Risk Factors Of New HBV Infection And Its Characteristics Of S Gene In High HBV Epidemic Area Of Northwest China

Background Hepatitis B was seriously harmful to people’s health in China. In recent years, with the broadly applications of hepatitis B vaccine and hepatitis B surface antigen(Hepatitis B virus surface antigen, HBs Ag) population died gradually, HBs Ag prevalence had dropped significantly, especially for children under five years old. According to hepatitis B serological survey data from Wuwei City, hepatitis B vaccination rate of children under 5 years old was near to 100%, but HBs Ag prevalence remained at around 1%; and according to Chinese CDC data in 2010, the national acute hepatitis B reported incidence rate was 5.6/ 100,000, at least 780000 persons were newly infected by HBV per year in China, and more than 95% of which were adults. The situation of the breakthrough HBV(hepatitis B virus, HBV) infection in children and new HBV infection in adults were still serious, and more attentions should be paid on the risk factors and the virus factors.Objectives To explore the risk factors, transmission route and genetic characteristics of the S region for breakthrough HBV infection of children and new HBV infection in adults in the Wuwei City, Gansu Province, which was the high HBV epidemic area in western China, and to provide a scientific basis for the development of new hepatitis B infection prevention strategies.Methods1. The study of children breakthrough HBV infection: The children born of HBs Ag-positive mothers, and preterm or low birth weight and their mothers between 2009 and 2012 were entried in three hospitals(Wuwei City People’s Hospital, Wuwei City Third People’s Hospital and Liangzhou Dsitrict People’s Hospital). Self-designed questionnaires were used to obtain demographic characteristics information of the children and their families and major risk factors(family members of hepatitis B, etc.). The sera of children and their mothers were collected, and tested for HBs Ag, anti-HBs, HBe Ag, anit-HBe, anit-HBc with ELISA kits, and HBV DNA was detected by real-time PCR method, and anti-HBs of children’s serum were quantified with Abbott reagent. The chi-square test or Fisher’s exact test was used to compare the frequency data, the t test was used to compared the mean data which was the normal distribution, the univariate and multivariate unconditional logistic regression were used for the risk factor analysis. 2. The study of new HBV infection in adults:The cases of the new HBV infection were recruited from six hospitals through healthy examination, outpatient and inpatient who were meet the inclusion criteria of new HBV infection in Wuwei City. The matched controls were selected in the same community, with the same sex, age difference was not more than 5 years of age and both HBs Ag and anti-HBs were negative, self-designed questionnaires were used to obtain their demographic characteristics, symptoms and major risk factors. The subjects’ sera were collected and tested for HBs Ag, anti-HBs, HBe Ag, anit-HBe, anit-HBc-Ig G and anti-HBc Ig M with ELISA kits, HBV DNA was detected by the real-time PCR method, ALT and AST were determined with velocity method. The chi-square test or Fisher’s exact test was use to compare the frequency data, the t test was used to compared the mean data which was the normal distribution, the univariate and multivariate unconditional logistic regression were used for the risk factor analysis, mutually exclusive risk hierarchy for risk factors was used to identify risk factors and infer transmission route. 3. The S gene characteristics of the new HBV infection:Subjects were selected from breakthrough HBV infection in children, HBV new infection adults and HBV asymptomatic carriers discovered firstly. The nested PCR was used to amplify S region.The software Mega6.0 was used to cut sequence alignment before the phylogenetic tree was constructed for gene typing; build standard consensus sequence of amino acids(amino acid, aa) according nucleotide sequence for different HBV genotypes, then each sequence compared with the consensus sequence, to describe each aa site mutation and the "a" determinant mutation; and to check reverse transcriptase region with common clinical drug resistance mutations corresponding to S region. The chi-square test or Fisher’s exact test was use to compare the frequency data.Results 1. A total of 339 mothers and 388 children were surveyed for breakthrough HBV infection. Of 388 children, 55.41%(215/388)were male; the average age was 2.95 ± 1.3 years; 7 cases were HBs Ag positive, and 11 cases were HBV DNA positive; the total infection cases were 13, and the breakthrough infection rate was 3.61%(95%CI: 1.56%-5.14%). The breakthrough HBV infection rate was 4.17%(95%CI: 1.82%-8.04%) for the children born to HBs Ag-positive mothers, and 2.55%(95%CI: 0.83%-5.85%) for the children born to HBs Ag negative mothers. Multivariate analysis showed that HBV DNA positive mothers(OR=4.49, 95%CI: 1.28-15.77) and other family members of hepatitis B history(OR=6.18, 95%CI: 1.73-22.08) were the independent risk factors for childhood breakthrough infection. According the risk factor constitution of 13 cases, 92.31%(12/13) breakthrough HBV infection was due to the spread in their family, which was the main route of transmission, and other ways were account for 7.69%(1/13), which was a secondary route of transmission. About 75%(8/12) within the family transmission was attributed to the mother, and other family members transmission was accounted for 25%(4/12). More than 70% of the children had the anti-HBs titer≥10m IU/ml. The children born to HBs Ag-negative and HBs Ag-positive mothers had geometric mean titer of anti-HBs 22.76 m IU/ml and 39.06 m IU/ml, and the difference between them was statistically significant(t=2.31, P<0.05). Regardless of the mother HBs Ag status, the child’s age was an important factor affecting the level of anti-HBs titer; the titer in 3-year-old group was higher than ≤3 years of age group, and the difference between them was statistically significant(P<0.05). 2. A total of 215 cases met the inclusion criteria for new HBV infection, and 567 cases met the inclusion criteria for control group. Of 215 cases, 142 were men(66.05%); male to female ratio was about 2:1; 30-39 age group was accounting for 40.47%. There were mainly two reasons for hepatitis B infection examination, which included healthy examination and symptom occurrence, accounting for 36.28% and 25.12%, respectively. The top three most common symptoms for new HBV infections were weakness, fatigue, loss of appetite, accounting for 61.86%, 51.16% and 44.65%, respectively. Dark urine and jaundice dominant had the lowest proportion, accounting for 20.93% and 13.49%, respectively. The proportion of ALT values >80U/L of the new HBV infections accounted for 49.77%; AST values>80U/L accounted for 29.30%; and AST/ALT>1 accounted for 13.49%(29/215). Abdominal B ultrasound results showed that 52.63% cases were normal, 10.53% for hepatosplenomegaly, 8.02% for fatty liver, and 7.52% for cirrhosis. 32 cases were diagnosed of "acute hepatitis B", accounting for 14.88% of new HBV infections. A total of 12 kinds of serum mode were found, of which the most two common modes were “HBs Ag, HBe Ag and anit-HBc positives” and “HBs Ag, anti-HBe and anit-HBc positives”, both of which accounted for 75.82% of all the serum modes; HBs Ag and anti-HBs co-exist positive rate was 8.85%. A total of 17.67% of new infection cases received antiviral treatment; 6.98% received Chinese traditional medicine treatment alone; 4.65% received anti-fibrosis treatment; one cases of young man progressed to acute liver failure and died. Multivariate analysis showed 1 protective factor: hepatitis B vaccination history(OR=0.07, 95%CI: 0.04-0.12); 10 independent risk factors: sexual life unusing condom(OR=2.00, 95%CI: 1.18-3.38), family history HBV intection(OR=2.85, 95%CI: 1.47-5.54), family history of cirrhosis or liver cancer(OR=4.65, 95%CI: 1.18-18.27), bleeding history when brushing teeth(OR=8.03, 95%CI: 4.76-13.55), migrant work history(OR=5.52,95%CI: 2.91-10.49), injection and infusion in village clinics and private clinics(OR=3.87, 95%CI: 2.11-7.1), surgery history(OR=5.27, 95%CI: 1.44-19.27), the history of oral diagnosis and treatment(OR=3.87, 95%CI: 1.88-7.98), sharing towels and toothbrushes(OR=4.35, 95%CI: 2.64-7.17), and often eating at unclean restaurant(OR=4.15, 95%CI: 2.4-7.19). According mutually exclusive risk hierarchy, currently 49.3% of new HBV infection in adult were attributed to close contact with family or other HBV carriers, which was the main route of transmission; 27.91% were attributed to iatrogenic transmission, in which sexual transmission accounted for 20.46%, and unexplained reson accounted for 2.33%. 3. There were 9 cases of successful sequencing from 13 children with breakthrough HBV infection, 172 cases from 215 adult with new HBV infections, and 84 cases from 121 cases of chronic asymptomatic carriers detected firstly. All of them were genotype B,C and D, but the genetype C and D accounted for the majority for 90.57%, in which genetype C and D type accounted for 59.62% and 30.94%, respectively. The genotype distribution differences among the three groups were not statistically significant(P>0.05). The aa mutations for the three types of infection among "a" determinant(aa124-aa147) region were as follows: 4 cases(44.44%) occurred 7 amino acid position mutations in 9 children with breakthrough HBV infection, and I126 T mutation was found in two cases, and G145 R mutation was found only in one case; there were 54 aa mutation sites and belong to 29 types in 27 cases with new HBV infection in adult, and the detection mutation rate was 12.56%, and the most common mutation was F134 Y detected in 10 cases, and I126 T detected in 9 cases; there were 28 aa sites mutations and belong to 12 types in 12 cases with chronic asymptomatic carriers, and the detection mutation rate of 11.90%, and the most common mutation I126 T were detected in 9 cases. The mutation rate of "a" determinants(aa124-aa147) in breakthrough HBV infection group was higher than the new HBV infection group and chronic asymptomatic carriers group(P<0.05). The drug resistance associated mutations were not detected in 9 breakthrough HBV infection in children, but it was 7.56%(95%CI: 4.09%-12.58%) among 172 new HBV infections and 11.90%(95%CI: 5.86%-20.81%) in chronic asymptomatic carriers.Conclusions1. The breakthrough HBV infection rate was still high among children in Wuwei City, which was a major obstacle to eliminate hepatitis B in the future. Breakthrough HBV infection was mainly come from the family, so active measures should be taken to prevent intrauterine infection in the late pregnancy for the HBV DNA positive mothers, hepatitis B vaccine plus HBIG should be vaccinated immediately after children were born, at the same time, the precation should be took to avoid horizontal transmission within the family. 2. The new HBV infections just showed some mild hepatic damage in adult, the early symptoms were not typical, common symptoms such as weakness, fatigue, etc., which took a major part of the symptoms and occurred early, but they were non-specific symptoms for identifying new HBV infection and had poor ability to identify HBV infection. It was difficult to diagnosis new HBV infection in early stage and deal with the severe cases for the health care workers in town or village clinics, so professional guidance and training should be strengthened. 3. Comprehensive measures should be taken to prevent new HBV infection in adult. Firstly, vaccine coverage should be improved in adult, and condom use should be promoted in young adults. Secondly, more attentions should be paid to personal oral hygiene, timely treatment for gingivitis to reduce blush bleeding, and do not share toothbrush and towels with others. Finally, the surveillance should be strengthened in the hospital operating room, private clinics, village clinics, the department of stomatology in hospital and dental clinics. 4. The "a" determinant aa mutation rate was higher in children breakthrough HBV infection, the reason might be the decreased binding force between antigen-antibody because of HBV "a" determinant mutation, thereby reduced the effects of hepatitis B vaccine. The "a" determinant mutation should be monitored in children and effectiveness of existing hepatitis B vaccines should be timely assessed. 5. The high aa mutations associated with drug resistance were detected in adult HBV new infections and chronic HBV carriers, these people might be potential patients hepatitis B in future, therefore the potent and a high genetic barrier to resistance drug should be used in untreated hepatitis B patients, or genetic resistance should be detected then the low-resistance drug was determined. Due to the "image change" between immune-escape mutations and NA resistance mutation, so a lot of NA drug resistant mutations occurrence might cause the effect of hepatitis B vaccine decreasing and be a threat for the current hepatitis B vaccine.