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

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