Seroepidemiological And Molecular Epidemiological Studies Of Virual Hepatitis B After 20 Years From The Introduction Of Hepatitis B Vaccine In Shandong Province

[Introduction] Hepatitis B is a serious infectious disease which could induce chronic hepatitis, cirrhosis and hepatocellular carcinoma. The risk of the development of chronic HBV carrier state is inversely related to the age at the time of infection. It is highest when the infection is acquired perinatally or in the early childhood and decreased by age. HBV infection is an important public health issue in Shandong province. Prior to the introduction of universal infants hepatitis B vaccine (HepB) immunization programme, the prevalence of hepatitis B virus (HBV) infection and hepatitis B surface antigen (HBsAg) was 50.00% and 6.4% respectively among the general populations, and was 45.63% and 8.00% respectively among the population under 15 years old in Shandong province. In China, HepB was first used in 1987, and was recommended for infants by the Ministry of Health in 1992. Since then, the universal infants HepB immunization programme has been implemented in China. HepB has been freely provided to the infants (but with a user fee of about US $ 1.10) since 2002 and infants have been vaccinated without any charges since 2005. In addition to the national programme, a massive HepB immunization campaign was carried out in Shandong province, in which children aged 1 to 14 years with incomplete HepB vaccination were required to obtain catch-up injections. The efforts had been also taken to improve the HepB coverage among the adults. The impacts of the long-time and wide use of HepB on HBV prevalence were verified in some other areas of the world. The last provincial HBV survey was carried out in Shandong province in 1992 and a new survey was urgly needed to find out the current station of HBV prevalence in the province. [Objective] The aims of the study are as following:To find out the prevalence rates, distribution characters of HBV seromarks in Shandong province after 20 years from the introduction of HepB.To clarify the HBV infection risk factors among youngsters and adults.To evaluate the HBV infection variation tendency and the effects of HBV control by comparing our results with those in 1992 survey.To known the distribution characters of HBV genotype and subtype.To find out the prevalence and distribution of HBV strain with S gene mutation and occult HBV infection in the general population of Shandong province.Part 1 Seroepidemiological Study on Viral Hepatitis B inShandong Province[Materials and methods] A community-based cross-section survey was carried out in Shandong province in 2006. 7601 target population for this study were selected by multi-stage random sampling from 12 counties of the province. The interviews were carried out using a standard questionare for all the study population. The blood samples were collected and detected for HBsAg, antibody to HBsAg (Anti-HBs) and antibody to core antigen (Anti-HBc) by enzyme-linked immunosorbent assay (ELISA) and those positive for HBsAg were furtherly detected for Hepatitis B e antigen (HBeAg) and antibody to HBeAg (Anti-HBe). The weighted prevalence of HBV seromarkers were analyzed and compared among the groups by age, gender, residential areas and geographical areas. The proportion ratios for different composite models of HBV seromarkers were also analyzed among the whole subjects and the women of child-bearing age. A case-control study was conducted to explore HBV risk factors among the youngsters and adults in Shandong Province. All the cases and controls were obtained from the above serosurvey. The case group was consisted of the subjects who were aged 15 to 59 years and positive for HBV infection and the control group was consisted of the subjects in the same age group but negative for HBV infection. The variables as the potential risk factors were gotten from the questionare. Logistic regression analysis was used to screening for the risk factors and OR and population attributable risk percent (PARP) were also calculated. [Results](1) Hepatitis B coverages. The complete HepB coverage was 27.07% (95%CI= 22.56%-31.59%) among the population aged 1 to 59 years and the coverage was significantly higher among children aged 1 to 4 years (99.66%) than that among those aged 5 to 14 years (89.95%) and those aged 15 to 59 years (13.21%). The complete HepB coverage was much lower among children aged 10 to 14 years (81.22%) than that among the children below 10 years of age (>95%). The complete HepB coverage among children aged 1 to 14 years was significantly higher in the western area than that in the middle area. The first dose of HepB (HepB1) timely coverage was 65.41% (95%CI=55.56%-75.25%) among children aged 1 to 14 years. It was significantly higher in the one-year age group than that in the other age groups. The significant higher HepB1 timely coverage was also found in children residing in urban areas, being born in hospitals when compared with those residing in rural areas and be born at home respectively.(2) The prevalence and distribution characters of HBV seromarkers. The weighted HBsAg prevalence rate was 3.39% (95%CI=2.51%-4.26%) among the population aged 1 to 59 years and was 0.92%, 1.53% and 3.81% among the population aged 1 to 4, 5 to 14 and 15 to 59 respectively. The HBsAg prevalence rate was significantly higher among the population aged 15 to 59 years than those aged 1 to 4 years and 5 to 15 years. In comparison with the 1992 survey, the HBsAg prevalence rate deceased by 47.03%, 88.38% and 80.95% among population aged 1 to 59 years, 1～4 years and 5 to 14 years. There were no significant differences in the HBsAg prevalence rate for different gender, occupation, residential area and geographic location. The wighted Anti-HBs prevalence rate was 44.96%(95%CI =41.34%-48.57%) among the population aged 1 to 59 years and was 72.14%, 66.71% and 40.04% among the population aged 1 to 4, 5 to 14 and 15 to 59 respectively. The Anti-HBs prevalence rate was significantly lower among the population aged 15 to 59 years than those aged 1 to 4 years and 5 to 14 years. In comparison with the 1992 survey, the Anti-HBs prevalence rate increased up to 99.82%, 2.97-fold and 2.44-fold among population aged 1 to 59 years, 1～4 years and 5 to 14 years. The weighted HBV prevalence rate was 24.26% (95%CI=21.94%-26.57%) among the population aged 1 to 59 years and was 2.18%, 5.18% and 28.46% among the population aged 1 to 4, 5 to 14 and 15 to 59 respectively. The HBV prevalence rate was significantly higher among the population aged 15 to 59 years than those aged 1 to 4 years and 5 to 15 years. There were no significant differences were observed in the HBV prevalence rate for different genders and geographic location. However, it was significantly higher among the population living in the urban areas than those in the rural areas. In comparison with the 1992 survey, the HBV infection prevalence rates were decreased by 51.48%, 94.95% and 88.83% among population aged 1 to 59 years, 1～4 years and 5 to 14 years. The weighted prevalence rate of HBV susceptibility was 48.25% (95%CI=45.05%-51.46%) among the population aged 1 to 59 years and was 26.79%, 31.51% and 52.07% among the population aged 1 to 4, 5 to 14 and 15 to 59 respectively. It was significantly higher among the population aged 15 to 59 years than those aged 1 to 4 years and 5 to 15 years. There were no significant differences were observed in the HBV prevalence rate for different genders, living areas and geographic location. However, among the population aged 15 to 59, the rate was significantly higher in the rural areas than the urban areas.(3) Comparision of prevalence of HBV seromarkers by Hepatitis B vaccination. The prevalence of Anti-HBs was significantly higher in the population vaccinated (65.51%) with hepatitis B than those unvaccinated (33.06%) and with unknown hepatitis B vaccination status (14.34%), while the HBV infection rate was much lower in those vaccinated than those unvaccinated (29.96%) and with unknown hepatitis B vaccination status (27.52%). Among the children aged 1 to 14 years and having received 3 doses of HepB during infancy, a significantly higher HBV prevalence and similar HBsAg prevalence were found in the group aged 10 to 14 years than those under 10 years of age.(4)The proportion ratio for different composite models of HBV seromarkers. Ten kinds of composite models of HBV seromarkers were detected in the survey. The model solely positive for Anti-HBs accounted for 43.69% of the subjects and the model negative for all of the five HBV seromarkers account for 37.68%. In the subjects of child-bearing age, 51.38% were negative for all of five HBV seromarkers.(5) Risk factors for HBV infection in youngsters and adults. The main risk factors in polation aged 14 to 59 years were living with the HBsAg positive person, age of≥30 years, acupuncture and moxibustion treatment and the gender of male, the OR value of these factors were 2.00, 1.57, 1.31 and1.20 respectively and the PARP of these factors were 2.01%,30.27%,2.43% and 7.70%。. HepB vaccination was the protective factor with the OR value of 0.78 and the PARP of 6.48%.[Conclusions] In comparison with the early stage of infants HepB immunization,the HBV infection was significantly abating and the immunity against HBV infectionwas improved in the population, especially in the children. A great success of HBVinfection control had been achieved. There were still many populations susceptible forHBV and further efforts should be made to control hepatitis B.[Creative points] It was the first study on the seroepidemiological characters ofhepatitis B in Shandong province 20 years after the introduction of hepatitis B vaccine.It was a comprehensive and systemic research with the high-quality design and theinnovitave analysisi method. The results were very useful for enhancing hepatitis Bcontrol in Shandong province.Part 2 Molecular Epidemiological Study on Hepatitis B Virus in Shandong Province[Materials and Methods] Total DNA was extracted from the stored serum positive for HBsAg obtained from the serosurvey. HBV S gene was amplified by Nested-PCR and PCR products were directly sequenced. Nucleotide sequences were multiply aligned by using the Bio-edit software program (version 5.0.9). Alignments were than fed into phylogenetic trees that were constructed for each subalignment implemented by the MEGA software program (version 3.1). The reference strains of A~H genotype were obtained from Genbank. The distribution of the HBV subtypes was deduced from amino acid sequences at positions 122, 160, 127, 134, 159, 177 and 178. The prevalence of S gene mutant was compared among the groups by age, gender, geographic location and HepB vaccination status. 485 samples were selected randomly from the stored serum negative for HBsAg in the serosurvey. Total DNA was extracted from the samples and S gene and C gene were amplified by Nested-PCR. Only those positive for both S gene and C gene were determined as OBI. The PCR products were sequenced and subjected to phylogenetic analysis. The HBV genotype, subtype and S gene mutation were also determined for the samples with OBI. The prevalence and distribution of OBI among the population negative for HBsAg were analyzed. Comparisons between groups were analyzed by the x~2 or Fisher's exact test as appropriate. P-values below 0.05 were considered significant. All statistical analyses were performed using the SPSS software(SPSS, Chicago, IL). [Results] HBV DNA was amplified from 102 serum samples positive for HBsAg. HBV genotype C and subgenotype Ce (C2) was identified in 101 (99.02%) samples and HBV subtype adrq+ was identified in 98 (96.08%) samples. The "a" determinant mutant was detected in 15 samples and the mutant rate was 14.70% (15/102). There was no significantly difference for the prevalence of "a" determinant mutant in the groups by age, gender, residential areas, geographic location and HepB status. 13 kinds of "a" determinant mutation were identified in the samples. Of these, I126S mutants were found in 4 samples, G145R and P127T in 2 samples, and all of the other kinds of mutants were detected only once. 4 of 485 (0.82%) serum samples were determined as OBI. The prevalence of OBI was significantly different among the population with various composite models of HBV seromarkers. 2 HBV strains with "a" determinant mutant were detected among the 4 OBI samples.[Conclusions] our study demonstrated that HBV genotype/subtype C/adrq+ was the predominant strain circulating in Shandong province, China. Further more, the "a" determinant mutant seemed to be uncommon in HBV carriers, and might not be attributed to vaccine-induced mutation. OBI could be found in a very small proportion of the community population negative for HBsAg.[Creative points] In our study, the predominant position of HBV genotype C and subtype adrq+ was firstly verified in the asympotomatic HBV carriers and the prevalence and distribution of HBV with S gene mutant was firstly clarified in the general population in Shandong province. Four new kinds of HBV S gene mutants were found in our study. It also was the first study on the prevalence of occult hepatitis B infection in the communitybased population in China.