Digital Droplet PCR for Detection and Quantitation of Hepatitis Delta Virus

Abstract

Introduction: 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.

Methods: 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.

Results: 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.

Discussion: 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.