丙型肝炎基因型、病毒量及肝损程度与α干扰素疗效关系

观察丙型肝炎基因型、病毒量及肝损程度对α干扰素治疗慢性丙型肝炎应答率的影响。对 41例慢性丙型肝炎用α干扰素 3MIU每天肌注一次 ,二周后改为隔日肌注一次 ,共 2 1周。治疗前用INNO -LIPA法作丙肝基因分型 ,并在治疗前、后每 2月及停药 6月时用PCR法检测HCVRNA含量和定性、每月检测肝功能。按国际标准判断干扰素的治疗末应答 (ETR)和持续应答 (SR)。结果显示 1b型HCVRNA的治疗末应答率占 82 8% (2 4/2 9) ;持续应答率占 78 9% (15 / 19)。ALT的应答率分别是 78 3 % (18/ 2 3)和 76 5 % (13/ 17)。HCVRNA含量显示≤ 5× 10 5copies/ml的持续应答率高于 >5× 10 5copies/ml,二组P <0 0 1。治疗前ALT活力高低与干扰素治疗后ALT的应答率影响不大。预测干扰素疗效的关键是治疗前HCVRNA在血清中的水平。

Quasispecies evolution in NS5A region of hepatitis C virus genotype 1b during interferon or combined interferon-ribavirin therapy

AIM: To evaluate the implication of substitutions in the hepatitis C virus (HCV) non-structural 5A (NS5A) protein in the resistance of HCV during mono-interferon (IFN) or combined IFN-ribavirin (IFN-R) therapy. Although NS5A has been reported to interact with the HCV RNA- dependent RNA polymerase, NS5B, as well as with many cellular proteins, the function of NS5A in the life cycle of HCV remains unclear. METHODS: HCV quasispecies were studied by clon- ing and sequencing of sequential isolates from patients infected by HCV genotype 1b. Patients were treated by IFN-α2b for 3 mo followed by IFN-α2b alone or com- bined IFN-R therapy for 9 additional months. Patients were categorized intro two groups based on their re- sponse to the treatments: 7 with sustained virological re- sponse (SVR) (quasispecies = 150) and 3 non-respond- ers (NR) to IFN-R (quasispecies = 106). RESULTS: Prior to treatment, SVR patients displayed a lower complexity of quasispecies than NR patients. Most patients had a decrease in the complexity of quasispe- cies during therapy. Analysis of amino acids substitu- tions showed that the degree of the complexity of the interferon sensitivity-determining region (ISDR) and the V3 domain of NS5A protein was able to discriminate thetwo groups of patients. Moreover, SVR patients displayed more variability in the NS5A region than NR patients. CONCLUSION: These results suggest that detailed mo- lecular analysis of the NS5A region may be important for understanding its function in IFN response during HCV 1b infection.

Hepatitis C virus genotypes in Serbia and Montenegro: The prevalence and clinical signifi cance

AIM： To investigate the prevalence of hepatitis C virus （HCV） genotypes in Serbia and Montenegro and their influence on some clinical characteristics in patients with chronic HCV infection. METHODS： A total of 164 patients was investigated. Complete history, route of infection, assessment of alcohol consumption, an abdominal ultrasound, standard biochemical tests and liver biopsy were done. Gene sequencing of 5＇ NTR type-specific PCR or commercial kits was performed for HCV genotyping and subtyping. The SPSS for Windows （version 10.0） was used for univariate regression analysis with further multivariate analysis. RESULTS： The genotypes 1, 2, 3, 4, 1b3a and 1b4 were present in 57.9%, 3.7%, 23.2%, 6.7%, 6.7% and 1.8% of the patients, respectively. The genotype 1 （mainly the subtype 1b） was found to be independent of age in subjects older than 40 years, high viral load, more severe necro-inflammatory activity, advanced stage of fibrosis, and absence of intravenous drug abuse. The genotype 3a was associated with intravenous drug abuse and the age below 40. Multivariate analysis demonstrated age over 40 and intravenous drug abuse as the positive predictive factors for the genotypes lb and 3a, respectively.CONCLUSION： In Serbia and Montenegro, the genotypes 1b and 3a predominate in patients with chronic HCV infection. The subtype lb is characteristic of older patients, while the genotype 3a is common in drug abusers. Association of the subtype lb with advanced liver disease, higher viral load and histological activity suggests earlier infection with this genotype and eventually its increased pathogenicity.

Autoantibodies and hepatitis C virus genotypes in chronic hepatitis C patients in Estonia

AIM: To determine the prevalence of several autoantibodies in chronic hepatitis C patients, and to find out whether the pattern of autoantibodies was associated with hepatitis C virus (HCV) genotypes. METHODS: Sera from 90 consecutive patients with chronic hepatitis C were investigated on the presence of anti-nuclear (ANA), anti-mitochondrial (AMA), anti-smooth muscle (SMA), anti-liver-kidney microsomal type 1 (LKMA1), anti-parietal cell (PCA), anti-thyroid microsomal (TMA), and anti-reticulin (ARA) autoantibodies. The autoantibodies were identified by indirect immunofluorescence. HCV genotypes were determined by a restriction fragment length polymorphism analysis of the amplified 5' noncoding genome region. RESULTS: Forty-six (51.1%) patients were positive for at least one autoantibody. Various antibodies were presented as follows: ANA in 13 (14.4%) patients, SMA in 39 (43.3%), TMA in 2 (2.2%), and ARA in 1 (1.1%) patients. In 9 cases, sera were positive for two autoantibodies (ANA and SMA). AMA, PCA and LKMAI were not detected in the observed sera. HCV genotypes were distributed as follows: 1b in 66 (73.3%) patients, 3a in 18 (20.0%), and 2a in 6 (6.7%) patients. CONCLUSION: A high prevalence of ANA and SMA can be found in chronic hepatitis C patients. Autoantibodies are present at low titre (1:10) in most of the cases. Distribution of the autoantibodies show no differences in the sex groups and between patients infected with different HCV genotypes.

New combination test for hepatitis C virus genotype and viral load determination using Amplicor GT HCV MONITOR test v2.0

AIM: To develop a new sensitive and inexpensive hepatitis C virus (HCV) combination test (HCV Guideline test) that enables the determination of HCV genotypes 1, 2 and 3, and simultaneous determination of HCV viral load using commercial Amplicor GT HOV MONITOR test v2.0 (microwell version). METHODS: The HCV Guideline test used the PCR product generated in commercial Amplicor GT HCV Monitor test v2.0 for viral load measurement using microwell plate version of Amplicor HCV Monitor and also captured on separate plates containing capture probes and competitive oligonucleotide probes specific for HCV genotypes 1, 2 and 3, The HCV genotype was subsequently determined using the biotin-labeled PCR product and five biotin-labeled HCV-specific probes. RESULTS: The sensitivity of the HCV Guideline test was 0.5 KIU/mL. Specificity of the HCV Guideline test was confirmed by direct sequencing of HCV core region and molecular evolutionary analyses based on a panel of 31 samples. The comparison of the HCV Guideline test and an in-house HCV core genotyping assay using 252 samples from chronic hepatitis C patients indicated concordant results for 97.2% of samples (59.5% genotype 1, 33.7% genotype 2, 6.0% genotype 3, and 0.8% mixed genotypes). Similarly, the HCV Guideline test showed concordance with a serological test, and the serological test failed to assign any serotype in 12.7% of the samples, indicating a better sensitivity of the HCV Guideline test. CONCLUSION: Clinically, both viral load and genotypes (1, 2 and 3) have been found to be major predictors of antiviral therapy outcome regarding chronic hepatitis C based on guidelines and they are, in normal circumstances, performed as separate stand-alone assays. The HCV Guideline test is a useful method for screening large cohorts in a routine clinical setting for determining the treatment regimen and for predicting the outcome of antiviral therapy of chronic hepatitis C.

Hepatitis C virus genotypes distribution and transmission risk factors in Luxembourg from 1991 to 2006

AIM： To analyze the Hepatitis C virus （HCV） genotype distribution and transmission risk factors in a population of unselected patients in Luxembourg.METHODS： Epidemiological information （gender, age and transmission risks） were collected from 802 patients newly diagnosed for hepatitis C and living in Luxembourg, among whom 228 patients referred from prison. Genotyping using 5＇noncoding （5＇NC） sequencing was performed. We compared categorical data using the Fisher＇s exact F-test and odds ratios （OR） were calculated for evaluating association of HCV genotype and risk factors.RESULTS： The sex ratio was predominantly male （2.2） and individuals aged less than 40 years represented 49.6% of the population. Genotype 1 was predominant （53.4%） followed by genotype 3 （33%）. Among risk factors, intravenous drug usage （IVDU） was the most frequently reported （71.4%） followed by medical-related transmission （17.6%） including haemophilia, transfusion recipients and other nosocomial reasons. Genotype 3 was significantly associated to IVDU （OR = 4.84, P 〈 0.0001） whereas genotype 1 was significantly associated with a medical procedure （OR = 2.42, P 〈 0.001）. The HCV genotype distribution from inmate patients differed significantly from the rest of the population （Chi-square test with four degrees of freedom, P 〈 0.0001） with a higher frequency of genotype 3 （46.5% vs 27.5%） and a lower frequency of genotype 1 and 4 （44.7% vs 56.8% and 5.3% vs 9.6%, respectively）. IVDU was nearly exclusively reported as a risk factor in prison.CONCLUSION： We report the first description of the HCV genotype distribution in Luxembourg. The repartition is similar to other European counties, with one of the highest European prevalence rates of genotype 3 （33%）. Since serology screening became available in 2992, IVDU remains the most common way of HCV transmission in Luxembourg.

Treatment of hepatitis C virus genotype 4 with peginterferon alfa-2a: Impact of bilharziasis and fibrosis stage

AIM： To evaluate pegylated interferon alpha2a （PegIFN-α2a） in Egyptian patients with HCV genotype 4, and the impact of pretreatment viral load, co-existent bilharziasis and histological liver changes on response rate. METHODS： A total of 73 nafve patients （61 with history of bilharziasis） with compensated chronic HCV genotype 4 were enrolled into： group A （38 patients） who received 180 mg PegIFN-alpha2a subcutaneously once weekly for a year and group B （35 patients） received IFN alpha-2a 3 MU 3 times weekly. Ribavirin was added to each regimen at a dose of 1200 mg. Patients were followed for 72 wk and sustained response was assessed. RESULTS： Significant improvement in both end of treatment response （ETR） （P 〈 0.002） and sustained response （SR） （P 〈 0.05） was noted with pegylated interferon, where ETR was achieved in 29 （76.3%） and 14 patients （40%） in both groups respectively, and 25 patients in group A （65.8%） and 9 （25.7%） in group B could retain negative viraemia by the end of follow up period. Sustained virological response （SVR） showed a significant negative correlation with age and positive correlation with pretreatment inflammation in patients receiving PegIFN. Viral clearance after 3 mo of therapy was associated with high incidence of ETR and SR （P 〈 0.001）, but without significant difference between both forms of interferon. Significant improvement in response was achieved in patients with high grade fibrosis （grade 3 and 4） with PegIFN-α2a, where SR was seen in 5 out of 13 patients in group A, but none in group B. There was no significant difference in response between bilharzial and non-bilharzial patients in both groups. In terms of safety and tolerability, neutropenia was the predominant side effect, both drugs were comparable. CONCLUSION： PegIFN-~2a combined with ribavirin results in improvement in sustained response in HCV genotype 4, irrespective of history of bilharzial infestation.

Interleukin-28 and hepatitis C virus genotype-4:Treatment-induced clearance and liver fibrosis

AIM:To investigate the association between interleukin-28B(IL28B) genotype and response to treatment and hepatic fibrosis in patients with hepatitis C virus(HCV) genotype 4.METHODS:Two hundred and one HCV-genotype 4 patients were included.All patients were treated with Peginterferon alph2a/Ribavirin for 48 wk.End of treatment response(ETR) was defined as loss of detectable serum HCV RNA at the end of treatment.Sustained viral response(SVR) was defined as loss of detectable serum HCV RNA at the end of 24 wk follow up.Genotyping of IL28B rs12979860 was performed using the TaqMan assay.We used logistic regression to estimate the adjusted odds ratio(aOR) and 95%CI.RESULTS:The study included 201 HCV-genotype 4 patients.The majority of patients were men(89.6%),with a median age of 47 years,inter-quartile range(40-51).Approximately 62.5% of patients had ETR,and 49.6% had SVR.Individuals who achieved SVR were more likely to be younger(χ 2 = 4.91,P = 0.027),and less likely to have fibrosis(χ 2 = 15.54,P < 0.0001),or inflammation(χ 2 = 7.58,P = 0.006).The genotype distribution of rs12979860 was 36.2%,49.0% and 14.8% for genotypes CC,CT,and TT,respectively.In these participants,rs12979860 genotype distribution did not differ by gender(P = 0.466),pretreatment viral load(P = 0.600),inflammation(P = 0.435),or fibrosis(P = 0.291).The frequencies of IL28B rs12979860 genotypes were TT(14.8%),CT(49.0%),and CC(36.2%).Compared to rs12979860 genotype TT,aORs(95%CI) for ETR and SVR were:CC genotype,[17.55(5.34-57.69) and 5.92(2.09-16.76),respectively];CT genotype,[5.15(1.80-14.78) and 2.48(0.94-6.52),respectively].In the current study,the patients who did not achieve ETR or SVR had a lower prevalence of rs12979860 CC(17.4% and 23.3%,respectively) than individuals who had ETR or SVR(47.9% and 47.2%,respectively).Individuals with rs12979860 CC genotype had approximately 6 times the odds of SVR compared to individuals with TT genotype(aOR = 5.92;95%CI:2.09-16.76).Similarly,patients with CT genotype had SVR more often than patients with TT genotype(aOR = 2.48;95%CI:0.94-6.52).Carrying at least one copy of the C allele(genotypes CT and CC) had almost 8 times the probability of ETR compared to those with genotype rs12979860 TT(aOR = 7.87;95%CI:2.84-21.82),and approximately 3 times the odds of SVR compared to those with genotype rs12979860 TT(aOR = 3.46;95%CI:1.37-8.74).In addition,data were consistent with a significant gene-dose relationship(aOR = 4.05/allele;95%CI:2.27-7.22).The association between rs12979860 genotype and SVR was similar among those who achieved and those who did not achieve SVR.CONCLUSION:In HCV-genotype 4 patients,rs12979860 is a sensitive predictor of viral clearance,independent of viral load,age,gender or fibrosis,with no similar relation to severity of fibrosis.

Human leukocyte antigen class II DQB1*0301, DRB1*1101 alleles and spontaneous clearance of hepatitis C virus infection: A meta-analysis

AIM： To assess the associations of human leukocyte antigen （HI_A） class Ⅱ DQB1＊0301 and/or DRB1＊1101 allele with spontaneous hepatitis C virus （HCV） clearance by meta-analysis of individual dataset from all studies published till date. METHODS： To clarify the impact of HLA class Ⅱ polymorphisms on viral clearance, we performed a metaanalysis of the published data from 11 studies comparing the frequencies of DQB1＊0301 and DRB1＊1101 alleles in individuals with spontaneous resolution to those with persistent infection. As we identified the heterogeneity between studies, summary statistical data were calculated based on a random-effect model. RESULTS： Meta-analyses yielded summary estimatesodds ratio （OR） of 2.36 [95%CI （1.62, 3.43）, P〈0.00001] and 2.02 [95%CI （1.56, 2.62）, P〈0.00001] for the effects of DQB1＊0301 and DRB1＊1101 alleles on spontaneous clearance of HCV, respectively. CONCLUSION： These results support the hypothesis that specific HLA class Ⅱ alleles might influence the susceptibility or resistance to persistent HCV infection. Both DQB1＊0301 and DRB1＊1101 are protective alleles and present HCV epitopes more effectively to CD4^＋T lymphocytes than others, and subjects with these two alleles are at a lower risk of developing chronic HCV infection. Large, multi-ethnic confirmatory and welldesigned studies are needed to determine the host genetic determinants of HCV infection.

Four-week pegylated interferon a-2a monotherapy for chronic hepatitis C with genotype 2 and low viral load:A pilot,randomized study

AIM:To assess the efficacy and advantages of 4-wk pegylated interferon a-2a(peg-IFN-a2a) monotherapy for chronic hepatitis C patients with strong predictors of sustained virologic response(SVR).METHODS:Patients(n = 33) with genotype 2 and low viral load(< 100 KIU/mL),who became HCV RNA negative after 1 wk of IFN treatment,were randomly allocated to receive a 4-or 12-wk treatment course at a ratio of 2:1,respectively,with a subsequent 24-wk follow-up period.Peg-IFN-a2a was administered subcutaneously at a dose of 180 μg or 90 μg once weekly.SVR was defined as absence of serum HCV RNA at the end of the follow-up period.RESULTS:All patients completed the treatment schedule,and more than half were symptom-free during the treatment.In the 4-wk treatment group,20 of 22(91%) patients achieved SVR.Two patients relapsed,but achieved SVR following re-treatment with peg-IFN-a2a alone.In the 12-wk treatment group,11 of 11(100%) patients attained SVR.CONCLUSION:Our results show that a 4-wk course of peg-IFN-a2a monotherapy can achieve a high SVR rate in "IFN-sensitive" patients,without negatively affecting outcome.

Clinical significance of"anti-HBc alone"in human immunodeficiency virus-positive patients

AIM： TO determine the prevalence and clinical relevance of isolated antibodies to hepatitis B core antigen as the only marker of infection （“anti-HBc alone”） among human immunodeficiency virus （HIV） type-1 infected patients. Occult hepatitis B infection frequency was also evaluated. METHODS： Three hundred and forty eight histories from 2388 HIV-positive patients were randomly reviewed. Patients with serological markers of hepatitis B virus （HBV） infection were classified into three groups： past hepatitis, ＂anti-HBc alone＂ and chronic hepatitis. Determination of DNA from HBV, and RNA and genotype from hepatitis C virus （HCV） were performed on ＂anti-HBc alone＂ patients. RESULTS： One hundred and eighty seven （53.7%） HIV-positive patients had markers of HBV infection： 118 past infection （63.1%）, 14 chronic hepatitis （7.5%） and 55 ＂anti-HBc alone＂ （29.4%）. Younger age [2.3-fold higher per every 10 years younger; 95% confidence intervals （Cl） 1.33-4.00] and antibodies to HCV infection [odds ratio （OR） 2.87; 95% CI 1.10-7.48] were factors independently associated with the ＂anti-HBc alone＂ pattern. No differences in liver disease frequency were detected between both groups. Serum levels of anti-HBs were not associated with HCV infection （nor viral replication or HCV genotype）, or with HIV replication or CD4 level. No ＂anti-HBc alone＂ patient tested positive for HBV DNA. CONCLUSION： ＂Anti-HBc alone＂ prevalence in HIM- positive patients was similar to previously reported data and was associated with a younger age and with antibodies to HCV infection. In clinical practice, HBV DNA determination should be performed only in those patients with clinical or analytical signs of liver injury,

Genetic diversity of the hepatitis C virus: Impact and issues inthe antiviral therapy

The hepatitis C Virus (HCV) presents a high degree of genetic variability which is explained by the combination of a lack of proof reading by the RNA dependant RNA polymerase and a high level of viral replication. The re- sulting genetic polymorphism defines a classification in clades, genotypes, subtypes, isolates and quasispecies. This diversity is known to reflect the range of responses to Interferon therapy. The genotype is one of the pre- dictive parameters currently used to define the antiviral treatment strategy and the chance of therapeutic suc- cess. Studies have also reported the potential impact of the viral genetic polymorphism in the outcome of antivi- ral therapy in patients infected by the same HCV geno- type. Both structural and non structural genomic regions of HCV have been suggested to be involved in the Inter- feron pathway and the resistance to antiviral therapy. In this review, we first detail the viral basis of HCV diversity. Then, the HCV genetic regions that may be implicated in resistance to therapy are described, with a focus on the structural region encoded by the E2 gene and the non- structural genes NS3, NS5A and NS5B. Both mechanisms of the Interferon resistance and of the new antiviral drugs are described in this review.

Hepatitis C virus infection down-regulates the expression of peroxisome proliferator-activated receptor a and carnitine palmitoyl acyl-CoA transferase 1A

AIM： To elucidate the role of the peroxisome proliferator-activated receptor α （PPARα） and its target gene camitine palmitoyl acyl-CoA transferase 2A （CPT1A） in the pathogenesis of hepatitis C virus （HCV） infection.METHODS： Liver samples were collected from the patients with chronic HCV infection and controls. HepG2 cells were transfected with vector pEF352neo carrying. Two independent clones （clone N3 and N4） stably expressing HCV core protein were analyzed. Total RNA was extracted from cells and liver tissues. PPARα and CPT1A mRNAs were quantified by real-time polymerase chain reaction （PCR） using SYBR Green Master. Total extracted proteins were separated by polyacrylamide gel electrophoresis, and electroblotted. Membranes were incubated with the anti-PPARα antibody, then with a swine anti-rabbit IgG conjugated to horseradish peroxidase for PPARα. Protein bands were revealed by an enhanced chemiluminescence reaction for PPARα. For immunohistochemical staining of PPARα, sections were incubated with the primary goat polyclonal antibody directed against PPARα at room temperature.RESULTS： Real-time PCR indicated that the PPARα level and expression level of CPT1A gene in hepatitis C patients lowered significantly as compared with the controls （1.8±2.8 vs 13±3.4, P = 0.0002; 1.1±1.5 vs 7.4±1, P = 0.004）. Western blot results showed that the level of PPARα protein in the livers of hepatitis C patients was lower than that in controls （2.3±0.3 vs 3.6±0.2, P = 0.009）. The immunohistochemical staining results in chronic hepatitis C patients indicated a decrease in PPARα staining in hepatocytes compared with those in the control livers. The in vitro studies found that in the N3 and N4 colon stably expressing HCV core protein, the PPARα mRNA levels were significantly lower than that in the controls.CONCLUSION： The impaired intrahepatic PPARα expression is associated with the pathogenic mechanism in hepatic injury during chronic HCV infection. HCV infection reduced the expression of PPARα and CPT1A at the level of not only mRNAs but also proteins. PPARα plays an important role in the pathogenesis of chronic HCV infection, but the impaired function of this nuclear receptor in HCV infection needs further studies.

Positional effect of mutations in 5'UTR of hepatitis C virus 4a on patients' response to therapy

AIM： To investigate the effects of mutations in domain Ⅲ of the hepatitis C virus （HCV） internal ribosome entry sequences （IRES） on the response of chronic HCV genotype 4a patients to interferon therapy.METHODS： HCV RNA was extracted from 19 chronic HCV 4a patients receiving interferon/ribavirin therapy who showed dramatic differences in their response to combination therapy after initial viral clearance. IRES domain Ⅲ was cloned and 15 clones for each patient were sequenced. The obtained sequences were aligned with genotype 4a prototype using the ClustaIW program and mutations scored. Prediction of stem-loop secondary structure and thermodynamic stability of the major quasispecies in each patient was performed using the MFOLD 3.2 program with Turner energies and selected constraints on base pairing.RESULTS： Analysis of RNA secondary structure revealed that insertions in domain Ⅲ altered WatsonCrick base pairing of stems and reduced molecular stability of RNA, which may ultimately reduce binding affinity to ribosomal proteins. Insertion mutations in domain - were statistically more prevalent in sustained viral response patients （SVR, n = 14） as compared to breakthrough （BT, n = 5） patients.CONCLUSION： The influence of mutations within domain Ⅲ on the response of HCV patients to combination therapy depends primarily on the position, but not the frequency, of these mutations within IRES domain Ⅲ.

Vitamin D improves viral response in hepatitis C genotype 2-3 nave patients

AIM: To examine whether vitamin D improved viral response and predicted treatment outcome in patients with hepatitis C virus (HCV) genotype 2-3. METHODS: Fifty patients with chronic HCV genotype 2-3 were randomized consecutively into two groups: Treatment group [20 subjects, age 48 ± 14 years, body mass index (BMI) 30 ± 6, 65% male], who received 180 μg pegylated α-interferon-2a plus oral ribavirin 800 mg/d (Peg/RBV), together with oral vitamin D3 (Vitamidyne D drops; 2000 IU/d, 10 drops/d, normal serum level > 32 ng/mL) for 24 wk; and control group (30 subjects, age 45 ± 10 years, BMI 26 ± 3, 60% male), who received identical therapy without vitamin D. HCV RNA was assessed by reverse transcription polymerase chain reaction. Undetectable HCV RNA at 4, 12 and 24 wk after treatment was considered as rapid virological response, complete early virological response, and sustained virological response (SVR), respectively. Biomarkers of in? ammation were measured. RESULTS: The treatment group with vitamin D hadhigher BMI (30 ± 6 vs 26 ± 3, P < 0.02), and high viral load (> 400 000 IU/mL, 65% vs 40%, P < 0.01) than controls. Ninety-fi ve percent of treated patients were HCV RNA negative at week 4 and 12. At 24 wk after treatment (SVR), 19/20 (95%) treated patients and 23/30 (77%) controls were HCV RNA negative (P < 0.001). Baseline serum vitamin D levels were lower at baseline (20 ± 8 ng/mL) and increased after 12 wk vitamin D treatment, to a mean level of (34 ± 11 ng/ mL). Logistic regression analysis identifi ed vitamin D supplement [odds ratio (OR) 3.0, 95% CI 2.0-4.9, P < 0.001], serum vitamin D levels (< 15 or > 15 ng/mL, OR 2.2, P < 0.01), and BMI (< 30 or > 30, OR 2.6, P < 0.01) as independent predictors of viral response. Adverse events were mild and typical of Peg/RBV. CONCLUSION: Low vitamin D levels predicts negative treatment outcome, and adding vitamin D to conventional Peg/RBV therapy for patients with HCV genotype 2-3 signifi cantly improves viral response.

Significance of hepatitis B virus surface antigen, hepatitis C virus expression in hepatocellular carcinoma and pericarcinomatous tissues

AIM： To investigate the correlation between hepatitis B virus surface antigen （HBsAg）, hepatitis C virus （HCV） expression in hepatocellular carcinoma （HCC）, the HAI score of the noncancerous region of the liver and the serum Alpha fetoprotein （AFP） level. METHODS： The patterns of HBsAg and HCV in 100 cases of HCC and their surrounding liver tissues were studied on paraffin-embedded sections with immunohistochemistry, the histological status was determined by one pathologist and one surgeon simultaneously using the hepatitis activity index （HAIl score, and AFP was detected by radioimmunity. The study included 100 consecutive patients who underwent curative resection for HCC. Based on HBsAg and HCV expression, the patients were classified into 4 groups： patients positive for HBsAg （HBsAg group）, patients positive for HCV （HCV group）, patients negative for both HCV and HBsAg （NBNC group） and patients positive for both HBsAg and HCV （BC group）. RESULTS： The BC group had significantly higher HAI scores than the other three groups. （BC 〉 HCV 〉 HBsAg 〉 NBNC）. HBV and HCV virus infection was positively correlated with HAI （rs = 0.39, P = 0.00011. The positive rate of AFP （85.7%） and the value of AFP （541.2 ng/mL） in the group with HBV and HCV co-infection were the highest among the four groups. The positive rate （53.3%） of AFP and the value of AFP （ 53.3 ng/mL） in the group with none-infection of HBV and HCV were the lowest. HBV and HCV virus infection was positively correlated with AFP（rs = 0.38, P = 0.0001）. CONCLUSION： The AFP increase in patients with liver cancer was positively correlated with the infection of HBV and HCV. The-serum AFP elevation by the infection of HBV and HCV is one of mechanisms which lead to hepatocarcinogenesis, and the antivirus intervening treatment of hepatitis is significant for the prognosis of liver cancer. From our Spearman＇s rank correlation analysis, we can conclude that the severity of virally induced inflammation is correlated with HBsAg and HCV expression in HCC tissues and noncancerous tissues. Prior co-infection of HBV in HCV patients may be an adverse risk factor for intrahepatic inflammation.

Hepatitis C virus genotype 3a infection and hepatocellular carcinoma: Pakistan experience

AIM: To assess the association between chronic hepatitis C virus (HCV) infection and hepatocellular carcinoma (HCC) in Pakistan, and the genotype distribution among these HCC patients.METHODS: One hundred and sixty-one subjects with HCC were included in this study. Liver biopsy was performed on 145 of the patients; sixteen were excluded because they failed to fulfill the inclusion criteria. Qualitative polymerase chain reaction (PCR) was performed for hepatitis B virus and HCV. Samples positive for HCV RNA were genotyped using genotype-specif ic PCR and conf irmed by HCV 5' noncoding region sequencing analysis. RESULTS: Chronic HCV infection was identified a major risk factor (63.44% of tested HCC patients) forthe development of HCC. The time from HCV infection to appearance of cancer was 10-50 years. In the HCC patient population, broader distributions of genotypes were present with genotype 3a as the predominant genotype. Using the type-specific genotyping method, we found HCV genotype 3a in 40.96%, 3b in 15.66%, 1a in 9.63%, and 1b in 2.40% of HCC tissue samples. About 28% of cases were found with mixed genotypes. Two cases were unable to be genotyped because of low viral load. Sixty-six percent of treated patients with cirrhosis had an end of treatment response, but unfortunately they relapsed quickly when the treatment was discontin-ued, and HCC developed during a median 3.8 years. CONCLUSION: There was a strong association between chronic HCV infection and HCC in Pakistan, and between HCV genotype 3a and HCC.

Vitamin D deficiency: Correlation to interleukin-17, interleukin-23 and PⅢNP in hepatitis C virus genotype 4

AIM: To assess vitamin D (Vit D) abnormalities in hepatitis C infected patients and their relationship with interleukin (IL)-17, IL-23 and N-terminal propeptide of type Ⅲ pro-collagen (PⅢNP) as immune response mediators. METHODS: The study was conducted on 50 Egyptian patients (36 male, 14 female) with hepatitis C virus (HCV) infection, who visited the Hepatology Outpatient Clinic in the Endemic Disease Hospital at Cairo University. Patients were compared with 25 ageand sexmatched healthy individuals. Inclusion criteria were based on a history of liver disease with HCV genotype 4 (HCV-4) infection (as new patients or under followup). Based on ultrasonography, patients were classified into four subgroups; 14 with bright hepatomegaly; 11 with perihepatic fibrosis; 11 with hepatic cirrhosis; and 14 with cirrhosis and hepatocellular carcinoma (HCC).Total Vit D (i.e., 25-OH-Vit D) and active Vit D [i.e., 1,25-(OH) 2 -Vit D] assays were carried out using commercial kits. IL-17, IL-23 and PⅢNP levels were assayed using enzyme linked immunosorbent assay kits, while HCV virus was measured by quantitative and qualitative polymerase chain reaction. RESULTS: Levels of Vit D and its active form were significantly lower in advanced liver disease (hepatic cirrhosis and/or carcinoma) patients, compared to those with bright hepatomegaly and perihepatic fibrosis. IL-17, IL-23 and PⅢNP levels were markedly increased in HCV patients and correlated with the progression of hepatic damage. The decrease in Vit D and active Vit D was concomitant with an increase in viral load, as well as levels of IL-17, IL-23 and PⅢNP among all subgroups of HCV-infected patients, compared to normal healthy controls. A significant negative correlation was evident between active Vit D and each of IL-17, IL-23 and PⅢNP (r = -0.679, -0.801 and -0.920 at P < 0.001, respectively). HCV-infected men and women showed no differences with respect to Vit D levels. The viral load was negatively correlated with Vit D and active Vit D (r = -0.084 and -0.846 at P < 0.001, respectively), and positively correlated with IL-17, IL-23 and PⅢNP (r = 0.951, 0.922 and 0.94 at P < 0.001, respectively). Whether the deficiency in Vit D was related to HCVinduced chronic liver disease or was a predisposing factor for a higher viral load remains to be elucidated. CONCLUSION: The negative correlations between Vit D and IL-17, IL-23 and PⅢNP highlight their involvement in the immune response in patients with HCV-4related liver diseases in Egypt.

Expression and immunoreactivity of HCV/HBV epitopes

AIM： To develop the epitope-based vaccines to prevent Hepatitis C virus （HCV）/Hepatitis B virus （HBV） infections. METHODS： The HCV core epitopes C1 STNPKPQRKTKRNTNRRPQD （residuals aa2-21） and C2 VKFPGGGQIVGGVYLLPRR （residuals aa22-40）, envelope epitope E GHRMAWDMMMNWSP （residuals aa315-328） and HBsAg epitope S CTTPAQGNSMFPSCCCTKPTDGNC （residuals aa124-147） were displayed in five different sites of the flock house virus capsid protein as a vector, and expressed in E. coli cells （pET-3 system）. Immunoreactivity of the epitopes with anti-HCV and anti-HBV antibodies in the serum from hepatitis C and hepatitis B patients were determined. RESULTS： The expressed chimedc protein carrying the HCV epitopes C1, C2, E （two times）, L3C1-I2E-L1C2- L2E could react with anti-HCV antibodies. The expressed chimeric protein carrying the HBV epitopes S, I3S could react with anti-HBs antibodies. The expressed chimeric proteins carrying the HCV epitopes C1, C2, E plus HBV epitope S, L3C1-I2E-L1C2-L2E-I3S could react with anti- HCV and anti-HBs antibodies. CONCLUSION： These epitopes have highly specific and sensitive immunoreaction and are useful in the development of epitope-based vaccines.

Hepatitis C virus in Pakistan:A systematic review of prevalence,genotypes and risk factors

In Pakistan more than 10 million people are living with Hepatitis C virus (HCV), with high morbidity and mortality. This article reviews the prevalence, genotypes and factors associated with HCV infection in the Pakistani population. A literature search was performed by using the keywords; HCV prevalence, genotypes and risk factors in a Pakistani population, in Pubmed, PakMediNet and Google scholar. Ninetyone different studies dating from 1994 to May 2009 were included in this study, and weighted mean and standard error of each population group was calculated. Percentage prevalence of HCV was 4.95% ± 0.53% in the general adult population, 1.72% ± 0.24% in the pediatric population and 3.64% ± 0.31% in a young population applying for recruitment, whereas a very high 57% ± 17.7% prevalence was observed in injecting drug users and 48.67% ± 1.75% in a multi-transfused population. Most prevalent genotype of HCV was 3a. HCV prevalence was moderate in the general population but very high in injecting drug users and multi-transfused populations. This data suggests that the major contributing factors towards increased HCV prevalence include unchecked blood transfusions and reuse of injection syringes. Awareness programs are required to decrease the future burden of HCV in the Pakistani population.