- Email: [email protected]
335 FACTORS INFLUENCING DETECTION OF LOW LEVELS OF HEPATITIS C VIRUS (HCV) GENOME AND ITS REPLICATION
05b: VIRAL HEPATITIS − b) HEPATITIS C − EXPERIMENTAL (VIROLOGY) able to recruit myofibroblast-like cells. These migratory capacities allows the progression of liver fibrosis promoted by HHSCs. 333 KINETICS OF HCV CORE ANTIGEN DURING ANTIVIRAL TREATMENT OF ACUTE AND CHRONIC HEPATITIS C AS DETERMINED BY A NOVEL CHEMILUMINESCENT MICROPARTICLE IMMUNOASSAY I. Mederacke1 , S. Ciesek2 , R. Raupach1 , K. Wursthorn1 , K. Deterding1 , E. Steinmann2 , M. Cornberg1 , E. Jaeckel1 , M.P. Manns1 , T. Pietschmann2 , H.L. Tillmann3 , H. Wedemeyer1 . 1 Hannover Medical School, 2 Twincore, and Hannover Medical School, Hannover, Germany, and Helmholtz Centre for Infection Research, Hannover, Germany; 3 Duke Clinical Research Institute, Durham, NC, USA E-mail: [email protected] Introduction: HCV-RNA testing is a very sensitive but rather cost intensive method with some weakness regarding stability. We have evaluated the performance of a new serological HCVcoreAg assay to assess its qualification for treatment monitoring of acutely and chronically infected HCV patients as well as for research purposes in supernatants of the in vitro HCVcc culture system. Methods: HCVcoreAg was quantified by a novel, fully automated chemiluminescent microparticle immunoassay (Architect HCV Ag, Abbott). For cell culture supernatants, HCVcoreAg was also tested by the Ortho HCV core Ag assay. HCV-RNA quantification was performed using the Cobas TaqMan Assay (Roche Diagnostics, Germany). Results: HCV-RNA and HCVcoreAg plasma levels showed very good correlation both in acutely viremic (N = 7; R2 =0.99) as well as in 46/49 chronically infected patients (N = 46, R2 =0.93) whereas highly discordant results were detected in the remaining three. Early kinetics of HCV-RNA and HCVcoreAg levels showed high correlations in three chronic hepatitis C patients studied during daily injections of consensus interferon and ribavirin on day 0, 1, 4, and 7 of treatment (R2 =0.87, 0.98, 0.98, respectively). As expected, HCVcoreAg was found negative in samples with very low viremia (9 samples of 8 patients with HCV-RNA between <15 and 350 IU/ml) but also in one patient with a flare during acute infection of 14,800 IU/ml. We also determined HCVcoreAg in cell culture supernatants of JFH-1-infected HUH7.5 cells and found very good correlations comparing the Abbott assay and the Ortho assay (N = 23, R2 = 0.99). This correlation held also true when supernatants with low (<100,000 fmol/L; R2 = 0.92) as well as high (>100,000 fmol/L; R2 = 0.99) HCVcoreAg concentration were compared. Conclusion: HCVcoreAg as determined by the Abbott CMIA assay is a robust and reliable marker of HCV replication demonstrating very good correlations with HCV-RNA both in acute and chronic hepatitis C despite weaker sensitivity. While the assay cannot replace sensitive viral load assays in patients with low viremia, it may offer an additional diagnostic tool in HCV infected patients. 334 MOLECULAR EVOLUTION OF THE E2 GENE OF HEPATITIS C VIRUS IN A PATIENT INFECTED AND RE-INFECTED WITH GENOTYPE 1A R. Molenkamp1 , K. Brinkman2 , S. Koekkoek1 , I. Spijkerman3 , J. Schinkel1 . 1 Medical Microbiology, Section of Clinical Virology, Academic Medical Center, 2 Internal Medicine, 3 Medical Microbiology, Onze Lieve Vrouwe Gasthuis, Amsterdam, The Netherlands E-mail: [email protected] Background: Hepatitis C virus (HCV) is emerging as a sexual transmitted infection among HIV-positive men having sex with men (MSM). Phylogenetic analysis of part of the NS5B gene of strains sent to our laboratory for genotyping suggested the existence of several monophyletic HCV clusters of genotypes 1a and 4d among these HIV-positive patients with acute HCV infection. We identified one patient, infected with genotype 1a, which after successful clearance by treatment with PEG-interferon and ribavirin, became re-infected with a genotype 1 a strain clustering with other 1a
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strains (patient X). Purpose of this study was to further characterize the 1a strains of both infection periods by carrying out a longitudinal phylogenetic analysis of the E2 gene, which contains the first hypervariable region (HVR1). Methods: 20−30 clones were amplified from four different time points during the first infection period, and three different timepoints during the second infection period. In addition, samples from other patients with NS5B sequences clustering with NS5B sequences from patient X were analyzed also. Phylogenetic trees were constructed to assess the phylogenetic relationship between the different strains at different timepoints. Results: Analysis of the HVR1 region of E2 confirmed the high phylogenetic signal of this region. Clonal sequences from the first and second infection period from patient X formed separate monophyletic clusters. No intermingling of sequences from the first and second infection period was observed. Conclusion: Clearance of infection does not necessarily result in protective immunity, even against infection with the same subtype. 335 FACTORS INFLUENCING DETECTION OF LOW LEVELS OF HEPATITIS C VIRUS (HCV) GENOME AND ITS REPLICATION T. Pham, T. Michalak. Molecular Virology and Hepatology Research Group, Faculty of Medicine, Memorial University, St. John’s, NL, Canada E-mail: [email protected] Background: Occult HCV infection (OCI), characterized by small amounts of HCV RNA in sera and replicating viral genomes in PBMC and liver, has been documented in individuals with clinically resolved hepatitis C and in those with elevated aminotransferases of unknown etiology. However, data seemingly opposing the existence of OCI have also been reported. This study identifies factors potentially reconciling these differences. Methods: Plasma and PBMC were collected from 84 patients with resolved hepatitis C (SVR duration 6−72 months). Sequential plasma and PBMC, collected at 6−12 month intervals, were obtained from 47/84 persons. Liver biopsies were available from 6. PBMC were left untreated or treated with mitogens before analysis. RNA was extracted using Trizol (Invitrogen) or, for comparative purposes, RNeasy kit (Qiagen) from 250 mL to 4 mL plasma, 1 x 107 PBMC or ~50 mg liver tissue. HCV RNA positive strand was evaluated by RT-PCR/NAH (sensitivity, 10 virus copies/mL; i.e., 5-fold higher than that of Bayer TMA), while the replicative strand by Tth-based RT-PCR/NAH (sensitivity, ~25 virus copies/rxn). HCV variants in PBMC were compared to those in plasma and liver by clonal sequencing. HCV NS5A protein was detected by confocal microscopy. Results: RNA recovery after Trizol extraction consistently superseded that from the RNeasy kit by 2−3 folds. HCV RNA was detectable in 48/84 (57%) cases at 250 mL plasma, although using up to 4 mL increased virus detection to 71%. HCV RNA in unstimulated PBMC was identifiable in 21% (18/84) cases, but mitogen stimulation led to HCV RNA detection in 67% of cells which had been negative when unstimulated. Three of 6 livers were HCV genome positive. Testing of sequential plasma samples increased HCV RNA detection by 10−15%. Virus negative strand was detectable in ~65% of PBMC and livers reactive for the positive strand. HCV NS5A was displayed by 0.02−0.1% of immune cells. Certain HCV variants occurred only in immune cells. Conclusions: The basis of reported differences in OCI occurrence is multifactoral. Variations in the type and number of samples analyzed, sample preparation, RNA extraction method, template amount, and assay sensitivity significantly influence the overall detection of low-level HCV.
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strains (patient X). Purpose of this study was to further characterize the 1a strains of both infection periods by carrying out a longitudinal phylogenetic analysis of the E2 gene, which contains the first hypervariable region (HVR1). Methods: 20−30 clones were amplified from four different time points during the first infection period, and three different timepoints during the second infection period. In addition, samples from other patients with NS5B sequences clustering with NS5B sequences from patient X were analyzed also. Phylogenetic trees were constructed to assess the phylogenetic relationship between the different strains at different timepoints. Results: Analysis of the HVR1 region of E2 confirmed the high phylogenetic signal of this region. Clonal sequences from the first and second infection period from patient X formed separate monophyletic clusters. No intermingling of sequences from the first and second infection period was observed. Conclusion: Clearance of infection does not necessarily result in protective immunity, even against infection with the same subtype. 335 FACTORS INFLUENCING DETECTION OF LOW LEVELS OF HEPATITIS C VIRUS (HCV) GENOME AND ITS REPLICATION T. Pham, T. Michalak. Molecular Virology and Hepatology Research Group, Faculty of Medicine, Memorial University, St. John’s, NL, Canada E-mail: [email protected] Background: Occult HCV infection (OCI), characterized by small amounts of HCV RNA in sera and replicating viral genomes in PBMC and liver, has been documented in individuals with clinically resolved hepatitis C and in those with elevated aminotransferases of unknown etiology. However, data seemingly opposing the existence of OCI have also been reported. This study identifies factors potentially reconciling these differences. Methods: Plasma and PBMC were collected from 84 patients with resolved hepatitis C (SVR duration 6−72 months). Sequential plasma and PBMC, collected at 6−12 month intervals, were obtained from 47/84 persons. Liver biopsies were available from 6. PBMC were left untreated or treated with mitogens before analysis. RNA was extracted using Trizol (Invitrogen) or, for comparative purposes, RNeasy kit (Qiagen) from 250 mL to 4 mL plasma, 1 x 107 PBMC or ~50 mg liver tissue. HCV RNA positive strand was evaluated by RT-PCR/NAH (sensitivity, 10 virus copies/mL; i.e., 5-fold higher than that of Bayer TMA), while the replicative strand by Tth-based RT-PCR/NAH (sensitivity, ~25 virus copies/rxn). HCV variants in PBMC were compared to those in plasma and liver by clonal sequencing. HCV NS5A protein was detected by confocal microscopy. Results: RNA recovery after Trizol extraction consistently superseded that from the RNeasy kit by 2−3 folds. HCV RNA was detectable in 48/84 (57%) cases at 250 mL plasma, although using up to 4 mL increased virus detection to 71%. HCV RNA in unstimulated PBMC was identifiable in 21% (18/84) cases, but mitogen stimulation led to HCV RNA detection in 67% of cells which had been negative when unstimulated. Three of 6 livers were HCV genome positive. Testing of sequential plasma samples increased HCV RNA detection by 10−15%. Virus negative strand was detectable in ~65% of PBMC and livers reactive for the positive strand. HCV NS5A was displayed by 0.02−0.1% of immune cells. Certain HCV variants occurred only in immune cells. Conclusions: The basis of reported differences in OCI occurrence is multifactoral. Variations in the type and number of samples analyzed, sample preparation, RNA extraction method, template amount, and assay sensitivity significantly influence the overall detection of low-level HCV.