| Objective:To explore the clinical significance of HBsAg and HBsAb coexistence and the possible related mechanism of HBV A1762T/G1764A mutation in serum coexistence of HBsAg and HBsAb.Methods:In the first part of the study,we collected 955 serum samples of chronic HBV-infected patients who visited a hospital in Yunnan province from February 2017 to November 2017 and recorded corresponding clinical information of all patients.Among them,250 patients with coexistence of HBsAg and HBsAb were assigned to an experimental group(double-positive group),and other 705 patients matched chronic HBV-infected patients with negative for HBsAb were a control group(single-positive group).Single Comparative analyses were conducted on the erum virological and biochemical clinical indicators of the two groups.In the second part of the study,we selected 183 patients with chronic HBV infection who visited a hospital in Yunnan Province from February 2017 to November 2017.Among them,97 patients with coexistence of HBsAg and HBsAb and HBV-DNA load more than or equal to 103 IU/ml were the experimental group.86 negative HBsAb patients with HBsAg positive and HBV-DNA load more than or equal to 103 IU/ml served as the control group.The patient's serum was collected and the serum HBV-DNA was extracted.The primers were designed and PCR was used to amplify the BCP region and theP region(overlapped with the S region).The PCR products were sequenced and analyzed.The sequencing results were analyzed using Mega6.0 and Bioedit 7.0.Finally,the SPSS21.0 sofware was used to conduct statistical analyses.The P values were two-sided tests,and there was a statistical difference when P<0.05.The differences in BCP double mutations(A1762T/G1764A dinucleotide substitutions)and surface antigen "a" determinants and serum virological,biochemical and other clinical indicators were compared between the two groups to investigate the possible mechanisms about the coexistence of serum HBsAg and HBsAb in chronic hepatitis B virus infection.Results:1.The first part of the study included 250 patients with coexistence of HBsAg and HBsAb(double-positive group)and 705 negative HBsAb patients with HBsAg positive(single-positive group).There was no statistical difference in age and sex between two groups.2.Clinical analyses showed that the positive rate of HBV DNA in double-positive group was higher than that of single-positive group,and the difference was significant(?2=32.69,P = 0.000).Among the CHB(chronic hepatitis B)patients with ALT>40U/L and ALT>80U/L,there were significantly more patients in double-positive group than in single-positive group(?2=12.86,P<0.001;?2=10.78,P=0.001).However,there was no statistical difference in HBeAg positive rate(?2=32.69,P =0.161).There was no statistically significant difference in DNA(lg)between the two groups patients with HBeAg positive,but there were significant differences in the four liver function indexes of ALT,AST,TBIL and DBIL.Among the HBeAg positive patients,the serum levels of ALT,AST,TBIL and DBIL of patients in the double-positive group were significantly higher than those in the single-positive group(P=0.0002,0.0003,0.0012,0.0002).3.The second part of the study enrolled 97 patients of double-positive group and 86 patients of single-positive group.There was no statistical difference in age and sex between the two groups.Two HBV genotypes were identified in the two groups:genotypes B and C.The major genotypes of double-positive group and single-positive group were C genotype,accounting for 70.1%(66/93)and 60.5%(46/76)respectively,with no statistical significance(?2=2.71,P=0.1).4.Analysis the mutations of "a" epitope determinants with C genotype indicated that the mutation rate of I126T/N/S in double-positive group(38.37%)was significantly higher than that of single-positive group(17.39%),with statistical significance(?2=6.16,P=0.013).The combined analysis of A1762T/G1764A mutations and I126T/N/S showed that the incidence of combination mutations in double-positive group was higher than that in single-positive group,with no statistical significance(?2=3.5,P=0.061).5.Separate analysis of A1762T or G1764A showed that the mutation frequencies of both A1762T and G1764A in double-positive group were significantly higher than that in single-positive group(?2=10.18,P=0.001;?2= 1.55,P=0.001).The combined analysis suggested that the rate of double mutations A1762T/G1764A(34.02%,33/97)in double-positive group was higher than that in single-positive group(15.12%,13/86),and the difference was statistically significant(?2=4.845,P=0.028).6.Combined with biochemical indicators,the serum levels of ALT,AST,TBIL and DBIL of patients with A1762T mutation in the double-positive group were significantly higher than those in patients without mutation(P=0.028,0.0005,0.001,0.044);The levels of AST and TBIL in patients with G1764A mutation were significantly higher than those without mutation(P=0.003,0.019).The ALT,AST,and TBIL indices were significantly higher in patients with A1762T/G1764A mutations than that of non-mutant patients(P=0.0204,0.0009,0.0014).In the single-positive group,there was a significant difference in ALT and AST between the 1762 mutation and non-mutation patients(P=0.041,0.011).Conclusions:1.The positive rate of HBV DNA in double-positive group was higher than that of single-positive group.Among CHB patients with ALT>40U/L and ALT>80U/L,there were significantly more patients in the double-positive group than in single-positive group.It indicated that patients in double-positive group encountered higher HBV activity and inflammation than in single-positive group.2.The mutation rate of the "a" epitope determinants of the C genotype in double-positive group was significantly higher than that of the single-positive group,which suggested that the conexistence of HBsAg and HBsAb was related to the immune escape of the virus.The combination incidence of A1762T/G1764A mutations and I126T/N/S in the double-positive group was higher than that in single-positive group.It is speculated that the linkage of A1762T/G1764A and I126T/N/S may be one of the possible influencing factors of the coexistence of HBsAg and HbsAb.3.Mutation frequencies of A1762T or G1764A alone and A1762T/G1764A in double-positive group were higher than that in the single-positive group,and the mutations were associated with the severe liver function.It suggested that A1762T/G1764A dual mutations may be associated with increased inflammatory response and impaired liver function while promoting the formation of the coexistence of HBsAg and HBsAb. |
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