Detection of hepatitis C markers--nucleocapsid protein, RNA and virus-specific antibodies in blood donor plasma

Comparative study of hepatitis C virus (HCV) markers (core protein, RNA, and virus-specific antibodies) was carried out in plasma samples from 80 donors. A method based on sandwich ELISA with monoclonal antibodies to recombinant protein was developed for measuring core protein. Nucleocapsid protein was detected after various treatments of precipitates obtained after concentration of virus-containing material from plasma samples. These treatments allowed differentiation of core protein in virions, free nucleocapsids, and immune complexes circulating in peripheral blood. The minimal detectable concentration was 5 pg/ml, maximal 850 pg/ml. The detection of core protein virtually coincided with the detection of HCV RNA: 94.4% RNA-positive samples contained the virus protein. Other parameters (activities of antibodies to HCV in ELISA and level of SGPT) did not allow differentiation of plasma samples by the presence of actively replicating virus. Assay of nucleocapsid protein in the plasma of subjects infected with HCV in various populations of virus particles is important from practical (for blood service) and theoretical viewpoints (for studies of virus pathogenesis mechanisms).     

Anti-HCV IgG avidity index in acute hepatitis C.

BACKGROUND: The diagnosis of acute hepatitis C (AHC) is based on seroconversion to positive anti-HCV, which is usually not clinically possible. OBJECTIVE: To determine if avidity of anti-HCV IgG can be used for the diagnosis of AHC infection. STUDY DESIGN: We enrolled 40 consecutive patients with AHC, 16 drug addicts (IVDA) with exacerbation of chronic hepatitis C (IVDA e-CHC group), 21 non-IVDA with exacerbation of chronic hepatitis C (IVDA-free e-CHC group) and 40 with chronic hepatitis C (CHC group). HCV avidity index (HCV-AI) was determined by ELISA on sera pre-diluted 1:10 with 1M guanidine. RESULTS: On admission, HCV-AI values were significantly lower in the AHC group (mean+/-S.D.: 0.50+/-0.30) than in IVDA-free e-CHC group (0.97+/-0.08, p<0.0001), IVDA e-CHC group (0.90+/-0.29, p<0.0001) or CHC group (1.06+/-0.20, p<0.0001). An HCV-AI lower than 0.7 obtained within the 8th day of illness distinguished patients with AHC infection from the IVDA-free e-CHC cases. An increase in HCV-AI was observed in 24 (72.7%) of 33 in AHC group, in none of 13 in IVDA-free e-CHC group and in 3 (27.3%) of 11 in IVDA e-CHC group. CONCLUSION: HCV-AI is useful in identifying AHC infection in patients observed within the 8th day from the onset of symptoms.     

Hepatitis C virus proteins in the liver cells in acute hepatitis C: their association with liver damage and with the outcome of the disease

The accumulation of individual hepatitis C virus (HCV) proteins in the liver cells of patients with acute hepatitis C (AHC) and their association with the course and outcome of the disease were studied. AHC protein expression in the cryostat liver sections from 20 patients with AHC was estimated by immunohistochemical assay using original monoclonal antibodies to 5 HCV proteins (core, NS3, NS4A, NS4B, and NS5A). The results of HCV detection in the patients were compared with their biochemical, clinical, and morphological findings. HCV proteins were totally revealed in the livers of all the patients, individual proteins were identified with a frequency of 89-95%, which is significantly more than those in patients with chronic hepatitis C. The hepatic expression of core protein was shown to inversely correlate with the duration of an acute period. There was a direct relationship between the accumulation of core, NS3, and NS5A proteins and the liver tissue damage caused by stepwise necrosis rather than intralobular necrosis. The presumed convalescence was ascertained to be associated with the larger count of hepatocytes containing the proteins NS4A and NS3 early after AHC manifestation.     

Steatosis and intrahepatic hepatitis C virus in chronic hepatitis.

Hepatic steatosis has been reported as one of the characteristics which discriminates hepatitis C from other forms of hepatitis, besides lymphoid follicles and bile duct damage. However, it is unclear whether or not the presence of hepatitis C virus (HCV) itself is associated with the development of steatosis. The possibility that the HCV itself is directly related to the development of steatosis was examined. The intrahepatic core protein levels, as a marker of the HCV load, were correlated with the presence of steatosis in 43 patients with chronic hepatitis C. Among 43 patients studied by Western blotting, the core protein was detected in the liver in 27 (62.8%). On the other hand, hepatic steatosis was observed in 21 (48.8%) of the 43 patients. Importantly, the core protein was detectable in 19 (90.4%) of the 21 patients with steatosis, while it was detected in only 8 (36.4%) of the 22 patients without steatosis (P = 0.008). However, serum HCV-RNA levels as determined by the Amplicor monitor were not significantly different between patients with and without steatosis. Multivariate analysis showed that the serum alanine aminotransferase level (P = 0. 013), body mass index (P = 0.038), and intrahepatic HCV core protein positivity (P = 0.038) were the independent parameters best predictive of steatosis. These results indicate a close relationship between intrahepatic HCV and the development of steatosis, and suggest a possible role of the HCV itself or core protein in the pathogenesis of steatosis in human chronic hepatitis C.     

Association of hepatitis C virus carrier state with the occurrence of hepatitis C virus core antibodies.

An enzyme immunoassay (EIA) was developed for the determination of antibodies against the "putative" core protein of hepatitis C virus (HCV). Antigens used were recombinant fragments (amino acids 6-77 or 6-143) of the HCV core protein, produced in Escherichia coli with truncated hepatitis B core (HBc) as fusion protein. Evaluation of 385 sera positive for HCV antibodies by first generation EIA, revealed 98 (25.4%) with HCV core antibodies. HCV-RNA, determined by the polymerase chain reaction (PCR), was exclusively found in the sera positive for HCV core antibodies (89 PCR positives). In random screening of 3,708 sera, 3 sera with HCV core antibodies were found PCR positive. Only 2 of these sera were positive in the first generation EIA. It is concluded that HCV core antibody determination is a reliable test for identifying HCV carriers among blood donors.     

Evaluation of antibodies to hepatitis C virus in a study of transfusion-associated hepatitis

BACKGROUND. The hepatitis C virus (HCV) is now known to be the chief cause of transfusion-associated non-A, non-B hepatitis, but the prevalence of HCV among blood donors and the frequency of transmission by blood transfusion are unknown. METHODS. To assess the sensitivity and specificity of a test for antibody to HCV, we tested serum samples from participants in a large study of transfusion-associated hepatitis. Samples were obtained prospectively from consecutive adults undergoing open-heart surgery in Spain, but were tested retrospectively, after the antibody enzyme immunoassay for anti-HCV became available. RESULTS. Of 280 transfusion recipients given a total of 1109 units of blood, 27 (9.6 percent) had transfusion-associated non-A, non-B hepatitis (mean follow-up, 52 weeks) and 24 of the 27 seroconverted to anti-HCV-positive, whereas only 2 (0.8 percent) of the remaining transfusion recipients seroconverted. Among the 1044 donor specimens available for testing, 16 (1.5 percent) had anti-HCV antibody. Only 1 additional seropositive donor was found when 44 implicated donors who had been seronegative were retested 9 to 12 months later. Of the 16 recipients of anti-HCV-positive blood, 14 (88 percent) had transfusion-associated hepatitis and seroconverted to anti-HCV-positive. The remaining two recipients had neither hepatitis nor anti-HCV antibody. Among 25 patients with non-A, non-B hepatitis for whom all transfused blood was tested, 14 had received blood positive for anti-HCV. CONCLUSIONS. About 90 percent of blood donors with antibody to HCV have infectious virus in their blood. The screening of blood donors for anti-HCV antibody should prevent about half the cases of transfusion-associated hepatitis, but the donors with infectious virus who are anti-HCV-negative may remain seronegative for prolonged periods.     

丙型肝炎病毒核心抗原检测用于献血者筛查价值的探讨

目的应用酶联免疫吸附技术筛查献血员中丙型肝炎病毒核心抗原（HCV—cAg）和抗体（HCV—Ab），了解丙型肝炎病毒核心抗原筛查献血员应用价值。方法对我站2004年8-12月间的3972份献血者血清标本进行抗-HCV初、复检和HCV—cAg ELISA检测，将ELISA法阳性的25份血清标本，再做RT-PCR检测证实。结果3972份血清标本检测中，有10份仅初检抗-HCV阳性样本，经HCVRNA检测阳性有l份，12份仅复检抗-HCV阳性样本，经HCVRNA检测阳性有1份，HCV—cAg检测阳性有3份，经HCVRNA检测阳性有2份。结论HCV—cAg ELISA检测技术的敏感性与HCVRNA技术类似，但成本明显降低，可与HCV抗体联合检测应用献血员筛查。     

丙型肝炎病毒核心抗原C33的测定

目的探索丙型肝炎病毒（ＨＣＶ）Ｃ３３抗原在正常人群及各类临床病人血清及肝组织中分布状况。方法用人工合成的ＨＣＶ核心抗原Ｃ３３肽（含３３个氨基酸）与小鼠血清白蛋白,经异型双功能交联剂ＳＰＤＰ连接后免疫Ｂａｌｂ／Ｃ小鼠,制成２株Ｃ３３肽单克隆抗体（ＭｃＡｂ）,分别用作包被抗体和酶标抗体,建立检测血清中Ｃ３３肽抗原的酶联免疫吸附试验（ＥＬＩＳＡ）,共检测了１２３６例各类病人。并用免疫荧光及固相免疫酶法检测肝活检组织中的ＨＣＶＣ３３肽抗原。结果急性丙型肝炎（ＨＣ）患者,Ｃ３３肽的阳性检出率为１４．０％（６／４３）;仅抗－ＨＣＶＩｇＧ阳性患者Ｃ３３肽的阳性检出率为８．８％（３５／３９６）;慢性丙型肝炎患者为６．５％（４／６２）;白血病患者为４．３％（１／２３）;血透病人为３．３％（１９／５７６）;普通住院病人为１．５％（２／１３６）;正常供血员为１．４％（６／４３８）。肝活检免疫荧光、固相免疫酶检测显示ＨＣＶＣ３３肽在细胞核、细胞质、细胞膜上均有表达,在胞质中或集中于全胞质一侧或弥散分布于胞质中。结论ＨＣＶＣ３３抗原测定,可以作为ＨＣＶ感染的标志物之一。     

徐州地区庚型肝炎病毒5′非编码区部分序列分析

自庚型肝炎病毒（ＨＧＶ）被报道以来,许多国家、地区相继分离出该病毒的基因序列,并发现ＨＧＶ基因序列存在一定变异,ＨＧＶ不同基因区以５′非编码区（５′－ｕＴＲ）变异较小,有学者根据该区序列的变异,认为ＨＧＶ至少存在三种基因型,以最初报道的美国株ＨＧＶ（...     

Serodiagnosis of hepatitis C virus infection by ELISA for antibodies against the putative core protein (p20c) expressed in Escherichia coli.

The putative core gene of hepatitis C virus (HCV) was incorporated into a plasmid vector (pCC5-J4), and expressed in Escherichia coli. The product of 180 amino acids (p20c) was purified by gel electrophoresis in the presence of sodium dodecyl sulfate, and used in enzyme-linked immunosorbent assay for antibodies against the putative core protein of HCV (anti-p20c). Anti-p20c was detected in 13 (1.5%) of 873 apparently healthy blood donors. It was detected in 205 (86.5%) of 237 patients with acute or chronic non-A, non-B (NANB) hepatic disease, significantly more frequently (p less than 0.01) than antibodies against the C100-3 protein encoded by nonstructural regions of HCV (anti-C100-3) that was found in 178 (75.1%). Anti-p20c developed in the circulation of a patient with acute NANB hepatitis much earlier than anti-C100-3. HCV RNA was detected by polymerase chain reaction in serum samples from blood donors positive for anti-p20c in high titers, one of which was negative for anti-C100-3. These results indicated that anti-p20c would be useful in complementing anti-C100-3 for the diagnosis of NANB hepatitis and further decreasing the incidence of posttransfusion NANB hepatitis.     

The diagnostic significance of an assay for 'total' hepatitis C core antigen.

A measurable serological response to hepatitis C infection is delayed on average until 70 days after infection. In addition, it may not occur in some immunocompromised people. Detection of free hepatitis C (HCV) core antigen in blood has enabled diagnosis in the pre-seroconversion period. The ability to detect 'total' HCV core antigen, both free and antibody bound, would widen its use for confirming anti-HCV antibody positive patients and monitoring a therapeutic response. This study has evaluated a prototype 'total' HCV core antigen immunoassay. Sera from 145 HCV negative blood donors gave a mean value of 54.9 (+/-46.2) pg/ml based on recombinant antigen standards. Using these figures, the HCV core antigen cut-off was set as 200 pg/ml. Two hundred blood donors sera with indeterminant (a single-band on recombinant immunoblot assay) HCV antibody statuses gave fully concordant HCV core antigen results compared to their polymerase chain reactions (PCRs)--three positive, and 197 negative. HCV core antigen and PCR results were compared for 59 sera from 19 HCV positive liver disease patients. The HCV core antigen results were in complete agreement with their PCRs for the nine patients always PCR positive and the three continuously negative. For six patients on antiviral therapy whose qualitative PCRs changed from positive to negative, the HCV core antigen results paralleled the PCR results. The only discrepant results were from one patient whose PCR results went from negative to positive. 'Total' HCV core antigen testing will greatly improve the scope of diagnostic tests for hepatitis C.     

Use of PCR for detection of positive (genomic) and negative (replicative) RNA chains of hepatitis C virus in the serum, lymphocytes, and liver of patients with chronic hepatitis C

The presence of viral RNA in liver tissue and peripheral blood serum and lymphocytes of patients with chronic hepatitis C (CHC) was studied by polymerase chain reaction with nested primers on the 5'-untranslated region of hepatitis C virus (HCV) genome. Positive (genome) RNA was more often detected in the liver (81% cases) than in the peripheral blood serum (55%) or lymphocytes (64%). Active replication of HCV (presence of negative RNA chains) was observed only in the liver (in 37% cases). Correlation between the frequency of HCV RNA detection in the liver, blood cells and sera and parameters of the inflammatory process activity (SGPT level, histologic activity index and sclerosis index) was investigated. No relationship between the studied parameters was revealed. Positive correlation between the presence of HCV genome RNA in lymphocytes and serum was detected. A tendency to a decrease in the incidence of replicative RNA of the virus in liver tissue with increase in the activity of chronic hepatitis C was observed.     

Comparative evaluation of the total hepatitis C virus core antigen,branched-DNA,and amplicor monitor assays in determining viremia for patients with chronic hepatitis C during interferon plus ribavirin combination therapy

An assay prototype designed to detect and quantify total hepatitis C virus [HCV] core antigen (HCV core Ag) protein in serum and plasma in the presence or absence of anti-HCV antibodies has been recently developed by Ortho-Clinical Diagnostics. The aim of the study was to evaluate the sensitivity, specificity, and reproducibility of the Total HCV core Ag assay in comparison with two quantitative assays for HCV RNA: Quantiplex HCV RNA 2.0 (bDNA v2.0) or Versant HCV RNA 3.0 (bDNA v3.0) assays and the Cobas Amplicor HCV Monitor version 2.0 (HCM v2.0) test. We have studied samples of a well-characterized panel and samples from patients with chronic hepatitis C treated with interferon alone or with ribavirin. We have also compared the kinetics of HCV core Ag and HCV RNA in the follow-up of treated patients. The HCV core Ag assay exhibited linear behavior across samples from different genotypes. The coefficients of variation for intra- and interassay performance were 5.11 and 9.95%, respectively. The specificity of the assay tested in blood donors was 99.5%. Samples from HCV-infected patients showed that the correlation between the HCV core Ag and the two HCV RNA quantitative assays (bDNA and HCM v2.0) was 0.8 and 0.7, respectively. This correlation was maintained across different genotypes of HCV (r(2) = 0.64 to 0.94). Baseline HCV core Ag values were significantly lower in sustained responders to interferon (IFN) than in other groups of patients (5.31 log(10) [10(4) pg/ml] versus 5.99 log(10) [10(4) pg/ml]; P < 0.001). In patients treated with IFN or combination therapy, we found an association between a decrease of more than 2 log IU/ml in viral load, undetectable HCV core Ag, and sustained response. Among sustained responders to IFN alone or combination therapy and among relapsers after IFN alone, 84 out of 101 (83.2%) had undetectable HCV core Ag, and 76 out of 96 (79.2%) had a viral load decrease of >/=2 log IU/ml, after 1 month of treatment. In conclusion, the Total HCV core Ag assay is a new useful test for the detection of HCV viremia and the monitoring of patients treated with IFN alone or in combination with ribavirin.     

The declining risk of post-transfusion hepatitis C virus infection.

BACKGROUND. The most common serious complication of blood transfusion is post-transfusion hepatitis from the hepatitis C virus (HCV). Blood banks now screen blood donors for surrogate markers of non-A, non-B hepatitis and antibodies to HCV, but the current risk of post-transfusion hepatitis C is unknown. METHODS. From 1985 through 1991, blood samples and medical information were obtained prospectively from patients before and at least six months after cardiac surgery. The stored serum samples were tested for antibodies to HCV by enzyme immunoassay, and by recombinant immunoblotting if positive. RESULTS. Of the 912 patients who received transfusions before donors were screened for surrogate markers, 35 seroconverted to HCV, for a risk of 3.84 percent per patient (0.45 percent per unit transfused). For the 976 patients who received transfusions after October 1986 with blood screened for surrogate markers, the risk of seroconversion was 1.54 percent per patient (0.19 percent per unit). For the 522 patients receiving transfusions since the addition in May 1990 of screening for antibodies to HCV, the risk was 0.57 percent per patient (0.03 percent per unit). The trend toward decreasing risk with increasingly stringent screening of donors was statistically significant (P less than 0.001). After we controlled for the method of donor screening, the risk of seroconversion was strongly associated (P less than 0.001) with the volume of blood transfused, but not with the use of particular blood components. CONCLUSIONS. The incidence of post-transfusion hepatitis C has decreased markedly since the implementation of donor screening for surrogate markers and antibodies to HCV. The current risk of post-transfusion hepatitis is about 3 per 10,000 units transfused.     

Seroprevalence of hepatitis C virus infection and its genotype in Lanzhou,Western China

The seroprevalence of hepatitis C virus (HCV) infection in Lanzhou, Western China was studied. HCV genotypes in 20 patients with HCV infection was determined by genotype-specific primer for polymerase chain reaction (PCR) based on HCV core region and compared with the genotype assigned by sequence comparison and molecular evolutionary analysis based on the same region. Antibody to HCV (anti-HCV) was present in 2.5% of volunteer blood donors and in 35.0% of paid blood donors (P < 0.01). HCV infection is uncommon in patients with liver disease who attended liver clinics in this locality; 4.0% with acute hepatitis and 4.0% with chronic hepatitis, 10.0% with liver cirrhosis, and none with hepatocellular carcinoma were seropositive for anti-HCV. Genotype 1b and 2a were both found to be prevalent. Together, they accounted for 19 of 20 (95%) patients with HCV infection. Sequencing of the HCV core region from two patients showed that the assignment of HCV genotype by genotype-specific primers for PCR matched well with the genotyping results based on sequence comparison and molecular evolutionary analysis. These data showed that HCV is present in Western China, HCV infection is more common in paid blood donors, and HCV genotypes 1b and 2a are both prevalent in Western China. © 1995 Wiley-Liss, Inc.     

Detection of antibodies to hepatitis C virus in U.S. blood donors.

An enzyme immunoassay (EIA) which utilizes a solid phase coated with a recombinant antigen (c100-3) derived from the hepatitis C virus (HCV) genome was evaluated for efficacy in the detection of antibodies to HCV (anti-HCV). The sensitivity of the antibody test was demonstrated by the detection of anti-HCV in a well-characterized panel of human specimens known to contain the infectious agent of non-A, non-B hepatitis. The specificity of the anti-HCV test was evaluated by testing 6,118 serum specimens from volunteer blood donors considered to be at low risk for exposure to HCV. The specificity of the anti-HCV EIA was demonstrated to be 99.56%, since 6,069 of 6,096 specimens from this low-risk group were nonreactive. A total of 49 (0.80%) of the 6,118 specimens were repeatedly reactive in the test, and 22 (46.81%) of the 47 specimens available for additional testing were confirmed as positive for antibodies to HCV c100-3. Among commercial plasma donors, 390 (10.49%) of 3,718 specimens were repeatedly reactive in the EIA. A total of 375 (97.40%) of the 385 specimens available for further testing were confirmed as positive. These limited data indicate that the prevalence of antibodies to HCV is 0.36% (22 confirmed positives among 6,118 specimens) among volunteer blood donors and 10.08% (375 confirmed positives among 3,718 specimens) among commercial plasma donors. The importance of confirmatory testing is discussed.     

Decreased level of intracellular cholesterol in peripheral blood mononuclear cells is associated with chronic hepatitis C virus infection

Peripheral blood mononuclear cells (PBMCs) constitute the main extrahepatic place of, hepatitis C virus (HCV) replication. We aimed to determine the impact of CHC infection and microRNA-, 122 expression on cholesterol expression in PBMCs. HCV RNA strand, intracellular cholesterol, HMGCoA, reductase and miR-122 expression in PBMC were determined in 54 CHC patients. The study shows that significant decrease of intracellular cholesterol level in PBMC (p = 0.000000), accompanied by serum hypocholesterolemia is the characteristic feature of chronic hepatitis C infection. Although, microRNA-122 expression was detectable in PBMCs of CHC patients (52.5%), the alteration of intracellular cholesterol level was independent of miR-122 expression.     

A chemiluminescent, magnetic particle-based immunoassay for the detection of hepatitis C virus core antigen in human serum or plasma

Hepatitis C virus (HCV) exposure in blood donors is determined serologically by the detection of anti-HCV antibodies in serum or plasma. However, a "window" period of 30-70 days after exposure exists where specific antibodies to HCV antigens are not detected. The use of nucleic acid testing for the detection of HCV RNA or antigen testing for the detection of HCV core protein have resulted in dramatic reductions in the pre-seroconversion window period. In this study, an automated HCV core antigen detection test was developed. This magnetic microparticle-based assay utilizes anti-HCV core monoclonal antibody to capture antigen present in human serum or plasma. Captured antigen is then detected using an anti-HCV core monoclonal antibody conjugated with a chemiluminescent compound. The specificity of this assay was established at 99% upon testing a population of normal volunteer blood donors. Sensitivity was determined by testing 16 commercially available HCV seroconversion panels representing genotypes 1a, 1b, 2b, and 3a. In each panel tested, HCV core antigen was detected prior to anti-HCV antibody, resulting in a reduction of the window period by greater than 23 days on average, and greater than 34 days on panels initially NAT negative. In addition, HCV core antigen was detected in >97% of HCV RNA positive/antibody negative specimens, exhibiting sensitivity nearly equivalent to nucleic acid testing in the pre-seroconversion window period for the panels examined.     

Hepatitis C viraemia in adults with type 2 autoimmune hepatitis.

Sera from 14 patients with type 2 autoimmune hepatitis (anti-LKM1 positive) were investigated for evidence of hepatitis C virus (HCV) infection. Antibodies to HCV were detected in 13 patients by both commercial and "in-house" ELISAs and also by a second generation recombinant immunoblot assay. Nine of the 13 (69%) anti-HCV positive patients were shown to be viraemic by a polymerase chain reaction-based assay for serum HCV RNA. Neither anti-HCV nor serum HCV RNA were detected in any of 6 controls with primary biliary cirrhosis or in 39 healthy blood donors. These findings strongly suggest a role for HCV in the pathogenesis of type 2 autoimmune hepatitis.