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, are 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 are analyzed. Results are compared with total HBV DNA levels. 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). HBV cccDNA can be detected in two of four international panels. Significant correlation is 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 can be detected. Mean level of cccDNA is 0.16 % of total HBV load. Plasma HBV cccDNA levels are higher in HBeAg-positive samples than in HBeAg-negative samples (p < 0.0001). Total HBV DNA levels and HBV genotype do not influence cccDNA detection.

Intrahepatic response markers in chronic hepatitis B patients treated with peginterferon alpha‐2a and adefovir

“…Amplification was performed with the Lightcycler 480 system (LC480) (Roche Diagnostics, Almere, The Netherlands). This assay was described and validated previously and had a lower limit of quantification of 150 copies/biopsy 20 …”

“…This assay was described and validated previously and had a lower limit of quantification of 150 copies/biopsy. 20 For quantification of total intrahepatic HBV DNA, genotype independent primers were used as described earlier. 21 The forward primer was HBV-SF-09 (5′-ACTCACCAACCTSYTGTCCT-3′ nt 333-352) and reverse primer was HBV-SR-09 (5′-GACAMAC GGGCAACATACCT-3′ nt 476-456).…”

Proposed model for the prediction of intrahepatic covalently closed circular DNA level in patients with chronic hepatitis B

“…Intrahepatic cccDNA levels were tested as reported elsewhere . For patients with complicating HCC ( n = 51), IH‐cccDNA levels were tested using non‐tumorous specimens.…”

“…Intrahepatic cccDNA levels were tested as reported elsewhere. 30 For patients with complicating HCC (n = 51), IH-cccDNA levels were tested using non-tumorous specimens. For HBsAg quantification, we measured hepatitis B e antigen (HBeAg) and HBV-DNA levels using commercial kits as described elsewhere.…”

One Case of Esophagitis Dissecans Superficialis

“…乙肝病毒生物标志物及其检测方法研究进展 中 60%的人受过 HBV 的感染，在某些人群中甚至高 达 79% [3] [19][20][21][22] ，表明肝组织内 cccDNA 的定量检测有 重要临床意义，可评价：①抗病毒药物疗效及持续应 答情况；②HBV 感染状态及其传染性指标 [23] ；③可 否引发肝癌；④是否肝外组织感染的客观指标 [24,25] ； ⑤肝移植后肝脏是否再感染的指标 [26][27][28]…”