Dynamic comparison between Daan real-time PCR and Cobas TaqMan for quantification of HBV DNA levels in patients with CHB.

BACKGROUND: Hepatitis B virus (HBV) DNA levels are crucial for managing chronic hepatitis B (CHB). It was unclear whether Daan real-time polymerase chain reaction test (Daan test) or COBAS TaqMan HBV DNA Test (Cobas TaqMan) was superior in measuring different HBV DNA levels in clinical specimens. METHODS: We enrolled 67 treatment-naïve, HBV surface antigen-positive CHB patients (high baseline viral levels) who received either lamivudine/adefovir or entecavir. Serum samples were tested at baseline and treatment week 24 using the Daan test and Cobas TaqMan. RESULTS: In the 67-baseline samples, the HBV DNA levels with the Cobas TaqMan (7.90 ± 0.73 log10 IU/mL) were significantly greater than those of the Daan test (7.11 ± 0.44 log10 IU/mL; P < 0.001). Of the 67 24-week samples (low viral levels), the Cobas TaqMan detected 59 (88.1%; 8 undetected); the Daan test detected 33 (49.3%; 34 undetected; P < 0.001). The Cobas TaqMan detected HBV DNA in 26 of 34 samples undetectable by the Daan test (range, 1.4-3.7 log10 IU/mL) or 38% of samples (26/67). The reductions in viral load after 24 weeks of oral antiviral treatment in the 33 samples that were positive for both the Daan test and the Cobas TaqMan test were significantly different (3.59 ± 1.11 log10 IU/mL versus 4.87 ± 1.58 log10 IU/mL, respectively; P = 0.001). Spearman correlation analysis showed positive correlation between results from two tests (rp = 0.602,P<0.001). The HBV genotypes and the anti-viral treatment did not affect the measurements of the HBV DNA by the Daan assay and the Cobas Taqman assay. CONCLUSION: The Cobas Taqman was more sensitive at low viral loads than the Daan test and the change from complete to partial virological response could affect clinical decisions. The Cobas Taqman may be more appropriate for detection of HBV DNA levels.

[A study on the situations of human L02 hepatocytes in genetically tolerized immunocompetent rats].

OBJECTIVE: To investigate whether human L02 hepatocytes could survive after implanting them into normal, immunocompetent rats. METHODS: Human L02 hepatocytes were injected through the uterine walls into the intraperitoneal cavities of fetal Sprague-Dawley rats to induce immune tolerance to human L02 hepatocytes. Human L02 hepatocytes stained with DiI were implanted into the spleens of the 2-week old rats. Immuno-fluorescent staining, SP immunohistochemistry, and DiI staining were used to detect human albumin and specific proliferating cell nuclear antigen (PCNA) in the rat livers. The distribution of human L02 hepatocytes was observed under the fluorescent microscope. RESULTS: Dynamic distribution of human L02 hepatocytes in the rat livers was observed from the 1st to the 10th week after the implantation. Human albumin was detected at 2, 4, 6 and 8 weeks, and at the 4th week it had the highest level. Specific human PCNA was detected in the rat livers from the 2nd to the 6th week after implantation. The PCNA positive cells were most abundant at the 4th week. CONCLUSION: Human L02 hepatocytes can survive and proliferate for 10 weeks after implanting them into genetically normal immunocompetent rats.

Association between HLA class II gene and susceptibility or resistance to chronic hepatitis B.

AIM: To investigate the association between the polymorphism of HLA-DRB1, -DQA1 and -DQB1 alleles and viral hepatitis B. METHODS: HLA-DRB1, -DQA1 and -DQB1 alleles in 54 patients with chronic hepatitis B, 30 patients with acute hepatitis B and 106 normal control subjects were analyzed by using the polymerase chain reaction/sequence specific primer (PCR/SSP) technique. RESULTS: The allele frequency of HLA-DRB1*0301 in the chronic hepatitis B group was markedly higher than that in the normal control group (17.31% vs 5.67%), there was a significant correlation between them (chi2=12.3068, Pc=0.0074, RR=4.15). The allele frequency of HLA-DQA1*0501 in the chronic hepatitis B group was significantly higher than that in the normal control group (25.96% vs 13.68%), there was a significant correlation between them (chi2=9.2002, Pc=0.0157, RR=2.87). The allele frequency of HLA-DQB1*0301 in the chronic hepatitis B group was notably higher than that in the normal control group (35.58% vs 18.87%), there was a significant correlation between them (chi2=15.5938, Pc=0.0075, RR=4.07). The allele frequency of HLA-DRB1*1101/1104 in the chronic hepatitis B group was obviously lower than that in the normal control group (0.96% vs 13.33%), there was a significant correlation between them (chi2=11.9206, Pc=0.0145, RR=18.55). The allele frequency of HLA-DQA1*0301 in the chronic hepatitis B group was remarkably lower than that in the normal control group (14.42% vs 30%), there was a significant correlation between them (chi2=8.7396, Pc=0.0167, RR=0.35). CONCLUSION: HLA-DRB1*0301, HLA-DQA1*0501 and HLA-DQB1*0301 are closely related with susceptibility to chronic hepatitis B, and HLA-DRB1*1101/1104 and HLA-DQA1*0301 are closely related with resistance to chronic hepatitis B. These findings suggest that host HLA class II gene is an important factor determining the outcome of HBV infection.

[Construction of the expression vectors of HDV ribozymes and their intracellular inhibiting activity against HCV RNA].

OBJECTIVES: To investigate whether HDV ribozymes can intracellularly inhibit HCV RNA. METHODS: The mammalian expression vectors, pC1-RzC1, pC1-RzC2 and pC1-RzC3, containing ribozymes cDNA of RzC1, RzC2, and RzC3, were constructed targeting different HCV-5' NCR-C RNA regions. Then the HCV-positive fetal hepatocytes were transfected with these plasmids using liposome-mediated method. The inhibitory effects of HDV ribozymes were evaluated by HCV RNA quantitation in cultured cells and the supernatants. RESULTS: (1) All the three HDV ribozymes were inserted into the expression vector. (2) Fetal hepatocytes were infected with HCV proven by RT-PCR and fluorescent quantitative PCR and expressed HCV NS3 and NS5 antigens by immunocytochemistry. (3) HDV ribozymes inhibited the activity of the target HCV RNA at expect positions in HCV-positive hepatocytes. At 0.5 micromol/L, the inhibitory rate of pC1-RzC1, pC1-RzC2, and pC1-RzC3 was 53.2%, 50.5 %, and 10.6% respectively. PC1-RzC1 was used continuously for one week, showing the inhibitory rate of 60.7%, 64.2%, 68.4%, 71.9%, 78.8% and 83.1% on the 2nd, 3rd, 4th, 5th, 6th and 7th day. CONCLUSION: The inhibitory activity of pC1-RzC1 (107-113nt) and pC1-RzC2 (268-274nt) is greater than that of pC1-RzC3 (345-351nt) in HCV-positive hepatocytes.

[Study on the polymorphisme of human leucocyte antigen-DRB1, -DQA1 and -DQB1 alleles in patients with hepatitis B].

OBJECTIVE: To investigate the association between the polymorphism of human leucocyte antigen (HLA)-DRB1, -DQA1 and -DQB1 alleles and viral hepatitis B. METHODS: HLA-DRB1, -DQA1 and -DQB1 alleles in 52 patients with chronic hepatitis B, 30 patients with acute hepatitis B and 106 normal control subjects were analysed, using the polymerase chain reaction/sequence specific primer (PCR/SSP) technique. RESULTS: The allele frequencies of HLA-DRB1 * 0301, -DQA1 * 0501 and -DQB1 * 0301 in the chronic hepatitis B group (17.31%, 25.96%, 35.58%) were markedly higher than that in the normal control group (5.67%, 13.36%, 18.87%), with statistical significance (chi(2)(1) = 12.3068, P(c1) = 0.0074; chi(2)(2) = 9.2002, P(c2) = 0.0157; chi(2)(3) = 15.5938, P(c3) = 0.0075). The allele frequencies of HLA-DRB1 * 1101/1104 and -DQA1 * 0301 in the chronic hepatitis B group (0.96%, 14.42%) were markedly lower than that in the acute hepatitis B group (13.33%, 30%), with significant correlation between them (chi(2)(1) = 11.9206, P(c1) = 0.0145; chi(2)(2) = 8.7396, P(c2) = 0.0167). CONCLUSION: HLA-DRB1 * 0301, -DQA1 * 0501 and -DQB1 * 0301 were closely associated with the susceptibility to chronic hepatitis B, while HLA-DRB1 * 1101/1104 and -DQA1 * 0301 closely associated with the resistance to chronic hepatitis B. These findings suggested that host HLA class II gene was an important factor determining the outcome of HBV infection.