Molecular Characterization Of Occult Hepatitis B Virus Infection In Infants Born To HBsAg Positive Mothers

BackgroundHepatitis B virus (Hepatitis B virus, HBV) infection is one of the most serious diseases all over the world which threatens the health of human’s. According to some reports, there are about 2 billion people nationwide who are infected with HBV, about 350 million people are diagnosed with chronic HBV infection. It is estimated that about 1 million people die each year due to HBV-related diseases caused by HBV infection, such as cirrhosis, liver failure and hepatocellular carcinoma (HCC). Our country is an high endemic area of HBV infection, it is estimated that the number of HBV carriers and chronic hepatitis B patients had reached 93 million and 25 million, respectively according to the 2006 national sero-epidemiological survey. It is obviously that the HBV infection is becoming one of the most important of the three major infectious diseases. Via molecular virology and clinical epidemiology studies, it indicates that HBV infection has been the most important reason for leading to HCC. Over the past decade, occult hepatitis B virus infection (OBI) has been paied more attention to widespread within transfusion medicine and clinical physicians. A latent form of infection is defined as the surface antigen negative, but HBV DNA positive. It has been proved that OBI is important reason for HBV infection in blood transfusion, organ transplantation and hemodialysis. Our country is a high prevalence of hepatitis B virus infection area, chronic hepatitis B infections are mainly and primarily caused by maternal transmission. As neonatal immune system is not yet mature, they are easily lead to be immunological tolerant and probably to be chronic infection with age growth in the development of cirrhosis and liver cancer. At the same time, not only in transfusion medicine OBI is paid attention to but also in MTCT OBI, there is a growing number of countries and researchers focused on this issue. OBI can occur in Anti-HBs/Anti-HBc positive samples, also may occur in Anti-HBs/Anti-HBc negative sample. Diagnosis of OBI depends on the reagent sensitivity of the detection of HBsAg and HBV DNA. Studies have shown that, the most common cause of OBI is mutations of HBV S gene particular region as well as pre-S region mutations, they can affect the expression of HBV protein, leading to HBsAg negative, or causing epitope conformation changes that affect antigen-antibody reaction. So it can be difficult to detect. Other causes includes other variant structures in regions, such as X area variation, pre-C and C mutations. What’s more, HBsAg secretion disorder can result in a large number of retention in the endoplasmic reticulum instead of secretion into the extracellular, as a result of HBsAg-negative. Co-infection with other virus can disturb the appearance of HBsAg such as other hepatotropic virus or HIV, which may also influence each other resulting in HBV replication, showing HBsAg-negative infection status. In either Pre-S/S region a mutation can lead to changes in HBsAg antigenicity and to inhibits Anti-HBs production. The first 124-147 amino acid sequence of this region contains"a"determinant and contains a response of anti-HBs dominant B cell antigen clusters. A replacement mutationion in this region can destroy anti-HBs cycle generation, leading to mutant infection after injected with hepatitis B vaccination, or the occurrence of OBI. In thr early stage of acute hepatitis B infection, hepatitis B virus can be free to form immune complexes. Subsequently, in HBsAg to anti-HBs after turning into immune complex, which indicates that in the serum HBV DNA and anti-HBs immune complex formation will cause OBI.ObjectiveThe purpose of this study is to follow up with infants whose mothers are HBsAg-positive and their mothers to explore the prevalence and characterization of OBI via the perenatal transmission though immuned with high titer hepatitis B immune globulin (Hepatitis B immunoglobulin, HBIG) and hepatitis B vaccine transmission. To provide theoretical guidance of OBI prevention, clinical, development and individualized treatment programs.MethodsIn this study, we collected hepatitis B surface antigen-positive mothers and their infants born at least 3 months or more than 3 months. Then we followed up these infants for a second time blood drawing. All the infants and their mothers samples were collected in department of paediatrics at Nanfang Hospital. And then the samples were using Abbott kit uses chemiluminescence (CMIA) in Nanfang hospital liver center to have HBsAg quantification (<0.05IU/ml is considered negative) and to quantify anti-HBs (<10mIU/ml is considered negative) and HBeAg (S/CO values <lnegative) tests. The quantification detection of anti-HBc was launched at Research Center for Infectious Diseases in diagnostic reagents and vaccine technology Engineering, Xiamen University (NIDVD).All samples were using EIA to test, standards came from the British NIBSC. We apply for the HBeAg antigen time-resolved immunofluorescence kit for quantitative HBeAg sample of the mothers which is produced by Guangzhou Darui company. We use Roche high-purity nucleic acid extraction kit to extract serum HBV DNA nucleic acids, according to the literature, to take the highly sensitive nested PCR and QPCR methods to amplify fragments to obtain HBV DNA, such as PreS/S, S region and BCP/PC area. In amplification of nested-PCR we use Taq enzyme (Takara’s rTaq and La-Taq enzyme). The PCR products were gel purified and cut by Roche recycling kit then sent to life-technology companies for Sanger sequencing, sequencing equipment is ABI3730. Seqman and MEGA6 sequence were applied by sequence analysis software to align and to have evolutionary analysis. We select B genotype, C genotype and D genotype HBV gene sequence from a wild strain of gene database Genbank reference sequence alignment analysis. Then translated into a protein to do the mutation analysis, using phylogenetic methods to obtain the genotype. For suspected samples, we did a T vector cloning and then analyzed. Real-time quantitative application (QPCR) of high sensitivity method in infant and maternal samples to quantify the viral load, the standards came from the British NIBSC, sensitivity is 5IU/ml.Results1. Population selection:The study included 77 infants and children, the age distribution is 3-96 months,92 samples were collected in total, of whom 62 had only one follow-up blood sample and there are 15 children obtained two follow-up blood samples. At the same time,68 serum samples were collected from 62 HBsAg-positive mothers,56 of whom had only one follow-up blood sample,and 6 were conducted for two follow-up blood samples. There are also 6 cord blood samples were collected whose mothers are HBsAg positive. Included mothers median age is 28 years. Hepatitis B vaccination rate of newborns involved in this study was 100%. Vaccination time is within 24 hours after birth, while in the inner 24 hours on different sides of the hips injected with high titer hepatitis B immune globulin (HBIG) for active immunization.2. Serological markers:(1) We found three cases of HBsAg positive infants (3.9%,3/77), the rest infants and children are HBsAg negative; 9 are positive with HBeAg (11.7%,9/77);2 cases are HBsAg negative/HBeAg positive/HBV DNA negative (2.6%,2/77); HBsAg negative/anti-HBs-negative/anti-HBc negative in 4 cases. A total of 11 cases of anti-HBs level are greater than 1000mIU/ml, of whom 10 cases (91%) were drawn blood after 6 months also after the third dose of hepatitis B vaccine to have detection, as well as anti-HBs in less than 6 months,6-12 month, more than 12 months of all ages were 169.8 (57.9-289.7) mIU/ml,387.7 (152.3-1301.2) mIU/ml,142.2 (49.1-498.8) mIU/ml,respectively. A total of74(80%,74/92) baby samples tested positive for anti-HBc. Anti-HBc levels were significantly reduced after 6 months, the level of anti-HBc has fallen to negative since 18 months after the general. (2) A total of 25 cases of mothers in the study (40.3%,25/62) are positive with HBeAg. (3) In 6 cord blood samples,1 case is weakly positive with HBsAg,3 cases are HBeAg positive, anti-HBc are all positive.3. HBV DNA testing:(1) With 89 HBsAg negative samples,20 amplified BCP/ PC segment, there is no 2nd follow-up blood samples to have amplified. The anti-HBs values are positive (>10mIU/ml), while there are 4 of the samples are positive for HBeAg (>1 S/CO).87 mutations were found,3 cases have A1762T/G1764A double mutations,8 cases have G1896A, but the8 cases are negative with HBeAg, and we also found G1752A, A1846T, T1858C mutations. (2) In HBsAg negative samples, there are 13 samples to amplify gene S, again without a 2nd follow-up blood sample, there are 8 cases simultaneously amplified BCP/PC and S 2 fragments. Anti-HBs are all positive (> 10mlU/ml), Anti-HBc are all positive, too.3 cases are HBeAg positive. No significant V39A, P41H, E64K, G145R mutation was found. Of S fragments were found in 8 samples amino acid mutations were G45E, T139L, T47A, S53L, C76S, Q101R, T125M and P127T. (3) HBV DNA positive samples (viral load, viral load, VL> 5IU/ml) in 92 samples detected in HBsAg-negative is 13 cases by QPCR, anti-HBs also are positive (>10 mIU/ml) and HBeAg are negative (<1 S/CO), there are 4 cases that BCP/PC, S and VL were positive, including BCP/PC or S-positive and VL-positive in 5 cases. (4) Of 6 cord blood samples,1 is positive with S fragment by nested PCR. (5) S gene sequence alignments between babies and mothers:3 cases of HBsAg positive samples are analyzed, of B75, there are 4 bases are different of S fragment with his mother’s, namely 357,381,480,636, resulting in 4 amino acids are inconsistent, respectively T68I, Y76C, Q109L, Y161F. The B74 and B76’s S fragment of their mothers are exactly the same.13 cases amplified by nested PCR to get S fragment analysis of 10 cases are genotype B,2 are genotype C,1 D genotype. While 12 pairs get the appropriate sequence. By alignment we found 6 pairs of high maternal sequence consistency,they have the same genotype, but the other six pairs of mother and child sequence does not match with each other, even in different genotype.ConclusionsTo conclude the survey in Nanfang hospital in Guangzhou, the HBsAg positive rate of newborns whose mothers are also positive is 3.9%(3/77), HBeAg-positive rate is 11.7%(9/77), the HBV DNA positive ratio is 36.3%(28/77). Because none of the 2nd follow-up blood samples had HBV DNA. In this study, all HBsAg negative infants with HBV DNA positive are defined as transient OBI. No persistant OBI is found. The hepatitis B vaccine and HBIG combination is very effective at current clinical condition to prevent more than 96% HBV transmission between HBsAg positive mothers to babies in China.