| Hepatitis B virus(HBV)infection is a major public health problem which affect the health of all human.Hepatitis B virus is a systemic infectious disease caused by liver damage caused by hepatitis B virus.Two billion people have serologically shown that they have been infected with HBV,and more than 350 million of them have developed chronic HBV carriers.Some patients with chronic hepatitis B(CHB)will die of cirrhosis and liver cancer caused by hepatitis B,which brings a heavy economic burden to patients,femilies and society.The prevalence of HBsAg in women of childbearing age in China is about 6.6%Currently,It is estimated that 1 million newborns still have a high risk of HBV infection after birth.The Global Health Sector Viral Hepatitis Strategy,released in 2016,proposed firstly a global goal to eliminate the threat of viral hepatitis to public health by 2030,and"Zero transmission of hepatitis B from mother to child" is an important part of achieving this goal The China Hepatitis Preventbn and Control Foundation established the "Zero transmission Project of hepatitis B from mother to child",and The Affiliated Hospital of Guizhou Medical University:has signed the 6th project unit in China.This topic will retrospectively study the pregnant women with high risk of hepatitis B in Guizhou(HB sAg,HBeAg double positive,HBV DNA(>106IU/ml),or the first child has been infected with HBV or the pregnant woman herself is infected by mother-to-child transmission of hepatitis B).Who were divided into drug intervention group during pregnancy,non-intervention group in outpatient clinic and control group(non-hepatitis B pregnant women)and their newborns/infants.To study the changes of HBV genotype and immune function during pregnancy and the effect of antiviral drugs in the intervention group,and to provide real clinical data for the mechanism and prevention and control of mother-to-child transmission of HBV infection after the combined standard immunization of hepatitis B vaccine and hepatitis B immunoglobulin.For mother-to-child transmission patients,through the extraction of virus DNA and fetal free DNA in maternal blood samples and second-generation sequencing analysis,the homology of the whole gene sequence of mother-to-child virus was determined and specific mutation sites with intrauterine transmission advantages were found.which provide real clinical research data for the occurrence mechanism and prevention and control of mother-to-child transmission of HBV All subjects were approved by the ethics comnittee of the affiliated hospital of guizhou medical university.The research content is divided into three parts.Objective:1.To observe the e ffects of antiviral blockade during pregnancy on HBV DNA and HBV RNA in pregnant women with different genotypes of high risk hepatitis B in Guizhou.2.To observe the maternal-infant outcomes of high-risk pregnant women with hepatitis B after maternal-infant transmission interruption in Guizhou3.To explore the non-invasive prenatal diagnosis of hepatitis B virus intrauterine infection.Methods:In the first part,we retrospectively collected the data from May 2016 to May 2018 in the Infection Department and Obstetrics Clinic of Affiliated Hospital of Guizhou Medical University.Diagnosis of selected patients conforms to the diagnostic criteria of the Guidelines for the Prevention and Treatment of Chronic Hepatitis B revised in 2015.Pregnant women diagnosed with CHB of chronic hepatitis B before 12 weeks of gestation.All patients are HBsAg(Hepatitis B surface antigen)positive for over 6 months,and B-ultrasound examination before 24 weeks of pregnancy suggests that the fetus is developing normally,and Serum hepatitis C virus(HCV),human immunodeficiency virus(HIV),Treponema pallidum antibody(RPR)markers were negative,and they had no history of threatened abortion.Exclusion criteria:?Husband has HBV infection;?patients with CHB were treated with interferon within half a year before pregnancy;?patients are suffered from other types of liver disease,diabetes,thyroid disease,hypertension,chronic kidney disease,tumor,mental illness and other chronic diseases during pregnancy;?patients were attacked by cirrhosis decompensated period.?prenatal B ultrasound suggests abnormal or abnormal fetal development.All patients were divided into two groups according to whether or not antiviral blockade was performed at 24-28 weeks of gestation:91 cases in the intervention group(T)and 259 cases in the non-intervention groiup(NT).During the same period,100 healthy non-hepatitis b pregnant women(Health control,HC,defined as HBsAg negative,normal liver function,no previous history of hepatitis)were selected as the control group(C).According to the antiviral guidelines for pregnant women with hepatitis B,the intervention group voluntarily started to receive tenofovir or tibifidine antiviral treatment until delivery at 24-28 weeks for the purpose of blocking.Demographic and clinical characteristics were investigated and blood samples were collected respectively at 12-24 weeks,28-32 weeks and 36-40 weeks.To detect genotype,HBsAg,HBeAg,HBVDNA,HBVRNA,liver function[alanine aminotransferase(ALT)\aspartate aminotransferase(AST)\total bilirubin(TBIL)\total bile acid(TBA)\cholinesterase(CHE)\alkaline phosphatase(ALP).and to detect the changes of T lymphocytes(CD3+,CD4+CD8+),NK cells(CD16+CD56)and B lymphocytes(CD19)for Control group,non-intervention group,intervention group before the use of drugs(12-24w)and before the intervention group(36-40 weeks).For the purpose of blocking,the intervention group started oral administration of tenofovir disoproxil fumarate tablets(TDF)300 mg/d at 24-28 weeks of gestation,purchased from GlaxoSmithKline(Tianjin)Co.Ltd.?? H20153090);or telbivudine(LDT)600 mg/d,purchased from Beijing Novartis Pharmaceutical Co.,Ltd.(National Medicine Zhunzi H20070028),antiviral treatment until delivery.Continuous variables were used to calculate mean standard deviation Classification variables were statistically analyzed at rates.In the second part,the study population included the pregnant women(259 cases in the non-intervention group(NT),91 cases in the intervention group(T),and 100 cases in the control group(C))and their infants(450 cases).All newborns/infants in the intervention group and the non-intervention group were given the national standard hepatitis b planned immunizaton(intramuscular injection of hepatitis b immunoglobulin 100 U within 6 hours of birth,and intramuscular injection of genetically engineered hepatitis b vaccine 10 g every time for 0,1 and 6 months).The control group received only the hepatitis b vaccine(0,1,6 months of intramuscular injection of 10 g of the genetically engineered hepatitis b vaccine).Adverse effects at gestational and gestational weeks were observed,the Apgar score for neonates was determined,and serum hepatitis B virus marker(HBVM)and HBV DNA were quantitatively detected at 7 months of age for infants.Mother-to-child transmission of HBV is defined as HBsAg positive or HBV DNA positive in a child born to a pregnant woman with hepatitis B after 7 months of age.Data statistics:pair-wise comparison was performed by LSD method,comparison between groups was performed by t test,efficacy analysis was performed by covariance analysis,and intra-group comparison was performed by symbolic rank sum test.In the third part,the study population was divided into pregnant women(91 cases in the intervention group and 259 cases in the non-intervention group)and their infants(350 cases).10ml blood was collected from HBV pregnant women during 16-24 weeks of pregnancy for high-throughput sequencing to predict whether the fetus would suffer from intrauterine infection(completed by the prenatal diagnosis center of guizhou province).Clinical information and blood samples have been collected.On the basis of fully considering the balance of the characteristics of maternal age and weeks of pregnancy between the infected group and the non-infected group,A salrple test set consisting of 128 pairs of maternal and infant samples was selected,among which 29 cases(all those without intervention in the first part)were confirmed to have intrauterine infection after postpartum follow-up,and 99 pairs of maternal and infant samples without intrauterine infection were confirmed.Through the extraction of virus DNA and fetal free DNA from maternal blood samples and the sequencing analysis by Illumina HiSeq 2000 platform,the homology of the whole gene sequence of maternal and infant viruses was determined and specific mutation sites with intrauterine transmission advantages were found.All subjects were approved by the ethics committee of our hospital.Statistical methods:t test was used for inter-group comparison,and signed rank sum test was used for intra-group conparison.RandonForest model was built with the "randomForest" toolkit provided by R software version 3.2.3.Results:In the first part,HBV genotyping was performed on 91 pregnant women with positive e antigen or high viral load and hepatitis B replication in the intervention group:57 cases of B type(57/91,62.63.18%)and 34 cases ofC type(34/91,37.36%),and no other genotypes were found.The intervention group did not differ in demographic and clinical characteristics between tenofovir(TDF)and tibifidine(LDT),including liver function tests[before TDF treatment:ALT21.30(9.01-362.49),AST 25.19(13.62-345.53),TBIL8.16(3.79-57.55),ALP78.44(31.00-232.52),TBA3.70(0.83-45.23),ALT21.30(8.66-98.10),AST 24.65(14.91-101.92)at the end of treatment.TBIL 8.78(3.1617.11)),ALP 148.05(53.98 470.61),3.07(0.92 74.83)]associates;Before treatment in LDT group:ALT22.56(6.65-117.00))AST23.02(16.37-62.00),TBIL7.81(5.48-18.27),ALP67.35(46.05-388.21),TBA3.24(1.06-42.58),ALT 21.40(8.14-62.05),AST 24.10(11.79-160.64)at the end of treatment.TBIL 7.28(3.50-18.40),ALP 162.40(47.30-423.96),TBA 3.76(0.69-28.41)].Log10(HBVDNA)and Log10(HBVRNA)[TDF group(before treatment:HBVDNA 4.84 2.01,HBVRNA6.351.10;at 4 weeks of treatment:HBVDNA3.99 0.79,HBVRNA 6.05 0.94;at the end of treatment:HBVDNA 3.06 0.66,HBVRNA 5.52 0.85;LDT group(before treatment:HBVDNA5.08 1.99,HBVRNA6.47 0.88;at 4 weeks of treatment:HBVDNA4.45 1.18,HBVRNA6.51 0.82;at the end of treatment:[HBVDNA3.51 1.20,HBVRNA6.13 0.66)],compared with before treatment(12-24 weeks of pregnancy),tenofovir and tibifidin significantly reduced Log10(HBVDNA)and Log10(HBVRNA)at the end of treatment(P<0.05).When other variables were excluded,HBV genotype had a certain impact on HBVRNA load,which was manifested in that HBVRNA of patients with genome C decreased by 0.54 units(log10)at the end of treatment compared with patients with genome B,with P value(0.01)<0.05.During the pregnancy,CD3+and CD19 cells did not change much in each group,but the egression of CD4+T cells and CD8+T cells in the unintervention group and the intervention group was significantly lower than that in normal pregnant women(P<0.05).The expression of CD4+T cells and CD8+T cells increased after treatment,but the increase of CD4+T cells was more obvious(F=9.38,P<0.01).There was no significant difference between the interventbn groip and the normal control grouP,but there was no statistically significant difference between the NK cells(CD16+CD56)and the control group before the treatment(P=8.72 P=0.014).In the second part,HBV maternal and child outcomes showed that hepatitis B infection occurred in infants in the unintervention group.The egression was HBsAg positive(29/259,11.19%),HBeAg positive(24/259,9.26%)and HBV DNA positive(27/259,10.24%).The prevalence of mother-to-child transmission of HBV was 11.19%(29/259).Furthermore,the relationship between infant HBV infection and Taternal HBV DNA load in the non-intervention groip was further analyzed.When maternal HBV DNA was more than 106 IU/ml,maternal to child transmission infection was up to 38.67%(29/75).In this study,maternal HBV DNA was greater than 106 IU/ml in the mother-to-child transmission infection group,among which 15 cases were positive for HBeAg and 14 cases were negative for HBeAg.Among the two pregnant women with HBV(the first was infected with HBV),1 case was infected with HBV in the non-intervention group.The reasons for not intervening were mainly due to the late visiting time of pregnant women.The visiting time was 38 weeks,and the pregnant women's HBV DNA>106 IU/ml was positive for HBeAg.The virus levels of the other two pregnant women in the non-intervention group were low,and their infants were not infected.In the intervention group,HBsAg,HBeAg and HBV DNA were all negative.The infection rate of mother-to-child transmission of HBV was zero,and the blocking rate of mother-to-child transmission of HBV was 100%.Moreover,there were differelt levels of hepatitis B protective antibodies,and the antibody titer was higher than that of the non-intervention group and the control group,and the difference was statistically significant(F=5.95,P<0.001).HBV time,gestational age and mode of delivery,neonatal Apgar score,birth weight,feeding method,gender,etc.were not statistically different,but there was a statistically significant difference with antiviral therapy during pregnancy.In the the third part,In29 patients with HBV mother-to-child vertical infectbn from mother to child,by the second generation sequencing technology,cell-free fetal DNA and hepatitis B virus DNA and HBVDNA sequence were Successfully extracted from pregnant women blood specimens.The analysis of hepatitis B virus gene sequences showed that all the mother and child to virus gene sequence homology in 99.4%to 100%,all 19 for mother and child(65.5%)virus sequence.According to different genotypes and sequences of B2 and C2 wild-type HBV strains,the B and C genotype mutant infected by the mother and the wild-type HBV strains can be transmitted through intrauterine transmission,and no specific mutation sites with intrauterine transmission advantages were found.Conclusions 1.Hepatitis B pregnant women in guizhou region are mainly B and C genotypes,with the purpose of blocking treatment.When tenofovir(TDF)or tibifidine(LDT)is used to block treatment in different genotypes of pregnant women,good efficacy can be achieved,but B genotype virus is easier to be controlled on the basis of continuous antiviral treatment.In addition to controlling HBV virus,TDF or LDT treatment in the middle and late pregnancy has no obvious adverse reactions,and can also regulate the immune function of T lymphocytes in pregnant women.The mechanism may be related to the improvement of CD4+T cell subpopulation reconstruction and subsequent increase of CD8+T cells.2.In this study,mother-to-child transmission of HBV oeeurred in the non-intervention group,with an incidence of 11.19%.The HBV DNA of infected mothers before delivery was greater than or greater than 106 IU/ml,including 15 cases with positive HBeAg and 14 cases with negative HBeAg.Further analysis was made on the relationship between infant HBV infection and maternal HBVDNA load in the non-intervention group.When maternal HBVDNA 106 IU/ml,maternal HBV infection was as high as 38.67%(29/75),suggesting that high viral HBVDNA replication was an independent risk factor for HBV transmission.3.The second-generation sequencing technology is used to monitor HBV gene and its mutation,and non-invasive prenatal diagnosis of intrauterine transmission of HBV,to provide a reliable basis for the design of accurate and individualized hepatitis B antiviral and antiviral programs,which may be an important prenatal diagnosis and evaluation technology for hepatitis B mother-to-child transmission.Therefore,for pregnant women with high risk of hepatitis b,prenatal HBV dynamic monitoring,drug intervention and postpartum standard combined with immune blockade are needed to truly achieve zero transmission of hepatitis b from mother to child clinically. |
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