丙型肝炎病毒E1抗体检测及临床应用

目的：研究丙型肝炎患者血清中HCV E1抗体，确定HCV E1抗原在抗体检测中的应用价值。方法：应用酶联免疫吸附测定（ELISA）方法检测80份卫生部第3代HCV血清参比品，821例职业献血员血清和720例临床肝炎患者血清中E1抗体。结果：用E1抗原单包板检查80份卫生部血清参比品的阳性符合率为70％、阴性符合率为100％；从821例职业献血员血清样品中检出E1抗体阳性率为1．9％；从720例临床肝炎患者血清中检出E1抗体阳性率为68％。大部分E1抗体阳性血清，同时也能和HCV的Core、NS3抗原及NS5A抗原呈阳性反应，但有个别血清只对E1抗原呈阳性反应。用市购HCV抗ELISA检测试剂盒检测为阴性的218例肝炎科门诊患者、813例献血员和848例一般人群血清，用E1抗原单包板复检，检出的阴性率分别为1．4％、1．1％和0．9％。在3例患者血清学转变的追踪研究中，HCV E1抗体都同现最早。结论：用E1工程蛋白检测E1抗体具有较高的灵敏度和特异性。E1抗体在HCV感染患者中普遍存在而且早出现，在临床诊断上是有意义的。     

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.     

High rate of false positives in blood donor screening for antibodies to hepatitis C virus

Summary Anti-hepatitis C virus antibody screening of blood donors in different countries revealed prevalences ranging from 0,4–1,4%. These results were obtained with an enzyme immunoassay based on a recombinant hepatitis C virus antigen. We applied a specific inhibition assay (neutralization assay) and a recombinant immunoblot assay to determine the specificity of positive reactions in the enzyme immunoassay.Of 2836 blood donor sera tested, 10 (0,35%) were reactive in the enzyme immunoassay, however, only 3 sera (0,1%) proved to be specifically anti-HCV positive in the inhibition assay. The recombinant immunoblot assay gave similar results. The prevalence of anti-hepatitis C virus antibodies among blood donors has been overestimated in recent publications. Furthermore the high rate of false positives in the enzyme immunoassay may explain reports claiming that only a minor part of EIA positive blood units transmitted the hepatitis C virus to recipients.The inhibition assay was also applied to sera of haemophiliacs and of patients with hepatopathy which had reacted positively in the anti-hepatitis C virus antibody enzyme immunoassay. The antihepatitis C virus specificity was confirmed for all sera from the haemophiliacs group (100%) and for 77% of the hepatopathy patients group. Thus, the anti-hepatitis C virus enzyme immunoassay has a high predictive value when it is used to screen groups with high risks of parenteral hepatitis C virus infection, however, its predictive value is very low when it is used for blood donor screening.Abbreviations EIA enzyme immunoassay - HCV hepatitis C virus - RIBA recombinant immunoblot assay - SOD superoxide dismutase     

Detection of hepatitis C virus sequences in sera with controversial serology by nested polymerase chain reaction.

The specificity of first-generation enzyme-linked immunosorbent assays (ELIAs) for antibody detection in individuals with hepatitis C virus (HCV) infection has been questioned in some pathological situations. We observed a surprisingly high prevalence of anti-HCV antibodies in alcoholic patients, and thus, false-positive reactions in anti-HCV tests were strongly suspected. The introduction of new epitopes, particularly a core protein, C22 (second-generation tests), seems to increase the sensitivity of anti-HCV detection. In order to study the specificity of the second-generation tests, 60 serum samples from alcoholic patients found to be positive by the first-generation anti-HCV ELISA (Ortho) were reexamined by a second-generation anti-HCV enzyme immunoassay (Abbott) and a recombinant immunoblot assay (RIBA II; Chiron). Fifteen serum samples gave contradictory results when they were tested by the two assays. We performed nested polymerase chain reactions (PCRs) to confirm that the discrepancies that we observed could be due to the presence of low levels of anti-HCV antibodies, which were detected by a more sensitive test, or to unspecific positive reactions. Nested PCR revealed the presence of HCV RNA sequences in all anti-HCV-positive sera or sera that were weakly positive by ELISA. Anti-HCV positive by RIBA II was always correlated with the presence of viral RNA in serum, but HCV RNA was detected in RIBA II-negative sera. These results indicate that the specificity of the second-generation tests is an important improvement but that an HCV infection can still persist without detectable antibodies. PCR remains the reference assay to clear up controversial serology results and to detect HCV infection in patients with no anti-HCV-detectable immune response.     

丙型肝炎病毒抗原检测方法的建立

目的以特异性单克隆抗体为基础建立丙型肝炎病毒(HCV)抗原检测的酶联免疫吸附(ELISA)法,探索从血浆或血清中检测HCV抗原的可能性.方法利用我们制备的抗-HCV核心及NS3区单克隆抗体(McAbs),进行多种交叉组合模式的分析,确立实验室模式,并测定348份义务献血员血样,确定此方法的Cutofff值,并分析146份抗-HCV阳性及225份抗-HCV阴性血浆,阳性结果用套式PCR试剂盒确证.结果构建了以抗-HCV核心区单抗C39及NS3区单抗C7-6为包被抗体,以C39及NS3区C7-57为标记抗体的夹心ELISA检测模型,以C7为抗原,其检出灵敏度为5ng/ml,其Cutoff值为阴性对照均值±0.25.146份抗-HCV阳性样本中,11份为抗原反应阳性,225份抗-HCV阴性样本中,16份为抗原阳性,这些阳性样本经PCR检测后,23份为HCVRNA阳性.结论用单抗构建的HCV抗原检测方法分别从抗-HCV阳性及阴性样本中检测出HCV抗原反应阳性样本,并经PCR确证,表明直接从血浆样本中检测HCV抗原是可能的,这将对于HCV和基础研究及控制HCV的传播有重要意义.     

Impact of screening donor blood for alanine aminotransferase and antibody to hepatitis B core antigen on the risk of hepatitis C virus transmission

The transfusion-related risk of transmission of hepatitis C virus (HCV) was evaluated in France for the periods before and after exclusion of donor blood units with the surrogate markers elevated alanine aminotransferase (ALT) levels and antibody to hepatitis B core antigen (anti-HBc). A total of 1,412 blood recipients undergoing surgery were followed up prospectively in the period from 1986 to 1989. The stored serum samples were tested for antibodies to HCV by an enzyme immunoassay (EIA) and the result in reactive sera confirmed by a recombinant immunoblot assay (RIBA). The risk of HCV transmission was estimated by the maximum likelihood method for a subpopulation of 892 recipients divided into three groups. Of 55 (3.9 %) EIA positive patients, 56.4 % were found to be positive prior to transfusion. HCV seroconversion (positive RIBA) occurred in 22 patients (1.6 %). The risk of HCV transmission per 1,000 transfused blood units decreased significantly from 4.11 in Group 1 (receiving non-screened blood) to 3.43 in Group II (receiving ALT screened blood) and to 1.40 in Group III (receiving ALT and anti-HBc screened blood). These results demonstrate that screening of donors for surrogate markers had reduced the risk of HCV transmission before the introduction of a systematic anti-HCV screening policy in France in March 1990.     

Comparison of serum hepatitis C virus RNA and core antigen concentrations and determination of whether levels are associated with liver histology or affected by specimen storage time

An enzyme immunoassay has recently been developed for the hepatitis C virus (HCV) core antigen. To evaluate the possible association between core antigen and HCV RNA levels with regards to the change in liver histology over time as well as study the effect of duration of storage on viral load results, sequential sera were analyzed from 45 patients with chronic HCV infection who had undergone two or more liver biopsies. A relatively strong association was found between the core antigen and HCV RNA concentrations (r(s) = 0.8), with a core antigen level of 1 pg/ml corresponding to approximately 1,000 IU/ml. All 42 sera with detectable HCV RNA at the time of the second biopsy had core antigen concentrations above 1 pg/ml, and the three sera without detectable HCV RNA had concentrations below 1 pg/ml. No association was found between HCV RNA or core antigen levels and the stage of fibrosis in biopsy samples, progression of fibrosis, necro-inflammatory grade, steatosis, genotype, alanine aminotransferase level, or alcohol consumption. A significant association was demonstrated between the storage time of the samples and both the HCV RNA and core antigen concentrations. The median log HCV RNA concentrations (international units/milliliter) were 3.92 for the sera obtained at the time of the first biopsy (median storage time, 13.0 years) and 4.41 for the sera obtained at the time of the second biopsy (median storage time, 6.6 years) compared to 5.96, the median for 102 different routine clinical patient samples.     

丙型肝炎IgM的检测及对丙型肝炎患者血清的分析研究

用丙型肝炎病毒(HCV)结构区(C区)和非结构区(Ms3区)制备固相抗原,建立了检测HCV IgM抗体的间接ELISA,并用此法检测了101份丙型肝炎病人血清。检测结果表明,急性输血后丙型肝炎病人和HCV抗体阳性伴ALT升高者,其抗C区IgM阳性者所占比例明显大于抗Ns3区。单独用C区抗原检测IgM抗体,其阳性率大于用C区和Ns3区抗原共同检测的结果。前者Cut off指数(COI)>2者占阳性例数的71.5％,后者仅占26％。因而显示了HCV结构区抗原在IgM检测中的重要意义以及抗C区IgM阳性有可能成为新近感染或活动性丙型肝炎的标志。     

Antigenic variation of core, NS3, and NS5 proteins among genotypes of hepatitis C virus.

Assays that detect antibody to hepatitis C virus (HCV) are used to screen blood donors and patients with hepatitis. Current enzyme-linked immunosorbent assay (ELISA)-based methods are invariably based upon antigens from expressed recombinant proteins or oligopeptides from HCV type 1. Some HCV antigens used in screening assays are coded by regions of the HCV genome that show extensive variability; therefore, HCV type 1-based assays may be less effective for the detection of antibody elicited by infection with other genotypes. In this study, we have measured antibody reactivity of sera from 110 hepatitis C patients infected with type 1b, 3a, or 4a to genotype-specific and cross-reactive epitopes present in recombinant proteins from HCV genotypes 1b (core, NS3, and NS5), 3a (NS3, NS5), and 4a (core, NS3), corresponding to those used in current third-generation screening ELISAs. By comparing the serological reactivities of sera to type-homologous and type-heterologous antigens, we detected a significant type-specific component to the reactivity to NS3 (61 to 77% of the total reactivity) and NS5 (60% of the total reactivity). Furthermore, despite the similarities in the amino acid sequences of the core antigens of type 1b and type 4a, we also found significantly greater reactivity to type-homologous antigens, with approximately 25% of reactivity being type specific. These findings are consistent with previous findings of fivefold weaker reactivity of sera from HCV type 2- and HCV type 3-infected blood donors in the currently used third-generation ELISAs and suggest that these assays are suboptimal for screening populations in which the predominant genotype is not type 1.     

Prevalence of hepatitis C in South Africa: detection of anti-HCV in recent and stored serum

The prevalence of anti-HCV was studied in a cohort of 2,072 South Africans. The results were compared in selected recently collected sera and in stored sera. The serum ALT and anti-HBc were also studied as surrogate markers in this population. The following groups were tested: (a) 498 urban, black blood donors (b) 500 white blood donors (c) 500 Asian blood donors (d) 216 rural hospitalized patients (e) 358 rural mineworkers. Sera found positive by the original ELISA were retested, and reproducibly positive tests in rural black men (group d) were confirmed both by recombinant immunoblot assay and by a second ELISA. An anti-HCV prevalence of 1.2%, 0.8%, and 0.6% in urban blacks, Asians, and whites was found. Antibodies to hepatitis B core antigen were found in 42.9%, 3.4%, and 1.2% of black, Asian, and white donors, respectively; 76% of donors positive for anti-HCV were anti-HBc negative. In rural African men, 17% of stored serum samples and 9.2% of recently collected serum samples were positive for anti-HCV. In this cohort 3.84% were positive by all three assays. These results suggest that the prevalence of anti-HCV in low and high-risk South African urban blood donors is comparable to high and low prevalence areas in Europe, the United States, and Japan, but indicates a relatively high degree of exposure to hepatitis C in rural African men. The reactivity of stored, frozen sera in this population requires further investigation. In South African urban blood donors, surrogate marker testing will not expedite HCV screening.     

Hepatitis C virus seroconversion by a third generation ELISA screening test in blood donors.

The significance of seroconversion as detected by an ELISA screening test for hepatitis C virus (HCV) antibody with a negative supplemental/confirmatory recombinant immunoblot assay (RIBA) result was investigated. Of 118,220 established West Midlands blood donors with at least one negative HCV antibody screen, 43 had seroconverted in 1994 according to the ELISA but had negative RIBA-3 results. The paired archive serum samples of the pre- and postseroconversion donations of 29 seroconverting donors were tested by nested polymerase chain reaction (PCR) for the detection of HCV RNA. All 58 samples were negative by PCR. The absence of detectable viraemia in all tested seroconverting donors suggests that HCV infection was not responsible for seroconversion by ELISA.     

检测丙型肝炎患者NS5区抗体的临床意义探讨

目的检测慢性丙型肝炎患者和１４例ＨＣＶ原发感染后ＮＳ５抗体长达一年半的动态变化，探讨ＮＳ５抗体的临床意义。方法应用重组ＮＳ５抗原，建立ＥＩＡ方法进行检测。结果慢性丙型肝炎患者抗－ＮＳ５抗体阳性率为６０．４８％，ＨＣＶ感染后一月抗－ＮＳ５抗体阳性率为１６．３５％，三月为７５％。结论抗－ＮＳ５抗体无早期诊断价值。抗－ＮＳ５抗体持续阳性者，血清ＡＬＴ多明显升高，抗－ＮＳ５抗体与肝脏疾病活动性相关。丙型肝炎患者中存在抗－Ｃ、抗－ＮＳ３、抗－ＮＳ４抗体阴性，而抗－ＮＳ５抗体单独阳性，表明检测抗－ＮＳ５抗体具有独特的诊断价值     

Utility of HCV core antigen ELISA in the screening for hepatitis C virus infection in patients on hemodialysis

An enzyme immuno assay for hepatitis C core antigen was recently developed and its performance was compared with that of the hepatitis C virus (HCV) RNA in the screening of HCV infection in patients on hemodialysis. One hundred and eleven chronic renal failure patients undergoing haemodialysis between May 2003 and October 2004 were included in the study. All the patients were tested for anti HCV antibody, core antigen and RNA. Fifteen patients were anti HCV antibody positive, three patients were positive for HCV core antigen and RNA, three patients were positive for HCV RNA, while two patients were positive only for core antigen but negative for RNA. In anti HCV antibody positive patients, the core antigen was negative while the viral RNA continued to be present. Hence, relying solely on a single HCV core antigen assay may not be useful for a definite diagnosis of early HCV infection. The sensitivity and specificity of the assay were 60% and 83% respectively, while the positive predictive value was 14.3%, negative predictive value was 97.7% and the efficiency was 81.9%.     

Screening of blood from potential organ and cornea donors for viruses

Prospective nucleic acid tests were carried out for human immunodeficiency virus (HIV) and hepatitis C virus (HCV) using the COBAS Amplicor HIV-1 and HCV tests (Roche Diagnostics, Meylan, France) on potential organ (n=113) and cornea (n=368) donors in France to evaluate their performance and suitability for use as a complement to routine serological tests. Blood samples were collected from organ donors with preserved cardiac function after verification of cerebral death. Blood samples were collected from cornea donors post-mortem within 48 hr after death. An internal control was added to the samples before extraction to monitor each individual polymerase chain reaction (PCR). The nucleic acid tests were always interpretable in organ donors and negative in all except in 2 anti-HCV positive patients. One had an indeterminate HIV p24 antigen but was negative for HIV RNA. HIV and HCV RNA were not found in cornea donors with a negative serology but indeterminate molecular results were frequent in this group (17.6%). Cornea donors also gave significantly more (14.4%) indeterminate serological results than organ donors (1.8%) (P<0.001). This was due to the poor quality of the blood samples collected post-mortem. However, there was no correlation between indeterminate results of serological and molecular tests. There were 16/19 (84%) indeterminate serological results for HIV and 4/4 (100%) for HCV that were negative by PCR. Thus, nucleic acid tests could be useful for qualifying a donor whose serological results are indeterminate. The extraction procedures on post-mortem specimens and/or blood collection must be changed to improve the performance of nucleic acid tests.     

Use of aminotransferase, hepatitis C antibody, and hepatitis C polymerase chain reaction RNA assays to establish the diagnosis of hepatitis C virus infection in a diagnostic virology laboratory.

Clinical and therapeutic decisions for hepatitis C virus (HCV) infection depend on factors that include documentation of past infection as well as identification of those who might benefit from antiviral chemotherapy with systemic interferon. To evaluate the ability of a diagnostic laboratory to accurately identify such patients, we compared results obtained with serum transaminase assays, two HCV antibody assays (enzyme immunoassay [EIA] and immunoblot), and a polymerase chain reaction (PCR)-based assay for HCV RNA using a group of consecutively submitted samples within our university-based diagnostic virology laboratory and sera from a population of random blood donors. One hundred percent of specimens with R values of greater than 3.0 in the HCV EIA were positive in the confirmatory immunoblot. However, 25% of specimens with EIA R values of between 1.0 and 3.0 were not confirmed by either recombinant immunoblot assay (RIBA) or RNA PCR assay (false-positive specimens). A significant correlation (P less than 0.01) between increasing reactivity in the RIBA and positivity in the RNA PCR assay was found. The incidence of HCV viremia, as determined by the RNA PCR assay, was 73% for confirmed seropositive specimens, 33% for seropositive specimens with indeterminate RIBA results, 12% for seronegative specimens obtained from infected patients, and 2.0% for seronegative specimens obtained from uninfected blood donors. In contrast, serum transaminase testing did not correlate with the RNA PCR assay for HCV. Use of the EIA and immunoblot assay followed by RNA PCR testing will identify most patients who are viremic with HCV.     

A sensitive PCR method for detecting HCV RNA in plasma pools,blood products,and single donations

Although current manufacturing processes appear to efficiently inactivate hepatitis C virus (HCV), it is possible that contaminated blood products may result from failure of some stage during manufacture or from virus overload of plasma pools used for preparation of products. While antibody screening probably removes the majority of HCV positive donations, some donations which are antibody-negative but HCV positive may be included in pools. The HCV RNA content of plasma pools from paid and voluntary donors was investigated by poly-merase chain reaction (PCR). A sensitive PCR method using a single pair of primers from the 5′ non-coding region of the HCV genome and a “hot-start” was established and shown to be as sensitive as the more conventional nested PCR (which uses two pairs of primers). The majority of pools from paid donors (prescreening) were HCV RNA positive, while all pools from voluntary donors were both antibody and RNA negative. Intravenous immunoglobulins prepared from contaminated pools were RNA negative despite having high antibody levels, indicating satisfactory clearance of the virus during manufacture. The virus load of the pools was at least a thousand-fold lower than that of single donations, possibly as a result of treatment during the production of the pools or the presence of factors in pools which reduce the sensitivity of some part of the PCR assay. The HCV content of a plasma donation was determined as 3.6 × 10⁶ genomes/ml by an end point dilution method. Thus a simple and sensitive PCR assay was established for detecting HCV RNA in plasma pools and blood products. © 1994 Wiley-Liss, Inc.     

Relevance of hepatitis B DNA detection in patient's serum

Detection of HBV-DNA showed a satisfactory sensitivity (6 pg/ml) and high specificity: 15 patients with hepatitis A, 6 with CMV, 7 with EBV infection, 81 with hepatitis NANB as well as 174 persons with isolated HBc antibodies and 9 hepatitis B vaccinees gave negative results. However, out of 118 persons with a history of hepatitis B (HBs and HBc antibodies positive), one repeatedly had a positive result. We followed 58 patients with chronic hepatitis B, 25 healthy antigen carries, and 16 HBV-infected dialysis patients over an extended period with multiple blood sampling. Most of those patients showed changes of detectability of HBV-DNA. Typically, periods with positive DNA tests were followed by intervals with negative tests which could last for up to a few years. 75% of dialysis patients were positive in all sera available, while 48% of healthy antigen carriers were consistently negative. Additionally, 9 patients with a history of recent hepatitis B and a positive HBs antibody test as well as 4 patients with simultaneous infection by A and B were tested for HBV-DNA. In none of these two groups, a variation from the pattern usually found in hepatitis B was established. A case of intrauterine infection with hepatitis B virus is reported. Because the blood of the mother was free from HBV-DNA and HBe antigen at delivery and because the hepatitis B immunoglobulin prophylaxis failed in the child who developed HBs antigen very soon after delivery, it has been concluded that the child was infected in utero. The highest values of HBV-DNA determined quantitatively were found in dialysis patients which explains the high risk of acquiring hepatitis B in the dialysis unit. The results from earlier studies on the prognostic relevance of HBV-DNA and the risk of medical personnel as a source of infection with hepatitis B are discussed briefly.     

A novel antigen detection immunoassay for field diagnosis of hepatitis C virus infection

The limitations of dominant methods-based on the detection of anti-HCV antibodies or HCV viremia currently used for the diagnosis of HCV infection enhance efforts to have a rapid, simple, sensitive, and specific alternative diagnostic approach to detect viral antigens. A highly reactive IgG antibody was raised to HCV-NS4 recombinant antigen. The produced antibody showed no cross-reactivity with the other HCV structural and nonstructural recombinant antigens (C1 + 2, C3 + 4, E2/NS1, NS3, NS5). The well established ELISA technique was adapted to detect the new target HCV-NS4 antigen in serum samples. Extremely high agreement was found between the results of ELISA and qualitative detection of HCV-RNA, using a RT-PCR test as a gold standard for the diagnosis of HCV infection. Based on these encouraging results, a novel enzyme immunoassay; dot-ELISA was developed for rapid (approximately 5 min) and simple qualitative detection of the target HCV antigen in serum. The developed method detected the HCV target antigen in 95% of serum samples from HCV infected individuals, with a specificity of 97% using sera of noninfected individuals in comparison with PCR test. The antigen detection method showed high predictive values of positive (99%) and negative (90%). Moreover, the dot-ELISA could detect the HCV target antigen in sera negative for anti-HCV Abs, but positive for HCV-RNA, and in sera of HCV infected individuals with low viremia, as well as those with high viremia, using quantitative RT-PCR. Accordingly, the developed highly sensitive and specific HCV antigen detection method could be applied for mass screening of HCV infection.