The significance of third-generation HCV RIBA-indeterminate, RNA-negative results in voluntary blood donors screened with sequential third-generation immunoassays

BACKGROUND: One of the problems associated with the use of anti-HCV immunoblot assays is the interpretation of indeterminate results without detectable HCV RNA. The purpose of this study was to examine the significance of third-generation RIBA (RIBA-3)-indeterminate, RNA-negative results in voluntary blood donors. STUDY DESIGN AND METHODS: Since June 2000 all Australian Red Cross Blood Service testing sites have used an anti-HCV sequential immunoassay testing strategy whereby donors who are reactive on the primary screening immunoassay are tested on a secondary immunoassay and if reactive on both assays, further tested by immunoblot. From the four testing sites that use RIBA-3, the result profiles of donors who were RIBA-3-indeterminate, HCV RNA-negative were analyzed. RESULTS: From 2,661,786 donations screened for anti-HCV during the study period, 102 RIBA-3-indeterminate, RNA-negative donors were identified, most of whom were reactive to either c33p (69.6%) or c22p (27.5%). The RIBA-3-indeterminate, RNA-negative donors showed a significantly higher screening immunoassay signal strength to assay cutoff (S/CO) distribution than those with biologic false-reactive (BFR) results (1.853 vs. 1.524, p < 0.05) but a significantly lower distribution than RIBA-3-positive, RNA-negative (1.853 vs. 4.546, p < 0.05) or RNA-positive (1.853 vs. 6.467, p < 0.05) donors. The RIBA-3-indeterminate, RNA-negative donors showed a similar distribution of c33c and c22p band intensities compared with RIBA-3-positive, RNA-negative donors but significantly lower distribution of band strengths compared to the RIBA-3-positive, RNA-positive group. Compared to the indeterminate donors with previous anti-HCV-negative or BFR results, the indeterminate donors not previously screened for anti-HCV showed higher immunoassay S/CO ratio distributions, a higher proportion with c22p reactivity (16.2% vs. 36.7%), and higher frequency of risk factors (46.4% vs. 75.0%). CONCLUSIONS: Our analysis suggests that a combination of indicators can be used to help clarify RIBA-3-indeterminate, RNA-negative results. Specifically, donors with high S/CO ratios on a screening immunoassay, RIBA-3 reactivity to c22p or c33c with band intensity of 2+ or greater, without a previous history of negative or BFR donations and with an identifiable risk factor, have a high probability of representing true anti-HCV rather than nonspecific reactivity.     

Recombinant immunoblot assay reaction patterns and hepatitis C virus RNA in blood donors and non-A, non-B hepatitis patients

To establish the value of the second-generation recombinant immunoblot assay (RIBA-2) and cDNA polymerase chain reaction (cDNA PCR) for confirmation of hepatitis C virus (HCV) infection, anti-HCV reaction patterns and the presence of HCV RNA were examined in 610 blood donors and 255 non-A, non-B hepatitis patients who were positive or indeterminate in RIBA-2. Of RIBA-2-positive donors (n = 191) and patients (n = 224), 75.4 and 89.7 percent, respectively, were HCV RNA positive. The most frequently observed anti-HCV recognition patterns in HCV RNA-positive donors and patients were c22, c33c, and c100 and/or 5- 1-1 (67.3%, 57.7%) and c22, c33c (24.8%, 29.3%). Among subjects with a RIBA-2-indeterminate result, HCV RNA was detected more often in patients (n = 31) than in donors (n = 419): 67.7 and 2.1 percent, respectively. In viremic persons with single-band reactivity in RIBA-2, this reactivity was always directed against either c22 or c33c. HCV RNA was detected by cDNA PCR in none of 162 persons with only anti-c100 and/or anti-5-1-1 reactivity. Therefore, RIBA-2 reactivity against c100 in combination with 5-1-1 should not be considered positive but indeterminate. In RIBA-2-indeterminate persons, HCV RNA detection is necessary for reliable confirmation of HCV infection.     

HCV RNA in blood donors with isolated reactivities by third-generation RIBA

BACKGROUND: The objective of this collaborative study was to learn the proportion of HCV RNA-positive samples obtained from a population of donors with isolated anti-HCV reactivities by third-generation RIBA (RIBA-3) (indeterminate results). STUDY DESIGN AND METHODS: During a 2-year period, 11 blood transfusion centers kept all samples with indeterminate RIBA-3 results to test them by PCR, using both local and commercial techniques. RESULTS: Of the 758 RIBA-3 indeterminate samples, 10 (1.3%) were positive for HCV RNA: 3. 3 percent (6/180) and 1.3 percent (4/317) of samples with anti-core or anti-NS3 reactivity, respectively, and none of the 52 and 209 samples with anti-NS4 or anti-NS5 reactivity, respectively. HCV RNA-positive donors with anti-core reactivity were infected with different subtypes (1 with HCV subtype 1b, 1 with 2, 1 with 2a/2c, 2 with 3a, and 1 with 5a), and a follow-up indicated a chronic-carrier state in two of the six donors. Acute hepatitis was diagnosed in three of the four donors with anti-NS3 reactivity alone. Two of these three were IV drug users and were infected with subtype 1a. CONCLUSION: HCV RNA-positive donors with indeterminate results in RIBA-3 are extremely rare, but they do exist. They were observed only when either anti-core or anti-NS3 was present. With such a RIBA-3 profile, PCR testing remains necessary to reveal an eventual acute or chronic HCV infection.     

Confirmation of hepatitis C infection: a comparison of five immunoblot assays

BACKGROUND: Recently, new immunoblot assays for the detection of antibodies to hepatitis C virus (HCV) became available. STUDY DESIGN AND METHODS: The performance of five confirmatory anti-HCV immunoblot assays was studied with samples with known HCV antibody and HCV RNA status. The assays were a third-generation strip recombinant immunoblot assay (RIBA-3, Chiron Corp., Emeryville, CA), a second-generation HCV blot (DB-2 blot, Diagnostic Biotechnology, Singapore), the Wellcozyme HCV Western blot (Murex blot, Murex Diagnostics, Dartford, UK), an immunodot HCV assay (Matrix, Abbott Laboratories, Chicago, IL), and the third-generation HCV line immunoassay (Liatek-III, Organon Teknika, Boxtel, The Netherlands). RESULTS: Sensitivity on samples from 48 HCV RNA-positive, second-generation RIBA (RIBA-2)-positive persons and specificity on samples from 31 low-risk donors was 96 percent or better for all assays. The sensitivity on 31 HCV RNA-positive, RIBA-2- indeterminate samples was as follows: Liatek-III, 94 percent; RIBA-3, 90 percent; Murex blot, 61 percent; Matrix, 55 percent; and DB-2 blot, 39 percent. In testing 39 HCV RNA-negative, RIBA-2-indeterminate donor samples, the percentage found to be negative was Liatek-III, 77 percent; RIBA-3, 67 percent; Murex blot, 49 percent; DB-2 blot, 33 percent; and Matrix, 15 percent. The order of sensitivity on four HCV seroconversion series was (from high to low): RIBA-3, Liatek-III, DB-2 blot, Murex blot, and Matrix; the differences were small. CONCLUSION: Detection of HCV antibodies was not refined by the addition of new HCV antigens (NS5, E2/NS1), but by improved classical antigens (core, NS3, NS4). Replacement of the commonly used RIBA-2 will resolve the status of a high proportion of RIBA-2-indeterminate samples.     

Long-term follow-up of and infectivity in blood donors with hepatitis C antibodies and persistently normal alanine aminotransferase levels

BACKGROUND: Little is known about the prevalence of serum hepatitis C virus (HCV) RNA in blood donors with HCV antibodies and persistently normal alanine aminotransferase (ALT) levels. STUDY DESIGN AND METHODS: Thirty-nine anti-HCV-positive donors with normal ALT on four determinations at 3-month intervals were further tested monthly for 6 months, and they had normal ALT values. The presence of HCV RNA was determined in these 39 donors. RESULTS: Serum HCV RNA was detected in 16 of 39 donors, 14 of 14 who reacted on second-generation recombinant immunoblot assay (RIBA-2) and 2 of 15 who were indeterminate. None of the 10 RIBA-2-nonreactive donors had evidence of viremia. The 15 RIBA-2- indeterminate samples were tested with third-generation RIBA (RIBA-3); the results showed reactivity in 5 (including the 2 HCV RNA positive), an indeterminate pattern in 7, and nonreactivity in 3 (all RNA negative). Among HCV RNA-positive subjects, mean age (p < 0.05), mean ALT (p < 0.001), signal-to-cutoff (S:CO) ratio on second-generation enzyme-linked immunosorbent assay (p < 0.001), and gamma globulin levels (p < 0.05) were higher than those among HCV RNA-negative subjects. During 6 additional months of ALT monitoring, completed by 36 of 39 donors, increased values were detected in 6 (5 HCV RNA positive). In 4 of those 6, however, ALT levels were less than 1.5-fold the upper normal limit. HCV RNA results were unchanged at the end of 1-year follow-up. CONCLUSION: Forty-one percent of anti-HCV-positive donors with persistently normal ALT had active HCV infection. Long-term ALT monitoring allowed the detection of significantly increased enzyme values in only 2 of 16 viremic donors. Reactivity on RIBA-2 or -3, greater age, mean ALT levels in the upper range of normal, higher S:CO ratio on second-generation enzyme-linked immunosorbent assay, and higher gamma globulin levels were predictive of viremia.     

Hepatitis C virus among blood donors: follow-up study

BACKGROUND: The exact significance of antibodies to hepatitis C virus (HCV) in blood donors remains unknown. Confirmatory tests of anti-HCV- reactive serum and HCV RNA by polymerase chain reaction (PCR) are used to refute a large proportion of false-positive results. STUDY DESIGN AND METHODS: Ninety-two blood donors who were anti-HCV reactive in a first-generation enzyme-linked immunosorbent assay (ELISA) were reevaluated 10 months later with a second-generation ELISA (ELISA-2) as well as with second-generation recombinant immunoblot assay (RIBA-2) and by PCR. RESULTS: Twenty-five (43.9%) of the 57 ELISA-2-positive donors were confirmed as positive by RIBA-2; of these, 84 percent were HCV RNA positive in PCR. Of the 57 who were still anti-HCV positive, 46 were followed up and tested again in the same manner 2 years after the first screening. At that time, the pattern was little changed: 94 percent of RIBA-2- and PCR-positive donors remained positive. Of RIBA-2- and PCR-positive blood donors, 62 percent had abnormal alanine aminotransferase levels in at least one of the three evaluations. Among the anti-HCV-positive donors confirmed by RIBA-2, 60 percent, versus 12.6 percent in the control group, had a significantly (p < 0.001) more frequent risk factor for HCV infection, due to parenteral exposure to blood. CONCLUSION: These data confirm a good correlation between RIBA-2 reactivity and the detection of HCV RNA in a population of anti-HCV- positive blood donors.     

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.     

Anti-HCV confirmatory testing of voluntary blood donors: comparison of the sensitivity of two immunoblot assays

BACKGROUND: In this study, the sensitivity of two commercially available anti-HCV immunoblot assays (HCV Western blot (Wellcozyme] and RIBA 3.0 SIA [RIBA-3, Chiron]) was compared in a voluntary blood donor population. STUDY DESIGN AND METHODS: Four groups of donor samples were retrospectively tested in this study. Groups 1 and 2 were donor samples that gave positive or indeterminate band patterns, respectively, when originally tested on the HCV Western blot between 1994 and 1998. These samples were tested on the RIBA 3.0. Donor samples in Groups 3 and 4 were originally tested on RIBA-3 during 1998 and 1999 and gave positive or indeterminate blot results, respectively. In this study these two groups were tested on the HCV Western blot. Samples with discrepant results on the two immunoblot assays were selected for genotyping or serotyping. RESULTS: The two immunoblots showed similar sensitivity to the core and NS5 proteins. However, of 35 samples positive on Western blot or RIBA-3, the Western blot failed to detect NS4 in 14 samples compared with only 5 for RIBA-3. As well, the Western blot failed to detect NS3 in 6 samples compared to 2 for RIBA-3. Five (27.8%) of 18 samples that were Western blot indeterminate due to core reactivity showed an additional NS3 band on RIBA-3. Of the samples with additional NS3 and/or NS4 reactivity on RIBA-3 that were genotyped or serotyped, all were HCV type 3. CONCLUSIONS: Western blot and RIBA-3 showed similar sensitivity to the HCV core and NS5 proteins. However, RIBA-3 showed greater sensitivity to both NS3 and NS4 compared to the Western blot. The reduced sensitivity of the Western blot to the NS3 and NS4 proteins was observed with HCV type 3 samples.     

A new HCV core antigen assay based on disassociation of immune complexes: an alternative to molecular biology in the diagnosis of early HCV infection

BACKGROUND: An EIA based on immune complex disassociation of nucleocapsid proteins of HCV has been developed to detect and quantify HCV core antigen. STUDY DESIGN AND METHODS: To evaluate whether this new assay (trak-C, Ortho Clinical Diagnostics) could be an alternative to NAT during the window period, its sensitivity in this context was assessed, and its performance was compared with that of a first-generation HCV core antigen assay dedicated to the blood screening (HCV core antigen ELISA). Studied populations included nine HCV RNA-positive, HCV antibody-negative blood donors and 23 hemodialysis patients who underwent an HCV seroconversion. From these individuals, 81 samples (23 HCV RNA-negative and 58 HCV RNA-positive) sequentially collected during the phase before seroconversion were tested. RESULTS: The nine blood donor samples were positive for the presence of HCV core antigen by the trak-C, and 6 of 8 tested were positive for the presence of HCV core antigen by blood screening ELISA. In the hemodialysis cohort, the 23 HCV RNA-negative samples were negative with the two HCV core antigen assays. Among the 58 HCV RNA-positive samples, 46 of 57 (80.7%) tested were positive for the presence of HCV core antigen with the blood screening assay, and 57 of 58 (98.2%) were positive for the presence of HCV core antigen with the trak-C. The mean delays in detecting HCV infection between trak-C and the appearance of HCV antibodies, between HCV RNA testing and trak-C, and between trak-C and HCV core antigen ELISA were 58.2, 0.24, and 3.33 days, respectively. CONCLUSION: Trak-C was more sensitive than the blood screening assay and had similar performance to HCV RNA assay in the window period. Trak-C could constitute an alternative to NAT for the diagnosis of HCV infection during the window period, especially when molecular biology procedures cannot be implemented.     

Third-generation recombinant immunoblot assay: comparison of reactivities according to hepatitis C virus genotype

BACKGROUND: Recombinant immunoblot assay (RIBA) is widely used as a supplemental test in hepatitis C virus (HCV) confirmatory algorithms. As this assay is based on HCV type 1, its performance was examined with the common European HCV genotypes (1, 2, and 3). STUDY DESIGN AND METHODS: A study was performed to retest in third-generation RIBA (RIBA- 3) all 146 second-generation RIBA (RIBA-2)-positive polymerase chain reaction-positive samples detected by second-generation enzyme-linked immunosorbent assays and having known HCV genotypes (74 HCV type 1, 21 type 2, 51 type 3). RIBA band intensities were examined according to HCV genotype. An additional 90 RIBA-3-confirmed PCR-positive samples (47 HCV type 1, 5 type 2, 38 type 3) detected by third-generation enzyme-linked immunosorbent assays were also examined. RESULTS: In the first group of 146 samples, the RIBA-3 NS4 (c100p) band showed a marked improvement in sensitivity for the detection of HCV types 2 and 3 over that of the c100 antigen of RIBA-2, but the mean band intensities of HCV types 2 and 3 remained significantly lower than those of type 1. Improved sensitivity of the NS3 band of RIBA-3 to HCV type 3 was also apparent, but, again, the mean band intensity measured was lower for type 3 than for either type 1 or type 2. The c22 band of RIBA-2 and RIBA-3 exhibited equal sensitivity for all HCV genotypes. These differences were also apparent when RIBA-3 was used in conjunction with third-generation enzyme-linked immunosorbent assays. CONCLUSION: The current RIBA-3 lacks sensitivity to the NS4 antibody for HCV types 2 and 3. The incorporation of type-specific components to other genotypes for NS4 (and NS3) antigens should be considered by the manufacturers.     

Unexplained hepatitis C virus antibody seroconversion in established blood donors

BACKGROUND: Understanding of the epidemiology and natural history of hepatitis C virus (HCV) infection is incomplete without reference to the early phase of infection. The prevalence of HCV infection is well documented in numerous reports. The seroconversion pattern in previously antibody-negative blood donors provides a model for the study of the incidence and transmission of HCV infection. STUDY DESIGN AND METHODS: Records of HCV antibody tests at the West Midlands Blood Transfusion Centre were reviewed to determine the seroconversion rate in 1994 among previously anti-HCV-negative blood donors. Seroconverting donors were counseled to investigate the possible routes of infection. RESULTS: In 1994, blood donations (n = 256,935) were collected from 149,370 donors; 24 donors (0.016%; 1/6224) were positive in the screening enzyme-linked immunosorbent assay (ELISA) and the third- generation recombinant immunoblot assay (RIBA-3). Two donors previously negative for HCV antibody in ELISA were positive in both tests in 1994. Four donors positive in ELISA and indeterminate in RIBA-3 in 1993 reacted positively in both tests in 1994. One donor negative for HCV antibody on previous screening reacted positively in ELISA and was indeterminate in RIBA-3 in 1994 and has become positive in both tests in 1995. A further 43 donors negative for HCV antibody on previous screening reacted positively in ELISA and were indeterminate in RIBA-3 in 1994. CONCLUSION: Documented seroconversion can take place in the absence of exposure to recognizable risk factors for the infection. The index donation or the donation immediately preceding seroconversion may be positive for HCV RNA in the polymerase chain reaction.     

The hepatitis C virus genotype and subtype frequency in hepatitis C virus RNA-positive, hepatitis C virus antibody-negative blood donors identified in the nucleic acid test screening program in Poland

BACKGROUND: Since 2002, blood donors in Poland have been tested not only for hepatitis C virus antibodies (anti-HCV) but also for HCV RNA or HCV core antigen. This screening program identifies asymptomatic, recently infected individuals with no anti-HCV (in the "window period"). The aim of this study was to compare HCV genotype and subtype distribution in window-period (wp) donors, anti-HCV-positive donors, and chronic hepatitis C (CHC) patients. STUDY DESIGN AND METHODS: A total of 2.37 million donors were investigated for HCV RNA, and 340,000 for HCV core antigen. HCV genotypes and subtypes were investigated in 50 HCV RNA-positive, anti-HCV-negative donors; in 70 anti-HCV-positive donors; and in 170 CHC patients. Re-questioning of wp donors for probable risk factors was introduced. RESULTS: HCV RNA was detected in 50 donors of 2.71 million (1:54,200) anti-HCV-negative blood donations. Of these 50 donors, 36 percent exhibited Subtype 1b, whereas Subtypes 3a and 4c/d were identified in 40 and 14 percent, respectively. In anti-HCV-positive donors and CHC patients, the frequency of Subtype 1b was significantly higher (75.7 and 85.3%, respectively); in both groups the lower frequency of Subtypes 3a (14.3 and 10.6%, respectively) and 4c/d (4.3 and 1.2%, respectively) was found. The probable source of infection was identified in 9 wp donors. CONCLUSIONS: The frequency of wp donors is 18.5 per 1 million. The unexpected high frequency of Genotype 4 and Subtype 3a and the low frequency of Subtype 1b was observed in wp donors compared to anti-HCV-positive individuals. Additional epidemiologic questioning introduced after HCV RNA detection may help to identify infection source.     

Comparison of two anti-hepatitis C virus enzyme-linked immunosorbent assays

BACKGROUND: Third-generation anti-hepatitis C virus (HCV) enzyme-linked immunosorbent assays (ELISA) are now implemented in most laboratories in Europe, but have not yet been fully implemented in the United States. STUDY DESIGN AND METHODS: Two ELISAs (Ortho 3.0 and Ortho 2.0, Ortho Diagnostics, Raritan, NJ) were compared by tests on various serum panels: A) blood donor samples (n = 530) that tested positive in first- or second-generation anti-HCV ELISA; B) samples from persons with chronic non-A, non-B hepatitis (n = 185); C) samples from multiply transfused patients (n = 79); D) samples from patients on hemodialysis (n = 473); and E) samples from Dutch random blood donors (n = 2153). RESULTS: In panels A, B, and C, 247 (100%) of 247 polymerase chain reaction (PCR)-positive and 278 (100%) of 278 second-generation recombinant immunoblot assay (RIBA-2)-positive specimens were detected by Ortho 2.0 and 3.0 (sensitivity, 100%). In the sera of panel D, used to represent a group of patients with a high risk for HCV, no additional positives were found by Ortho 3.0. In panel E, of 2153 blood donor samples, 2 (0.1%) were positive in Ortho 2.0 and 8 (0.4%) in Ortho 3.0. Two samples that were positive in both Ortho 2.0 and 3.0 were also positive in RIBA-2; one was positive on PCR. From the 6 remaining Ortho 3.0-positive (Ortho 2.0-negative) samples, 1 was positive in RIBA-2 (isolated anti-c100) and 3 were positive in third- generation RIBA (1/3 isolated anti-c100, 2/3 isolated NS5). All 6 samples were PCR negative. In first-time donors, no difference in specificity was found. CONCLUSION: The sensitivity and specificity of the Ortho 3.0 ELISA are comparable to those of the Ortho 2.0 ELISA.     

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)     

Improved serologic detection of hepatitis C virus with a paramagnetic microparticle assay using multiple antigenic sequences

A paramagnetic microparticle (MP) assay for antibody to hepatitis C virus (anti-HCV) was developed, in which the probe for antibody consisted of synthetic peptides corresponding to HCV capsid and nonstructural c-100 regions, as well as a recombinant protein corresponding to the nonstructural c33c region. Assay performance was evaluated by testing serum from 108 geographically diverse patients with non-A, non-B hepatitis (NANBH). The frequency of anti-HCV reactivity detected with the MP assay and with an enzyme-linked immunosorbent assay (ELISA) for c-100 was 91 and 70 percent, respectively. All c-100 HCV ELISA-reactive specimens also reacted on the MP assay. In addition, anti-HCV seroconversion in three plasma donors was detected one to two blood collection dates earlier by the MP assay than by the c-100 HCV ELISA and at similar blood collection dates by the MP assay and a second-generation anti-HCV ELISA. Serologic responses to the three distinct antigenic regions of HCV in NANBH patients varied: reactivity to all three antigens was most common (49%), reactivity to both capsid and c33c (40%) was next most common, and single-antigen reactivity was rare (4%). MP assay reactivity of 825 volunteer donors was 0.1 percent. These results demonstrate both the utility of additional HCV antigens for an effective anti-HCV screening assay and the application of paramagnetic MP technology to serologic testing for HCV infection.     

Detection of hepatitis C virus infection with recombinant immunoblot assay,synthetic immunoblot assay,and polymerase chain reaction

The newly developed immunoblot assay, RIBA SIA (recombinant and synthetic polypeptide immunoblot assay), Chiron, Calif., was compared with the commercially available second generation recombinant immunoblot assay (RIBA-2) for the detection of antibody to hepatitis C virus (anti-HCV). The two immunoblot tests were also compared with the polymerase chain reaction (PCR) for the detection of HCV RNA. Ninety-one percent of samples reactive by RIBA-2 were positive for anti-HCV by RIBA SIA. A total of 31% of RIBA-2 indeterminate samples became reactive by RIBA SIA, 24% became non-reactive, and 45% remained the same. Samples reactive by RIBA-2 or SIA from different risk groups, were mostly positive (67-100%) by PCR for HCV RNA. All indeterminate samples from hemophiliacs and intravenous drug users were PCR positive. RIBA SIA is more sensitive and specific than RIBA-2 and correlates well with PCR results © 1993 Wiley-Liss, Inc.     

Combination HCV core antigen and antibody assay on a fully automated chemiluminescence analyzer

BACKGROUND: HCV exposure among blood donors is serologically determined by detection of antibodies to HCV (anti-HCV); however, the recent development of an assay for the detection of HCV core antigen identifies infection before anti-HCV development. Simultaneous detection of HCV core antigen and anti-HCV would shorten the window period before seroconversion over conventional HCV antibody screening assays. STUDY DESIGN AND METHODS: A prototype chemiluminescent immunoassay was developed for simultaneous detection of HCV core antigen and anti-HCV in human sera and plasma. The assay was performed on a single-channel instrument representing an automated serologic analyzer (PRISM, Abbott Laboratories) system. Sensitivity and specificity were evaluated by testing 23 HCV seroconversion panels and plasma or sera from volunteer blood donors. RESULTS: The prototype HCV core antigen and antibody combination assay detected 80 of 89 (89.9% ) HCV RNA-positive and antibody-negative specimens from 23 panels, thereby reducing the seroconversion window period by an average of 34.3 days compared to PRISM HCV antibody detection. All PRISM HCV antibody-positive specimens were detected by the combination assay for a relative sensitivity of 100 percent. The repeatedly reactive rate was 0.20 percent based on testing of 3017 screened anti-HCV-negative sera and plasma. CONCLUSIONS: The prototype combination assay was shown to detect HCV core antigen and anti-HCV simultaneously and significantly closed the time gap between the initial detection of HCV RNA and the first appearance of detectable antibodies to HCV.     

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.     

Prevalence of antibodies against hepatitis B and C virus among pregnant women and female blood donors in Sweden

The prevalence of elevated alanine aminotransferase (ALT) levels and antibodies against hepatitis B and C virus (HBV and HCV) among 755 pregnant women attending a prenatal clinic was studied. Consecutive female presumptive blood donors (n = 649) served as serological controls. Among the pregnant women 85 (11%) had elevated serum ALT levels. Antibodies against HCV (anti-HCV) were detected in six (0.8%) pregnant women in a screening enzyme-linked immunosorbent assay (ELISA) and with consecutive confirmatory test (RIBA-2). Antibodies against HBV (anti-HBc and/or anti HBs) was found in 63 (8.3%) pregnant women, of whom three (0.4%) were HBsAg positive. Among the blood donors anti-HCV was detected in seven (1%) of whom one (0.15%) was positive in the confirmatory test (RIBA-2). None of the blood donors had any serological marker of HBV exposure as measured by HBsAg and anti-HBc. The anti-HCV prevalence among pregnant women was low and did not differ significantly from that of healthy blood donors. Prenatal screening for hepatitis is concluded not to be justified without having a history of risk exposure or risk behaviour.