Molecular Biological Study On Mother-to-Infant Transmission Of Hepatitis B Virus

Objective Chronic hepatitis B virus (HBV) infection remains one of the most severe global public health problems. Globally, there are over 350 million carriers of HBV. In areas where persistent HBV infection is endemic, vertical transmission of HBV is the major reason for high HBV carrying rate. The aim of our study is to explore the molecular biological mechanism on mother-to-infant transmission of hepatitis B virus (HBV). It was carried out through three aspects: ① To research the relationship between HBV carrying status of pregnant women and mother-to-infant transmission. ② To study preliminary on HBV genotypes of pregnant women and mother-to-infant transmission. ③ To investigate weather or not breast feeding was feasibility and its role in the transmission of HBV to infants born to HBV carring mothers if immunization is carried out.Methods Enzyme-linked immunosobent assay (ELISA) was used to detect the HBV-makers including hepatitis B surface antigen (HBsAg) , hepatitis B surface antibody (anti-HBs), hepatitis B e antigen (HBeAg) , hepatitis e antibody (anti-HBe) and hepatitis B core antibody (anti-HBc) in blood of pregnant women who were attended prenatal care at Qilu Hospital of Shangdong University, Jinan Maternity and Infant Health Institute of Shangdong province in the first or the second trimester of pregnancy from September 2001 through November 2003, serum alanine transaminase (ALT) levels were examined simultaneously. If the gravida's HBsAg waspositive and ALT normal, HBV deoxyribonucleic acid (HBV DNA) was detected by fluorescence quantitative polymerse chain reaction (FQ-PCR) , other hepatitis markers including hepatitis A virus (HAV), hepatitis C virus (HCV), hepatitis D virus (HDV) and hepatitis E virus (HEV) were tested by ELISA with permission. The gravidas whose other hepatitis markers were normal and whose husband's HBV-makers was negative were recruited into the research. HBV-makers and ALT were tested again when the women were admitted to the hospitals for delivery. Umbilical blood HBV DNA was detected at birth. All recruiting gravidas were accorded with the asymptomic HBV carrier diagnosing criteria made at the country hepatitis symposium in 2000. The newborns whose gestational age equals or less than 37 weeks, birthweight less than 2500 grams or whose mothers had the histories of threstened abortion or antepartum hemorrhage were excluded. Women whose HBV DNA and their baby's umbilical blood DNA were positive were included as a studying group; while ones whose baby's umbilical blood DNA was negative as a control group. HBV genotypes were detected by PCRmicroplate sandwich hybridization—ELISA.All babies were administered hepatitis B immunogluobin within 12 hours after birth and at 14 days of age. The hepatitis B recombinant vaccine was given within 24 hours after birth and at 1 and 6 months of age. Infants were then followed up to 7~12 months of age and tested for HBV-makers and HBV DNA. Information on breast-feeding and its duration was affirmed. Infants who were breast-fed more than one month were included as a breast-feeding group; while ones who were never breast-fed as a bottled-feeding group; breast-fed less than one months were excluded.The presence of HBsAg and/or HBV DNA indicated HBV infection and immunoprophylaxis failure. Uninfected infants with negative anti-HBs also indicated immunoprophylaxis failure and were given repeat vaccine.Results1. HBV DNA was all positive in pregnant women with HBsAg, HBeAg and anti-HBc positive, the positive rate was 100% (49/49); HBV DNA positive rate was also 100% (16/16) in ones with HBsAg and HBeAg positive; 54.17% (13/24) incases with HBsAg, anti-HBc positive; HBV DNA positive rate was lower in ones with HBsAg, anti -Hbe and anti-HBc, it was 4.05% (3/74) . No one was HBV DNA positive in HBsAg or HBsAg and anti-HBe positive ones.2. The positive rate of umbilical blood HBV DNA was highest for the newborns whose mothers were HBsAg, HBeAg, anti-HBc, it is 18.37%(9/49). The positive rate of umbilical blood HBV DNA was 12.50%(2/16) for ones whose mothers were HBsAg, HBeAg positve; 12.50% (3/24) for HBsAg, anti-HBc positive; only 1.33 % (1/75 ) for HBsAg, anti-HBe, anti-HBc positive. No one was HBV DNA positive in HBsAg or HBsAg and anti-HBe positive ones. There was significant difference in positive rates of umbilical blood HBV DNA the two groups between HBsAg, HBeAg, anti-HBc positive and HBsAg, anti-HBe, anti-HBc positive groups (x2=9.415> P=0.002 ) . There was significant difference in positive rates of umbilical blood HBV DNA between HBsAg, anti-HBc positive and HBsAg, anti-HBe, anti-HBc positive groups. There were no significant differences in the others.3. 11 in 15 babies whose umbilical blood HBV DNA was positive were born to the mothers who were HBeAg positive, the positive rate was 16.92% (11/65), only 4 born to the mothers who were HBeAg negative, the positive rate was 3.77% (4/106). There was significant difference in positive rate of umbilical blood HBV DNA between infants whose mothers were HBeAg positive or negative (x2 =8.706 > P=0.003).4. All babies whose umbilical blood HBV DNA was positive were born to the women who were positive for HBV DNA, no one was born to mothers who were negative for HBV DNA. There was significant difference in positive rate of umbilical blood HBV DNA between infants whose mothers were HBV DNA positive or negative groups (x2 =18.269, P=0.001). The positive rate of umbilical blood HBV DNA was increased following the mother's HBV DNA rise. The positive rate of umbilical blood HBV DNA in the group whose mother's HBV DNA was 1.00xl08~5.87xl09 is higher than that in the group whose mother's HBV DNA was less than l.OOxlO6. There was significant difference between the two groups(x2=27.522, P=0.001) .5. Mother-to-infant transmission of HBV occurred only to mothers whose HBV DNA was positive when followed-up babies to 12 months. The vertical transmission rate was 16.36 % (9/55 ) in HBV DNA positive mothers.6. Of the 170 HBsAg carrying pregnant women, 81 ones were HBV DNA positive. Of the 81 cases, 32 ones (39.51 %, 32/81) were mixed type of HBV genotype C and D, 31 (38.27 %, 31/81) were genotype C. 8 (9.88 %, 8/81) were mixed type of HBV genotype B and C, 6 (7.41%, 6/81) were genotype D, 2 (2.47%, 2/81) were mixed type of HBV genotype B and D. There was no statistical significance in HBV genotype between gravidas who transmitted HBV to their neonates and did not transmitted HBV to their neonates.7. The rate of mother-to-infant transmission was higher in mixed types. 9 of 32 mothers who were mixed genotype C and D transmitted HBV to their neonates, while only 3 of 31 ones who were genotype C transmitted HBV to their neonates. There was increasing tendency of mother-to-infant transmission in pregnant women infected with mixed types although there was no statistical significance. Mother and infant had the same HBV genotype.8. Two infants of the 171 babies (one twin) were excluded because breast-fed last only two weeks. One infant who became HBV DNA positive was excluded the study in braest-fed group because he failed to complete whole immunoprophylaxis. The infant's mother was HBV DNA positive. 119 infants had been followed up to 12 months, 76 breast-fed infants and 43 bottle-fed infants. There were no differences in newborn's gestational age and birthweight between the two groups. The positive rates of maternal blood HBV DNA and umbilical blood HBV DNA were comparable between the two groups. The positive rates of HBV DNA of the followed-up infants at the age of 7 and 12 months were 6.76 % (5/74) and 6.58 % (5/76 ) in braest-fed group, 6.98% (3/43) and 6.98% (3/43) in bottle-fed group. There were all no differences between the two groups (P= 1.000). The positive rates of anti-HBs of the followed-up infants at the age of 7 and 12 months were 87.84 % (65/74) and 93.42% (71/76)in breast-fed group, 86.05% (37/43) and 93.02%(40/43) in bottle-fed group. There were all no differences between the two groups. (P =0.338 and 1.000 respectively)9. Four (5.41%) breast-fed infants and three (6.98%) bottle-fed ones were both HBV DNA and anti-HBs negative at the age of 7 month. All was given additional vaccine and seroresponded at the age of 12 month. The difference was not significant Cx2=0.119, P=0.707).Conclusions1. The HBV carrying status of the pregnant women is associated with mother-to-infant transmission. It is one of the important factors for vertical transmission that the serum HBeAg and/or HBV DNA positive in pregant women. HBV DNA positivity can reflects fatalness of maternal infant transmission more accurate than HBeAg positive does. The higher the HBV DNA load, the more dangerous for maternal infant transmission.2. There are no infants born to mothers who were negative for HBV DNA infecting HBV. Although we cannot conclusively say that there are no risk of mother-to-infant transmission for mothers whose HBV DNA was negative, our results indicate that the risk of mother-to-infant transmission is less in women who HBV DNA was negative.3. With appropriate immunoprophylaxis, combining passivity and active immunity, no babies were infected HBV after delivery in spite of their mothers' HBV status. Maternal infant transmission occurs mainly in intra-uterus. Although only one infant who failed to complete whole immunoprophylaxis became HBV DNA positive after delivery, it also states that HBV may be transmitted through osculation living contact. The most important thing is to follow-up the infants who born to HBV carrying mothers and to make sure their immune status. Follow-up the infants can not only know the vaccine administering condition to avoid HBV infection due to failing to complete whole immunoprophylaxis, but also detect immunoprophylaxis failure as early as possible.4. The immunoprophylaxis failure is not only due to intrauterine transmission of HBV, but also due to infant's response to HBV vaccine, the later can been improved through additional doses of vaccine.