Predictive Value Of Intrahepatic Hepatitis B Virus Covalently Closed Circular DNA And Serum HBsAg In Children With Chroic Hepatitis B Receiving Anti-viral Treatment

To detect quantitatively intrahepatic covalently closed circular DNA(cccDNA)and serum HBsAg,and analyze their relationship with serum HBV DNA and HBsAg as wellas HBcAg expression in thehepatocyte,and investigate the application value of serum HBsAg titer andintrahepatic cccDNA in evaluating therapy response in children with HBeAg-positive CHB.A total of 63 childrenwith HBeAg-positive chronic hepatitis B who had formalin fixed paraffin-embedded liver biopsy samples after routine pathology were enrolled,and the serum sample was stored at the same time.Intrahepatic cccDNA was measured by plasmid-safe ATP dependent Danes(PSAD)digestion in combination with rolling circle amplification(RCA)and gap-spanning selective real-time PCR assay;serumHBsAg and hepatitis B virus serological markers(HBV-M)were measured by Electro-Chemiluminescence Immunoassay;serum HBV DNA and HBV genotype were quantified by real-time TaqMan PCR;the serum ALT and AST were detected using automatic biochemistry analyzer.The inflammation and fibrosis stage of liver tissue were assessed by pathological staining;HBsAg and HBcAg expression was identified by immunostaining of formalin-fixed,paraffin-embedded specimens.Taking the HBsAg seroconversion occurred after 48 weeks of antiviral therapy as antiviral efficacy criteria,single factor was analyzed using t test and chi-square test;several factors were entered in logistic regression analysis;pearson's or spearman's correlation was used to describe the correlation among quantity of intrahepatic cccDNA and serum HBsAg level andother clinical parameters;area under the receiver operating characteristic(ROC)curve analysis was performed to predict the likelihood of achieving HBsAg seroconversion in the children with CHB after 48 weeks of antiviral therapy;the difference between intrahepatic cccDNA and serum HBsAg in the predictive value of achieving HBsAg seroconversion was compared using paired chi-square test.We found that HBsAg seroconversion was achieved in 13 of 63(20.3%)in children with CHB,age was younger in Responders(R)than non-responders(NR)(1.68±0.71 vs 6.47±3.16,P=0.000),serum AST(1.85±0.15 vs 1.98±0.31,P=0.04),intrahepatic cccDNA(6.38±0.79 vs 6.79±0.66,P=0.04),serum HBsAg(3.48±1.22 vs 4.14±0.69,P=0.009),and the expression intensity of HBsAg in the hepatocytes(P=0.012)were lower in R than NR;but there was no statistical difference in baseline serum ALT,HBV DNA,gender,HBV genotype,the inflammation and fibrosis stage of liver tissue and the expression intensity of HBcAg in the hepatocytes between the two groups(P>0.05).The multivariate analysis revealed that serum HBsAg,intrahepatic cccDNA and age had a significant association with achieving HBsAg seroconversion after 48 weeks of antiviral treatment.The intrahepatic cccDNA was positively correlated with serum HBsAg(r=0.464,P=0.000);and the two parameters were positively correlated with serum HBV DNA(r=0.665,P=0.000;r=0.512,P=0.000),but they had no correlation with the serum ALT,AST level,HBV genotype,inflammation and fibrosis stage of liver tissue(P>0.05);there was no correlation between the intrahepatic cccDNA and the expression intentsity of HBsAg in the hepatocytes(r=-0.024,P=0.854),but there was positive correlation between the HBsAg titer and the expression intentsity of HBsAg in the hepatocytes(r=0.258,P=0.041);there was statistical difference in intrahepatic cccDNA level between the different expression intentsity of HBcAg in the hepatocytes(P=0.003),but we the difference in serum HBs Ag titer between the different expression level of HBcAg in the hepatocytes was not abserved(P>0.05).Using log10 cccDNA at 0.08copies/cell equivalent as cutoff,the sensitivity,specificity,and positive and negative predictive values of predicting the likelihood of achieving HBsAg seroconversion were 92.0%,46.2%,86.8% and 60.0% respectively;using log10 HBsAg at 3.68IU/mL equivalent as cutoff,the sensitivity,specificity,and positive and negative predictive values of predicting the likelihood of achieving HBsAg seroconversion were 86.0%,69.2%,91.2% and 56.3% respectively;there was no significant statistical difference between the serum HBsAg and intrahepatic cccDNA in predicting the likelihood of achieving HBsAg seroconversion(P=0.146).Serum HBsAg,intrahepatic cccDNA and age were associated with antiviral efficacy in children with HBeAg-positive CHB.Baseline intrahepatic cccDNA was positively correlated with serum HBsAg level,there was positive correlation between the two parameters and serum HBV DNA;the two parameters combined with serum ALT,AST and liver tissue inflammation activity,degree of fibrosis can evaluate children with CHB condition changes,comprehensively.Compared with serum HBsAg level,the intrahepatic cccDNA level can more accurately reflect the replication of HBV in the hepatocytes,serum HBsAg can reflect the state of HBV infection,but they had potential in predicting sussessful therapeutic efficacy in children with HBeAg-positive CHB.To some extent,serum HBsAg could be used as a surrogate maker of intrahepatic cccDNA in children with HBeAg-positive CHB,combined with the serum HBV DNA,ALT,AST,inflammation and fibrosis stage of liver tissue,and it plays a role in guiding clinical treatment.