Risk Factors And Immune Molecular Mechanisms Of Hepatitis B Virus Reactivation In Community-based Population

Hepatitis B virus(HBV)infection can result in a series of different disease outcomes,among which HBs Ag carriers are very common in chronic HBV infection modes.They usually have no clinical symptoms and abnormal liver function,but there is latency of HBV in the body.HBV in these people may suddenly activate again,which is called HBV reactivation.At present,most studies on HBV reactivation are focused on clinical patients,especially those who received chemotherapy,radiotherapy and organ transplantation.However,few studies about HBV reactivation based on community population have been reported,which poses a challenge to the prevention and treatment of chronic HBV infection.Objective: This study aims to identify the risk factors associated with HBV reactivation through a nested case-control study,and to explore the molecular mechanism of HBV reactivation through immunological detection,so as to provide scientific basis for evaluating the timing of antiviral therapy for HBs Ag carriers.Methods: 1.The HBs Ag carriers identified by community health examination in the demonstration area of prevention and treatment of hepatitis B in Wuwei City in 2010 were selected as the research cohort.Four times of follow-up were conducted in the cohort in 2012,2014,2016 and 2018 respectively.Combining with the criteria of HBV reactivation,the subjects with HBV reactivation during the follow-up period were included in the case group,and those without HBV reactivation were randomly selected in the control group.A self-designed questionnaire was used to investigate the risk factors related to HBV reactivation.Serum samples were collected for HBV-related serological markers and real-time fluorescence quantitative detection.HBV DNA was extracted,and its S-region sequence was amplified.HBV genotypes and amino acid mutations were analyzed by sequence alignment.2.Three groups of subjects were divided by people who have HBV reactivation,HBs Ag carriers and chronic hepatitis B patients(CHB).We collected their basic information and serum samples,detected the levels of Th1 and Th2 cytokines in the serum of the subjects by ELISA,and analyzed the balance relationship between Th1 and Th 2 by comparing the ratio of Th1/Th 2 of the three groups,and analyzed the immune molecular mechanism of HBV reactivation.3.All data were analyzed by SPSS23.0 software.Measuring data obeying normal distribution were analyzed by t-test,counting data by chi-square test and multivariate analysis by logistic regression.The measurement data of skewed distribution were expressed by(M,Q1-Q3).Kruskal-Wallis H test was used for comparison among three groups.Spearman rank correlation analysis was used to analyze the correlation between data.P < 0.05 was statistically significant.Results: 1.A total of 119 cases of HBV reactivation were collected among follow-ups.Among them,76 cases of HBV DNA changed from undetectable to detectable,and 43 cases of HBV DNA increased more than 10 times.2.Univariate analysis showed that smoking(OR = 1.905,95% CI: 1.132-3.207),cold occurred many times in one year(OR = 2.090,95% CI: 1.292-3.381),out-migrantion for work(OR = 2.007,95% CI: 1.113-3.553),and negative life events(OR = 2.413,95% CI: 1.490-3.905)were significantly associated with HBV reactivation.Other factors showed no significant difference,which included alcohol consumption,operation history,diabetes history,hypertension history,tuberculosis history,autoimmune disease history and others.By analyzing the baseline HBV DNA load levels of the two groups,it was found that there was a significant correlation between HBV DNA load ≥ 10IU/ml and HBV reactivation(OR= 1.951,95% CI: 1.118-3.405).3.Multivariate logistic regression analysis showed that smoking(OR = 2.279,95% CI: 1.196-4.342),cold occurred many times in one year(OR = 2.105,95% CI: 1.229-3.606),negative life events(OR = 2.168,95% CI: 1.257-3.742)and HBV DNA load ≥ 10IU/ml(OR= 2.282,95% CI: 1.248-4.173)were still significantly associated with HBV reactivation.4.We randomly extract case group and control group for HBV DNA sequencing,71 cases were successfully sequenced in case group and 59 cases in control group.All sequences amplified their S region genes and constructed phylogenetic trees.It was found that the D genotype was the most(56.3%)in the case group,followed by C genotype(32.4%)and B genotype(11.3%).The D genotype was the most(61.0%)in the control group,followed by C genotype(23.7%)and B genotype(15.3%).There was no significant difference in the distribution of the three genotypes between the two groups(P=0.506).5.Comparing the reference sequence of HBV S region gene and corresponding genotype between the reactivated group and the carrier group,the results showed that there was no significant difference in the mutation rate of a antigen determinant of genotype B,C and D between the reactivated group and the carrier group(P > 0.05).There was no significant difference in the mutation rate of amino acids in MHR region between B,C and D genotypes.Further analysis of mutation sites showed that 126,143 and 161 amino acids in S region of genotype B were prone to mutation,114 and 126 amino acids in S region of genotype C were prone to mutation,while 122,130,131 and 143 amino acids in S region of genotype D were prone to mutation,and the mutation sites were different among genotypes.6.TNF-α concentration showed skewed distribution among people who have HBV reactivation,HBs Ag carriers and CHB patients,and there was significant difference among the three groups(P=0.011);IL 10 concentration in the three groups was skewed distribution,and there was no significant difference among the three groups.Further comparing the levels of TNF-α,we found that there was a significant difference in the levels of TNF-α between the HBV reactivation group and the CHB group(P=0.016).The level of TNF-α in the HBV reactivation group was higher than that in the CHB group.7.Spearman correlation coefficient of correlation analysis between TNF-α and ALT level in HBs Ag carriers group was 0.304(P=0.017),there was a positive correlation between TNF-α and ALT level in HBs Ag carrier group.Spearman correlation coefficient of correlation analysis between TNF-α and AST level in HBV reactivation group was 0.404(P=0.005),which showed that there was a positive correlation between TNF-α and AST level in HBV reactivation group.8.By comparing Th1/Th2 ratios among HBV reactivators,HBs Ag carriers and CHB patients,it was found that there were significant differences in Th1/Th2 ratios among the three groups(P < 0.05).Conclusion: 1.Smoking,cold occurred many times in one year,negative life events and HBV DNA ≥ 10IU/ml are important risk factors of HBV reactivation in HBs Ag carriers in community.2.TNF-α participates in hepatocyte injury during HBV reactivation.The shift of Th1/Th2 balance toward Th1 type may be one of the immune molecular mechanisms related to HBV reactivation.Therefore,if HBs Ag carriers have the characteristics of smoking,susceptibility to get cold and experiencing negative life events when HBV DNA is detectable,the risk of HBV reactivation will increase.It is suggested the follow-up management for those kind of HBs Ag carriers in community should be increased from one time every six months recommended by Guidelines to every three months,so as to prevent adverse outcomes early.