| According to the data from the nationwide seroepidemiological survey in 2006,the prevalence of hepatitis B surface antigen(HBsAg) carriers in general population, whose age were ranged from 1 to 59,is lower to 7.18%than 9.75%in 1992.There are 93 million HBsAg carriers in China according to the calculation by present prevalence of HBsAg carriers.These people accounts for about a quarter of the people who are chronicly infected by HBV worldwide.There are 20 million to 30 million patients with chronic hepatitis B(CHB) in China.It is estimated that over 300,000 chronic HBV carders die each year from diseases related to CHB.So the management of individuals who are chronicly infected by HBV is an important and difficult task.In the condition of mature immunity,immune tolerance is destroyed and hepatitis B e antigen(HBeAg) are cleared by immunity and then hepatitis B e antibody (anti-HBe) presents.This process is called as the HBeAg seroconversion.After HBeAg seroconversion,about 67%to 80%subjects can keep low or undetectable levels of HBV DNA and persistently normal ALT levels and inactive liver histology with a usually minimal fibrosis.These persons are called inactive HBsAg carriers (HBsAg-IaCs).This state can be achieved spontaneously or after antiviral therapy with nucleot(s)ide analogue or alpha-interferon.Majority of HBsAg-IaCs with good prognosis is an ideal state for persons who are infected by HBV.However there is potential risk of inflammation activity in liver because of the exist of cccDNA in hepatic cellular nucleus,which cannot be cleared by antivial treatment.In condition of immune suppression,immune deficiency or HBV mutations,virus replication could be reactivated.And then obvious necroinfiammation can be present in liver.It is reported that more than 20%of HBsAg-IaCs progress to HBeAg negative CHB.And HBeAg negative CHB could act repeatedly and progress to cirrhosis and hepatocellular carcinoma.About 23%and 4%HBeAg negative CHB progress to cirrhosis and hepatocellular carcinoma respectively.To prevent progression from HBsAg-IaC to HBeAg negative CHB, recognizing the possible differences of host and viral characteristics between HBsAg-IaC and HBeAg negative CHB are very important.HBeAg positive hepatitis B patients with complete response to nucleot(s)ide analogue treatment(NCR-e +CHB) could achieve and keep HBsAg-IaC state with the effect of nucleot(s)ide analogue(NA)and the host's immunity,which is different from the spontaneous state in formation process.To explore the relationship between antiviral effect of NA and host and virological characteristics,realizing the differences of host and virological characteristics of the two group is very important.For resolving the two questions above two researches have been done successively.1.1 ObjectiveTo explore the risk factors for HBsAg-IaC progressing to HBeAg negative CHB, compare the possible differences of genotypes and viral mutations at nt1896 and nt1762/1764 between HBsAg-IaC and HBeAg negative CHB.1.2 MethodsAccording to the Chinese Guidline for Treatment Chronic Hepatitis B,HBsAg-IaCs (n=187) and HBeAg negative CHB(n=99) were consecutively collected in nanfang Hospital.HBV genomic DNA specimens were extracted from serum samples of 187 HBsAg-IaCs and 99 HBeAg negative CHB patients using QIAgen blood DNAkit(QIAGEN GERMANY).The CX fragment(nt1643-nt1974) was amplified by half nested PCR.The primers of first round were P9 and P35.And the primers of second round were P9 and CSP.For the positive samples of CX fragment,which cover the mutations at nt1896 and nt1762/1764 were analyzed by direct sequencing (nt1643-1974) with primer of CSP using ABI3730 sequencer.HBV genotypes were determined with polymerase chain reaction-restriction fragment length polymorphism assay(PCR-RFLP) in combination with direct sequencing.The result of sequencing were analyzed by Cluster X and affirmed manually.1.3 Results113 HBsAg-IaCs have low level viraemia were detected by nested-PCR,and then genotypes were determined.And direct sequencing was successfully performed in 103 cases.The prevalences ofHBV genotype B(HBV/B) infection(84/113 vs. 46/99,74.3%vs 46.5%,P=0.000) and G1896Amutation(69/103 vs.39/99,67.0% vs 39.4%,P=0.000) was higher in HBsAg-IaC than that in HBeAg negative CHB.The prevalence of A1762T/G1764Amutations was lower in HBsAg-IaC than that in HBeAg negative CHB(38/103vs.65/99,P=0.000).Male(OR=7.519, 95%CI=2.693-20.995,P=0.000),older than 40(OR=25.369,95%CI=5.323-120.913, P=0.000) and genotype C(HBV/C) infection(OR=2.309,95%CI=1.028-5.186, P=0.043) were associated with HBeAg negative CHB.The occurrences of A1762T/G1764A mutations(OR=2.017,95%CI=0.958-4.247,P=0.065) and 1896 mutations(OR=0.565,95%CI=0.265-1.205,P=0.140) were not associated with HBeAg negative CHB.1.4 ConclusionsViral characteristics,including HBV genotypes distribution,occurrences of mutations as G1896Aand A1762T/G1764A were different between HBsAg-IaC and HBeAg negative CHB.And the male,older than 40 and HBV/C infection are the risk factors for HBsAg-IaCs progressing to HBeAg negative CHB.2.1 ObjectiveTo realize the difference of stability of HBsAg-IaC state and factors for influencing the therapeutic effect of nucleot(s)ide analogues between NCR-e+CHB and HBsAg-IaCs,investigate the difference of host's characteristics and virological characteristics between these two groups.2.2 MethodsAccording to the Chinese Guidline for Treatment Chronic Hepatitis B,NCR-e +CHB(case group) and HBsAg-IaC(control group) were consecutively collected in nanfang Hospital.HBV genomie DNA specimens were extracted from serum samples of 117 NCR-e+CHB patients and 58 HBsAg-IaCs using QIAgen blood DNAkit(QIAGEN GERMANY).And the S gene fragments were amplified by nested PCR.The primers of first round were BS1 and POL2.And the primers of second round were YS1 and YS2.HBV genotypes were determined with polymerase chain reaction-restriction fragment length polymorphism assay.The PC and BCP genic fragments were amplified by nested PCR respectively.For the positive samples of PCR mutations at nt1896 and nt1762/1764 were determined with PCR-RFLP.The samples amplified negatively for PC or BCP fragments were amplified again with another PCR system.Then the positively amplified PCR product was analyzed by direct sequencing(nt1600-nt1974).The results of sequencing were analyzed by Cluster X and affirmed manually.2.3 ResultsThe ratio of male in NCR-e+CHB group is higher than that in HBsAg-IaC group (77.8%vs 58.6%,P=0.008).The average age of the patients in NCR-e+CHB group is older than that in HBsAg-IaC group(t=2.2.67,P=0.025).The positive rate of amplification of S fragment in NCR-e+CHB is lower than that in HBsAg-IaC group(52.1%vs 79.3%,P=0.001).No statistical difference is found in the constituent ratio of the main two HBV genotypes between these two groups(χ~2= 0.154,P=0.694).Prevalence of A1762T/G1764A and G1896Awere significantly lower in the case groupthan that in the control group(9.8%vs 41.4%,P=0.002) (14.0%vs 72.4%,P=0.000).2.4 ConclusionsThe potential HBV level is lower in NCR-e+CHB than that in HBsAg-IaC group.No statistical difference is found about the constituent ratio of HBV genotypes between these two groups,but genotype B is the main genotype in the two groups.Prevalences of G1896A and A1762T/G1764A mutations of NCR-e+CHB are lower than those of HBsAg-IaCs respectively. |
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