 20%；然而，ddPCR 的检测率分别为 0%、33.33%、30.77%和 60%。
我们建立了一种基于 ddPCR 的高灵敏度和特异性定量 HDV RNA 检测方法。乙型肝炎病毒相关终末期肝病，尤其是肝功能衰竭，与 HDV 感染率显著增高相关。
