Clinical and molecular characterization of Wilson's disease in China: identification of 14 novel mutations.

BACKGROUND: Wilson's disease (WND) is a rare autosomal recessive disorder. Here we have evaluated 62 WND cases (58 probands) from the Chinese Han population to expand our knowledge of ATP7B mutations and to more completely characterize WND in China. METHODS: the coding and promoter regions of the ATP7B gene were analyzed by direct sequencing in 62 Chinese patients (58 probands) with WND (male, n = 37; female, n = 25; age range, 2 ~ 61 years old). RESULTS: neurologic manifestations were associated with older age at diagnosis (p < 0.0001) and longer diagnostic delay (p < 0.0001). Age at diagnosis was also correlated with urinary copper concentration (r = 0.58, p < 0.001). Forty different mutations, including 14 novel mutations, were identified in these patients. Common mutations included p.Arg778Leu (31.9%) and p.Pro992Leu (11.2%). Homozygous p.Arg778Leu and nonsense mutation/frameshift mutations were more often associated with primary hepatic manifestations (p = 0.0286 and p = 0.0383, respectively) and higher alanine transaminase levels at diagnosis (p = 0.0361 and p = 0.0047, respectively). Nonsense mutation/frameshift mutations were also associated with lower serum ceruloplasmin (p = 0.0065). CONCLUSIONS: we identified 14 novel mutations and found that the spectrum of mutations of ATP7B in China is quite distinct from that of Western countries. The mutation type plays a role in predicting clinical manifestations. Genetic testing is a valuable tool to detect WND in young children, especially in patients younger than 8 years old. Four exons (8, 12, 13, and 16) and two mutations (p.Arg778Leu, p.Pro992Leu) should be considered high priority for cost-effective testing in China.

Sustained response of hepatitis B e antigen-negative patients 3 years after treatment with peginterferon alpha-2a.

BACKGROUND & AIMS: Patients with hepatitis B e antigen (HBeAg)-negative chronic hepatitis B treated with peginterferon alpha-2a with or without lamivudine achieve significantly higher 6-month posttreatment rates of response compared with those treated with lamivudine alone. The durability of

Characterization of hepatitis B virus reverse transcriptase sequences in Chinese treatment naive patients.

BACKGROUND AND AIMS: The hepatitis B virus (HBV) reverse transcriptase (RT) plays an important role in viral replication. The aim of the present study was to characterize profiles of the RT region and to construct a database for further studies. METHODS: Serum samples were obtained from 328 treatment naive patients chronically infected with HBV in five Chinese cities. Mutation status, genotypes and deep sequence analysis were carried out by amplifying and sequencing the RT region. RESULTS: The base usage in the RT region differed at the mono- and dinucleotide level and thymidine dominated. The higher the variability of the strain was, the more it replicated. No significant clustering was found between our HBV RT sequences and those isolated 10 years ago (achieved from genebank). Nucleotide analogue resistance related mutants exist. The M204V/I mutation was found in 1.8% of the strains, 1.2% had L180M+ M204V/I, 0.6% had A181T/V, and only one had all three mutations. Minor strain mutants were found in 9.3% of the samples studied. The genotype B patients made up 36.6% (88.7% B2) and were mostly found in southern China, 63.4% (92.2% C2) were genotype C, and only one was genotype D. The average age of HBeAg positive genotype B patients was 29.5 +/- 10.4 years, for genotype C it was 36.1 +/- 10.9 (P < 0.001). CONCLUSION: Primarily antiviral resistance related mutant strains do exist in treatment naïve patients. Without antiviral pressure, HBV strains evolved at a normal speed. In depth sequence analysis implied that viral replication might be correlated with its variability, which needs to be further investigated.

Assessment of specific antibodies to F protein in serum samples from Chinese hepatitis C patients treated with interferon plus ribavarin.

The hepatitis C virus (HCV) alternate reading frame protein or F protein of the HCV 1b genotype is a double-frameshift product of the HCV core protein. In order to assess the presence of antibodies specific for F protein and their clinical relevance in sera from HCV patients, we produced recombinant F protein and core protein of the HCV 1b genotype in Escherichia coli. An enzyme-linked immunosorbent assay was developed using purified recombinant HCV core, F protein, and a 99-residue synthetic F peptide (F99). The seroprevalences of anticore, anti-F protein, and anti-F99 synthetic peptide were 95%, 68%, and 36%, respectively, in 168 HCV patients. The prevalence of anti-F antibodies did not correlate with viral load, genotype, or alanine aminotransferase level. Interferon combination therapy induced a decline in the level of anti-F antibodies in 55 responders (P < 0.01). Thirteen responders (24%) lost their anti-F recombinant protein antibodies, and 17 (31%) lost their anti-F synthetic peptide antibodies, whereas no decrease was observed for the 17 nonresponders. These changes were significant between responders and nonresponders (P < 0.05). Meanwhile, no change was found in the anticore antibody titer of the 72 treated patients. The percentage of anti-F-protein-negative patients (15/15 [100%]) who achieved a sustained virological response (SVR) was higher than that of the anti-F-positive patients (70%) (P < 0.05). Based on these findings, HCV F protein elicits a specific antibody response other than the anticore protein response. Our data also suggest that the presence and level of anti-F antibody responses might be influenced by the treatment (interferon plus ribavirin) and associated with an SVR in Chinese hepatitis C patients.

MxA induction may predict sustained virologic responses of chronic hepatitis B patients with IFN-alpha treatment.

The objective of this study was to find potential biomarkers for predicting sustained virologic responses to interferon-alpha (IFN-alpha) treatment in chronic hepatitis B (CHB) patients. A total of 101 CHB patients were treated with pegylated IFN-alpha2a for 48 weeks and followed up for 24 weeks, including 34 IFN responders (IFN-Rs) and 67 IFN nonresponders (IFN-NRs). After peripheral blood mononuclear cells (PBMCs) and Epstein-Barr virus-transferred B (EBV-B) cell lines were treated with different concentrations of IFN-alpha in vitro, activated IFN-stimulated gene factor3 (ISGF3) and IFN-gamma-activation factor (GAF) were measured by EMSA, and MxA, OAS1, and PKR mRNA were measured by real-time PCR. Polymorphisms in the MxA promoter were genotyped to find the possible association. IFN-alpha-activated ISGF3 and GAF levels were similar between IFN-NRs and IFN-Rs. However, MxA mRNA induction in IFN-Rs was higher than that in IFN-NRs, and such discrepancy increased when highly concentrated IFN was used to stimulate. The OAS1 and PKR mRNA induction have a similar pattern between IFN-Rs and IFN-NRs. In addition, frequency of the MxA-88G/T genotype was significantly different between IFN-Rs and IFN-NRs, and this polymorphism was also functional because MxA mRNA induction in patients with GG genotype was lower than those with GT genotype. Regression analysis showed that MxA mRNA induction after 10,000 IU/mL IFN stimulation could serve as an independent factor for predicting IFN-alpha, with an area under curve (AUC) of 0.838, a positive predictive value of 68% for IFN-Rs, and a negative predictive value of 89% for IFN-NRs. MxA mRNA induced by IFN-alpha might predict sustained virologic responses to IFN-alpha treatment in CHB patients.

Peginterferon alfa-2a alone, lamivudine alone, and the two in combination in patients with HBeAg-negative chronic hepatitis B.

BACKGROUND: Available treatments for hepatitis B e antigen (HBeAg)-negative chronic hepatitis B are associated with poor sustained responses. As a result, nucleoside and nucleotide analogues are typically continued indefinitely, a strategy associated with the risk of resistance and unknown long-term safety implications. METHODS: We compared the efficacy and safety of peginterferon alfa-2a (180 microg once weekly) plus placebo, peginterferon alfa-2a plus lamivudine (100 mg daily), and lamivudine alone in 177, 179, and 181 patients with HBeAg-negative chronic hepatitis B, respectively. Patients were treated for 48 weeks and followed for an additional 24 weeks. RESULTS: After 24 weeks of follow-up, the percentage of patients with normalization of alanine aminotransferase levels or hepatitis B virus (HBV) DNA levels below 20,000 copies per milliliter was significantly higher with peginterferon alfa-2a monotherapy (59 percent and 43 percent, respectively) and peginterferon alfa-2a plus lamivudine (60 percent and 44 percent) than with lamivudine monotherapy (44 percent, P=0.004 and P=0.003, respectively; and 29 percent, P=0.007 and P=0.003, respectively). Rates of sustained suppression of HBV DNA to below 400 copies per milliliter were 19 percent with peginterferon alfa-2a monotherapy, 20 percent with combination therapy, and 7 percent with lamivudine alone (P<0.001 for both comparisons with lamivudine alone). Loss of hepatitis B surface antigen occurred in 12 patients in the peginterferon groups, as compared with 0 patients in the group given lamivudine alone. Adverse events, including pyrexia, fatigue, myalgia, and headache, were less frequent with lamivudine monotherapy than with peginterferon alfa-2a monotherapy or combination therapy. CONCLUSIONS: Patients with HBeAg-negative chronic hepatitis B had significantly higher rates of response, sustained for 24 weeks after the cessation of therapy, with peginterferon alfa-2a than with lamivudine. The addition of lamivudine to peginterferon alfa-2a did not improve post-therapy response rates.

[Factors associated with HBeAg seroconversion in chronic hepatitis B patients treated with interferon alfa-2a].

OBJECTIVE: The aim of this paper was to investigate the factors associated with viral response and HBeAg seroconversion and the relationship between them at different stages of interferon treatment in HBeAg-positive chronic hepatitis B patients. METHODS: PEG-IFN alfa-2a was injected subcutaneously in doses of 180 microg once a week for 48 weeks to HBeAg-positive chronic hepatitis B patients, and the patients were followed for another 24 weeks after the treatment. The serum HBV DNA load was measured by real-time quantitative PCR assay. Microparticle enzyme immunoassay analysis (MEIA) was then carried out by an automatic enzyme immunoassay analysis instrument to measure HBeAg and anti-HBe. Virological response and HBeAg seroconversion rates, and the factors associated with them were analyzed. RESULTS: The differences in ALT baselines between viral responding and non-responding groups were significant at treatment time and at the end of the follow-up period. These differences were also significant in patients with HBeAg seroconversion at 12 weeks and at the end of the follow-up period compared with the non-conversion group. No significant difference of HBV DNA baseline was observed between the HBeAg seroconversion and non-conversion group. At 12, 24 and 48 weeks, in patients with viral response during the treatment, their HBeAg seroconversion rates were 43.8%, 21.4% and 18.9% respectively; their respective HBeAg seroconversion rates remaining at 72 weeks were 42.9%, 33.3% and 27.6%. HBeAg seroconversion was related to HBV DNA negativity at 48 weeks treatment in the multivariate analysis (OR=2.15, 95.0% CI=1.744-2.664, P less than 0.01). CONCLUSIONS: Viral response and early and sustained HBeAg seroconversion were associated with pretreatment ALT levels. HBeAg seroconversion was related to viral response during IFN treatment, but not to the baseline HBV DNA load.

[A double-blind, randomized, lamivudine-controlled clinical trial of DAIDING (adefovir dipivoxil) for lamivudine-resistant patients with chronic hepatitis B].

OBJECTIVE: To investigate the efficacy and safety of adefovir dipivoxil (ADV, DAIDING) for Chinese chronic hepatitis B patients with lamivudine (LAM) resistance. METHODS: This study was a multicenter, double-blind clinical trial. 209 chronic hepatitis B patients with LAM resistance were randomly put in an ADV, DAIDING or a LAM group. After 24 and 48-weeks of treatment, serum HBV DNA levels were measured by quantitative PCR and liver function tests; HBV serology and safety assessments were also conducted. RESULTS: The mean reduction of HBV DNA from baseline at 24 and 48 weeks was significantly greater in the ADV group compared with that in the LAM group (2.40 log10 vs 0.94 log10, P < 0.01; 2.71 log10 vs 1.07 log10, P < 0.01). In the ADV group, the virological response and ALT normalization at 24 and 48 weeks were significantly higher than those in the LAM group. There was no significant difference between the two groups in the portion of HBeAg reduction, HBeAg seroconversion and incidence of adverse events. There was no severe adverse event related to the investigational product, DAIDING, in this trial. CONCLUSION: DAIDING (ADV) is effective and safe for the treatment of chronic hepatitis B patients with LAM resistance.

[Retreatment with interferon of relapsed chronic hepatitis C patients].

OBJECTIVE: To evaluate the efficacy and investigate the influencing factors of the interferon (IFN) retreatment for patients with chronic hepatitis C relapsed after a previous IFN treatment. METHODS: A retrospective study was designed to analyze the retreatment with IFN of 60 relapsed chronic hepatitis C patients. All patients were from a randomized, opened and multi-center clinical trial about the efficacy and security of PEG-IFNalpha-2a compared to CIFNalpha-2a in the treatment of chronic hepatitis C in China. There were 35 patients treated with PEG-IFNalpha-2a and 25 with CIFNalpha-2a. The main parameter to evaluate the efficacy was sustained viral response (SVR) rate. The influence of viral concentration in serum, genotype and drug categories on the responses to IFN were analyzed. RESULTS: For all the patients, the end of treatment virus response (ETVR) and SVR rates were 55.00% and 35.00% respectively. ETVR rate of PEG-IFNalpha-2a was significantly higher than that of CIFNalpha-2a (74.29% and 28.00% respectively, P < 0.01). SVR rate of PEG-IFNalpha-2a was also markedly higher than that of CIFNalpha-2a (45.71% and 20.00% respectively, P < 0.05). However, there was no significant difference between the high and low viral load groups. Among the patients with genotype 1, ETVR and SVR rates of PEG-IFNalpha-2a (75.00%, 45.83%) were significantly higher than those of CIFNalpha-2a (22.22%, 11.11%), (P < 0.01, P < 0.05 respectively), but in patients with genotype non-1, there were no such differences between the two groups. CONCLUSION: Some relapsed patients were not responsive to the IFN retreatment. The efficacy of PEG-IFNalpha-2a was superior to CIFNalpha-2a. The conventional IFN was not suggested to be used in the relapsed cases with genotype 1. The viral load was not associated with the efficacy of IFN retreatment.

[The factors associated with viral relapse after interferon treatment in chronic hepatitis C patients].

OBJECTIVE: To investigate the relationship between hepatitis C virus (HCV) genotype, serum viral load and ALT levels, and the factors associated with the viral relapse after IFN treatment in patients with chronic hepatitis C. METHODS: The HCV RNA levels were determined with Cobas Amplicor Monitor Test, version 2.0, and HCV genotypes were examined by means of PCR products of 5' NTR digested with restriction endonucleases. The patients with chronic hepatitis C were treated with PEG-IFN alpha -2a and Roferon-A for 24 weeks. Those with a viral response after 24 week treatment were followed for an additional 24 weeks. The association of clinical characteristics, such as sex, age, the way of the HCV infection, IFN treatment history and platelet counts, and the HCV genotype, virus load and medicine used for the viral relapse after IFN treatment were analyzed. RESULTS: Of the 208 chronic hepatitis C patients, the ALT levels were not related to HCV RNA levels (r = 0.093, P > 0.05). No difference of ALT levels between HCV genotypes was found, and the HCV RNA load was also of no difference between HCV genotype 1 patients and non 1 patients. Of the 119 patients with viral response after 24 week treatment, 58 cases (48.7%) relapsed after another 24 week's follow-up. Relapse was not significantly related to the clinical characteristics, such as sex, age, mode of the infection, treatment history of IFN, AST/ALT ratio, platelet counts and the baseline viral load. Among patients with genotype 1 virus, the relapse rate was significantly higher than those patients with non-genotype 1 virus (54.5% vs 32.1%, P=0.039). The relapse rate after PEG-IFN alpha -2a treatment was lower than that of Roferon-A treatment (47.0% vs. 52.8%), but not significantly. CONCLUSION: The viral relapse of chronic hepatitis C patients after IFN treatment was significantly associated with the genotypes of the HCV.

Predictive factors for sustained response to interferon treatment in patients with chronic hepatitis C: a randomized, open, and multi-center controlled trial.

BACKGROUND: This study was undertaken to investigate the predictive factors of sustained viral response in roferon-A or pegasys treated chronic hepatitis C patients after logistic regression analysis of the factors that might be associated with the therapeutic effects of interferon (IFN). METHODS: All patients enrolled into this randomized, open and multi-center controlled trial were divided into two groups randomly and treated with pegasys and roferon-A for 24 weeks, then followed up for another 24 weeks. Before treatment, hepatitis C virus (HCV) genotype was determined, and HCV-RNA in serum was detected before and at the end of treatment and follow-up. HCV-RNA turning negative was considered the major index for evaluating the therapeutic effect. The clinical characteristics including gender, age, infection route of HCV, treatment with IFN, platelet count, AST/ALT ratio and treatment drugs were analyzed by logistic regression. RESULTS: Intention to treat (ITT) and per-protocol (PP) population groups have 208 and 197 patients respectively. In the PP group, after treatment for 24 weeks, the response rates of female patients aged less than 50 years, infected through non-transfusion, relapsed after IFN treatment, and presented with a AST/ALT ratio/=1, virus load equal or more than 8 x 10(5) IU/ml, and genotype 1 infection, and treated finally with roferon-A. But, at the end of follow-up, the patients with a AST/ALT ratio>/=1 and virus load more than 8 x 10(5) IU/ml had a higher rate of sustained response than did those with a AST/ALT ratio

[Establish a transgenic mice model harboring structural genes of hepatitis C virus].

OBJECTIVES: To establish an animal model of HCV transgenic mice to elucidate the pathogenesis of hepatitis C virus infection and function of the viral structural proteins. METHODS: Structural gene of HCV were amplified and recombined into eukaryotic expression vectors, pcDNA4HisMax and pMT/BiP/V5-His A, after their expressive activity was confirmed to detect the structural protein in the transfected COS7 and S2 cells by Western blot. The fertilized expression element, which contained CMV or pMT promoter, structural gene of HCV and polyadenylation signal sequence, was microinjected into 1736 C57BL/6 mouse fertilized ova. The ova were then replanted into the oviducts of 69 pseudopregnant recipient mice. RESULTS: Twenty-five recipient mice were impregnated and later produced 105 newborns; 49 of them died from unknown causes and 57 survived. After the specific HCV structural genes were identified by PCR and Southern blot hybridization, 26 founders were obtained; among them 10 were stable expression mice and 16 were the inducible ones. The rate of founders developed from implanted embryos was only 1.50%. Through hybridization with normal mice, 58 hybrid mice have been obtained at present. CONCLUSION: Two kinds of different transgenic mice of HCV were developed; one is of stable expression, and the other is inducible. This transgenic mice model may create an opportunity for studying the function of the structural gene of HCV and elucidate its pathogenicity.

Development of the diagnostic immunoassay to detect anti-PreS1(21-47aa) antibody--a marker suggesting the health improvement of hepatitis B patients.

BACKGROUND: A new immunoassay has been developed for the detection of the anti-PreS1(21-47aa) antibody in sera of hepatitis B virus (HBV)-infected patients. Anti-PreS1(21-47aa) antibody involves virus neutralization and is a new marker for diagnosing acute and chronic B hepatitis. METHODS: The expression plasmids pGEXS I and pGEXS II, which expressed glutathione S-transferase (GST) fusion proteins containing a copy of PreS1(21-47aa) peptide and two orderly joined copies of PreS1(21-47aa) peptide, were constructed. The soluble expression products were purified by affinity chromatography. RESULTS: The two PreS1(21-47aa) fusion proteins were both successfully applied in the immunoassay based on biotin-protein A and streptavidin-HRP, and could detect the anti-PreS1(21-47aa) antibody with high sensitivity in sera from hepatitis B patients. The anti-PreS1(21-27aa) antibody was detected during the recovery phase of acute hepatitis B patients, but it was found only in few of the chronic carriers by the established conventional system. CONCLUSIONS: The follow-up study suggested that the presence of the anti-PreS1(21-27aa) antibody correlated well with the recovery of patients from hepatitis and the improvement in health.

Expression of hepatitis C virus hypervariable region 1 and its clinical significance.

AIM: To explore the properties of hypervariable region 1 (HVR1) in the envelope 2 gene of hepatitis C virus by analyzing the reactivity of HVR1 fusion proteins from different Chinese HCV strains with sera of patients with chronic hepatitis C and by comparing their reactivity between interferon therapy responders and non-responders. METHODS: Gene fragments of HVR1 of four HCV strains (three genotype 1b and one genotype 2a) were amplified from pGEMT-E2 plasmids and sub-cloned into pQE40 vectors respectively to construct recombinant expression plasmids which expressed HVR1 fused downstream to DHFR in Escherichia coli strain TG1. The purified DHFR- HVR1 proteins were then used to detect the anti-HVR1 antibodies in 70 serum samples of patients with chronic hepatitis C. RESULTS: Four DHFR- HVR1 fusion proteins were successfully expressed in E.coli (320-800 ug fusion proteins per 100 ml culture). Each fusion protein (SH1b, BJ1b, SD1b and SD2a) reacted with 72.8 % (51/70), 60 % (42/70), 48.6 % (34/70), and 58.6 % (41/30) of the anti-HCV positive patients' sera respectively by ELISA. 57 % (4/7) of non responders reacted with all four HVR1 fusion proteins, while only 15.3 % (2/13) of responders reacted with all of them. The O.D. values of sera from IFN therapy responders were significantly higher than those of non responders (P<0.05). CONCLUSION: The selected HVR1 fusion proteins expressed in E. coli can broadly react with HCV-infected patients' sera. The intensity and/or quality of the immune response against HCV may be a critical factor determining the response to interferon treatment. With the evolution of virus strains, anti-HVR1 antibodies can not neutralize all the quasispecies. A polyvalent and high immunogenic vaccine comprising a mixture of several HVR1 sequences that cover the reactivity of most HCV isolates may be useful.

Detection of anti-preS1 antibodies for recovery of hepatitis B patients by immunoassay.

AIM: To establish a convenient immunoassay method based on recombinant antigen preS1(21-119aa) to detect anti-preS1 antibodies and evaluate the clinical significance of antibodies in hepatitis B. METHODS: The expression plasmid pET-28a-preS1 was constructed, and a large quantity of preS1(21-119aa) fragment of the large HBsAg protein was obtained. The preS1 fragment purified by Ni(2+)-IDA affinity chromatography was used as coated antigen to establish the indirect ELISA based on streptavidin-biotin system for detection of the anti-preS1 antibodies in sera from HBV-infected patients. For follow-up study, serial sera were collected during the clinical course of 21 HBV-infected patients and anti-preS1 antibodies, preS1 antigen, HBV-DNA and other serological HBV markers were analyzed. RESULTS: preS1(21-119aa) fragment was highly expressed from the plasmid pET-28a-preS1 in a soluble form in E.Coli (30mg.L(-1)), and easily purified to high purity over 90% by one step of Ni(2+)-IDA-sepharose 6B affinity chromatography. The purity and antigenicity of the purified preS1(21-119aa) protein was determined by 150g.L(-1) SDS-PAGE, Western blot and a direct ELISA. Recombinant preS1(21-119aa) protein was successfully applied in the immunoassay which could sensitively detect the anti-preS1 antibodies in serum specimens of acute or chronic hepatitis B patients. Results showed that more than half of 19 acute hepatitis B patients produced anti-preS1 antibodies during recovery of the disease, however, the response was only found in a few of chronic patients. In the clinical follow-up study of 11 patients with anti-preS1 positive serological profile, HBsAg and HBV-DNA clearance occurred in 6 of 10 acute hepatitis B patients in 5-6 months, and seroconversion of HBeAg and disappearance of HBV-DNA occurred in 1 chronic patients treated with lavumidine, a antiviral agent. CONCLUSION: The high-purity preS1(21-119aa) coated antigen was successfully prepared by gene expression and affinity chromatography. Using this antigen, a conveniently detective system of anti-preS1 antibodies in sera was established. Preliminarily clinical trial the occurrence of anti-preS1 antibodies in acute hepatitis B patients suggests the clearance of HBV from serum in a short-term time, and anti-preS1 positive in chronic patients means health improvement or recovery from the disease.

Expression, Purification and Preliminary Clinical Use of Recombinant HBsAg GST-PreS1(21--47 aa) Fusion Proteins.

Expression plasmids pGEXSI and pGEXSII containing one copy and two orderly joined copies of PreS1(21--47 aa) DNA fragment, respectively, were constructed. GST-PreS1(21--47 aa) and GST-2xPreS1(21--47 aa) fusion proteins were highly expressed in E.Coli TG1, induced by IPTG. The expression level of GST-PreS1(21--47 aa) was about 30% of total soluble proteins in the lysate of expression bacteria, and GST-2xPreS1(21--47 aa) was about 15% of total soluble proteins, asestimated by SDS-PAGE. 50 mg GST-PreS1(21--47 aa) or 20 mg GST-2xPreS1(21--47 aa) with purity over 90% was obtained, respectively, from 1 L culture by using affinity chromatography of glutathione-Sepharose 4B. Direct ELISA results showed that antigenicity of GST-2xPreS1(21--47 aa) was better than GST-PreS1(21--47 aa) and synthetic peptide. Using GST-2xPreS1(21--47 aa) as coated antigen, a sensitive indirect ELISA for detection of anti-PreS1(21--47 aa) antibody, based on protein A-biotin and streptavidin-HRP, was established. The results from 99 sera samples of hepatitis B patients showed that anti-PreS1(21--47 aa) antibody was detected in nearly half of acute hepatitis B patients during recovery, but it was detected only in a few chronic hepatitis patients. Clinical follow-up study suggested that appearance of anti-PreS1(21--47 aa) was related to the course of the disease and recovery of patients. Detection system established in the study is promising for clinical application.

Impact of virus genotype on interferon treatment of patients with chronic hepatitis C: a multicenter controlled study.

BACKGROUND: Some factors have been reported to be associated with a greater likelihood of sustained viral response (SVR) in the interferon (IFN) treatment of chronic hepatitis C. The factors include HCV genotype, HCV RNA level in serum, state of liver disease, baseline body weight, age, sex, and race. The aim of this trial was to investigate the influence of HCV genotype on the IFN treatment of patients with chronic hepatitis C. METHODS: The genotypes of HCV virus were determined in the patients with chronic hepatitis C from several hospitals of China enrolled into the randomized, opened and controlled trial of Peg-IFN alpha-2a (pegasys) treatment, controlled with IFN-alpha-2a (roferon-A). The serum ALT levels and HCV RNA concentrations of the patients were detected before and at the end of treatment and during the follow-up. The influence of HCV genotype on the IFN treatment of patients with chronic hepatitis C was analyzed in intention-to-treat (ITT) population. RESULTS: The HCV genotypes of 202 patients were determined. Of these patients, 158(78.22%) were infected with genotype 1 HCV and 44(21.78%) with genotype non-1. The viral response at the end of treatment (ETVR) and sustained viral response (SVR) rates were 53.80% and 25.32% respectively in patients with genotype 1 HCV, but they were 61.36% and 43.18% in patients with genotype non-1. The difference of SVR between patients with genotype 1 HCV and those with genotype non-1 was significant (P=0.021). After being grouped by the used drugs, the ETVR rates of patients infected with genotype 1 and non-1 HCV were 76.83% and 80.95% in the patients treated with pegasys (P=0.686); but their SVR rates were 35.37% and 66.67% (P=0.01). The viral relapse rate of genotype 1 HCV (55.56%) was significantly higher than that of genotype non-1 HCV (23.53%) (P=0.02). In roferon-A group, the ETVR and SVR rates of patients with genotype 1 HCV were 28.95% and 14.47% respectively, which were lower but not more significant than those of patients with genotype non-1 HCV (43.48% and 21.74%). Moreover, the viral relapse rate of genotype 1 HCV (72.73%) was higher but not more significant than that of genotype non-1 HCV (50.00%) (P=0.21). CONCLUSION: HCV genotype could affect the efficacies, mainly sustained responses, of IFN treatment in patients with chronic hepatitis C, and the effects of IFN are related to drugs and therapeutic course.

[The pegasys using and HCV genotype were the independent predictors of the response to antiviral therapy in chronic hepatitis C].

OBJECTIVE: To investigate the predictors of IFN therapy in patients with chronic hepatitis C through making the multivariate logistic regression analysis. METHODS: The patients in the opened, randomized and controlled trial were enrolled into two group, pegasys and Roferon-A group, and were given 24 weeks of pegasys (injection of 180 microg a week), and Roferon-A (injection three times of Roferon-A 3 MU a week) therapy, and followed 24 weeks. The HCV RNA content was determined at the time before, end of treatment and at the followed-up. The association of the response to the treatment with the clinical characteristics including age, gender, way of HCV infection, history of IFN treatment, planet count, AST/ALT ratio, HCV RNA level, HCV genotype and treatment drugs was made trough multivariate logistic regression analysis. RESULTS: The PP population containing 197 cases was analyzed. After controlling for age, gender, way of HCV infection, history of IFN treatment, planet count, AST/ALT ratio, HCV RNA level and treatment, the HCV genotype was not predictor of the end of treatment viral response (ETVR) to IFN therapy (OR 0.604, 95% CI 0.271-1.349, P = 0.219), but was the independent predictor of sustained viral response (SVR) (OR 0.408, 95% CI 0.189-0.881, P = 0.023). After controlling for other characteristics, the treatment drug was the predictors of ETVR (OR 0.105, 95% CI 0.052-0.212, P < 0.001) and SVR (OR 0.255, 95% CI 0.123-0.529, P < 0.001). CONCLUSION: The pegasys using and HCV genotype were the independent predictors of the response to antiviral therapy in chronic hepatitis C.

[The influence of HCV genotype on the IFN treatment of patients with chronic hepatitis C].

OBJECTIVE: To investigate the influence of HCV genotype on the IFN treatment of patients with chronic hepatitis C. METHODS: The genotypes of HCV virus were determined in the patients enrolled into the Randomized, opened and controlled trial of Peg-IFN alpha-2a (Pegasys) treatment, controlled with IFN-alpha-2a (Roferon-A), on chronic hepatitis C patients in China. The serum ALT levels and HCV RNA concentration of the patients were detected in the time of before treatment, the end of therapy and follow-up. The influence of HCV genotype on the IFN treatment of patients with chronic hepatitis C was analyzed in intention to treat (ITT) population. RESULTS: The HCV genotypes of 202 cases were determined. 158 (78.2%) cases infected with genotype 1 HCV and 44 (21.8%) cases with genotype non-1. For overall patients, the viral response at the end of treatment (ETVR) and sustained viral response (SVR) rates were 53.8% and 25.3% respectively in patients with genotype 1 HCV, but in genotype non-1 patients those was 61.4% and 43.2%, and the difference of SVR between genotype 1 and non-1 was significant (P=0.021). After grouped by the used drugs, in the patients given Pegasys treatment, the ETVR rates of patients with genotype 1 and non-1 HCV infection were 76.8% and 81.0%, the difference was not significant (P=0.686), but the difference of SVR rates, which were 35.4% and 66.7%, of the patients was significant (P=0.01). The viral relapse rate of genotype 1 was 55.6%; it was significant higher than that of genotype non-1 (23.5%) (P=0.02). In Roferon-A group, the ETVR and SVR rates of patients with genotype 1 HCV were 29.0% and 14.5%, which were lower, but not significant, than those of patients with genotype non-1 (43.5% and 21.7%). The viral relapse rate of genotype 1 was 72.7% and higher, but not significant, than that of genotype non-1 also (50.0%) (P=0.21). CONCLUSION: HCV genotype could affects the efficacies, mainly the sustained responses, of IFN treatment of patients with chronic hepatitis C, and the effects of IFN were related to the kinds of drugs and therapeutic course.

Hepatitis B surface antigen: association with sustained response to peginterferon alfa-2a in hepatitis B e antigen-positive patients.

PURPOSE: Patients with hepatitis B e antigen (HBeAg)-positive chronic hepatitis B, who achieve HBeAg seroconversion 6 months after completing 48 weeks of peginterferon alfa-2a therapy, have an increased chance of clearing hepatitis B surface antigen (HBsAg) during long-term treatment-free follow-up. This analysis aimed to determine whether HBsAg quantification during treatment could be used to identify posttreatment response. METHODS: Patients (n = 399) treated with peginterferon alfa-2a (180 µg/week) alone or in combination with lamivudine (100 mg/day) for 48 weeks during a large, randomized study were included in this retrospective analysis. Receiver-operating characteristic analyses were used to identify baseline and on-treatment HBsAg levels associated with response (HBeAg seroconversion 6 months posttreatment). RESULTS: Baseline HBsAg levels were lower in patients achieving posttreatment response than in nonresponders (3.97 and 4.21 IU/mL, respectively, p = 0.039). Two baseline HBsAg cutoff levels (5,000 and 50,000 IU/mL) provided a positive predictive value of 42% and a negative predictive value of 77%. HBsAg decline was significantly greater during and posttreatment in responders than in nonresponders (p < 0.0001). HBeAg seroconversion rates 6 months posttreatment were significantly higher in patients with HBsAg < 1,500 IU/mL at weeks 12 and 24 (56.7 and 54.4%, respectively) versus patients with HBsAg 1,500-20,000 IU/mL (32.3 and 26.1%, respectively) or HBsAg < 20,000 IU/mL (16.3 and 15.4%, respectively) (all p < 0.0001 and <0.0001). CONCLUSIONS: HBsAg levels at baseline strongly associated with posttreatment response were not identified. Low HBsAg levels during peginterferon alfa-2a therapy were associated with high rates of posttreatment response. On-treatment HBsAg quantification may, therefore, help guide patient management in the future.