Comparison of HCV core antigen and anti-HCV with HCV RNA results

Background

The measurement of anti-HCV antibodies using immunological methods and the confirmation of viral nuclear acid based on molecular methods is important in diagnosis and follow-up of the HCV infection.

Objectives

In this study, we aimed to analyse HCV core Antigen positivity among anti-HCV antibody positive sera to determine the significance of testing of HCV core Ag for the laboratory diagnosis of HCV infection, by considering the correlation between serum HCV core Ag and HCV RNA levels.

Methods

115 patients suspected of having hepatitis C and who were positive for anti-HCV antibody were investigated using chemiluminescent and molecular methods. Anti-HCV antibody, HCV core Ag and HCV RNA levels were detected by the Vitros ECiQ immunodiagnostic system, Architect i2000 system and RT-PCR, respectively.

Results

The sensitivity, specificity, positive and negative predictive values and accuracy rate of HCV core Antigen assay were detected as 86.5%(83/96), 100%(19/19), 100%(83/83), 59.4%(19/32), 88.7%(102/115) respectively.

Conclusion

HCV core Ag assay could be used for diagnosis of HCV infection as it is easy to perform, cost-effective, has high specificity and positive predictive value. However, it should be kept in mind that it may have lack of sensitivity and negative predictive value.     

Reduction in intracellular HCV RNA and virus protein expression in human hepatoma cells following treatment with 2'-O-methyl-modified anti-core deoxyribozyme

HCV RNA is gaining greater consideration as a principal target for newer HCV antivirals because its destruction has the potential of eliminating the virus. These newer antivirals include deoxyribozymes (Dz), which are small single-stranded DNA molecules that cleave homologous RNA targets. Using a liver cell model containing functional genomic-length HCV-1b RNA we tested whether 2'-O-methyl-modified Dz, designed to recognize a highly-conserved RNA sequence located within the core-E1 coding region, could recognize and cleave its target sequence in the structural context of a functional HCV RNA molecule. Dz858-4-OMe contains four 2'-O-methyl nucleotide derivatives consecutively located on the distal ends of its two annealing arms. Intracellular HCV RNA, core protein and HCV antigen expression were reduced by 63%, 87% and 84%, respectively, when HCV RNA was challenged 6 h post-transfection with Dz858-4-OMe. The observed reduction of intracellular HCV RNA and protein by Dz858-4-OMe suggests that it may constitute an attractive HCV antiviral.     

Loss of serum HCV RNA at week 4 of interferon-α therapy is associated with more favorable long-term response in patients with chronic hepatitis C

To determine the virological factors associated with a favorable long-term response to interferon-α (IFN) therapy in chronic hepatitis C virus (HCV) infection, 61 Japanese patients with chronic HCV infection were treated with IFN for 24 weeks (780 million units in total) and followed for 8 to 16 months after cessation of therapy. Ten patients dropped out because of severe side effects. Of the 51 patients who completed IFN therapy, 23 showed complete and sustained response (CR→SR), 13 complete response with early relapse (CR→Rel), and 15 no response to IFN (NR). For the pretreatment serum HCV RNA level, 20/23 who had CR→SR had <l0⁶ eq/ml compared to 3/13 CR→Rel and 1/15 NR (P< 0.01). Serologically defined HCV type 2 infection was also associated with a better opportunity to develop CR→SR compared to CR→Rel of NR (P<0.01). Loss of serum HCV RNA at week 4 of IFN therapy was also associated with a more favorable long-term response [17/19 CR→SR were HCV RNA negative compared to 3/11 CR→Rel (P<0.01)and2/13NR(P<0.01)]. n contrast, normalization of serum alanine ami-notransferase (ALT) levels at week 4 was found in 9/19 CR→SR compared to 8/11 CR→Re1 (P= NS), and 0/13 in NR (P<0.01). Six months after cessation of IFN therapy, 3/25 CR→SR patients were HCV RNA positive despite normalization of serum ALT levels. These data indicated that in addition to pretreatment serum HCV RNA levels and HCV type, the kinetics of response to IFN (at week 4) were also predictive of subsequent long-term response to IFN in patients with chronic HCV infection. © 1995 Wiley-Liss, Inc. © 1995 Wiley-Liss, Inc.     

HCV核心蛋白抑制SFRP1基因启动子活性

 目的 探讨HCV 核心蛋白对Wnt信号通路抑制分子SFRP1启动子活性的影响。方法 以人肝癌细胞系Huh7基因组为模板,扩增SFRP1启动子区不同片段(-407～-27bp, -837～-27bp, -1202～-27bp, -1619～-27bp, -2029～-27bp),以pGL3-Basic为载体分别构建SFRP1启动子区截短报告质粒。将构建好的质粒与pRL-TK共转染HEK293细胞,确定启动子区活性最强区域;分别用编码HCV 核心蛋白的腺病毒或GFP对照腺病毒感染已转染重组报告质粒的SK-Hep1细胞,观察AdCore对SFRP1启动子活性的调控作用。结果 SFRP1启动子区域-407～-27bp片段活性最强;与pGL3-Basic对照组相比较,相对荧光素酶活性增加了37.31±4.45倍(P<0.01),pGL3-S2～S5的活性分别增加了28.74±2.47、13.56±2.52、12.97±0.87和8.29±0.09倍(P<0.01);HCV Core蛋白能够抑制SFRP1基因启动子区活性,其对pGL3-S1的抑制作用最强,与GFP对照组相比较,抑制率为63.8%±1.0% (P<0.01)。结论 HCV核心蛋白通过抑制SFRP1启动子区活性下调SFRP1基因的表达,可能参与Wnt/β-catenin信号通路的活化,并与原发性肝癌的发生密切相关。     

Attenuated dengue 2 viruses with deletions in capsid protein derived from an infectious full-length cDNA clone

A full-length cDNA clone (pD212) of dengue virus type 2 isolated in China (DEN2-43) was constructed. Based on this, we constructed several mutants with deletions in capsid protein C using fusion PCR. These deletions removed part or almost all of the internal stretch of hydrophobic amino acid residues that is probably involved in virion assembly. We thus obtained viable mutant viruses. The propagation capacity of the mutant viruses in cell culture was impaired in parallel with the increasing size of the deletion, and the infectivity of mutant C(Delta42-59), from which all of helix III of capsid protein C was removed, was completely abolished. More importantly, the mutant viruses were highly attenuated in suckling mice but induced high levels of antibodies in adult mice. This study indicates that the structural and functional flexibility of capsid protein C make it a candidate target for the attenuation of dengue virus, which could open a promising new avenue for the development of live attenuated dengue vaccines.     

Effect of genotypes on the quantification of hepatitis C virus (HCV) RNA in clinical samples using the amplicor HCV monitor test and the quantiplex HCV RNA 2.0 assay (bDNA)

The Amplicor HCV Monitor test and the Quantiplex HCV RNA 2.0 (bDNA) assay are two commercially available assays for the quantification of hepatitis C virus (HCV) RNA in clinical samples. A direct comparison of the two assays was carried out using sera frozen previously from patients known to be chronically infected with HCV. Overall, 61 samples from 51 patients were tested simultaneously by the two methods: 67% (28/42) of the patients were infected by HCV genotype/serotype 1, 10 % (4/42) with type 2, and 24% (10/42) with type 3. When the absolute value from each assay was examined, the Quantiplex assay gave a consistently higher reading and the mean logarithmic difference between the two assays was 1.4 (1.0 in type 1, 2.0 in type 2, and 2.2 in type 3). When analyzed according to genotype, strong correlation was observed between the two assays for type 1 (r = 0.83, 95% CI 0.63–0.93, P < 0.01), but not for nontype 1 samples. Despite the difference in absolute level reported by the two assays, there was a consistent trend of change in HCV RNA concentration by both assays in patients whose consecutive samples were analyzed and the differences between the two assays in consecutive samples were within 0.4 log of each other. The results suggested that with samples containing genotype 1, the Amplicor assay was more sensitive than the Quantiplex assay by about one log. However, the sensitivities of the two assays with nontype 1 samples were much closer probably due to the failure of the Amplicor assay to quantify nontype 1 genotypes effectively. J. Med. Virol. 55:191–196, 1998. © 1998 Wiley-Liss, Inc.     

Analytical performance of the VERIS MDx system HCV assay for detecting and quantifying HCV RNA

BackgroundThe diagnosis of HCV relies on the detection of viral RNA.ObjectiveTo evaluate the performance of the VERIS/MDx System HCV Assay, a new automated system for quantifying HCV RNA, and to compare with the COBAS^® Ampliprep/COBAS^® Taqman™ (CAPCTM) HCV Test version 2.0.Study designThe limit of detection was determined by Probit analysis with the 3rd International WHO HCV standard and precision by assaying in duplicate control samples with HCV RNA concentrations of 7.9; 5.0; 3.4; 1.6 and 0 log IU/ml over 20 days. Analytical specificity was assessed by assaying 180 samples from negative anti-HCV and HCV RNA blood donors and linearity with replicates of serial dilutions of a clinical plasma (6.4–0.6 log IU/ml). We compared the VERIS MDx HCV and CAPCTM HCV assays by testing 209 samples.ResultsThe limit of detection was 6.1 IU/ml [CI 95%: 5.0–8.3] and the precision, given by the standard deviation, was ≤0.11 log IU/ml. Specificity was 100%. The linearity ranged from 1.5 to 6.4 log IU/ml. Passing-Bablok regression analysis gave: VERIS log IU/ml = −0.33 + [1.04× CAPCTM] log IU/ml, with biases for the 25th, 50th, 75th percentiles of 0.18, −0.10 and −0.06 log IU/ml. The two assays were well correlated (ρ = 0.92, p < 0.001) and Bland-Altman analysis gave biases of 0.12, log IU/ml for genotype 1, −0.19 for genotype 2, −0.26 for genotype 3, and −0.77 for genotype 4.ConclusionThe VERIS MDx HCV assay performed well. But, we observed an under-quantification of the genotype 4 samples.     

耐核糖核酸酶内含HCV RNA病毒样颗粒的表达

目的 构建可以表达耐核糖核酸酶(RNase)的内含HCV RNA病毒样颗粒的载体质粒。方法 将表达载体pI NCCL用Hind Ⅲ酶切后，与用相同内切酶酶切的HCV RNA非编码区(5′-UTR)扩增产物，在T4 DNA连接酶的存在下连接，构建一新的表达载体pI NCCL-HCV RNA 5′-UTR，再转化BL21-DE3 E．coli进行原核表达。结果 成功构建得到了新的表达载体pI NCCL-HCV RNA5′-UTR，经原核表达为耐RNase的内含HCV RNA病毒样颗粒。结论 得到的pI NCCL-HCV RNA 5′-UTR表达载体及原核表达系统，可作为一个耐RNase的HCV RNA标准品和质控品的构建和制备表达载体平台。     

Comparison of three quantitative HCV RNA assays in samples from HCV genotype 1- or 4-infected patients treated with the NS3/4A protease inhibitor simeprevir

BackgroundMonitoring HCV RNA levels during treatment is an important tool for managing protease-inhibitor-based regimens, and different assays used in clinical practice can impact treatment decisions.ObjectivesThe concordance of three HCV RNA assays was determined, and their impact on treatment decisions assessed using samples from HCV genotype (GT) 1- and GT4-infected patients treated with the NS3/4A inhibitor simeprevir in combination with pegylated interferon-α/ribavirin.Study designPlasma samples collected during the simeprevir Phase III studies QUEST-1 and QUEST-2 (GT1), and RESTORE (GT4) were analyzed with the Roche High-Pure-System COBAS^® TaqMan^® HCV v2.0 (HPS), the Roche AmpliPrep COBAS^® TaqMan^® HCV v2.0 (CAP), and the Abbott RealTime HCV (ART) assay.ResultsIn GT1, of the 440 samples, 81% were undetectable (rapid virological response; RVR) by HPS at Week 4, 76% by CAP and 44% by ART. In GT4 (103 samples), RVR rates were 67% by HPS and 24% by ART. HCV RNA <25 IU/mL at Week 4 was observed for 95–96% and 92% GT1 samples and 86% and 74% GT4 samples by HPS/CAP and ART, respectively. At Week 12, assay concordance for undetectability was high in GT1 and GT4, (95–98% and 93%, respectively).ConclusionsWhile different HCV RNA assays can lead to substantially different RVR rates, a good concordance was observed with a cut-off of 25 IU/mL. Sustained virologic response rates among GT1 patients achieving RVR or <25 IU/mL at Week 4 were high and similar between assays used. At later time points, when viremia is low, assay concordance was high.     

Performance of ARCHITECT HCV core antigen test with specimens from US plasma donors and injecting drug users

BackgroundHepatitis C virus (HCV) core antigen is a serological marker of current HCV infection.ObjectivesThe aim of this study was mainly to evaluate the performance characteristics of the ARCITECT HCV core antigen assay with specimens from US plasma donors and injecting drug users.Study designA total of 551 serum and plasma samples with known anti-HCV and HCV RNA status were tested for HCV core antigen using the Abbott ARCHITECT HCV core antigen test.ResultsHCV core antigen was detectable in 100% of US plasma donor samples collected during the pre-seroconversion phase of infection (anti-HCV negative/HCV RNA positive). Overall sensitivity of the HCV core antigen assay was 88.9–94.3% in samples collected after seroconversion. The correlation between HCV core antigen and HCV RNA titers was 0.959.ConclusionsHCV core antigen testing may be reliably used to identify current HCV infection.     

Peginterferon alfa-2a and peginterferon alfa-2b combined with ribavirin in patients with genotype 1 chronic hepatitis C: Results of a prospective single-centre study

PurposeThis prospective, randomized, single-centre study compared peginterferons alfa-2a and alfa-2b, combined with ribavirin, in treating patients infected with hepatitis C virus (HCV) genotype 1.Material/methodsHundred-and-one patients received 48 weeks of open-label treatment with peginterferon alfa-2a (180 μg/week) and 111 patients received peginterferon alfa-2b (1.5 μg/kg/week). All patients received the same dose of ribavirin 1000/1200 mg/day, depending on weight. The primary efficacy endpoint was sustained virologic response (SVR), defined as undetectable HCV RNA (<50 IU/mL) 24 weeks after the end of treatment.ResultsEarly virologic response (EVR), defined as at least 2 log₁₀ IU/mL reduction of viral load at 12 weeks, was more common in patients treated with peginterferon alfa-2a (88% vs. 74.8%; p = 0.04). However, the difference in SVR was not statistically significant (49.5% vs. 44.1%; p = 0.43).ConclusionsPeginterferon alfa-2a treated patients were also more likely to be HCV RNA negative at the end of treatment (67.3% vs. 57.7%), but this difference did not reach statistical significance. Multivariate logistic regression analysis found that SVR was associated with low fibrosis stage (F1–2 by Scheuer; p = 0.001) and low serum HCV RNA level (<400,000 IU/L; p = 0.023). While both forms of peginterferon showed similar efficacy as measured by SVR, use of peginterferon alfa-2b could lower the number of patients receiving unnecessary treatment beyond 12 weeks.     

Influence of HCV genotype 1 subtypes on the virus response to PEG interferon alpha-2a plus ribavirin therapy

New factors that influence the viral response in HCV non-genotype 2/3 patients must be identified in order to optimize anti-HCV treatment. This multicenter prospective study evaluates the influence of HCV variability and pharmacological parameters on the virological response of these patients to pegylated interferon α2a (peg-IFN-α2a: 180 μg/week) and ribavirin (RBV; 800-1,200 mg/day) for 48 weeks. HCV subtypes were identified by sequencing the NS5B region. Serum RBV and peg-IFN-α2a concentrations were measured at weeks 4 and 12. The 115 patients (67 men; median age = 49, range 31-76) included 64 who had never been treated and 27 co-infected with HIV. The mean baseline HCV RNA was 6.30 ± 0.06 log IU/ml and the HCV genotypes were: G1 (n = 93) with 1a (n = 37) and 1b (n = 50), G4 (n = 20) and G5 (n = 2). Most patients (79/108; 73%) had an early virological response. Independent predictors of an early virological response were interferon naive patients (OR= 2.98, 95% CI: 1.15-7.72) and RBV of >2,200 ng/ml at week 12 (OR = 3.41, 95% CI: 1.31-8.90). Forty of 104 patients (38%) had a sustained virological response. The only independent predictors of a sustained virological response were subtype 1b (OR = 6.82, 95% CI: 1.7-26.8), and HCV RNA <15 IU/ml at week 12 (OR = 25, 95% CI: 6.4-97.6). Thus a serum RBV concentration of >2,200 ng/ml was associated with an early virological response and patients infected with HCV subtype 1b had a better chance of a sustained virological response than did those infected with subtype 1a.     

The entire core protein of HCV JFH1 is required for efficient formation of infectious JFH1 pseudoparticles

The vast majority of hepatitis C virus (HCV) strains cannot be grown in cell culture. Therefore, tests for neutralizing antibodies have relied heavily on retrovirus pseudoparticles displaying the envelope glycoproteins of HCV on their surface (HCVpp). Unfortunately, the envelope proteins of some strains, especially of JFH1, did not efficiently form functional HCVpp. We have manipulated the length and composition of the HCV core gene in the HCVpp expression vectors for three strains of HCV in an attempt to obtain more efficient production of pseudoparticles. The results demonstrated that the truncated core region included in the HCV expression plasmids of the classic pseudoparticle system was optimal for formation of strain H77pp, suboptimal for strain J6pp, and insufficient for strain JFH1pp. Efficiency of JFH1pp formation increased 20‐fold when the truncated core gene was replaced with the entire core gene. The full core from J6 and HK had modest effect on the production of infectious J6 and HKpp. The data suggested that pairs of HCV glycoproteins differ inherently in their ability to associate into functional heterodimers and that the core protein, provided in cis as the beginning of the polyprotein product, can in some cases facilitate this process, possibly by increasing the rate of proper folding of the glycoproteins. J. Med. Virol. 82: 783–790, 2010. Published 2010 Wiley‐Liss, Inc.     

Assessment of early virological response to antiviral therapy by comparing four assays for HCV RNA quantitation using the international unit standard: implications for clinical management of patients with chronic hepatitis C virus infection

WHO International Standards for nucleic acid tests are used widely to compare the different assays used in HCV RNA quantitation. The aim of the study was to assess the impact of the international unit standard for measuring HCV RNA in the management of patients with chronic hepatitis C virus (HCV) infection. Twenty‐seven naïve patients infected chronically by HCV were treated with ribavirin plus PEG‐interferon‐alfa‐2b for 48 weeks. SVR was obtained for 16 patients (the other were non‐responders). For HCV RNA quantitation, four assays were undertaken: Versant HCV RNA 3.0 (Bayer), Real time PCR (TaqMan, Roche), LCx HCV RNA (Abbott), and Cobas Amplicor‐Monitor v2 (Roche). Considering a 2‐log decline at Week 12 after the beginning of therapy, discordant results were found with the four HCV RNA methods in predicting SVR or non‐response. At Week 4 and Week 12, significant differences were observed between Versant HCV RNA 3.0 versus PCR HCV Taqman, Versant HCV RNA 3.0 versus LCx HCV RNA, Cobas Monitor Amplicor HCV 2.0 versus LCx HCV RNA, and Cobas Monitor Amplicor HCV 2.0 versus PCR HCV Taqman (P < 0.001). The HCV RNA cutoff, given a 100% negative predictive value at Week 4 and Week 12, differed with the assays used to quantify HCV RNA, despite the use of the IU/ml units. Eighty‐nine percent of serum values for HCV RNA were concordant by the IU standard. All assays, however, failed to detect HCV RNA in some cases. Despite the use of the IU standard HCV‐infected patients might be monitored with only one assay. J. Med. Virol. 78:208–215, 2006. © 2005 Wiley‐Liss, Inc.