Validation of a sensitive and specific real‐time PCR for detection and quantitation of hepatitis B virus covalently closed circular DNA 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 persistence of HBV infection. The origin and significance of cccDNA in plasma however, is not well understood. A sensitive, specific, and reproducible real-time PCR for detection and quantitation of cccDNA in plasma of chronic hepatitis B patients was developed and validated. Four HBV DNA reference panels, and 96 plasma samples of chronic hepatitis B patients were analyzed. Results were compared with total HBV DNA levels, individual ALT levels and the Histology Activity Index (HAI). This cccDNA assay had a lower limit of detection at 15 copies/PCR, a lower limit of quantitation at 91 copies/PCR and a correlation coefficient (R) of 0.98 (P < 0.0001). cccDNA was detected in two of four international panels. Significant correlation was found between cccDNA and total HBV DNA levels in both panels (R = 0.96, and R = 0.43) and in samples of the chronic hepatitis B patients (R = 0.88, P < 0.0001). In 57% of these samples cccDNA was detectable. Mean level of cccDNA was 0.16% of 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 total HBV DNA and HBV genotype did not influence cccDNA detection. ALT levels and HAI-score were not correlated with plasma cccDNA levels. These findings suggest that cccDNA levels in plasma are not the result of increased hepatocyte degeneration, but indicate that other mechanisms might be responsible.

DNA double-strand breaks, potential targets for HBV integration

“…The mark of productive virion infection and genome replication, HBV covalently closed circular DNA (cccDNA), was detected using selective PCR technology [11] . Experimental processes and amplified conditions were performed in accordance with references [12,13] . About 10 μg cell DNA was digested with 1 μL Mung bean nuclease (MBN; New England Biolabs, England) enzyme.…”

Nucleos(t)ide analogue treatment reduces apoptotic activity in patients with chronic hepatitis B

“…A major mechanism of HBV persistence during antiviral treatment with Nuc is persistence of cccDNA in infected cells. 11,12 A reduced cell death rate of infected cells could explain why HBV persists during effective antiviral treatment. 13 HBs-Antigen decline during antiviral treatment indicates reduction of HBV infected cells.…”

Detection of HBV Covalently Closed Circular DNA

“…Some reports have shown that cccDNA is present in patients’ sera and that it is a marker of off-treatment virological relapse [11,26,32,34,114], whereas others have refuted the existence of cccDNA in serum and have used DNA extracted from CHB patients’ sera as a negative control in the quantification of intracellular cccDNA [39,42,63]. Do some non-liver cells, such as PBMCs, support HBV infection and formation of cccDNA?…”

“…To further increase the sensitiveness and accuracy, Singh et al applied two hybridization fluorescence resonance energy transfer (FRET) probes in LightCycler TM (Roche Diagnostic, Mannheim, Germany) real-time PCR [41] which were able to maintain a cccDNA:rcDNA specificity ratio greater than 1:10,000. Due to the inherent speed, simplicity and sensitivity of qPCR, subsequent efforts to optimize general real-time PCR conditions [11,12,13,26,32,33,34,38,54,63,69,85,86,87,88,89] (including primers, probe design and sample handling) to effectively distinguish cccDNA from other HBV DNA forms have been made without sacrificing the high amplification efficiency and sensitivity of the method.…”

“…The widely accepted method for cccDNA detection is Southern blotting, which is insensitive, complex, time-consuming and not suitable for high-throughput drug screening. To resolve this problem and facilitate research on cccDNA, many new methodologies, including polymerase chain reaction (PCR)-based methods, Invader assays, in situ hybridization, and surrogates, have recently been applied to detect and quantify cccDNA [20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46]. In this review, we summarize and compare currently available approaches for cccDNA detection.…”