This study examined a signal amplification assay, the Invader assay, for the quantitation of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in liver biopsies and sera. DNA was extracted from liver biopsy and serum samples were collected from 16 hepatitis B e antigen (HBeAg)-positive and 36 antibody-to-HBeAg-positive (anti-HBe-positive) chronic hepatitis B patients. The amount of total HBV DNA and cccDNA was measured using the Invader assay. Anti-HBe-positive patients had lower median total intrahepatic HBV DNA (P < .001) and intrahepatic cccDNA levels (P ‫؍‬ .001) than HBeAg-positive patients. Intrahepatic cccDNA correlated positively with the total intrahepatic HBV DNA (r ‫؍‬ 0.950, P < .001). However, the proportion of intrahepatic HBV DNA in the form of cccDNA was inversely related to the amount of total intrahepatic HBV DNA (r ‫؍‬ ؊0.822, P < .001). A small amount of cccDNA was detected in 39 of 52 (75%) serum samples. Anti-HBe-positive patients had lower median serum cccDNA levels than HBeAg-positive patients (P ‫؍‬ .002). Serum HBV DNA correlated positively with intrahepatic total HBV DNA (r ‫؍‬ 0.778, P < .001) and intrahepatic cccDNA (r ‫؍‬ 0.481, P ‫؍‬ .002). In conclusion, the Invader assay is a reliable assay for the quantitation of cccDNA. vitro studies have shown that lamivudine has a profound effect on relaxed circular DNA (rcDNA) while having little or no effect on cccDNA. 2,3 This is one possible reason for the rebound of HBV DNA to pretreatment levels often seen after lamivudine withdrawal. 4 Another nonreplicative form of HBV DNA is the double-stranded linear (DL) form produced by in situ priming during HBV replication. 5 DL DNA is a possible precursor to HBV DNA integration. 6 -8 It can also form cccDNA through nonhomologous recombination at its ends via a process called illegitimate replication. 9,10 cccDNA monitoring and the development of an accurate quantitative assay for cccDNA are becoming important in the understanding of the natural history and management of chronic hepatitis B (CHB). Most attempts for the quantitation of cccDNA have been made with liver biopsies from ducks or woodchucks. [11][12][13][14][15][16] Quantitation of cccDNA in human peripheral blood mononuclear cells and liver biopsies has been performed. [17][18][19][20] In these studies, primers spanning across the gap in the minus strand and corresponding to the variable region on the plus strand were used to amplify across noninterrupted cccDNA. It should be noted that, even with selective polymerase chain reaction (PCR) methods,

The role of hepatitis B virus (HBV) genotypes, core promoter (CP) and precore mutants on hepatocellular carcinoma (HCC) is still controversial. We aimed to determine their role on the development and clinical features of HCC. HBV genotypes and CP/precore mutations were determined in 90 HCC patients and 180 matched control patients. In the 90 HCC patients, 22 (24.4%) and 68 (75.6%) had subtype Ba and genotype C, respectively. The prevalence of genotype C and CP mutations was significantly higher in HCC patients compared with controls (75.6 versus 57.8%, P = 0.004; 90.9 versus 74.8%, respectively, P = 0.007). Among carriers of genotype C, 91.8% of the HCC patients and 88.8% of controls had CP mutations. Among carriers of subtype Ba, HCC patients had a higher prevalence of CP mutations compared with controls (88.2 versus 54.5%, respectively, P = 0.02). By logistic regression analysis, the only factor associated with HCC was a mutation of the CP region (P = 0.032). There were no differences in the clinical features on presentation, the chance of receiving treatment and the cumulative survival rate for chemoembolization-treated patients between patients with subtype Ba and genotype C. There was too small a number of CP wild-type to do a similar comparison with CP mutants. In conclusion, there was a significantly higher prevalence of both genotype C and CP mutations in patients with HCC. The association between HBV genotype C and HCC was probably not genuine but was due to the high percentage of CP mutations in patients with genotype C.

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