HCV clearance patterns in saliva and serum of patients with chronic HCV infection under interferon plus ribavirin therapy

This is the peer reviewed version of the article which has been published in final form at Wiley Online Library. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for self-archiving[Abstract] Statements of the problem:  Hepatitis C virus (HCV)-RNA is often present in saliva of HCV-infected patients, with plasma viral load being the only known predictable factor. Interferon plus ribavirin therapy yields a sustained reduction in HCV viremia. This study aimed to assess the presence of HCV in saliva and serum specimens from patients undergoing this combination therapy (CT). Method of study:  Paired serum and saliva specimens were collected from 44 chronic HCV-infected patients at basal time, 4 and 12 weeks after CT onset, at the end of treatment and 6 months latter. Serum HCV-RNA levels were determined by the polymerase chain reaction (PCR) Amplicor system. Presence of HCV-RNA in saliva was tested by a highly sensitive non-commercialized nested-PCR. Results:  The HCV-RNA was detected in 26 saliva specimens at basal time (59.1%). In 34.1% of cases, a concordance viral clearance pattern in serum and saliva was observed in both responders (pattern 1a) and non-responders (pattern 1b). In pattern 2 (13.6% of cases), HCV was detected longer during CT in serum than in saliva (pattern 2a) or in saliva than in serum (pattern 2b). In 11.3% of patients, viral clearance was corroborated either in their serum (pattern 3a) or in their saliva (pattern 3b), but not in both fluids. Of the eight primary responders with 1a clearance pattern, seven were sustained responders. None of the patients with 2a clearance pattern was a sustained responder. Of the two primary responders showing the 3b salivary pattern, one had already relapsed in the first 6 months of follow up. Conclusions:  The present results suggest that the monitoring of salivary levels of HCV would be a helpful means of determining sustained antiviral effects of interferon and ribavirin in the treatment of HCV disease

Hepatitis C virus transmission during organ transplantation

Background . There is a high prevalence of liver disease among the recipients of organs from donors with antibodies to hepatitis C virus (HCV). We undertook a study to determine the frequency of persistent HCV infection, as indicated by the presence of HCV RNA, among both cadaveric organ donors positive for antibodies to HCV (anti-HCV) and the recipients of organs from these donors. Methods . Serum samples from donors and recipients were tested for HCV RNA with the reverse transcriptase polymerase chain reaction, with use of primers from the 5′ untranslated region of the HCV genome, and for anti-HCV with the first-generation enzyme-linked immunosorbent assay (ELISA) and two second-generation tests. Results . HCV RNA was detected in 9 of 11 organ donors (82 percent) with a positive first-generation ELISA for anti-HCV. Among the organ recipients, the prevalence of HCV RNA increased after transplantation: 7 of 26 patients (27 percent) had positive samples before transplantation, as compared with 23 of 24 patients (96 percent) after transplantation (P < 0.001). Among 13 recipients who were HCV RNA–negative before receiving organs from the nine HCV RNA–positive donors, HCV infection was detected in all 13 after transplantation, and anti-HCV developed in 8 (62 percent). On the basis of a positive test for HCV RNA, the maximal sensitivity of the three anti-HCV tests was 57 percent (positive in 4 of 7 patients with end-stage organ failure) before transplantation and 70 percent (positive in 16 of 23 patients) after transplantation. Conclusions . Nearly all the recipients of organs from anti-HCV–positive donors become infected with HCV. The current tests for anti-HCV antibodies underestimate the incidence of transmission and the prevalence of HCV infection among immunosuppressed organ recipients. Objective: To determine the prevalence of antibodies to hepatitis C virus (anti-HCV) and HCV RNA among cadaver organ donors and to correlate these results with donor liver histologic abnormalities and evidence for transmission of disease through organ transplantation. Design: Retrospective testing of stored serum samples from cadaver organ donors for anti-HCV and HCV RNA. Setting: Transplantation service of the University of Miami/Jackson Memorial Medical Center and other cooperative medical centers furnishing follow-up data. Subjects: Of 1096 cadaver organ donors harvested between 1 January 1979 and 28 February 1991, 484 had stored serum samples available for analysis. Recipients of organs from recombinant immunoblot assay (RIBA)–positive donors for whom adequate follow-up was available were also included in the analysis. Measurements: Samples were tested for anti-HCV by enzyme-linked immunosorbent assay (ELISA). Confirmatory testing was done using a second-generation RIBA. Hepatitis C viral RNA was detected in serum using the polymerase chain reaction. Liver biopsies were obtained from the organ donor and interpreted blindly by a pathologist unaware of the clinical data. Liver chemistry profiles and serum sample analysis for HCV RNA were done for transplant recipients. Results: From the 484 cadaver organ donors, 89 samples (18%; 95% CI, 15% to 21%) were reactive by ELISA. Of these, 33 (6.8%; CI, 4.6% to 9%) were RIBA seropositive. Hepatitis C viral RNA sequences were detected in 50% of the RIBA-positive serum samples tested. Liver tissue was available from 24 of the 33 RIBA-positive donors and showed chronic active hepatitis in 16, chronic persistent hepatitis in 2, and no abnormality in 6. Among the 46 recipients of a kidney from a RIBA-positive donor, 13 (28%; CI, 15% to 41%) developed post-transplant liver disease, of which only 4 cases were highly suggestive of viral transmission from the donor. Little morbidity and no mortality could be attributed to liver disease in this cohort of recipients. Conclusions: These data suggest that HCV transmission by organ transplantation is low and that the consequences of infection are small. If the medical condition of the potential recipient is so serious that other options no longer exist, the use of an organ from an anti-HCV-seropositive donor should be considered.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38389/1/1840170127_ftp.pd

Genetic Diversity of Near Genome-Wide Hepatitis C Virus Sequences during Chronic Infection: Evidence for Protein Structural Conservation Over Time

Infection with hepatitis C virus (HCV) is one of the leading causes of chronic hepatitis, liver cirrhosis and end-stage liver disease worldwide. The genetics of HCV infection in humans and the disease course of chronic hepatitis C are both remarkably variable. Although the response to interferon treatment is largely dependent on HCV genotypes, whether or not a relationship exists between HCV genome variability and clinical course of hepatitis C disease still remains unknown. To more thoroughly understand HCV genome evolution over time in association with disease course, near genome-wide HCV genomes present in 9 chronically infected participants over 83 total study years were sequenced. Overall, within HCV genomes, the number of synonymous substitutions per synonymous site (dS) significantly exceeded the number of non-synonymous substitutions per site (dN). Although both dS and dN significantly increased with duration of chronic infection, there was a highly significant decrease in dN/dS ratio in HCV genomes over time. These results indicate that purifying selection acted to conserve viral protein structure despite persistence of high level of nucleotide mutagenesis inherent to HCV replication. Based on liver biopsy fibrosis scores, HCV genomes from participants with advanced fibrosis had significantly greater dS values and lower dN/dS ratios compared to participants with mild liver disease. Over time, viral genomes from participants with mild disease had significantly greater annual changes in dN, along with higher dN/dS ratios, compared to participants with advanced fibrosis. Yearly amino acid variations in the HCV p7, NS2, NS3 and NS5B genes were all significantly lower in participants with severe versus mild disease, suggesting possible pathogenic importance of protein structural conservation for these viral gene products

Portal Hypertensive Gastropathy in Chronic Hepatitis C Patients with Bridging Fibrosis and Compensated Cirrhosis: Results from the HALT-C Trial

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72863/1/j.1572-0241.2006.00461.x.pd

Variants in interferon-alpha pathway genes and response to pegylated interferon-Alpha2a plus ribavirin for treatment of chronic hepatitis C virus infection in the hepatitis C antiviral long-term treatment against cirrhosis trial

Combination treatment with pegylated-interferon-alpha (PEG IFN-Α) and ribavirin, the current recommended therapy for chronic hepatitis C virus (HCV) infection, results in a sustained virological response (SVR) in only about half of patients. Because genes involved in the interferon-alpha pathway may affect antiviral responses, we analyzed the relationship between variants in these genes and SVR among participants in the Hepatitis C Antiviral Long-Term treatment Against Cirrhosis (HALT-C) trial. Patients had advanced chronic hepatitis C that had previously failed to respond to interferon-based treatment. Participants were treated with peginterferon-Α2a and ribavirin during the trial. Subjects with undetectable HCV RNA at week 72 were considered to have had an SVR. Subjects with detectable HCV RNA at week 20 were considered nonresponders. We used TaqMan assays to genotype 56 polymorphisms found in 13 genes in the interferon-alpha pathway. This analysis compares genotypes for participants with an SVR to nonresponders. The primary analysis was restricted to European American participants because a priori statistical power was low among the small number (n = 131) of African American patients. We used logistic regression to control the effect of other variables that are associated with treatment response. Among 581 European American patients, SVR was associated with IFNAR1 IVS1-22G (adjusted odds ratio, 0.57; P = 0.02); IFNAR2 Ex2-33C (adjusted odds ratio, 2.09; P = 0.02); JAK1 IVS22+112T (adjusted odds ratio, 1.66; P = 0.04); and ADAR Ex9+14A (adjusted odds ratio, 1.67; P = 0.03). For the TYK2 -2256A promoter region variant, a borderline association was present among European American participants (OR, 1.51; P = 0.05) and a strong relationship among African American patients; all 10 with SVR who were genotyped for TYK2 -2256 carried the A variant compared with 68 of 120 (57%) nonresponders ( P = 0.006). Conclusion: Genetic polymorphisms in the interferon-Α pathway may affect responses to antiviral therapy of chronic hepatitis C. (H EPATOLOGY 2009.)Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63061/1/22877_ftp.pd

A novel duplex real-time reverse transcriptase-polymerase chain reaction assay for the detection of hepatitis C viral RNA with armored RNA as internal control

<p>Abstract</p> <p>Background</p> <p>The hepatitis C virus (HCV) genome is extremely heterogeneous. Several HCV infections can not be detected using currently available commercial assays, probably because of mismatches between the template and primers/probes. By aligning the HCV sequences, we developed a duplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay using 2 sets of primers/probes and a specific armored RNA as internal control. The 2 detection probes were labelled with the same fluorophore, namely, 6-carboxyfluorescein (FAM), at the 5' end; these probes could mutually combine, improving the power of the test.</p> <p>Results</p> <p>The limit of detection of the duplex primer/probe assay was 38.99 IU/ml. The sensitivity of the assay improved significantly, while the specificity was not affected. All HCV genotypes in the HCV RNA Genotype Panel for Nucleic Acid Amplification Techniques could be detected. In the testing of 109 serum samples, the performance of the duplex real-time RT-PCR assay was identical to that of the COBAS AmpliPrep (CAP)/COBAS TaqMan (CTM) assay and superior to 2 commercial HCV assay kits.</p> <p>Conclusions</p> <p>The duplex real-time RT-PCR assay is an efficient and effective viral assay. It is comparable with the CAP/CTM assay with regard to the power of the test and is appropriate for blood-donor screening and laboratory diagnosis of HCV infection.</p

Novel Evolved Immunoglobulin (Ig)-Binding Molecules Enhance the Detection of IgM against Hepatitis C Virus

Detection of specific antibodies against hepatitis C virus (HCV) is the most widely available test for viral diagnosis and monitoring of HCV infections. However, narrowing the serologic window of anti-HCV detection by enhancing anti-HCV IgM detection has remained to be a problem. Herein, we used LD5, a novel evolved immunoglobulin-binding molecule (NEIBM) with a high affinity for IgM, to develop a new anti-HCV enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase-labeled LD5 (HRP-LD5) as the conjugated enzyme complex. The HRP-LD5 assay showed detection efficacy that is comparable with two kinds of domestic diagnostic kits and the Abbott 3.0 kit when tested against the national reference panel. Moreover, the HRP-LD5 assay showed a higher detection rate (55.9%, 95% confidence intervals (95% CI) 0.489, 0.629) than that of a domestic diagnostic ELISA kit (Chang Zheng) (53.3%, 95% CI 0.463, 0.603) in 195 hemodialysis patient serum samples. Five serum samples that were positive using the HRP-LD5 assay and negative with the conventional anti-HCV diagnostic ELISA kits were all positive for HCV RNA, and 4 of them had detectable antibodies when tested with the established anti-HCV IgM assay. An IgM confirmation study revealed the IgM reaction nature of these five serum samples. These results demonstrate that HRP-LD5 improved anti-HCV detection by enhancing the detection of anti-HCV IgM, which may have potential value for the early diagnosis and screening of hepatitis C and other infectious diseases