O.1.1 Development of a new real-time PCR for the detection of covalently closed circular DNA (cccDNA) in plasma of chronic hepatitis B patients

Abstract

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) serves as a template for viral replication and plays a role in the persistence of HBV infection. The origin and significance of cccDNA in plasma however, is not well understood. We developed and validated a sensitive, specific, and reproducible real-time PCR for detection of cccDNA in plasma of chronic hepatitis B (CHB) patients. We analyzed 4 international HBV DNA standards, and 96 plasma samples of CHB patients. Results were compared with HBV relaxed circular DNA (rcDNA) levels, individual ALT levels and the Histology Activity Index (HAI). Our cccDNA assay had a lower limit of detection at 15 copies/PCR and a correlation coefficient (R) of 0.98 (p<0.0001). Plasma cccDNA was detected in 2 of 4 panels. We found a significant correlation between cccDNA and HBV rcDNA levels in both panels (R = 0.96, and R= 0.43) and in plasma samples of CHB patients (R = 0.88, p< 0.0001). In 57% of these samples cccDNA was detectable (only when HBV rcDNA was 2×106 copies/mL). The mean level of cccDNA was 0.16% of the total HBV load. Plasma cccDNA levels were higher in HBeAg positive samples than in HBeAg negative samples (4.91 log copies/mL vs 3.88 log copies/mL, p< 0.0001). Levels of HBV rcDNA and HBV genotype did not influence cccDNA detection. The ALT levels and HAI-score were not correlated with plasma cccDNA levels. These findings suggest that cccDNA levels in plasma is not the result of increased hepatocyte degeneration, but indicate that other mechanisms are responsible.