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      Automated RIBA TM HCV Strip Immunoblot Assay: A Novel Tool for the Diagnosis of Hepatitis C Virus Infection in Hemodialysis Patients

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          Abstract

          Hemodialysis (HD) patients remain a high-risk group for hepatitis C virus (HCV) infection. Serological assays (enzyme-linked immunosorbent assays, ELISAs) are the only tests currently approved by the Food and Drug Administration in the United States for the diagnosis of HCV. The RIBA<sup>TM</sup> HCV Strip Immunoblot Assay (SIA) is an established method for supplemental testing of repeat reactive hepatitis C ELISA patients on HD. However, the current manual procedure is labor intensive, requiring subjective band scoring and result interpretation. Recently, the automated CHIRON<sup>®</sup> RIBA<sup>TM</sup> HCV Processor System has been designed to perform RIBA supplemental testing. The CHIRON RIBA HCV Processor System consists of a bench-top instrument that provides objective evaluation of the RIBA immunoblot strips, by measuring the light differentially reflected from the developed bands and white background, creating a density of reflectance. The CHIRON RIBA HCV Processor System assesses the intensity of each of the reactive bands in relation to the intensity of the internal control bands on each RIBA HCV strip. Comparison between processor and manual protocols was performed using a large (n = 200) cohort of ELISA 3.0 HCV negative and positive patients on maintenance HD. The test characteristics of RIBA HCV 3.0 SIA were identical with manual and automated runs. The relative intensity values of antigenic bands by the CHIRON RIBA HCV 3.0 Processor System between anti-HCV positive and negative patients were significantly different; only 15 of 784 (1.9%) antigenic bands had borderline reactivities. The correlation of test results between manual and automated runs was very high (kappa value 0.989). Among positive results by RIBA HCV 3.0 SIA, there was a strong concordance between manual and automated runs with regard to the pattern of reactivity (kappa value 0.943). The discordant results between manual and automated protocols were attributable to increased variability of antigen scores close to the cutoff value for both tests. In conclusion, the CHIRON RIBA HCV 3.0 Processor System is capable of performing RIBA HCV 3.0 SIA in the HD population accurately with minimal operator involvement. The test characteristics of RIBA HCV 3.0 SIA were identical by manual and automated runs. There was a strong correlation between the results of the manual and automated runs; the few discordant results between the two procedures were mostly due to increased variability of antigen scores close to the cutoff value for both tests. The Centers for Disease Control and Prevention in the USA have recently included chronic HD patients among those persons for whom routine HCV testing is recommended; HCV-infected patients on HD often have a high rate of indeterminate results by manual RIBA technology which is operator dependent for band scoring and result interpretation. The CHIRON RIBA HCV 3.0 Processor System may be very useful for supplemental anti-HCV testing of ELISA repeat reactive specimens in clinical practice within dialysis units.

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          Most cited references 7

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          Hepatitis C virus infection in French hemodialysis units: a multicenter study.

          The aims of the study were: (i) to evaluate the prevalence of hepatitis C virus (HCV) antibodies (third generation tests) and RNA (standardized ultrasensitive RT-PCR assay) in a large cohort of hemodialysis patients, and (ii) to correlate HCV markers with bioclinical features and alanine-aminotransferase (ALT) activity. Antibodies were assayed by two methods in 1,323 patients (60% men, median age 65 years, median hemodialysis duration 3 years) attending 25 French hemodialysis centers including 9 self-care units. RNA was assayed using the Cobas Amplicor 2.0 method in pooled samples from 10 anti-HCV(-/-) patients and on individual samples from the other patients. Of the 16.3% patients (range 0-44%) tested (+/+) for HCV antibodies (anti-HCV), 2.3% tested (+/-) and 81.4% tested (-/-). 70% of the anti-HCV(+/+) patients and 3% of the HCV(+/-) patients were RNA(+). Pooled analysis revealed that 5/1077 anti-HCV(-/-) patients (0.5%) were RNA(+); all 5 displayed subsequently an increase in ALT and became anti-HCV(+/+). Mean ALT was higher (multiple of normal) in anti-HCV(+/+) RNA(+) patients than in anti-HCV(+/+) RNA(-) patients (0.46 +/- 0.08 vs. 0.22 +/- 0.07, P < 0.0001) and similar in all the RNA(-) patients, whatever their HCV antibody status. Multivariate analysis demonstrated that HCV status was linked to hemodialysis duration, previous kidney transplantation and positive anti-HBc. To summarize, the determination of the RNA status of anti-HCV(+/-) patients may have clinical relevance if a policy of isolation is contemplated. Standardized ultrasensitive RT-PCR assay combined with a pooling strategy is a promising method for use in epidemiological studies. Copyright 2000 Wiley-Liss, Inc.
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            Quantitative Assessment of HCV Load in Chronic Hemodialysis Patients: A Cross-Sectional Survey

            Recent evidence has been accumulated showing that chronic hemodialysis (HD) patients have a very high prevalence of antibodies to hepatitis C virus (HCV). In contrast, there is little information addressing the virological characteristics of HCV infection in this population. Aim: To measure HCV viral load and to correlate this with demographic, biochemical, and clinical features of a large cohort of HCV-infected patients on chronic HD. Methods: 394 chronic HD patients were tested by branched-DNA signal amplification assay, anti-HCV enzyme-linked immunosorbent assay 2.0, and on the basis of the aspartate aminotransferase/alanine aminotransferase (AST/ALT) activity. Multivariate analysis by ordinal logistic regression model was performed: age, gender, race, time on HD, allocation of the patients among the HD units, etiology of end-stage renal disease, HBsAg status, anti-HCV positivity, HCV genotype, and AST/ALT levels were independent factors, and viremic levels of HCV in serum were assumed as dependent variables. Results: 88 (22.3%) patients showed serological and/or virological signs of HCV infection. 59 (15%) out of 394 had detectable HCV RNA in serum, the mean HCV load was 19.4 × 10 5 (95% CI, 6.06 × 10 7 to 6.2 × 10 4 ) Eq/ml. According to the criteria suggested by others [J Infect Dis 1994;169:1219–1225], there were 8 (13.5%) individuals with high-titer viremia (>1 × 10 7  Eq/ml) in the subset of viremic patients. A small subset (8/394 or 2%) of individuals was seronegative, but viremic; 29 (7%) out of 394 were seropositive without detectable HCV RNA in serum. Univariate analysis showed that the frequency of anti-HCV positivity was significantly higher in viremic patients as compared with individuals with no detectable HCV viremia: 51/59 (86%) vs. 29/335 (8.6%), p = 0.0001. Serum AST and ALT levels were significantly higher in viremic patients than in individuals with no detectable HCV RNA in serum: 23.8 (95% CI 60.8–9.3) vs. 17.1 (95% CI 50.4–5.8) U/l (p = 0.009) and 14.4 (95% CI 48.9–4.3) vs. 9.8 (95% CI, 37.3– 2.5) U/l (p = 0.008). Logistic regression analysis showed an association between HCV viremia and anti-HCV positivity (p = 0.00001) and ALT activity (p = 0.01). Conclusions: Hepatitis C virus infection is highly prevalent in the HD population; the viral load is relatively low, and it was associated with elevated hepatic enzyme levels and anti-HCV positivity. No other clinical characteristics were associated with HCV RNA levels. Seronegative but viremic patients were also found. Longitudinal studies with long follow-up periods are necessary to evaluate the course of HCV load over time in this population.
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              Identification of hepatitis C virus seroconversion resulting from nosocomial transmission on a haemodialysis unit: implications for infection control and laboratory screening.

               D Irish,  L Burnapp,  I Abbs (1999)
              Hepatitis C virus (HCV) seroconversion was detected by routine screening in a haemodialysis patient, Patient 1. Serological investigations were undertaken over the following 3 months to determine if further transmission to other patients on the unit had occurred. No additional cases were identified. Twenty-two haemodialysis patients known to have HCV infection were investigated using molecular epidemiological methods to determine if transmission between patients had occurred. HCV viraemia was demonstrated by polymerase chain reaction in 19 of 22 patients (86%). Genotyping showed that eight patients were infected with genotype 1, three with genotype 3 and eight, including Patient 1, with genotype 2. Phylogenetic analysis of viral sequences from the eight patients with genotype 2 revealed three, including Patient 1,with a novel subtype of HCV type 2, and revealed close similarity between viral sequences from patient 1 and those from one other patient, suggesting transmission. This was consistent with haemodialysis histories. Among other patients with genotype 2, there were two with subtype 2a and three others with three separate novel subtypes, as yet undesignated. With the exception of patient 1, all patients infected with novel subtypes were of Afro-Caribbean origin. The HCV prevalence among patients on the haemodialysis unit was high (14%), which may reflect the ethnicity of our haemodialysis population. This case emphasises the risk of nosocomial transmission and the importance of infection control procedures on haemodialysis units, and highlights the usefulness of molecular epidemiological techniques for the investigation of outbreaks of HCV infection. Copyright 1999 Wiley-Liss, Inc.
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                Author and article information

                Journal
                AJN
                Am J Nephrol
                10.1159/issn.0250-8095
                American Journal of Nephrology
                S. Karger AG
                0250-8095
                1421-9670
                2001
                April 2001
                07 May 2001
                : 21
                : 2
                : 104-111
                Affiliations
                aDivision of Digestive Diseases, UCLA School of Medicine, Los Angeles, Calif., and bChiron Corporation, Emeryville, Calif., USA; cNephrology and Dialysis Division, Maggiore Hospital, IRCCS, Milan, Italy
                Article
                46232 Am J Nephrol 2001;21:104–111
                10.1159/000046232
                11359017
                © 2001 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 2, Tables: 3, References: 48, Pages: 8
                Product
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/46232
                Categories
                Clinical Study

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