Epidemiological, biological and histological characterization of patients with indeterminate third-generation recombinant immunoblot assay antibody results for hepatitis C virus (original) (raw)
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Reliability of the third-generation recombinant immunoblot assay for hepatitis C virus
Transfusion, 1995
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 RNAnegative 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 clOO (RIBA-2: 94.3% and 62.6%; RIBA-3: 99.5% and 88.6%). Replacement of the c22 and c 100 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%, pO.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). Conclusions: 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, twoband reactivity in anti-HCV enzyme-linked immunosorbent assay-reactive blood donors.
Third-generation hepatitis C virus tests in asymptomatic anti-HCV-positive blood donors
Journal of Hepatology, 1994
This study evaluated the performance of third-generation anti-HCV assays in blood donors who were positive by secondgeneration anti-HCV, and assessed any possible relationship between antibody patterns, HCV replication and liver damage. Fifty-two second-generation enzyme immunoassay-positive asymptomatic Italian blood donors were retested for anti-HCV by third-generation enzyme immunoassay and recombinant immunoblot assay (Ortho third-generation enzyme immunoassay, third-generation recombinant immunoblot assay), utilising recombinant C33c and NS5 and synthetic peptide C100 and C22 antigens, and for HCV-RNA by "nested" polymerase chain reaction with 5' region primers. Alanine aminotransferases were tested monthly for 6 months. Two out of 52 second-generation enzyme immunoassay-positive donors were third-generation enzyme immunoassay, third-generation recombinant immunoblot assay and HCV-RNA negative. Among 50 third-generation enzyme immunoassay-positive cases, two had a third-generation enzyme immunoassay optical density-<l: one was third-generation recombinant immunoblot assay and HCV-RNA negative, and the other was third-generation recombinant immunoblot assay "indeterminate" and HCV-RNA-positive. The remaining 48 cases had third-generation enzyme immunoassay optical density>l: six were third-generation recombinant immunoblot assay negative (one HCV-RNA+ve), eight "indeterminate" (two HCV-RNA+ve) and 34 positive (22 HCV-RNA+ve). All "indeterminate" subjects reacted only to C22. HCV-RNA was positive in 22/34 cases with positive third-generation recombinant immunoblot assay (two or more Ags), 3/9 "indeterminate" and 1/11 negative. Alanine amino-transferases were abnormal in 13 cases with positive third-generation recombinant immunoblot assay, one was "indeterminate" and three were negative. There was, however, a significant relation between C33c positivity and raised alanine aminotransferases (p<0.023). Reactivity to C33c was strongly related to viraemia; 21/29 C33c-positive cases were also HCV-RNA positive, as opposed to 5/25 C33c negative. Viraemia was often but not always associated with liver damage, alanine aminotransfoase levels being abnormal in 13/26 (50%) HCV-RNA positive and 4/28 (14%) HCV-RNA negative subjects (p<0.005). Third-generation HCV tests can reduce the number of second-generation enzyme immunoassay positive donors with a negative HCV-RNA. The concordance between third-generation enzyme immunoassay and third-generation recombinant immunoblot assay is high (96°/,,). Some third-generation enzyme immunoassay/third-generation recombinant immunoblot assay positive, HCV-RNA-negative subjects might have latent viraemia. An "indeterminate" third-generation recombinant immunoblot assay pattern or even a negative third-generation recombinant immunoblot assay does not exclude active infection, since HCV-RNA is often present. © Journal of Hepatology.
Journal of Medical Virology, 1994
Fifty-seven sera with indeterminate results by the second generation RIBA (RIBA 2) for confirmation of hepatitis C virus (HCV) enzyme linked immunoassay (ELISA) reactivity were tested by the new third generation RlBA (RIBA 3). Thirty three (57.9%) displayed reactivity for a t least one other band and were therefore classified as positive; two became negative and 22 (38%) remained indeterminate. The incidence of HCV viremia, as determined by the RNA polymerase chain reaction (PCR), was 75% for the latter sera.
Transfusion, 2012
BACKGROUND-A solid phase recombinant-immunoblot-assay(RIBA) is often used to determine the specificity of antibody to hepatitis-C-virus(anti-HCV). The RIBA result is recorded as positive, negative or indeterminate. The interpretation of RIBA indeterminate reactivity and its significance to patients and blood donors are unclear. We attempted to address the clinical relevance of RIBA-indeterminate reactions in the context of the natural history of HCV infection in a prospectively followed cohort of anti-HCV positive blood donors. STUDY-DESIGN AND METHODS-Donor demographics, HCV exposure history, humoral and cell-mediated immunity(CMI) to HCV were compared in 15 RIBA-indeterminates, 9 chronic-HCV-carriers and 8 spontaneously-recovered subjects. Serum samples were tested for the presence of anti-HCV by a liquid phase Luciferase-Immunoprecipitation-System(LIPS) assay. CMI was assessed by IFN-γ-ELISpot assay. RESULTS-In the quantitative LIPS assay, the sum of antibody responses to 6 HCV-antigens showed significant (p<0.001) step-wise diminution progressing downward from chronic-carriers to spontaneously-recovered to RIBA-indeterminates. CMI responses in RIBA-indeterminates were similar to spontaneously-recovered subjects, and greater than chronic-carriers and negative controls (p<0.008). A parenteral risk factor was identified in 13% of RIBA-indeterminates as compared with 89% of chronic-carriers and 87% of spontaneously-recovered subjects. On average, donors in the RIBA-indeterminate group were older than the other groups. CONCLUSION-The combined CMI and LIPS results suggest that persistent RIBAindeterminate reactions generally represent waning anti-HCV responses in persons who have recovered from a remote HCV infection. In such cases, detectable antibody may ultimately
Revista da Sociedade Brasileira de Medicina Tropical, 2014
Introduction: Hepatitis C virus (HCV) infection is diagnosed by the presence of antibodies and is supplemented by confi rmatory testing methods, such as recombinant immunoblot assay (RIBA) and HCV-RNA detection. This study aimed to evaluate the effi cacy of RIBA testing to diagnose HCV infection in blood donors positive for anti-HCV antibodies. Methods: A total of 102 subjects positive for anti-HCV determined by enzyme-linked immunosorbent assay (ELISA) at the Hematology and Hemotherapy Foundation of Bahia (HEMOBA) were later assessed with new samples using the Abbott Architect anti-HCV test (Abbott Diagnostics, Wiesbaden, Germany), the RIBA III test (Chiron RIBA HCV 3.0 SIA, Chiron Corp., Emeryville, CA, USA), the polymerase chain reaction (PCR; COBAS® AMPLICOR HCV Roche Diagnostics Corp., Indianapolis, IN, USA) and line probe assay (LiPA-Siemens, Tarrytown, NY, USA) genotyping for HCV diagnosis. Results: Of these new samples, 38.2% (39/102) were positive, 57.8% (59/102) were negative and 3.9% (4/102) were indeterminate for anti-HCV; HCV-RNA was detected in 22.5% (23/102) of the samples. RIBA results were positive in 58.1% (25/43), negative in 9.3% (4/43) and indeterminate in 32.6% (14/43) of the samples. The prevailing genotypes were 1 (78.3%, 18/23), 3 (17.4%, 4/23) and 2 (4.3%, 1/23). All 14 samples with indeterminate RIBA results had undetectable viral loads (detection limit ≤50 IU/mL). Of these samples, 71.4% (10/14) were reevaluated six months later. Eighty percent (8/10) of these samples remained indeterminate by RIBA, and 20% (2/10) were negative. Conclusions: In this study, individuals with indeterminate RIBA results had no detectable HCV-RNA.
Lack of correlation between different hepatitis C virus screening and confirmatory assays
Journal of Virological Methods, 1996
2nd and 3rd generation screening and confirmatory assays for the detection of anti-HCV antibodies have been introduced on the international market. The aim of the present study was to compare the performance of five different commercially available screening assays and four 'confirmatory' assays in a panel of serum samples that had tested positive or borderline with a 2nd generation EIA (Abbott HCV EIA 2nd generation). Considerable discrepancies were observed between the different screening assays and confirmatory tests. The antigens from the putative 'core' region of HCV were recognized most frequently by the confirmatory assays. By considering the reactivity to either NS5 (RIBA III and Inno-LIA) or E2/NSl antigens (Inno-LIA Ab III) no sample could be identified as anti-HCV positive that would otherwise have been regarded as borderline or negative according to its banding pattern with core, NS3 and NS4 proteins. All 24 HCV-RT-PCR positive samples were anti-HCV reactive by the screening EIAs but only 18 and 21 samples were confirmed anti-HCV positive with the RIBA II and III, respectively.
Annals of Hepatology, 2002
The most practical screening test for hepatitis C virus antibodies are second and third-generation enzyme immunoassays. We evaluated the usefulness of the third generation microparticle enzyme immunoassay (MEIA) in predicting HCV viraemia in anti-HCV positive patients. Serum samples from 106 patients with positive anti-HCV were obtained. To evaluate the diagnostic value of the MEIA test in predicting HCV viraemia, anti-HCV positive patients were categorized in two groups according to the presence or absence of serum HCV-RNA. Among the 106 patients, 26 had non detectable serum HCV-RNA and 80 had detectable HCV-RNA by PCR. The assay automatically calculates a result based on the ratio of sample rate to the cut-of rate for each sample and control (S/CO). When the means of S/CO values for patients with detectable and non detectable HCV-RNA were analyzed, a statistically significant difference was found, (79.3 SD 22.2 vs. 8.2 SD 6.4, respectively) (p 0.0001). We further analyzed the best cutoff value of the S/CO in differentiating viremic from non viremic patients. The S/CO value of 26 showed a sensitivity of 99% and a specificity off 96% in discriminating both categories of HCV infected patients. In conclusion, our data demonstrate that viremic HCV patients had higher S/CO values in the MEIA test in comparison with non viremic patients. Hence, this assay may be used to predict HCV viraemia in anti-HCV positive individuals.
Journal of Clinical Microbiology, 2019
An estimated 41,200 people were newly infected with hepatitis C virus (HCV) in 2016 in the United States. Screening tests for antibodies to HCV may generate up to 32% false positivity in low-risk populations. Current Centers for Disease Control and Prevention (CDC) screening recommendations do not require confirmatory testing of a screening anti-HCV-positive test; however, confirmation is valuable for surveillance in the absence of HCV RNA testing. A recombinant immunoblot assay (RIBA) was used as a confirmatory assay for anti-HCV-reactive samples but was discontinued in 2013. Another anti-HCV confirmatory assay, INNO-LIA, is commercially available in Europe but is not approved by the Food and Drug Administration (FDA) in the United States. We report the development of an anti-HCV assay that was performed on an automated immunoblot platform using a fourth-generation HCV recombinant fusion protein. Based on testing of 70 well-characterized samples, of which 40 were HCV RNA and anti-HCV positive, 15 were HCV RNA positive/anti-HCV negative, and 15 were HCV RNA and anti-HCV negative, the specificity and sensitivity of the HCV-WES assay were 100% and 95%, respectively. Concordance between INNO-LIA and HCV-WES was determined by testing 205 HCV RNA-negative/ anti-HCV-positive samples, of which 149 (72.7%) were positive by HCV-WES, while 146 (71.2%) were positive by INNO-LIA. We have shown proof of concept for the use of this test for confirmation of screened anti-HCV results. The HCV-WES assay has advantages over manual Western blot assays and INNO-LIA, including ease of use, lower cost, and reduced hands-on time. KEYWORDS automated immunoassay, hepatitis C virus, serology H epatitis C virus (HCV) is a major public health problem worldwide with an estimated 71 million individuals living with chronic HCV infection in 2015 (1). In the United States from 2003 to 2010, there were an estimated 4.6 million Americans infected with HCV, 3.5 million of whom were estimated to have current infection (2). An estimated 41,200 people were newly infected with HCV in 2016 in the United States (3). Furthermore, it is estimated that more than half of the people infected with HCV may not be aware of their infection status (4). Testing data from a large U.S. commercial laboratory suggest that an estimated one in four HCV-infected people in the United States have significant liver disease and could benefit from treatment (5). The Centers for Disease Control and Prevention (CDC) recommends determining current HCV infection status by screening for HCV antibodies (anti-HCV), followed by testing for HCV RNA if the HCV antibody test is reactive (6), to facilitate linkage to care and treatment with currently available highly efficacious direct-acting antiviral agents (DAAs). Anti-HCV assays have a lower positive predictive value in low-prevalence populations. A recent study showed up to 32% anti-HCV false positivity in a national U.S.-based surveillance study (7). Although positive results for anti-HCV and HCV RNA