Molecular Epidemiological Analysis Of Occult Hepatitis B Virus Infection In Hemodialysis Patients

Objectives To Investigate the prevalence of occult HBV infection among HBsAg-negative isolated from renal patients undergoing hemodialysis. To evaluate the correlation between gender, age, HBV serological markers, HCV status, time of dialysis etc. and the prevalence of occult HBV infection among hemodialysis patients. At the same time,the hemodialysis patients with HBsAg-positive are sellected as controls at random to explain the possible molecular mechanism of occult hepatitis B infection from S gene mutation.Methods①Collection of cases and sample: From december 2009 to april 2010 sera of 102 HBsAg-negative hemodialysis patients who attend some dialysis units in HeFei were collected. The hemodialysis patients with HBsAg-positive were sellected as controls at random.②The quantitiative of HBV serological markers were tested with Abbott Architect I 2000R electrochemiluminescence instrument.③Treatment of HBV DNA:The classic proteinase K digestion-phenol-chloroform method was uesed.④To check the occult HBV infection by nested PCR: according to literatures home and abroad as well as HBV reference sequences we designed a pair of nested primers located at S,C and X gene respectively. At least two regions were positive, the patient can be defined as occult HBV infection. Comparing gender, age, HBV serological markers, HCV status, time of dialysis etc. of patients with occult HBV infection, with that of patients without occult HBV infection.⑤According to HBV reference sequences we designed a pair of primers in the S gene of HBV for amplifying and sequencing to find the possible mutations related to occult HBV infection and analyze genotype and mutations in"a"determinant of HBsAg.At the same time, the HBsAg-positive hemodialysis patients were aslo sequenced and compared with occult HBV infections.Results①The HBV serological markers of 102 HBsAg-negative sera that detected by ELISA were confirmed by ECLIA. After detected by nested PCR we found 8 were positive for two regions of HBV at least.It means HBV DNA was detected in 8 samples among the whole 102 cases. The prevalence of occult HBV infection among HBsAg-negative hemodialysis patients was 7.8%.②8 occult HBV infection patients were compared with 94 HBsAg negative isolates.We found that gender, age and the time of dialysis can not distinguish between occult HBV infection and non-infected isolates. The incidence of occult HBV infection was 16.7% in HCV Ab-positive hemodialysis patients while was 6.7% in HCV Ab-negative hemodialysis isolates. There were no significant differences in statistics of HCV status, HBV serological markers between patients with and without occult HBV infection, with an exception of anti-HBc-only serological marker. In hemodialysis patients, the incidence of occult HBV infection in anti-HBc-only(30.8%,4/13) was significantly higher than other patterns of serological marker(4.5%,4/89)(χ~2 = 7.504,P<0.01), indicating that occult HBV infection was more prone to find in patient who has anti-HBc-only serological marker.③2 of 6 occult HBV infection isolates who were successfully sequenced were genotype B and the other 4 were genotype C while 7 HBsAg-positive hemodialysis patients were all genotype B.The two group's homology of nucleotide and amino acid were analyzed by comparison with HBV reference sequences respectively.The mutation rate of S region in occult HBV infections was from 0.15% to 4.26% at DNA level while it was from 0.73% to 1.17% in HBsAg-positive controls. At the aa level the mutation rate of S region was from 0.44% to 9.73% in occult HBV infections while from 1.77% to 2.65% in HBsAg-positive controls.④Specific aa substitutions happened in aa124-aa147 in 3 samples of 6 occult HBV infections. 2 joint mutation were found in C genotype. One was Q129P/T131N/S143T/G145S, the other was Q129P/T131N/D144V/G145S. However, there was no mutation in"a"determinant sequences of 7 HBsAg-positive hemodialysis patients.Conclusions①In this geographic area the incidence of occult HBV infection in HBsAg-negative hemodialysis patients is about 7.8% and has a higher incidence(30.8%) in patients with isolated anti-HBc.②There is significant correlation between occult HBV infection and the presence of isolated anti-HBc while HCV positivity is not a contributing factor to occult HBV infection in hemodialysis patients. Testing for probable occult HBV infection from hemodialysis patients, especially from the patients with isolated anti-HBc have important clinical significance.③The mutation in S region, especially in"a"determinant of HBsAg may be the molecular basis of occult HBV infection.