【作者】 张丽；

【导师】 李立明；

【作者基本信息】 山东大学 ， 流行病与卫生统计学， 2009， 博士

【摘要】 【背景】乙型病毒性肝炎（以下称乙肝）是由乙肝病毒（Hepatitis B virus,HBV）感染引起的以肝脏损害为主的传染病,可发展成为慢性肝病、肝硬化和肝癌,是我国重要的公共卫生问题。多年来,山东省乙肝控制工作一直采取以“新生儿乙肝疫苗（Hepatitis B vaccine,HepB）接种”为主的策略;1987年开始引入HepB,1992年开始在新生儿普及接种,但需自费接种;2002年将HepB纳入儿童计划免疫,免费为新生儿提供HepB,仅收取少量注射费用;2005年开始新生儿接种HepB完全免费。山东省还于2004年和2006年分别开展了学龄前儿童和15岁以下儿童HepB查漏补种工作,同时在成人中积极推广使用HepB。山东省曾于1992年新生儿普种HepB初期开展了全省乙肝血清学调查,HBV流行率高达50.00%,人群慢性乙肝病毒表面抗原（Hepatitis B surface antigen,HBsAg）携带率为6.4%,其中儿童HBV流行率和慢性HBsAg携带率分别为45.63%和8.00%,此后未再开展全省大样本调查工作。HepB的长期、广泛使用可能对人群HBV的流行强度和流行特征产生影响。为了解HepB接种20年后山东省HBV血清流行病学和分子流行病学现状及特征,于2006年9月开展本研究。【研究目的】了解HepB使用20年后山东省社区人群HBV血清学指标流行率、分布特征、感染模式构成以及青少和成人HBV感染的主要危险因素;与新生儿普种HepB初期比较,探讨山东省乙肝流行病学特征的变化趋势,对防治效果进行初步评价。了解山东省社区人群中HBV基因型、血清学业型、HBV S基因变异株和HBV隐匿性感染（Occult hepatitis B viruS infection,OBI）的流行状况、分布特点以及病毒株的分子生物学特点,为完善乙肝控制策略提供参考。第一部分乙型病毒性肝炎血清流行病学研究【材料与方法】根据人群HepB免疫策略调整情况,将研究对象分为新生儿普种HepB前出生（15～59岁）、新生儿HepB自费接种时期出生（5～14岁）和HepB计划免疫时期出生（1～4岁）3个年龄组,分别计算样本量;通过多阶段随机抽样在山东省12个县（市、区）随机抽取1～59岁调查对象7601人。对所有调查对象进行问卷调查和血标本采集;使用酶联免疫吸附试验（Enzyme-linkedimmunosorbent assay,ELISA）方法,检测调查对象血清HBsAg、乙肝病毒表面抗体（Antibody to HBsAg,Anti-HBs）、乙肝病毒核心抗体（Antibody to core antigen,Anti-HBc）,HBsAg阳性者进一步检测乙肝病毒e抗原（Hepatitis B e antigen,HBeAg）和乙肝病毒e抗体（Antibody to hepatitis B e antigen,Anti-HBe）。采用抽样权重对不同地区、不同人群HepB接种率,HBsAg、Anti-HBs流行率,HBV流行率和HBV易感率进行点值估计,采用泰勒级数线性法估计率的方差,然后构建点值估计的置信区间,通过比较置信区间进行率的统计学检验;计算调查人群和育龄期妇女中不同HBV血清学感染模式构成比;以以上调查中青少年和成人HBV感染者为病例组,非HBV感染者为对照组,以调查问卷中HBV感染可能相关因素作为备选变量,采取成组病例对照研究方法,通过单因素和多因素非条件logistic回归分析,筛选出HBV感染危险因素,计算各危险因素的比值比（OR值）和人群归因危险度百分数（Population attributable risk percent,PARP）。【结果与分析】（1）人群HepB接种情况。山东省1～59岁人群HepB全程接种率为27.07%（95%CI=22.56%～31.59%）,1～4岁人群HepB全程接种率（99.66%）显著高于5～14岁（89.95%）和15～59岁人群（13.21%）;10～14岁人群HepB全程接种率（81.22%）显著低于10岁以下各年龄组（均＞97%）。全省1～14岁人群首针HepB（First dose of hepatitis B,HepB₁）及时接种率为65.41%（95%CI=55.56%～75.25%）;1岁组HepB₁及时接种率（93.07%）显著高于8岁以上各年龄组（＜75%）,城市地区HepB₁及时接种率（86.20%）显著高于农村地区（61.92%）;在家出生者（23.67%）显著低于在乡镇级（70.70%）和县级以上医院（75.17%）出生者。（2）HBV血清学指标流行率和分布特征。全省1～59岁人群HBsAg流行率为3.39%（95%CI=2.51%～4.26%）,15～59岁人群HBsAg流行率（3.81%）显著高于1～4岁（0.92%）和5～14岁（1.53%）人群;东、中、西部和城乡之间,以及不同性别人群之间HBsAg流行率无显著差异;与新生儿普及HepB初期相比,1～59岁人群HBsAg流行率下降47.03%,而1～4岁和5～14岁儿童分别下降88.38%和80.95%。全省1～59岁人群Anti-HBs流行率为44.96%（95%CI=41.34%～48.57%）,15～59岁人群Anti-HBs流行率（40.04%）显著低于1～4岁（72.14%）和5～14岁（66.71%）人群;不同地区和不同性别人群Anti-HBs流行率无显著差别;与新生儿普及HepB初期相比,1～59岁人群Anti-HBs流行率上升99.82%,1～4岁和5～14岁儿童分别上升2.97和2.44倍。全省1～59岁人群HBV流行率为24.26%（95%CI=21.94%～26.57%）,15～59岁人群HBV流行率（28.46%）显著高于1～4岁（2.18%）和5～14岁（5.18%）人群;城市地区HBV流行率（34.38%）显著高于农村地区（22.43%）;与新生儿普及HepB初期相比,仝人群HBV流行率下降51.48%,1～4岁和5～14岁儿童分别下降94.95%和88.83%。全省1～59岁人群中HBV易感率为48.25%（95%CI=45.05%～51.46%）,其中15～59岁人群HBV易感率（52.07%）显著高于1～4岁（26.79%）和5～14岁（31.51%）人群;农村地区人群HBV易感率（49.65%）显著高于城市地区（40.53%）。（3）不同HepB免疫史人群HBV流行率。有HepB免疫史人群Anti-HBs流行率（65.51%）显著高于无免疫史（33.06%）和免疫史不详（38.77%）人群,HBV流行率（14.34%）显著低于无免疫史（29.96%）和免疫史不详（27.52%）人群;未发现不同HepB免疫史人群HBsAg流行率存在显著差别。在婴儿期完成3剂次HepB的1～14岁人群中,10～14岁组人群HBV流行率（8.76%）显著高于10岁以下人群（＜3%）。（4）HBV血清学指标检出模式。调查对象共检出10种HBV血清学指标组合模式,其中第8种感染模式（即Anti-HBs单阳性）检出率最高,占调查对象的43.69%;第10种感染模式（即乙肝血清学五项指标全阴者）次之,占调查对象的37.68%;不同年龄、HepB免疫史人群HBV血清学感染模式不同。育龄期妇女（20～49岁）中共检出9种感染模式,其中第10种模式所占比例最高,为51.38%;其次为第7种（Anti-HBc和Anti-HBs同时阳性）和第8种模式,分别占22.34%和18.07%,以上3种模式合计占调查育龄期妇女的91.79%;第4种和第6种（即HBsAg和HBeAg双阳性）占调查育龄期妇女的1.58%。（5）青少年及成人HBV感染危险因素分析。15～59岁人群HBV感染的主要危险因素按照OR值大小依次为:长期一起生活的人HBAg阳性、年龄≥30岁、有针灸史、男性和乙肝疫苗接种史（为保护因素）其OR值分别为2.00,1.57,1.31,1.20,0.78;其人群归因危险百分比（PARP）分别为2.01%、30.27%、2.43%,7.70%和6.48%。【结论】山东省目前仍为乙肝中度流行区,青少年和成人中HBV流行强度显著高于儿童,不同地区间流行差异依然存在。与新生儿普种HepB初期相比,山东省HBV流行强度减弱,儿童时期流行高峰消失;人群特别是儿童HBV免疫水平明显提高,山东省实施以“新生儿HepB接种”为主的乙肝控制策略取得了显著成效。目前山东省社区人群特别是成人中仍有大量HBV易感人群,应进一步加强乙肝控制工作。【创新点】首次对山东省HepB使用20年后乙肝血清学流行状况开展全省范围大样本研究。研究内容系统全面,设计实施科学严谨,分析方法具有创新,研究结论翔实可靠,为进一步加强山东省乙肝控制工作提供了科学的依据。第二部分乙型病毒性肝炎分子流行病学研究【材料与方法】从本研究第一部分乙肝血清学研究保留的HBsAg阳性血清标本中提取总核酸（DNA）,采取巢氏聚合酶链反应（Nested polymerase chainreaction,Nested-PCR）方法扩增HBV S基因区,对扩增产物进行双向测序;用BioEdit Sequence Alignment Editor software 5.0.9软件进行核苷酸序列排列;用MEGA 3.1软件按照邻接法（Neighbor-joining）构建系统发生树（Phylogenetictree）,在进化树上靠近的标准株基因型即为该标本病毒株的基因型。使用MEGA3.1软件将核苷酸序列翻译成氨基酸联排序列并进行序列比较,根据S基因中122位、160位、127位、134位、159位、177位和178位氨基酸种类确定HBV血清学亚型,通过氨基酸序列比较确定S基因变异位点。计算不同地区和人群各HBV基因型和血清型的构成比;分析HBV S基因变异株变异位点、变异形式和检出率;利用χ²检验或Fisher’s精确概率法,对不同地区、不同年龄、性别、HepB免疫史和血清学感染模式人群“α”抗原决定簇变异株检出率的差异进行统计学检验。按照系统抽样方法,从HBV血清学研究保留的HBsAg阴性血清标本中随机抽取485份,提取总DNA,使用Nested-PCR方法扩增HBV S基因和C基因,两次扩增两基因均为阳性者确定为OBI;利用以上相同方法确定OBI病毒株的基因型、血清型和S基因、C基因变异情况;计算不同地区、不同年龄、性别和血清学感染模式人群OBI检出率,利用χ²检验或Fisher’s精确概率法对OBI检出率差异进行统计学检验。【结果与分析】（1）基因型和血清型分布。对102份HBsAg阳性血清标本进行了基因型、血清学亚型和HBV S基因变异的检测。检出B型和C型两种基因型,其中99.02%（101/102）为C基因型且全部为C2亚型,仅1株为B基因型B2亚型;检出adrq+、adw2和ayr 3种血清型,其中adrq+型占96.08%（98/102）,adw2型占2.94%（3/102）,ayr型占0.98%（1/102）;3株adw2血清型和1株ayr血清型毒株均来自山东省中部地区。（2）S基因变异株流行情况。102株HBV中共检出15株“α”抗原决定簇变异株病毒,变异株检出率为14.70%（15/102）;检出11个“α”抗原决定簇aa位点的13种突变形式,其中9种突变形式既往有文献报道,包括I126S、I126N、P127T、Q129L、G130R、M133T、F134S、P142S和G145R;4种突变形式既往未见文献报道,包括P135S、P135L、C137L和S143T;15株“α”抗原决定簇变异病毒株除4株发生I126S变异,各有2株发生P127T变异和G145R变异外,其余变异形式均仅在1株病毒中检出;不同地区和不同年龄、性别、HepB免疫史人群间“α”抗原决定簇变异株检出率均无显著性差异。102株病毒中,43.14%被检测出前S区变异,21.57%发生在前S1区,15.69%（16/102）发生在前S2区,5.88%同时发生在前S1和前S2区;尤以前S1区的前端突变较为集中。（3）隐匿性乙肝病毒感染（OBI）检出率及分布情况。485份HBsAg阴性标本中4份检出OBI,检出率为0.82%;不同HBV血清学指标人群中OBI的检出率有显著差别。OBI毒株均为C基因型、adrq+血清学亚型;2例（0.41%）出现“α”抗原决定簇变异（1例为G145R突变,1例为S143T突变）,2例出现“α”抗原决定簇以外突变（均为T47K突变）。【结论】山东省社区人群中HBV以C型基因、adrq+血清型为主;少量的adw2和ayr血清型主要集中在中部地区;社区人群中HBV S基因变异株的发生率较低,变异位点比较分散,野毒株仍是优势毒株;aa126点是HBV“α”抗原决定簇最常见的变异位点。山东省社区人群中OBI检出率较低;其检出率与HBV血清学感染指标有关。【创新点】本研究首次从无症状HBV携带者中证实C基因型和adrq+血清型在山东省HBV流行的优势地位。在国内首次从有代表性的社区人群中对S基因变异株特别是“α”抗原决定簇变异株的流行率、分布特征和分子生物学特点进行研究,发现了4种新的“α”抗原决定簇变异形式。本研究是国内近20年来首次在社区人群中开展的OBI流行现状研究。更多还原

【Abstract】 [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.更多还原