Impact of HCV 3.0 EIA relative to HCV 2.0 EIA on blood-donor screening

BACKGROUND: In 1996, the Ortho HCV Version 3.0 ELISA Test System (HCV 3.0 EIA) was licensed in the United States for donor screening but was neither mandated nor universally implemented. Data from two studies comparing the differential performance of HCV 3.0 EIA and HCV 2.0 EIA are presented. The first study evaluated the differential performance in a cross-section of screened whole-blood donors after implementation of HCV 3.0 EIA; the second study evaluated the differential performance of HCV 3.0 EIA in plasma donors acutely infected with HCV, identified during routine Abbott HCV 2.0 EIA and HCV NAT (using Roche Ampliscreen plate assay) donor screening. STUDY DESIGN AND METHODS: The first study evaluated HCV 3.0 EIA repeat-reactive donations from four US blood centers, identified during the first 5 months of HCV 3.0 EIA implementation. HCV EIA repeat-reactive donations confirmed by RIBA HCV 3.0 SIA were retested using both Ortho HCV Version 2.0 ELISA Test System and Abbott HCV 2.0 EIA. All EIA-discordant donations were tested by polymerase chain reaction (PCR). In the second study, Abbott HCV 2.0 EIA-nonreactive, HCV PCR-positive donors were enrolled in a follow-up study in which the index and follow-up samples were re-evaluated by HCV 3.0 EIA. RESULTS: In the first study, of 292,459 donations, 501 (0.17%) confirmed HCV 3.0 EIA-reactive donations were identified; 15 (0.005%) were nonreactive by Ortho HCV 2.0 EIA and were all HCV RNA negative. In the second study, Ortho HCV 3.0 EIA retesting of Abbott HCV 2.0 EIA-nonreactive, RNA-positive index donations identified 16 (23%) as 3.0 EIA reactive. In 42 panels with a discordant time of seroconversion, HCV 3.0 EIA sero-conversion preceded HCV 2.0 EIA in all cases (p < 0.001). Two donors with HCV 3.0 EIA-reactive index donations never seroconverted by HCV 2.0 EIA during 160 to 180 days of follow-up. CONCLUSION: These studies demonstrate that HCV 3.0 EIA compared to HCV 2.0 EIA can better detect 1) remote nonviremic HCV infections, 2) acute infection, and 3) HCV antibodies in cases of atypical seroconversion.     

A comparison of two supplemental procedures for confirmation of antibody to hepatitis C virus c100-3 antigen in Louisiana blood donors

In a pilot study designed to evaluate the performance of supplemental hepatitis C virus (HCV) tests, 146 consecutive HCV enzyme immunoassay (EIA)-reactive samples (0.98% of 14,949 donors) were comparatively evaluated with two sets of supplemental tests: HCV antibody neutralization/c100-3 peptide EIA and the first-generation HCV recombinant immunoblot assay (RIBA). Of these samples, 68.5 percent were positive and 17.8 percent were negative on both supplemental assays. Nineteen samples were discordant. Eleven samples were positive on one assay (9 on neutralization/peptide, 2 on RIBA) and negative or indeterminate on the alternate supplemental test, but reacted with two additional HCV antigens outside the c100-3 region in a second-generation dot immunoblot assay. The dot immunoblot assay was used as a reference and reactive samples were considered confirmed. The remaining eight discordant samples were indeterminate or negative on either assay and did not react on the dot immunoblot assay. These data indicate a 0.74-percent prevalence of HCV exposure detected by reactivity with the c100-3 antigen in blood donors in southern Louisiana.     

A pattern of 5-1-1 and c100-3 only on hepatitis C virus (HCV) recombinant immunoblot assay does not reflect HCV infection in blood donors

Current criteria for a reactive (positive) interpretation on hepatitis C virus (HCV) recombinant immunoblot assay (RIBA) require > or = 1+ reactivity to at least two of the four HCV antigens present in the assay. Given that 5-1-1 is a subcomponent of c100-3, there is concern that donor samples reacting only with these two antigens (and not with c22-3 or c33c) could be incorrectly classified as positive on the basis of limited reactivity to only one HCV gene product. It is determined that 0.23 to 0.44 percent of HCV enzyme immunoassay-repeatably reactive donor sera demonstrate a pattern of 5-1-1 and c100-3 only on RIBA. Evaluation of six such donor sera using peptide enzyme immunoassays spanning the c100-3 antigen showed highly restricted reactivity to the 5-1-1 N-terminal region of c100-3, in contrast to broad 5-1-1 and c100- 3 C-terminal peptide reactivity observed in the majority of donor sera with other positive RIBA patterns. HCV polymerase chain reaction and follow-up serologic evaluations of four of these donors indicated the absence of viremia or evolving seroconversion in all cases. It is concluded that, in the blood donor setting, a pattern of only 5-1-1 and c100-3 reactivity is typically not indicative of HCV infection. To avoid overinterpretation, it is recommended that RIBA grading criteria be revised to require reactivity to two or more HCV-encoded gene products.     

Antibodies to hepatitis C virus in autologous blood donors

Hepatitis C virus (HCV) is the major cause of posttransfusion hepatitis. Two anti-HCV enzyme immunoassay (EIA) kits and one recombinant immunoblot assay (RIBA) were used to test serum samples of 1476 donations from 692 autologous blood donors to assess the prevalence of anti-HCV and its relationship to transfusion history. Of all autologous blood donations, 23 (1.6%) reacted when tested with one EIA kit and 29 (2.0%) reacted when tested by the other EIA kit. Of the autologous donors, 12 (1.78%) reacted by the first EIA kit and 14 (2.02%) by the second. Discrepancies in the EIA results from different donations by the same donor were seen in seven donors. The RIBA was positive or indeterminate in 33 percent of the EIA-reactive donations and in 41 percent of EIA-reactive donors. All RIBA-positive and -indeterminate samples reacted with both EIA kits. There was no significant difference in the EIA-reactive rates of autologous and first-time homologous blood donors. Previously transfused autologous blood donors had a higher anti-HCV EIA-reactive rate than nontransfused autologous donors, but the difference was not significant. In regard to hepatitis C, the use of autologous blood for homologous transfusion appears to be as safe as the use of blood from first-time homologous donors. Universal testing of previously transfused patients for hepatitis C appears premature at this time. Discrepant anti-HCV EIA results from different donations from the same individual have implications regarding donor deferral.     

The use of a recombinant immunoblot assay in the interpretation of anti- hepatitis C virus reactivity among prospectively followed patients, implicated donors, and random donors

Samples from prospectively followed recipients, their respective donors, and a cohort of random donors were used to evaluate the specificity and efficacy of a recombinant immunoblot assay (RIBA) as an adjunct to anti-hepatitis C virus (HCV) testing by enzyme immunoassay (EIA). RIBA reacted (RIBA+) in 100 percent of patients who developed hepatitis associated with anti-HCV seroconversion documented by EIA and in 100 percent of the EIA-positive (EIA+) donors implicated in these cases. In contrast, RIBA reacted in none of 10 recipients who were EIA+ but did not develop hepatitis, in none of 7 EIA+ patients with hepatitis B or cytomegalovirus infection, in 33 percent of EIA+ donors who were not implicated in hepatitis transmission, and in 37 percent of EIA+ random donors. Hence, the vast majority of EIA+ individuals who have ancillary evidence of HCV infection react on RIBA, whereas the majority of EIA+ individuals in low-risk settings do not react (RIBA-negative, or RIBA-). There was a strong association between RIBA reactivity and the presence of a surrogate marker (elevated alanine aminotransferase [ALT] and/or antibody to hepatitis B core antigen); 43 percent of RIBA+ implicated donors had a surrogate marker as compared to none of 14 EIA+, RIBA- donors. Among EIA+ random donors, 77 percent of those with a surrogate marker were RIBA+, as compared with 29 percent of those without a surrogate marker. In addition, in EIA+ donors, RIBA reactivity correlated with the extent of ALT elevation; 86 percent of those with an ALT greater than 135 IU per L were RIBA+ compared with 18 percent of those with an ALT less than 30 IU per L.(ABSTRACT TRUNCATED AT 250 WORDS)     

Routine HCV PCR screening of blood donations to identify early HCV infection in blood donors lacking antibodies to HCV

BACKGROUND: Detection of early hepatitis C infection of blood donors is still a major problem for blood transfusion. Common anti-HCV screening assays show differences in sensitivity and specificity. The often mild symptoms of acute hepatitis C also cause difficulties in the identification of early HCV infection. The feasibility and efficacy of routine screening of blood donations for HCV RNA were investigated. STUDY DESIGN AND METHODS: Blood donations (n = 251,737) were screened for HCV RNA over 4 years. RNA extraction, amplification, and detection were done by two commercial HCV PCR kits (HCV Cobas Amplicor and HCV Cobas Amplicor 2.0, Roche Diagnostics). Screening was done by pool testing with a maximum pool size of 40 serum samples. RESULTS: Three donations out of 251,737 were HCV RNA positive and anti-HCV negative. ALT levels of these donations were 271, 32, and 10 U per L. The HCV infection of a fourth HCV RNA-positive donor could not be identified by routine, second-generation HCV EIA (Abbott Diagnostika). In this case, two previous donations were also HCV RNA positive, and three second-generation test systems (Abbott) could not detect anti-HCV, whereas third-generation anti-HCV screening assays detected antibody with different sensitivity. The first HCV RNA-positive donation was identified only by the HCV ELISA 3.0 (Ortho Diagnostic Systems). The results of confirmatory assays like RIBA HCV 3.0 (Ortho) and Matrix (Abbott) indicate a restricted immune response to NS3 only. CONCLUSION: HCV RNA detection by PCR can be carried out routinely in blood donor screening without significant delay of release of the components. The residual risk of transmission can be reduced by identification of early infection, which can lead to an improved safety of blood components. RNA screening can also be advantageous in cases of incomplete or lack of antibody response to HCV.     

Comparative yield of HCV RNA testing in blood donors screened by 2.0 versus 3.0 antibody assays

BACKGROUND: Two HCV antibody tests (EIA 2.0 [EIA2], Abbott; and the Version 3.0 ELISA [EIA3], Ortho) are currently licensed for screening of US blood donors. Testing of donors for HCV RNA allows comparison of the sensitivities of the two antibody-screening assays. STUDY DESIGN AND METHODS: All allogeneic blood donations at 13 US test sites were screened for HCV RNA by testing plasma minipools using an investigational assay (COBAS AmpliScreen HCV test, v2.0, Roche Molecular Systems). Some sites screened for HCV antibody by EIA2 and some used EIA3. The frequency of RNA-positive and antibody-negative (RNA-pos and Ab-neg) donations among donors screened by each antibody assay was compared. Antibody appearance was assessed in a donor follow-up study. RESULTS: A total of 5.51 x 10(6) donations were screened for HCV RNA. Of these, 2.27 million were screened for antibody by EIA2, and 3.24 million by EIA3. Twenty-three donations were HCV RNA-pos and Ab-neg. The frequency of RNA-pos and Ab-neg donations was higher among donations screened by EIA2 (1 in 134,000), compared to those screened by EIA3 (1 in 540,000) (p = 0.001). Of the 17 RNA-pos and Ab-neg donations identified by test sites that used EIA2, 14 were retested by EIA3 and 10 (71%) were reactive. Most RNA-pos and Ab-neg donors appear to be in the process of seroconversion. Donors that were initially EIA2-negative and EIA3-reactive showed a more prolonged pattern of seroconversion compared to those that were initially nonreactive by both antibody assays. Four donors were EIA2-negative, EIA3-reactive, and RIBA-indeterminate (c33c) for at least 90 days, 1 for more than 317 days. CONCLUSION: EIA3 would have detected the majority of RNA-positive donations missed by EIA2. Some RNA-positive donors are EIA2-negative and EIA3-reactive for a prolonged period of time.     

Integration of nucleic acid amplification test results into hepatitis C virus supplemental serologic testing algorithms: implications for donor counseling and revision of existing algorithms

BACKGROUND: The routine use of hepatitis C virus (HCV) nucleic acid amplification testing (NAT) donor screening assays has provided an opportunity for revision of the current HCV supplemental testing algorithm, which requires that recombinant immunoblot assay (RIBA) be performed on every HCV enzyme immunoassay (EIA)-repeat-reactive donation. The FDA has approved variance requests to use a new algorithm that eliminates the need to perform RIBA when HCV NAT results are reactive. Data are provided in support of this new algorithm. STUDY DESIGN AND METHODS: HCV EIA (including signal-to-cutoff optical density ratio), RIBA, and NAT data were compiled from 33.2 million donations screened over an approximately 4-year period by the American Red Cross and Blood Systems Laboratories. Further, donations having specific combinations of HCV EIA, RIBA, and minipool (MP) NAT results were evaluated, with more sensitive individual-donation (ID) NAT, to construct improved counseling messages for donors. RESULTS: Of 47,041 EIA-repeat-reactive donations, 49.3 percent were RIBA-positive, 17.1 percent RIBA-indeterminate, and 33.5 percent RIBA-negative. NAT-reactive rates were 79.2, 2.5, and 0.18 percent for RIBA-positive, -indeterminate, and -negative donations, respectively. The new algorithm classified an additional 1 percent of donations as HCV-infected while at the same time detecting all infections classified as HCV-infected under the current algorithm. An additional 2.4 percent of RIBA-positive, MP NAT-nonreactive donations were reactive when a frozen-thawed aliquot was retested by ID NAT. CONCLUSION: Integrating HCV NAT results with RIBA results for purposes of donor notification allows more appropriate counseling messages to be given to EIA-repeat-reactive donors. The new HCV supplemental algorithm is an acceptable alternative to the current algorithm because it provides equivalent or superior accuracy in formulating donor counseling messages and may also result in reduced costs and more timely notification of infected donors.     

Investigation of an algorithm for anti HCV EIA reactivity in blood donor screening in Turkey in the absence of nucleic acid amplification screening

Purpose

In this study we aimed to propose an algorithm for initial anti HCV EIA reactive blood donations in Turkey where nucleic acid amplification tests are not yet obligatory for donor screening.

Reliability of the third-generation recombinant immunoblot assay for hepatitis C virus

BACKGROUND: In a confirmatory laboratory, the second-generation recombinant immunoblot assay (RIBA-2) was replaced by the third- generation RIBA (RIBA-3) in March 1993. The aim of this validation study was to compare the sensitivity and specificity of RIBA-2 and RIBA- 3 in a routine setting, by using a validated hepatitis C virus (HCV) RNA polymerase chain reaction to establish plasma viremia. STUDY DESIGN AND METHODS: RIBA-2 testing was performed (March 1991-March 1993) in 593 HCV RNA-positive and 1498 HCV RNA-negative subjects. RIBA-3 testing was performed (March 1993-May 1994) in 220 HCV RNA-positive and 530 HCV RNA-negative subjects. All samples reacted for anti-HCV in enzyme- linked immunosorbent assay. RESULTS: In HCV RNA-positive individuals, the sensitivity of RIBA-3 was significantly higher than that of RIBA-2 (99.5% vs. 93.3%, p = 0.0005). This was not caused by inclusion of the NS5 antigen, but by a higher sensitivity of the antigens c33 and c100 (RIBA-2: 94.3% and 62.6%; RIBA-3: 99.5% and 88.6%). Replacement of the c22 and c100 recombinant proteins by synthetic peptides significantly reduced nonspecific reactivity against these antigens (p < 0.0001). Unfortunately, increased nonspecific reactivity against the modified c33 antigen and the new NS5 antigen canceled out this effect. Two-band reactivity occurred more often in nonviremic persons than in viremic persons (32.7% vs. 8.2%, p < 0.0001). Risk factors for HCV infection were less frequently observed in 11 blood donors with two-band reactivity than in 6 blood donors with other positive RIBA-3 patterns (18% vs. 83%, p = 0.03). CONCLUSION: The higher sensitivity of RIBA-3 significantly reduced the number of indeterminate test results in HCV RNA-positive persons. Confirmatory laboratories must be aware of the frequent occurrence of nonspecific, isolated reactivity and even nonspecific, two-band reactivity in anti-HCV enzyme-linked immunosorbent assay-reactive blood donors.     

Evaluation of clinical and laboratory aspects of antibody tests for detection of hepatitis C virus infection in blood donors and recipients from a low-risk population

BACKGROUND: When the first-generation enzyme immunoassay (EIA) for detection of antibody to hepatitis C virus (anti-HCV) was approved in May 1990, blood banking agencies recommended testing of all components in inventory. In many cases, one or more components from these units had already been transfused. STUDY DESIGN AND METHODS: Donors that reacted in first-generation EIAs and recipients of their components were identified, and anti-HCV test methods (including first-generation EIA, second-generation EIA, and recombinant immunoblot assay [RIBA]) were evaluated. RESULTS: Of 66 donors identified as anti-HCV-positive by first-generation EIA, 17 were positive in second-generation EIA. Of these 17, 9 reacted in RIBA; 6 of these showed evidence of HCV infection in polymerase chain reaction (4) and/or probable transmission of HCV to a transfusion recipient (3). Of the 48 specimens that were positive in first-generation EIA and negative in second-generation EIA, only 1 was positive in RIBA; serum was not available for polymerase chain reaction testing, and there were no living transfusion recipients in whom to assess evidence of transmission of HCV. CONCLUSION: This study documents the low predictive value of EIAs for anti-HCV in a low- prevalence blood donor population and emphasizes the need for additional testing to confirm the specificity of samples that react in the screening tests.     

Serological and histological features of anti-HCV-positive individuals without serum HCV RNA.

Antibody to hepatitis C virus (anti-HCV) in patients who are negative for HCV RNA in serum may indicate a memory of past infection of HCV. However, their clinical features have not been well understood. Fourteen anti-HCV-positive but HCV RNA-negative individuals were examined for serological and histological features. As a result, it was found that they had only antibody to HCV core antigen C22-3 with or without antibody to nonstructural viral antigen C33c on a recombinant immunoblot assay (RIBA), and that an concentration of anti-C22 was low. Liver biopsy showed two having no evidence of an obvious hepatic injury, two having a minimal change, and two having portal fibrosis. HCV RNA was not found in the liver. These results corroborate an idea that the anti-HCV in HCV RNA-negative individuals implies a past infection of HCV. Furthermore, it is suggested that a combination of an appearance pattern of antibody to HCV antigens on RIBA and anti-C22 titer are an useful marker to distinguish anti-HCV-positive individuals without viremia from those with viremia.     

Predictive markers for hepatitis C antibody ELISA specificity in Australian blood donors

The hepatitis C antibody reactivity rate in 91,748 blood donors tested using the ORTHO HCV C-100 ELISA system was 0.51%. Specificity of ELISA positive reactions was measured using a recombinant immunoblot assay (RIBA). The aim of this study was to identify markers in ELISA positive donors which were predictive of a RIBA positive result. Samples from 430 ELISA positive donors were tested by the first generation RIBA, RIBA-1, which incorporates two HCV peptides C-100 and 5-1-1. Fifty-five per cent (236) were positive and 19% (83) indeterminate. Multivariate analysis of gender, age, HCV ELISA OD ratio, alanine aminotransferase (ALT) status and hepatitis B core antibody (anti-HBc) status identified age, magnitude of HCV ELISA OD ratio and anti-HBc status as the only independent predictors of a positive RIBA-1 result. The relative odds of being RIBA-1 positive were 4.6-fold (95% CI 1.3-16.4) higher among donors aged 25-34 years compared with donors less than 25 or greater than 44; 6.1-fold (2.1-17.9) higher if the donor was anti-HBc positive and 273.4-fold (30.9-2417) higher if the HCV ELISA OD ratio was greater than 5.98 compared to those with a ratio less than 1.77. Seventy-eight of the 83 RIBA-1 indeterminates were tested on the second generation RIBA, RIBA-2, which includes two additional HCV peptide, C22 and C33c. Thirty-one per cent (24) were positive and 41% (32) were negative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anti-hepatitis C virus (anti-HCV) seroconversion in patients undergoing hemodialysis: comparison of second- and third-generation anti-HCV assays

BACKGROUND: The results obtained in sequential specimens from recently infected subjects generally provide the best means of comparing the sensitivity of assays. STUDY DESIGN AND METHODS: The sensitivity of second- and third-generation assays for antibody to hepatitis C virus (HCV) was compared on sequential specimens, generally collected at monthly intervals from 45 patients undergoing hemodialysis who seroconverted for HCV between 1980 and 1990. RESULTS: Fifteen patients (33%) were positive earlier in the third-generation enzyme-linked immunosorbent assay (ELISA), with a mean difference of 17 days (range, 7–30) between the last negative and the first positive specimens. At the first rise in alanine aminotransferase, and at its peak, 63 and 91 percent of the patients, respectively, were anti-HCV positive in the third-generation ELISA. Third-generation recombinant immunoblot assay (RIBA) reacted at the same time as third-generation ELISA. Of the first specimens that were positive in second-generation ELISA, 44 percent reacted and 56 percent were indeterminate in third-generation RIBA, while 10 percent reacted, 72 percent were indeterminate, and 18 percent did not react in second-generation RIBA. From the beginning to the end of the follow-up, antibody to c33c was the most prevalent, followed in descending order by antibody to c22-3, antibody to c100-3, and antibody to NS5: 56, 54, 26, and 18 percent, respectively, at time 0, and 100, 86, 83, and 31 percent, respectively, 12 months later. CONCLUSION: Third-generation assays (both ELISA and RIBA) were more sensitive than second-generation assays in the diagnosis of HCV infection, in that positive results were obtained earlier and a higher proportion of specimens were confirmed positive in RIBA testing.     

Evaluation of the persistence and characteristics of indeterminate reactivity against hepatitis C virus in blood donors

BACKGROUND: A substantial number of individuals are excluded from blood donation due to indeterminate results in screening tests for hepatitis C virus antibody (anti-HCV). Disclosure of the characteristics of the indeterminate serologic pattern could optimize the testing and the management and care of blood donors.
STUDY DESIGN AND METHODS: Ninety-two former blood donors deferred from blood donation due to consistent reactivity in anti-HCV enzyme immunoassay and indeterminate HCV recombinant immunoblot assay (RIBA) results were retested for anti-HCV to examine the extent of disappearance of reactivity. In addition, they were screened for selected viral, immunologic, and inflammatory variables to detect possible causes of the test reactivity pattern.
RESULTS: After a median observation time of 75 months, 66 of 92 individuals had persistent indeterminate HCV RIBA results. Reactivity against the nonstructural NS5 antigen was the most frequent finding. A significant association was detected both between indeterminate reactivity in HCV RIBA and against the NS5 antigen independently and detectable antibody against adenovirus. Novel indeterminate reactivity showed increased prevalence during autumn and winter months.
CONCLUSION: Indeterminate HCV RIBA reactivity in blood donors persists over years. Increased prevalence during autumn and winter and association to detection of adenovirus antibody indicate that viral infection and cross-reactivity are etiologic factors in indeterminate HCV RIBA reactivity. Further prospective studies are needed to confirm the results.     

Hepatitis C virus (HCV)‐specific T‐cell responses among recombinant immunoblot assay‐3–indeterminate blood donors: a confirmatory evidence of HCV exposure

BACKGROUND: Blood donors are routinely screened for hepatitis C virus (HCV) infection. Some show weak anti‐HCV responses, often restricted to a single antigen on confirmatory immunoblot (recombinant immunoblot assay [RIBA]) testing. The aim of this study was to investigate the extent to which such RIBA‐indeterminate donors had previously been exposed to HCV. STUDY DESIGN AND METHODS: T‐cell responses to HCV recombinant proteins (core, NS3, and NS3 helicase) were analyzed using an interferon‐γ (IFN‐γ) enzyme‐linked immunospot (ELISpot) assay and quantification of cytokines in culture supernatants in 27 RIBA‐indeterminate donors, 60 RIBA‐confirmed donors (48 with and 12 without HCV RNA), and 30 RIBA‐negative donors. RESULTS: HCV‐specific T‐cell responses were identified in 13 (48%) RIBA‐indeterminate donors, 33 (55%) RIBA‐confirmed donors, and 4 (13%) RIBA‐negative controls (p = 0.008 and p < 0.001, respectively). The magnitude of the T‐cell response among indeterminate donors was similar to that of RIBA‐confirmed donors for all HCV antigens and the specificity of the ELISpot results was confirmed by antigen‐specific cytokine production (interleukin‐2 and IFN‐γ) in short‐term culture supernatants. CONCLUSIONS: These findings confirm that approximately half of RIBA‐indeterminate donors have resolved a previous HCV infection and suggest that ELISpot might be a useful tool to clarify the status of such donors and help in their counseling and management.     

Immunologic events during the incubation period of hepatitis C virus infection: the role of antibodies to E2 glycoprotein. Multicentre Hemodialysis Cohort Study on Viral Hepatitis

BACKGROUND: The study of the sensitivity of screening assays is greatly facilitated by testing the sequential changes in seroconverting individuals. The aim of this study was to investigate the early immunologic response after hepatitis C virus (HCV) infection and to evaluate whether HCV envelope (E2) recombinant antigen would provide a significant increase in sensitivity for detection of anti-HCV. STUDY DESIGN AND METHODS: Twenty hemodialysis patients who were seroconverting to anti-HCV were included in this study. They were followed up for a mean period (+/− SD) of 10.5 +/− 3.3 months, in which 13 to 46 serum samples per case were collected. Each sample was tested for anti-HCV by second- and third-generation enzyme immunoassay (EIA-2 and EIA-3) and recombinant immunoblot assay (RIBA-3). E2 antibodies were tested by a prototype EIA in which E2 was expressed as a recombinant antigen in Chinese hamster ovary cells. RESULTS: Alanine aminotransferase elevation was observed in 18 of 20 cases. Reactivity against c100, c33c, c22, NS5, and E2 was detected in 15 (75%), 19 (95%), 15 (75%), 2 (10%), and 17 (85%) patients, respectively; c33c was the most immunogenic antigen, followed in descending order by E2, c22, c100, and NS5. E2 antibody reactivity resolved the two RIBA-3- indeterminate cases. However, there was no case in which E2 reactivity preceded all other HCV antigens. Anti-E2 was found to react in all patients of genotypes 1a, 1b, and 3a but in only 2 of 4 patients of genotype 4a. CONCLUSION: In this group of seroconverting individuals, E2 antigen was shown to be highly immunoreactive and did resolve some RIBA-3-indeterminate samples as being positive, on the basis of reactivity to multiple antigens, but it did not improve early detection of seroconversion.