Distinguishing Acute from Chronic Hepatitis C Virus (HCV) Infection Based on Antibody Reactivities to Specific HCV Structural and Nonstructural Proteins

Abstract

Currently available serological assays for detection of antibodies to hepatitis C virus (HCV) cannot reliably discriminate acute from chronic HCV infection. We developed a multiplexed, flow-cytometric microsphere immunoassay to measure anti-HCV-IgG reactivities to the core, NS3, NS4, and NS5 HCV recombinant proteins and applied it to 99 serum samples from 24 anti-HCV seroconverters and 141 anti-HCV-IgG and HCV RNA-positive plasma specimens from chronically infected people. Differences in the geometric means or means of signal/cutoff ratios between the two sample sets were statistically significant for all the antigens tested. A multivariate logistic regression model correctly classified the samples in two groups, with a cross-validation accuracy of 90.8% for the acute group and 97.2% for the chronic group. The immunoassay described has the potential to distinguish acute from chronic HCV infection.The diagnosis of acute hepatitis C virus (HCV) infection is based on the detection in serum or plasma of HCV RNA, anti-HCV IgG, and elevation of alanine aminotransferase levels (5). However, none of these markers alone or in combination can be used to identify acute infection, since they may also be detectable during the chronic phase of infection. Further, distinguishing acute from chronic infection on the basis of clinical history, epidemiological risk factors, and symptoms can be difficult because for most patients acute infection is asymptomatic (12, 3). Several approaches, which include detection of anti-HCV IgM (4, 2), measurement of the anti-HCV IgG avidity index (9), and observation of serial changes in viral load (10), have been proposed as indicators of acute HCV infection. The usefulness of anti-HCV IgM as a marker of acute infection remains controversial (4, 2). The recently published approach of using viral load fluctuations to identify acute HCV infection requires serial testing of samples (10). We report here the development of a high-throughput microsphere immunoassay, which simultaneously detects anti-HCV IgG responses to multiple structural and nonstructural HCV recombinant proteins, and its application to serum and plasma samples collected from people in the acute and chronic phases of HCV infection. The assay has the potential to discriminate between the acute and chronic phases by testing of single specimens. MATERIALS AND METHODSStudy serum specimens. This study was performed using unlinked anonymous serum or plasma specimens and specimens obtained commercially from blood donor anti-HCV seroconversion panels. Ninety-nine plasma samples were obtained from 24 donor panels; the number of samples per panel varied between 1 and 11. Ten panels were acquired from Zeptometrix (Buffalo, NY) (batch numbers 6211, 6212, 6213, 6214, 6215, 9041, 9047, 9054, 9055, and 9058), 5 from NABI (Boca Raton, FL) (batch numbers 10, 20, 30, 40, and 60), 4 from Serologicals (Clarkston, GA) (batch numbers 4812B, 4813, 4814, and 4814B), 3 panels (batch numbers 908, 920, and 921) from BBI (West Bridgewater, MA), and 2 from Prof...

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