| HBV infection was highly prevalent in China. Some strategies have been used to interrupt HBV transmission in recent years, and the crowd of HBV infection has been gradually under the control. The prevalence of HBs Ag in children has decreased significantly, but new infection continues to happen. Hepatitis B virus infection in children can lead to even carry for lifetime, and HBV transmission from mother-to- infant is the common and major mode of acquiring chronic HBV infection at this age. The reported incidence of hepatitis B is much higher than the national level in Wuwei city of Gansu province, a national HBV prevention and control demonstration area. The actual community of HBs Ag positive rate was 7.19%, higher than the national average level. To reduce the incidence of hepatitis B infection in the area, it is very important to take prevention and control measures for children. Thus it is necessary to find out the status of hepatitis B infection and vaccination implementation in children. With the rapidly development of molecular biology and the application of science of technology in virus detection, occult hepatitis B infection has been suffered more and more concern, but the situation of occult hepatitis B infection in community children is still unclear. Our study will investigate HBV infection in community children under 12 years old, to find HBV infection status and its distribution characteristics, and to analyze the HBV S gene-related mutations between occult HBV infection and HBs Ag positive. This work was provided a reliable basis for the prevention and blocking HBV infection in children.Methods: 1. Children under 12 years old from different communities of Wuwei city were selected as study subjects who participated the whole crowd hepatitis B survey in 2010. Epidemiological investigations were conducted in the parents or guardians of children for collecting the information of HBV infection and vaccination. Serum samples of children were collected and used for detection of HBs Ag and anti-HBs by ELISA, and only the HBs Ag positive samples were tested for ALT, HBe Ag and hepatitis B viral load(HBV DNA). 2. HBs Ag negative children under 12 years old were selected as other parts of study subjects. Using magnetic beads method extractes DNA in serum, and nested PCR and real-time quantitative were used to detect HBV DNA. PCR products of the S regions of HBV DNA were sequenced, if it is positive that can be difined as occult HBV infection. Those children would be followed up and to observe their seroepidemiological features. 3. We recruited the occult HBV infection children as case group, the HBs Ag positive children as the control group. HBV S gene variation and genotype were analyzed by phylogenetic tree with the reference sequence of HBV genotype(B, C, D). The amino acids at position 122, 127, 134, 159, 160, 177 and 178 were used to predicte the serotypes of the HBV S gene. The differences of the amino acid sequence of the HBV S gene were compared with the amino acid sequence of the reference strain. Two groups of HBV S gene sequences entry into PAML 4.7 CODEML program in software package, to analyze the selective pressure by codon-based maximum likelihood method(CODEML). 4. Epidata 3.1 software was used for data double entry. SPSS 19.0 statistical software was used for statistical analysis. T test was used to analyze quantitative variables. The Chi-square test or Fisher’s exact was used to analyze categorical variables. P-Value of <0.05 was considered as significant. The Mann-Whitney U test was used to analyze HBV DNA levels and the S gene nucleotide substitution rate.Results: 1. In our survey, 4243 children under 12 years old were enrolled from four communities, and of them 4159(98.02%) acquire epidemiological investigation information and serum specimens. Among the 4159 children, 52(1.25%) HBs Ag positive were determined. No significant differences were found for positive rate of HBs Ag in different age groups(χ2=2.90, P=0.407). The coverage rate of HBV vaccination was 98.68%(4104/4159), and anti-HBs positive rate was 59.05%(2456/4159). The Chi-square test showed that anti-HBs positive rates were different among different age groups(χ2=216.81, P<0.001). The highest anti-HBs positive rate was 73.60%(1116/1517) in 10~12 age group. Of the 52 HBs Ag positive children, 8(15.38%), 15(28.90%) and 27(51.92%) were positive for anti-HBs, HBe Ag and HBV DNA, respectively. And 3(5.80%) were found of abnormal alanine aminotransferase(ALT) levels(greater than 40 U/L). 2. Selected 886 subjects from HBs Ag negative children, 9 cases of occult HBV infection were detected by nested PCR, and the incidence of occult HBV infection was 1.01%(9/892). Of them there are 6 male cases, 3 femal cases, and the youngest age is 4, the biggest 12. The coverage rate of HBV vaccination was 77.78%(7/9), and the anti-HBs positive rate was 33.33%(3/9). Among those of 9 occult HBV infection children, 3 children(33.33%) were followed up after 5 years, and their HBs Ag, HBe Ag, anti-HBe, anti-HBc and HBV DNAwere all negative, but anti-HBs was positive for all, and ALT was in the normal range. 3. The children with occult HBV infection and HBs Ag positive have more genotype C of 55.56%(5/9)and 50.50%(8/16), and ayr is the main serotype. However, there was no different genotype distribution(χ2=0.365, P=0.488), and there was no different of serotype in two groups(χ2=2.824,P=0.108). Comparison of children with occult HBV infection and HBs Ag positive HBV S gene function region, there were no different in amino acid mutation rates, which included Sc(aa41-49), MHR(aa100-169), “a” determinant(aa124-147), the first loop(aa124-137), the second loop(aa139-148), pre-‘a’ region(aa100-123), after-‘a’ region(aa149-169) of CTL epitope. We found 3 amino acid mutation of P127 S, I140 T, S143 L and 5 amino acid mutation of P127 T, T131 P, S143 L D144R, G145 R in the children with occult HBV infection and HBs Ag positive, which maybe impact HBs Ag “a” determinant. The selection models of M2, M3 and M8 significantly favored over the neutral models of M0, M1 and M7 in children with occult HBV infection(P<0.05). While the selection models of only M3 in the children with HBs Ag positive is significantly higher than neutral model. This result show that is different between sites under selection pressure. Only 1 site was identified in occult HBV infection group, and it located on site 180.Conclusions: 1. HBs Ag positive rate was 1.25%(52/4159) in those communities, significantly higher than the national average rate(0.94%), which means we still face a severe HBV infection status in children in the area. Some infection children have high HBV DNA load, so we should strengthen surveillance and follow-up work. Anti-HBs positive rate in 4~6 years old children decreased obviously, which suggests a booster vaccination of hepatitis B vaccine in those children. 2. There is a certain proportion(1.02%) occult HBV infection in community population, although it is lower than HBs Ag positive rate, the situation of children with occult HBV infection could not be ignored, and we should strengthen follow-up and research work. 3. There were no obvious differences in HBV genotype, serotype, mutation sites, and positive selection sites between occult HBV infection and HBs Ag dominant infected children, which suggests the occult HBV infection of children may be a stage in the HBV infection progress, and it has no obvious correlation with the characteristics of the virus itself. |
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