Rapid quantification of semen hepatitis B virus DNA by real-time polymerase chain reaction

AIM： To examine the sensitivity and accuracy of real-time polymerase chain reaction （PCR） for the quantification of hepatitis B virus （HBV） DNA in semen. METHODS： Hepatitis B viral DNA was isolated from HBV carriers＇ semen and sera using phenol extraction method and QIAamp DNA blood mini kit （Qiagen, Germany）. HBV DNA was detected by conventional PCR and quantified by TaqMan technology-based real-time PCR （quantitative polymerase chain reaction （qPCR））. The detection threshold was 200 copies of HBV DNA for conventional PCR and 10 copies of HBV DNA for real time PCR per reaction. RESULTS： Both methods of phenol extraction and QIAamp DNA blood mini kit were suitable for isolating HBV DNA from semen. The value of the detection thresholds was 500 copies of HBV DNA per mL in the semen. The viral loads were 7.5×10^7 and 1.67×10^7 copies of HBV DNA per mL in two HBV infected patients＇ sera, while 2.14×10^5 and 3.02×10^5 copies of HBV DNA per mL in the semen. CONCLUSION： Real-time PCR is a more sensitive and accurate method to detect and quantify HBV DNA in the semen.

Development of Fok-I based nested polymerase chain reaction-restriction fragment length polymorphism analysis for detection of hepatitis B virus X region V5M mutation

AIM: To develop a Fok-I nested polymerase chain reaction(PCR)-restriction fragment length polymorphism analysis(PRA) method for the detection of hepatitis B virus X region(HBx) V5 M mutation.METHODS: Nested PCR was applied into DNAs from 198 chronic patients at 2 different stages [121 patients with hepatocellular carcinoma(HCC) and 77 carrier patients]. To identify V5 M mutants, digestion of nested PCR amplicons by the restriction enzyme Fok-I(GGA TGN9↓) was done. For size comparison, the enzymetreated products were analyzed by electrophoresis on 2.5% agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator.RESULTS: The assay enabled the identification of 69 patients(sensitivity of 34.8%; 46 HCC patients and 23 carrier patients). Our data also showed that V5 M prevalence in HCC patients was significantly higher than in carrier patients(47.8%, 22/46 patients vs 0%, 0/23 patients, P < 0.001), suggesting that HBx Ag V5 M mutation may play a pivotal role in HCC generation in chronic patients with genotype C infections.CONCLUSION: The Fok-I nested PRA developed in this study is a reliable and cost-effective method to detect HBx Ag V5 M mutation in chronic patients with genotype C2 infection.

Locked nucleic acid real-time polymerase chain reaction method identifying two polymorphisms of hepatitis B virus genotype C2 infections,rt269L and rt269I

BACKGROUND The presence of two distinct hepatitis B virus(HBV)Pol RT polymorphisms,rt269L and rt269I,could contribute to the unique clinical or virological phenotype of HBV genotype C2.Therefore,a simple and sensitive method capable of identifying both types in chronic hepatitis B(CHB)patients infected with genotype C2 should be developed.AIM To develop a novel simple and sensitive locked nucleic acid(LNA)-real timepolymerase chain reaction(RT-PCR)method capable of identifying two rt269 types in CHB genotype C2 patients.METHODS We designed proper primer and probe sets for LNA-RT-PCR for the separation of rt269 types.Using synthesized DNAs of the wild type and variant forms,melting temperature analysis,detection sensitivity,and endpoint genotyping for LNA-RT-PCR were performed.The developed LNA-RT-PCR method was applied to a total of 94 CHB patients of genotype C2 for the identification of two rt269 polymorphisms,and these results were compared with those obtained by a direct sequencing protocol.RESULTS The LNA-RT-PCR method could identify two rt269L and rt269I polymorphisms of three genotypes,two rt269L types[‘L1’(WT)and‘L2’]and one rt269I type(‘I’)in single(63 samples,72.4%)or mixed forms(24 samples,27.6%)in 87(92.6%sensitivity)of 94 samples from Korean CHB patients.When the results were compared with those obtained by the direct sequencing protocol,the LNA-RT-PCR method showed the same results in all but one of 87 positive detected samples(98.9%specificity).CONCLUSION The newly developed LNA-RT-PCR method could identify two rt269 polymorphisms,rt269L and rt269I,in CHB patients with genotype C2 infections.This method could be effectively used for the understanding of disease progression in genotype C2 endemic areas.

Nested real-time quantitative polymerase chain reaction assay for detection of hepatitis B virus covalently closed circular DNA

Background Successful treatment of hepatitis B can be achieved only if the template for hepatitis B virus （HBV） DNA replication, the covalently closed circular HBV DNA （cccDNA） can be completely cleared. To date, detecting cccDNA remains clinically challenging. The purpose of this study was to develop a nested real-time quantitative polymerase chain reaction （PCR） assay for detecting HBV cccDNA in peripheral blood mononuclear cells （PBMCs） and bone marrow mononuclear cells （MMNCs）. Methods Based on the structural differences between HBV cccDNA and HBV relaxed circular DNA （rcDNA）, two pairs of primers were synthesized as well as a downstream TaqMan probe. Blood and bone marrow samples were collected from hepatitis B patients and healthy controls. To remove rcDNA, samples were incubated with mung bean nuclease and the resultant purified HBV cccDNA was then amplified by nested real-time fluorescence quantitative PCR. The cccDNA levels were calculated using a positive standard. Results The nested real-time fluorescence quantitative PCR method for HBV cccDNA was successful, with a linear range of 3.0×10^2 copies/ml to 3.9×10^8 copies/ml. Of the 25 PBMC samples and 7 MMNC samples obtained from chronic hepatitis B or liver cirrhosis patients, 3 MMNC samples and 9 PBMC samples were positive for HBV cccDNA, while all of the 21 PBMC samples from healthy controls were negative. Conclusion The nested real-time fluorescence quantitative PCR may be used as an important tool for detecting cccDNA in hepatitis B patients.

Optimization of competitively differentiated poiymerase chain reaction in detection of HBV basal core promoter mutation

AIM:To improve competitively differentiated polymerase chain reaction (CD-PCR) in detection of HBV basal core promoter mutation. METHODS: Recombinant plasmid of double point mutation A1762T/G1764A in basal core promoter of HBV constructed by site-directed mutagenesis was used as mutant control. To reveal the deficiency mechanism of CD-PCR, relationship between the circle number of PCR and the increased speed of products of each competitive primer was comparatively studied. Diversified amount of dNTPs and mutual primer of the competitive primers were tried to optimize CD-PCR. Optimized CD-PCR was evaluated by detecting A1762T/G1764A mutation in recombinant plasmids and clinical sera from patients with HBV infection. RESULTS: The deficiency mechanism of CD-PCR was that the products of mismatched competitive primer grew fast when the amplification of matched primer entered into plateau stage, which led to decrease in or disappearance of the difference in the amount of their products. This phenomenon could be eliminated by reducing dNTPs to 10μmol/L and mutual primer to about 100μmol/L Optimized CD-PCR could detect both mutant and wild strain independent of the amount of templates and the number of PCR cycles. Its detection limit was 103 copies/mL, about 50 copies/reaction. About 10% of mutant DNAs among wild type DNAs could be detected. A1762T/G1764A mutant was detected in 41.8% (51/122) of patients with HBV infection, but not detected in controls with negative HBsAg. CONCLUSION: Optimized CD-PCR can detect mutation independent of the amount of initial templates and the number of PCR cycles.

Recent advances in molecular diagnostics of hepatitis Bvirus

Hepatitis B virus(HBV)is one of the important global health problems today.Infection with HBV can lead to a variety of clinical manifestations including severe hepatic complications like liver cirrhosis and hepatocellular carcinoma.Presently,routine HBV screening and diagnosis is primarily based on the immuno-detection of HBV surface antigen(HBsAg).However,identification of HBV DNA positive cases,who do not have detectable HBsAg has greatly encouraged the use of nucleic acid amplification based assays,that are highly sensitive,specific and are to some extent tolerant to sequence variation.In the last few years,the field of HBV molecular diagnostics has evolved rapidly with advancements in the molecular biology tools,such as polymerase chain reaction(PCR)and real-time PCR.Recently,apart of PCR based amplification methods,a number of isothermal amplification assays,such as loop mediated isothermal amplification,transcription mediated amplification,ligase chain reaction,and rolling circle amplification have been utilized for HBV diag-nosis.These assays also offer options for real time detection and integration into biosensing devices.In this manuscript,we review the molecular technologies that are presently available for HBV diagnostics,with special emphasis on isothermal amplification based technologies.We have also included the recent trends in the development of biosensors and use of next generation sequencing technologies for HBV.

Prevalence of occult hepatitis B virus infection in haemodialysis patients from central Greece

AIM:To assess the hepatitis B virus(HBV)-DNA and the prevalence of occult HBV infection in end-stage renal failure(ESRF)patients from Central Greece. METHODS:Sera from 366 ESRF patients attending five out of six dialysis units from Central Greece were investigated for HBV-DNA by real-time polymerase chain reaction.Only serum samples with repeatedly detectable HBV-DNA were considered positive.IgG antibodies to hepatitis C virus(anti-HCV)were tested by a third generation enzyme linked immunosorbent assay(ELISA),while IgG antibodies to hepatitis E virus (anti-HEV)were tested by two commercially available ELISAs.RESULTS:HBV-DNA was detected in 15/366 patient (4.1%)and HBsAg in 20/366(5.5%).The prevalenc of occult HBV infection was 0.9%(3/346 HBsAg negative patients).Occult HBV was not associate with a specific marker of HBV infection or anti-HCV o anti-HEV reactivity.There was no significant differenc in HBV-DNA titres,demographic and biochemica features,between patients with occult HBV infectio and those with HBsAg-positive chronic HBV infection. CONCLUSION:In central Greece,4%of ESRF patient had detectable HBV-DNA,though in this setting,th prevalence of occult HBV seems to be very low(0.9%).

TT virus and hepatitis G virus infections in Korean blood donors and patients with chronic liver disease

AIM:To determine the prevalences of TTV and HGV infections among blood donors and patients with chronic liver disease in Korea,to investigate the association of TTV and HGV infections with blood transfusion,and to assess the correlation between TTV and HGV viremia and hepatic damage. METHODS:A total of 391 serum samples were examined in this study.Samples were obtained from healthy blood donors(n=110),hepatitis B surface antigen(HBsAg)-positive donors(n=112),anti-hepatitis C virus(anti-HCV)-positive donors(n=69),patients with type B chronic liver disease (n=81),and patients with type C chronic liver disease(n=19). Trv DNA was detected using the hemi-nested PCR.HGV RNA was tested using RT-PCR.A history of blood transfusion and serum levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were also determined. RESULTS:TTV DNA was detected in 8.2%of healthy blood donors,16.1%of HBsAg-positive donors,20.3%of anti- HCV-positive donors,21.0%of patients with type B chronic liver disease,and 21.1%of patients with type C chronic liver disease.HGV RNA was detected in 1.8%of healthy blood donors,1.8%of HBsAg-positive donors,17.4%of anti-HCV-positive donors,13.6%of patients with type B chronic liver disease,and 10.5%of patients with type C chronic liver disease.The prevalence of TTV and HGV infections in HBV- or HCV-positive donors and patients was significantly higher than in healthy blood donors(P<0.05), except for the detection rate of HGV in HBsAg-positive donors which was the same as for healthy donors.There was a history of transfusion in 66.7%of TTV DNA-positive patients and 76.9%of HGV RNA-positive patients(P<0.05).No significant increase in serum ALT and AST was detected in the TTV or HGV-positive donors and patients. CONCLUSION:TTV and HGV infections are more frequently found in donors and patients infected with HBV or HCV than in healthy blood donors.However,there is no significant association between TTV or HGV infections and liver injury.

Hepatitis C and B Virus Infection in Chinese Patients with Extrahepatic Bile Duct Carcinoma

Objective In China, the incidence of extrahepatic bile duct carcinoma (EBDC) tends to increase over the past decades. The etiology of the noted increase in EBDC is not identified. Approximately, in a half of the overall Chinese patients with EBDC, the causative factors in the development of EBDC have not been demonstrated.There is a high prevalence of hepatitis C virus (HCV) or hepatitis B virus (HBV) in China, both of which can induce malignant transformation of infected cells and strongly associated with hepatocellular carcinoma (HCC). In this study, EBDC tissues from Chinese patients were examined for the presence of HCV and HBV infection to investigate further the potential causes of EBDC.Methods HCV NS5 protein and HBsAg were detected by labeled streptavidin biotin (LSAB) method; HCV RNA and HBV DNA were detected by in situ polymerase chain reaction (IS-PCR) in formalin fixed, paraffin embedded specimens from 51 Chinese patients with EBDC. HCV RNA and HBV DNA were detected by IS-PCR in 34 Chinese patients with specimens of benign lesions of hepatobiliary tract (control group) .Results In 51 case tissue sections of EBDC, NS5 protein was detected in 14 (27.5%), and HBsAg in 5 (9.8%), HCV RNA in 18 (35.4%) and HBV DNA in 8 (15.9%) .respectively, of which HCV and HBV co-infection was detected in 2 (3.9%). In 34 case tissue sections of the control group, HCV RNA was detected in 2 (5.9 % ), and HBV DNA in 3 (8.8%).Conclusion In this study using standard histochemical and PCR techniques, HCV and HBV genomes and their encoding proteins were detected in the tissues of EBDC. The data show that there is a higher than expected incidence of HCV and HBV presence in EBDC tissues than would be expected on serologic grounds. The detectable rate of HCV RNA in EBDC tissues was significantly higher than in control group (x2 = 9.808, P = 0.002). As a result, this study indicates that there is a correlation between the presence of HCV infection and EBDC, and HCV infection has possible etiologic significance in the development of EBDC in China. While HBV DNA was detected in EBDC tissues with the difference in the detectable rate of HBV, DNA being not significance between EBDC tissues and the control group (x2 = 0.853, P = 0.356) . Further research is necessary to determine the presence of a causal relationship between HCV/HBV infection and the development of EBDC.

Investigation on Hepatitis C and B Virus Infection in Carcinoma of the extrahepatic bile duct in CHINA

Objective: The incidence of carcinoma of extrahepatic bile duct tends to increase during recent decade in China, but its cause is unclear. This study is to investigate the hepatitis C virus (HCV) and Hepatitis B virus (HBV) infection in the tissues of carcinoma of extrahepatic bile duct and study their correlation. Methods: HCV RNA and HBV DNA was detected by in situ polymerase chain reaction (IS-PCR) in sections of 51 cases of carcinoma of extrahepatic bile duct and 34 cases of control group. Results: Of 51 carcinoma of extrahepatic bile duct, HCV RNA was detected in 18 (35.4%), HBV DNA in 8 (15.9%). In 34 cases of control group, HCV RNA was detected in 2 (5.9%), and HBV DNA in 3 (8.8%). Conclusion:The prevalence of hepatitis C and B viralinfection in the tissues of carcinoma of extrahepatic bile duct was significantly higher than in control group. The findings suggest a correlation between HCV, HBV infection and carcinoma of extrahepatic bile duct, inferring HCV and HBV might be involved in the development of carcinoma of extrahepatic bile duct.

Establishment of transgenic mouse harboring hepatitis B virus (adr subtype) genomes

INTRODUCTIONHepatitis B virus (HBV) belongs to the group ofhepatovirus, a major pathogen of human acute andchronic hepatitis B[1 4], which has a very closeassociation with human hepatocellular carcinoma(HCC)[5-8], For example, a statistical data from ahospital in Shanghai showed that 80% of HCCpatients were positive for HBsAg ( personalcommunication).

Differentially expressed genes in hepatocellular carcinoma induced by woodchuck hepatitis B virus in mice

INTRODUCTIONHepatocellular carcinoma(HCC)is one of the major causes of death in the word.The mechanism of carcinogenesis is unknown,although it is widely accepted that HBV and HCV are clsely related to liver cancer[1-5[1-5].Previously,a variety of studies have described the differences in gene expression which distinguished tumor from nontumor[6-11].Cloning of the genes,especially the genes associated with HBV and HCV,is still very important to account for the development of liver cancer.

Research on Hepatitis B virus Genotypes and Subgenotypes among Bai Nationality in Dali, Yunnan Province

To investigate the distribution of hepatitis B virus (HBV) genotypes and subgenotypes among the Bai nationality in Dali, a total of 100 serum samples from patients with chronic HBV-infection were collected for the detection of HBV genotypes and subgenotypes by genotype-specific primers and restriction fragment length polymorphism (RLFP), respectively. Among the 100 samples, the proportions of genotype B, C and mixed genotype (B+C) were 41%, 25% and 34%, respectively. All the genotype B strains belonged to subgenotype Ba. In genotype C, 84% were Subgenotype Cs and 12% were subgenotype Ce. The distribution of genotypes B, C and B+C showed no significant difference between male and female patients (P=0.182) and among the age groups of patients (P=0.812). The rates of HBeAg/HBeAg positivity were no significantly different among genotypes B, genotype C and mixed genotype (B+C) (P=0.077/P=0.663). In Dali, genotypes B, B+C and C existed among Bai nationality with chronic HBV-infection, and genotype B was the major genotype. Subgenotypes Ba and Cs were the predominant strains in patients with HBV genotype B/C infection. The most prominent characteristic was the higher prevalent rate of mixed genotype (B+C) in patients.

Follow up of infection of chacma baboons with inoculum containing a and non-a genotypes of hepatitis B virus

AIM: To determine whether one genotype (A or non-A genotypes of HBV) predominated over the other during the course of HBV infection.METHODS: Four baboons were inoculated with HBV. DNA was extracted from serum obtained at monthly intervals postinoculation for 52 weeks and HBV DNA was amplified using primers specific for the core region containing an insert characteristic of genotype A (nt 2 354-2 359, numbering from the EcoRI site). The amplicons were cloned into PCRScriptTM and a minimum of 15 clones per time point were sequenced in both directions.RESULTS: Both genotypes persisted for the entire followup period of 52 weeks. Genotype non-A predominated in two baboons and genotype A in one baboon. Neither genotype predominated in the fourth baboon, as shown at a 5 % level of testing.CONCLUSION: No conclusions concerning the dominance of either genotype or the natural progression or replication rates of HBV could be drawn because the pattern of the genotypes found may have been caused by sampling fluctuations at the time of DNA extraction and cloning as a result of the very low viral loads in the baboon sera.

Interleukin-10 promoter polymorphisms in patients with hepatitis B virus infection or hepatocellular carcinoma in Chinese Han ethnic population

BACKGROUND: Since single nucleotide polymorphisms (SNPs) can serve as gene markers, polymorphism profiles may help scientists to identify the full collection of genes that contribute to the development of complex diseases such as cancer. The distribution of interleukin-10 (IL-10) promoter polymorphisms in Chinese Han ethnic patients with hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC) was investigated in this study. METHODS: The polymorphisms of IL-10 promoter region were detected by pulymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing. Sixty-six health controls, 42 patients with HBV infection, 30 HCC patients, and cell line SMMC-7721 were examined this way. RESULTS: Polyrnorphisms of T/C or T/N on-872 site occurred frequently in Han ethnic population. Pulyrnorphisms were detected in HBV and HCC patients and cell line SMMC-7721. The hotspot among the pulymorphisms was inserting base A between-1058 and-1057. CONCLUSION: Polymorphisms of IL-10 promoter in HBV and HCC patients may be associated with HBV infection and HCC development.

Therapeutic effectiveness of interferon alpha on hepatitis B virus DNA in peripheral blood mononuclear cells

Six patients with chronic hepatitis B were investigated for hepatitis B virus (HBV) DNA both in serum and in peripheral blood mononuclear cells (PBMC) by polymerase chain reaction (PCR) before ,during and after interferon alpha (IFN-a)treatment.All patients responded to IFN therapy with remission of serum alanine aminotransferase (ALT).HBV DNA disappeared in serum of 6 and in PBMCs of 5 patients during the treatment with IFN-a. The average elimination period was 5 weeks (range 3-10 weeks) in serum and 15 weeks (range 12-20weeks) in PBMCs. Relapse of serum ALT was seen in one patient with HBV DNA in PBMCs persistently positive during and after treatment with IFN-a. The results showed that clearance of HBV DNA was more difficult in PBMCs than that in serum,and that the persistent appearance of HBV DNA in PBMCs during and after treatment with IFN-a may imply the failure of IFN therapy.

Detection of pre－C region mutants of hepatitis B virus from HBeAg negative patients in Xi＇an area of China

HBV-DNA extracted from forty-seven sera of HBeAg negative and anti-HBe positive hepatitis B patients was used as template for polymerase chain reaction(PCR) with pre-C region primer. Amplified products of hepatitis B virus DNA were digested with EcoRI and the resultant gene fragment of 312 base pairs(bp) containing the precore region was cloned into PUC18 plasmid and then transformed into E. coli JM109. After being identified by PCR and digested with restriction endonuclease, the nucleotide sequence of the precore region was determined by the dideoxy-chain termination method. The results demonstrated that hepatitis B virus (HBV) mutation was present in the precore region in the clones of two patients. A point mutation from G to A at 1896 site of HBV precore region might convert Trp 28(TGG) to a stop codon(TAG), which is the commonest mutation observed so far.The discovery of mutants of HBV precore region in Xi'an area of China could be of value for further investigation of the pathogenesis of hepatitis B.

抗柯注射液治疗柯萨奇B病毒性心肌炎及其机理研究

目的：探讨抗柯注射液治疗柯萨奇Ｂ病毒性心肌炎作用机理。方法：以苦参中抗柯萨奇病毒的有效成分制成纯度为９９．４３％的抗柯注射液（下称ＲＳＦ）治疗７６例血中柯萨奇Ｂ病毒核酸（ＣＢＶ－ＲＮＡ）持续阳性的病毒性心肌炎患者（Ａ组），并与用葡萄糖加胰岛素加氯化钾治疗的５０例患者（Ｂ组）进行对照。结果：ＲＳＦ对血中ＣＢＶ－ＲＮＡ的清除率与所用药物剂量及血中药物浓度有相关性；患者心悸、胸闷气短的总有效率（９６．０％）、抗。Ｃ律失常的总有效率（１００．０％，４０／４０例）明显优于Ｂ组（６２．５％，２５／４０例）。ＲＳＦ能使射血分数、心脏每搏输出量、心排血量以及心脏指数等心功能指标明显上升（Ｐ＜０．０１），左室重量及重量指数较治疗前明显下降（Ｐ＜０．０５）；ＲＳＦ治疗（５例）５个月后中和抗体恢复正常，ＮＫ细胞活性上升（Ｐ＜０．０１）。结论：ＲＳＦ对逆转病毒性心肌炎的病理状态效果明显。

氧化苦参碱治疗慢性乙型病毒性肝炎疗效及其与HBV负荷的关系探讨

目的 :研究氧化苦参碱治疗慢性乙型病毒性肝炎疗效及其与HBV负荷的关系。方法 :4 4例慢性乙型肝炎患者 ,随机分为治疗组 (给予氧化苦参碱和一般护肝药物 ) 2 3例 ,对照组 (仅给予一般护肝药物 ) 2 1例。以聚合酶链反应 (PCR)法定量测定血清HBV水平 ,分析不同疗效与不同血清HBV负荷的关系。结果 :氧化苦参碱肌肉注射慢性乙型肝炎 3个月 (0 4g/d) ,HBVDNA及HBeAg转阴率均为 4 3 4 7% ,明显优于对照组 (P <0 0 5 )。治疗后HBVDNA定量水平从 10 6 83± 1 2 7copy/ml降至 10 3 35± 3 0 8copy/ml,治疗后HBeAg转阴的患者 ,其治疗前血清HBVDNA定量水平 (10 6 30± 1 4 2 copy/ml) ,明显低于未转阴者 (10 7 2 3± 1 2 3copy/ml)。结论 :氧化苦参碱治疗慢性乙型肝炎有效 ,治疗前血清HBVDNA水平较低者疗效较好。

慢性HBV感染前C区变异与病毒复制水平关系

探讨HBV前C基因变异与病毒复制水平的关系在慢性HBV感染者中的意义。应用错配聚合酶链反应 (PCR) -限制性片段长度多态性 (RFLP)分析与荧光定量聚合酶链反应检测HBVDNA相结合 ,对 30例HB sAg(+ )、抗 -HBe(+ )及抗 -HBc(+ )慢性HBV感染者 ,其中无症状携带者 (AsC) 9例、慢性乙型肝炎 (CHB) 12例及慢性重症肝炎 (CHF) 9例进行前C区基因变异与HBVDNA水平关系进行分析。AsC组 3例 (33 33 % ) ,CHB组9例 (75 % )及CHF组 8例 (88 89% )有A83(nt 1896 )变异。荧光定量PCR结果表明HBVDNA含量在CHF组中最高。HBV前C变异在HBV不同感染状态中都可见 。