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Background Chronic carriers of hepatitis B virus (HBV) usually show hepatitis B surface antigen (HBsAg) in their sera, which is considered the marker for acute and chronic HBV infection. however,in some individuals, this antigen can not be detected by routine serological assays despite the presence of virus in liver and peripheral blood, that is, occult hepatitis B virus infection, is defined as HBsAg negative in their serum, HBV DNA positive in serum or liver tissue. This special form of infection may occur in anti-HBs or anti-HBc positive patients,but is also found in individual with all markers of hepatitis B negative. At present, there is not definitive conclusion on the incidence of occult hepatitis B virus infection in different populations, with respect to the incidence in family members of chronic HBV infection patients is also inconsistent.viral repliation and gene expression levels of this particular form of infection were usually very low, but rise to the reasions for this are mostly unclear.Various attempts have been made to clarify the mechanism(s) responsible for this serological pattern. Two possibilities are discussed: either the antigen is indeed absent from the peripheral blood or it is present but not detectable. The first alternative, absence of HBsAg, could be due to mutations that block the export of the antigen, as described for certain pre-S deletions. The second alternative, HBsAg is present but not detectable by standard enzyme immunoassay techniques, could be ascribed to the presence of circulating immune complexes between HBsAg and anti-HBs, which can worsen or even inhibit completely the detection of both the antigen and the antibody. Many studies suggest that low levels of virus replication is the reason for HBsAg negative in peripheral blood,however, others consider that genen mutation,pariticularly S gene mutation of HBV is the main cause for occult hepatitis B virus infection.china is a high incidence of hepatitis B, so we should pay more attention to occult HBV infection.Objectives To evaluate the relationship between the prevalence of occult hepatitis B infection and age,gender, test results of biochemics and HBV markers in family members of chronic hepatitis B infection. At the same time,the first degree relatives with HBsAg positive of occult hepatitis B infection are as controls, from S gene mutation to explain the possible molecular mechanism of occult hepatitis B infection.Methods 1) Collection of cases and sample: the criteria of case selection are, the patients were diagnosed with chronic hepatitis B infection, all family members have not been vaccinated with hepatitis B vaccine. From december 2007 to december 2009 we collected a total of 89 patients with chronic hepatitis B infection at inpatient and outpatient departments of the first affiliated hospital of anhui medical university and Lu’an people’s hospital, asked their history and collected serum samples stored for inspection. 2)Treatment of viral DNA template:the classic proteinase K digestion-phenol-chloroform method was uesed. 3)Primers design:according to literatures home and abroad and HBV reference sequences we designed a pair of nested primers located at S、C and X gene respectively. 4)To check the occult HBV infection by nested PCR, at least two regions were positive, the patient can be defined occult hepatitis B virus infection, and calculate the incidence of occult hepatitis B infection in family members of chronic HBV infection . 5) According to HBV reference sequences we designed a pair of primers in the S gene of HBV for sequencing, and found the possible mutations related to occult hepatitis B virus infection.at the same time, the HBsAg-positive first-degree relatives of occult HBV infection were aslo sequenced,and compared with occult HBV infection. Results 1) In family memebers of 89 patients with chronic hepatitis B infection we found a total of 220 HBsAg-negative subjects, of which 151 were anti-HBs(+), 44 anti-HBc(+), 33 anti-HBs and anti-HBc(+) and 56 anti-HBs and anti-HBc(-). All the 220 HBsAg(-) subjects were detected by nested PCR, of which 5 were positive for three regions of HBV, 20 were positive for two regions of HBV, in 3 patients who were S positive alone. Because in the follow-up sequencing experiment,the 3 subjects are successful, So the incidence of occult HBV infection in family memebers of chronic hepatitis B infection was about 12.7%. 28 occult hepatitis B infection patients were compared with 192 HBsAg negative subjects, we found that HBV markers, age, gender and the results of biochemistry can not distinguish occult HBV infection and non-infected subjects, moreover, we also found that occult HBV infection has a higher incidence in anti-HBc positive subjects, indicating that occult HBV infection has a higher incidence in patients who has a history of HBV infection. 2)A sample was positive for C, X region, so only 27 occult HBV infection patients were sequenced. 2 genetypes (HBV/B, HBV/C) were detected in occult HBV infection patients and controls, in which HBV/B were dominant, but the distribution of genotype is not statistically significant between the two groups (χ2=1.975, P=0.16). By sequencing, we found that some occult HBV infection (29.6%) did not exist mutation in the small S gene, but some have more mutations (≥3, 25.9%),and there are two cases who form terminant codon in advance with a storted amino acid sequence. Compared to occult HBV infection, the majority of controls (90%) have gene mutations, and most of these mutation sites were inconsistent. Moreover,we found that the distribution of gene mutation of B genotypes and C genotypes occult HBV infection were significant differences. In B genotypes, gene mutation of occult HBV infection were more concentrated in the middle of S gene, and the controls focused on both side of S gene, W56L mutation were more common (35.71%)in occult hepatitis B infection. However, in C genotypes, that is different from B genotypes, their genetic variation distributed throughout the small S gene, while the controls are relatively concentrated, I68T (30.77%) and I126T (30.77%) are more common in occult hepatitis B infection.Conclusions The incidence of occult HBV infection in family members of chronic hepatitis B infection patients is about 12.7%, and has a higher incidence(25.0%) in anti-HBc-positive subjects. There are not gene mutations in part of occult HBV infection, and most of controls have gene mutations. The significantly differences of ditribution between the two groups indicated that genetic mutation may relate to occult HBV infection in family members of chronic HBV infection patients.
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