Study Of Effect Of Treating And Observing By Quantitative Assay Of Hepatitis B Virus Covalently Closed Circular DNA Of Hepatic Tissue In Chronic Hepatitis B Patients

Objective:With the knowledge gained from the molecular biology of HBV, it was shown that HBV covalently closed circular DNA(cccDNA) take on all-important meaning versus HBV copy and foundation of infective posture, it played a key role in HBV persistence, repeat of state of an illness after anti-virus treatment withdrawal and drug resistance. To date , our knowledge about cccDNA has been primarily from animal models and cell models.The woodchuck and duck and transfer gene mice is mostly studies by way of experimental animals. At present there is report about quantitative assay of hepatitis B virus covalently closed circular DNA of hepatic tissue.The aim of this study is to examine the relationship between the quantification of liver tissue HBVcccDNA, the quantification of the liver and serum HBVDNA, serum HBsAg titer, and liver damage, and then to evaluate the value of the quantification of liver tissue HBVcccDNA by real time polymerase chain reaction(PCR) in the diagnosis and treatment of chronic hepatitis B (CHB).Method:We screened 85 patients with CHB (including 60 men and 20 women)aged 21-54years old(averaged32)who were admitted to Shandong Provincial Hospital between May 2005 and December 2005.All patients were diagnosed according to Proposal of Prevention and Treatment for Viral Hepatitis emended in the Tenth National Academic Conference for Viral Hepatitis in 2000,hepatitisA,C,D,E and hepatic lesion by other factors excluded. All patients failed to receive antiviral treatment six months before hospitalization. According to the laboratorial examination results of HBeAg and anti-HBeAg, all patients were divided into two groups: CHB with HBeAg-positive and CHB with HBeAg-negtive. There were no significant differences between the two groups with regard to age, sex, and course of disease. There were 11 non-CHB patients in the control group, including 2 patients with chronic hepatitis C,l patients with autoimmune hepatitis,4 patients with fatty liver,2 patients with alcoholic fatty liver, 2 patients with unidentified hepatic lesion. Quantificative PCR were used to detect HBVcccDNA in liver biopsies and HBVDNA in serum and in liver biopsies.Hepatic function(index such as ALT) was detected using automatic biochemisty analyzer. HBsAg titers in serum were quantified using Electric ECL-Technology. Liver tissue inflammatory activity grading(G) and degree of fibrosis(S) were evaluated.Results:1: HBVcccDNA was detected in liver tissue of all patient with CHB, quantified 1.45×10~3copy/mg～3.14×10~6copy/mg(logarithm value: 4.54±1.32).Quantification of liver tissue HBVDNA was 3.21×10~4copy/mg～2.35×10~7copy /mg(logarithm value: 4.90±1.33). Quantification of serum HBVDNA was 3.92×10~4copy /ml ～ 55.53×10~8copy/ml(logarithm value: 5.10±1.34).2:Correlation between quantification of hepatic tissue HBVcccDNA and quantification of hepatic tissue and serum HBVDNA.There was a strong correlation between the HBVcccDNA and HBVDNA in the hepatic tissue.(logarithm value: 4.54±1.32 and 4.90± 1.33, r=0.738, p<0.001).There was a also positive correlation between the HBVcccDNA in the hepatic tissue and HBVDNA in the serum(logarithm value: 4.54± 1.32 and 10±1.34, r=0.436, p<0.005).3: There was a positive correlation between the HBVcccDNA in the hepatic tissue and HBsAg titers in serum, The average value of serum HBsAg titers was 769±211.32COI (r=0.721, p<0.005) .4:The quantification of hepatic tissue HBVcccDNA(logarithm value: 5.32±1.20), quantification of hepatic tissue HBVDNA(logarithm value: 6.13±1.26),and serum HBVDNA (logarithm value: 6.51±1.54) in patients with HBeAg-positive were higher than in patients with HBeAg-negtive(logarithm value : 3.55±1.27, 4.30±1.33,4.35±1.65respectively).5:There was no correlation between quantification of hepatic tissue HBVcccDNA and hepatic lesion index such as ALT, TBIL,and PT(p>0.05).6:There was no correlation between quantification of hepatic tissue HBVcccDNA and hepatic tissue inflammatory activity grading (G)and degree of fibrosis(S).7: After treated 48 weeks by lamifudine 45 patients with HBeAg-positive received many examinations. HBeAg of 18 patients becamed negtive. HBeAg of 27 patients not becamed.Before treatment and after treatment result of serum HBeAg and hepatic tissue HBVcccDNA, hepatic tissue HBVDNA, serum HBVDNA are on table4. Before treatment and after treatment result of serum HBeAg and serum examinations are on table5.Conclusion:1 :The quantification of hepatic tissue HBVcccDNA nicely quantified and may reflect directly infection and duplication in the body. And it is more sensitive to and reliable than quantification HBVDNA and HBeAg. HBVcccDNA is very important index for evaluating HBV duplication, persistence of infection, diagnosis, effect of antiviral drug treatment and clinical recovery, especially when level of serum HBVDNA is low enough not to be detected.2:There was a positive correlation between the HBVcccDNA in the hepatic tissue and HBVDNA in the serum. The dynamic detection of HBVDNA in the serum in patients with CHB may reflect viral duplication in the hepatic tissue. It helps us to understand changes of quantification of hepatic tissue HBVcccDNA by way of choice of anti-virus treatment and estimation clinical effect. However we can not guess the quantification of hepatic tissue HBVcccDNA from serum HBVDNA since the correlation between quantification of hepatic tissue HBVcccDNA and serum HBVDNA is not strong. Especially when level of serum HBVDNA is low enough not to be detected hepatic tissue HBVDNA still keep to a certainty.3 :There was a positive correlation between the hepatic tissue HBVcccDNA and HBsAg titers in serum. The quantification of serum HBsAg titers may reflect quantification of hepatic tissue HBVcccDNA to some extent.4: The quantification of hepatic tissue HBVcccDNA,quantification of hepatic tissue HBVDNA,and serum HBVDNA in patients with HBeAg-positive were higher than in patients with HBeAg-negtive. HBeAg is also one of the indicative markers virus highly duplication in the body. Anti-HBeAg-positive shows weaken of virus duplication and depression of infectivity. But HBVcccDNA and HBVDNA can still be detected in patients with anti-HBeAg-positive.Patients with anti-HBeAg-positive still highly infectious and have virus duplication and persistence of liver injury in the body. It suggests a tendency of chronicity and cirrhosis.5:There was no correlation between quantification of hepatic tissue HBVcccDNA and hepatic tissue inflammatory activity grading(G)and degree of fibrosis(S)and hepatic lesion index such as ALT, TBIL, and PT, which suggests HBVcccDNA can not as a predictive index of pathologic changes degree of hepatic tissue in patients with CHB.Therefore, non-cytolytic mechanism may be involved in the removal of HBVcccDNA.6: After treated 48 weeks by lamivudine We find that lamivudine is no effect of the HBVcccDNA in the hepatic tissue and has obvious restraining effect of the HBVDNA in the hepatic tissue and HBVDNA in the serum but hepatic tissue HBVDNA still keep to a certainty of lever(Especially when level of serum HBVDNA is low enough not to be detected). We can not guess the quantification of hepatic tissue HBVcccDNA from serum HBVDNA since the correlation between quantification of hepatic tissue HBVcccDNA and serum HBVDNA is not strong.7: After treated 48 weeks by lamivudine It has better effect of the serum ALT in patients with HBeAg turning negative than in patients with HBeAg contining postive. It has to a certainty effect of the serumTBIL. It is no effect of the serum PT. HBeAg of 18 patients becamed negtive in the 45 patients with HBeAg-positive.It is relate to less patients and less stylebook.