Clinical evaluation of a single reaction, diagnostic polymerase chain reaction assay for the detection of hepatitis C virus RNA

doi:10.1016/S0168-8278(96)80183-8

Journal of Hepatology

Volume 24, Issue 1, 1 January 1996, Pages 33-37

https://doi.org/10.1016/S0168-8278(96)80183-8 Get rights and content

Abstract

Backgrounds/Aims:In the past few years the detection of HCV-RNA by polymerase chain reaction has become a well-established diagnostic tool for patients with chronic hepatitis C. However, the lack of reproducible results between laboratories and the relatively high proportion of false-positive results, has indicated the need for a standardized and reliable polymerase chain reaction assay. In the present study we have analyzed the performance of a commercial, HCV-RNA polymerase chain reaction assay based on a single, combined reverse transcription and amplification reaction and on the use of Uracil-N-glycosilase to prevent carry-over contamination (Amplicor HCV, Roche Molecular Systems).

Methods:In this assay the amplification products are detected in microwell plates using biotinylated primer and the HRP avidin colorimetric system. Serum samples collected from 446 patients, including 181 with chronic active hepatitis C, 50 with autoimmune chronic hepatitis, 117 in hemodialysis, 30 asymptomatic carriers of anti-HCV and 68 with indeterminate serology (RIBA indeterminate results), as well as from 121 controls were tested with the commercial, single-step assay and with nested polymerase chain reaction. Both techniques use primers located within the 5′ non-coding region of the HCV genome.

Results:In all cases a good concordance was observed between the commercial, single-step assay and nested polymerase chain reaction which, for patients with chronic active hepatitis, showed a sensitivity and specificity of 100% and 99.3% for the former and of 98.8% and 100% for the latter, when compared to clinical diagnosis taken as the gold standard. Most of the 11 discordant samples were seen in the group of RIBA-indeterminate cases and in patients with chronic active hepatitis C. Further analysis of these cases, based on repeat testing and clinical data showed that 64% and 36% of the discrepancies were due, respectively, to nested polymerase chain reaction and Amplicor inconsistent reactions. In hemodialyzed patients, patients with autoimmune hepatitis and asymptomatic carriers of anti-HCV, both assays produced results which were consistent with the clinical diagnois. In the former group, polymerase chain reaction was able to identify the presence of active viral replication in some antibody negative samples.

Conclusions:Taken together, these results indicate that the commercial, single-step polymerase chain reaction assay has the same clinical sensitivity and specificity as nested polymerase chain reaction and that, because of its simplified procedures and fast turn-around time, it may be a valuable test for routine diagnostic applications.

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We evaluated the cost-effectiveness of a hepatitis C (HCV) screening and active linkage to care intervention in US methadone maintenance treatment (MMT) patients using data from a randomized trial conducted in New York City and San Francisco.
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The control strategy resulted in a projected 35% linking to care within 6 months and 31% achieving sustained virologic response (SVR). The intervention resulted in 60% linking and 54% achieving SVR with an ICER of $24,600/QALY compared to no intervention from the healthcare sector perspective and was a more efficient use of resources than the control strategy. The intervention had an ICER of $76,500/QALY compared to the alternative strategy. From a societal perspective, the intervention had a net monetary benefit of $511,000–$975,600.
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We previously reported on a chimpanzee immunized with both putative envelope glycoproteins (E1 and E2) of hepatitis C virus (HCV), strain HCV-N2, and synthetic peptides of hypervariable region 1 (HVR1) of a different isolate, HCV-#6. The chimpanzee showed complete protection against HCV-#6 infection only when the titer of anti-HVR1 increased, suggesting that an immune response to the HVR1 is more essential in protecting a chimpanzee from HCV infection than an immune response to E1 and E2. In this study, we immunized this chimpanzee with only synthetic HVR1 peptides after anti-E1 and anti-E2 antibody levels dropped and then rechallenged with 10 infectious chimpanzee doses of HCV. The immunized animal was protected, and neutralization of HCV with the antiserum from the protected animal was achieved by inoculating another chimpanzee with HCV preneutralized by this antiserum mixture. Epitope analysis of HVR1 by Pin-ELISA using this antiserum seemed to demonstrate that the antibody response was directed mainly against the C terminus of HVR1. Moreover, our results showed that, if a part of the sequences was conserved, a broad cross-reactivity of the antiserum could be observed, even if amino-acid sequences in this epitope were substituted for those of other HCV strains.
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Background/Aims: Hepatitis C virus (HCV) infection becomes chronic in most cases, with only 10–20% of those infected not developing persistent viraemia. The immune response to HCV may be an important determinant of disease resolution and can be influenced by a number of host factors. The aim of this study was to assess the role of host HLA class II type in influencing viral clearance or susceptibility to chronic HCV infection.
Methods: We have compared the distribution of HLA DRB1, DQA1 and DQB1 alleles in 49 patients with spontaneous clearance of HCV infection (HCV antibody positive but persistently HCV RNA negative), with 55 chronically infected patients and 134 racially matched controls.
Results: Three alleles were found significantly more frequently in patients with spontaneous viral clearance compared to those with chronic infection-DRB1*04 (pc=0.0022, odds ratio OR= 4.52), DQA1*03 (pc= 0.0012, OR= 4.69) and DQB1*0301 (pc=0.0078, OR= 5.09). DQB1*0302 was found at reduced frequency in all HCV-antibody-positive patients compared to controls (pc=0.0063).
Conclusions: DRB1*04, DQA1*03 and DQB1*0301 are associated with spontaneous clearance of HCV viraemia, with the primary association likely to be with DQB1*0301 and the associations with DRB1*04 and DQA1*03 being due to linkage. In addition, DQB1*0302 is associated with protection from HCV infection. These findings suggest that host HLA class II genotype is an important factor in determining the outcome of infection with hepatitis C virus.
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Results: HBsAg and anti-HCV were detected in 19% and 40.1% of the patients, respectively. Serum and liver HBV DNA were detected in 82% and 91% of the HBsAg positive subjects. HBV DNA was also detected in the serum and liver of 33% and 47% of HBsAg negative patients. In this group, serum HBV DNA was more prevalent in anti-HBs and/or anti-HBc patients (47.9%), compared to those without any HBV marker (25.1%). HCV RNA was detected in 89% and 7% of anti-HCV positive and negative cases, respectively, HCV 1b being the most prevalent genotype (80%). Coinfection with HBV and HCV was shown in 20.4% of patients, while only 29% had neither HBV nor HCV. GBV-C/HGV RNA was detected in only $457$ sera.
Conclusions: This study offers the first large analysis of HCC in Europe, based on both serology and molecular tests. It demonstrates the major impact of HBV and HCV, but not of GBV-C/HGV, in liver carcinogenesis in Northern as well as Southern Europe. It also stresses the need to use viral genome detection in epidemiological studies when serological tests are negative.
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1997, Journal of Hepatology
Background/Aims: So far, there are no reliable parameters that can predict the long-term sustained response to treatment with interferon-α in patients with chronic hepatitis C. In this study, we have developed a semi-quantitative method to determine the viral load per liver cell and have correlated this factor with the outcome of hepatitis C patients treated with interferon-α.
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Results: Long-term sustained responders had a significantly lower viral load per liver cell (median: 5 vs. 650 copies/1000 liver cells, p-value 0.0001), lower age (median: 32 vs. 54 years, p-value: 0.006) and lower percentage of geno- or serotype 1 (46% vs. 91%). Regarding viral load in serum and peripheral blood mononuclear cells, alanine aminotransferase levels, γ-globulin levels and histological changes, no statistically significant differences were observed.
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Methods: Eighteen selected chronic HCV patients received three doses of 3 or 6 MU interferon-α2a weekly for 6 to 12 months and were followed up for 6 to 60 months. Anti-HCV antibody levels were serially measured either in end-point diluted sera with the Matrix-Assay or with quantitative anti-HC34-IgG and -IgM ELISA. Circulating immune complexes were assessed by flow cytometry and the results were correlated with histology, quantitative HCV-RNA levels and genotypes.
Results: Nine complete responders (CR; genotypes 1a n=4; 1b n=1; 2a n=1; 3a n=3) showing sustained virus elimination and ALT normalisation had low HCV-RNA pretreatment levels (mean 14x10³copies/ml) compared to six nonresponders and three partial responders (NR/PR; genotypes 1a n=2; 1b n=7) who had significantly higher HCV-RNA pretreatment levels (mean 254x10³copies/ml; p<0.01). In untreated NR/PR the HC34 core-antigen was most immunogenic, in CR the NS3-derived HC29-antigen. Pre-treatment levels of anti-HC 34-IgG and -IgM antibody levels in NR/PR were higher than in CR (IgM/IgG p=0.05, n.s.) and these differences became significant during or after therapy (3 months therapy: IgM p<0.02/IgG p<0.07; end of therapy: IgM 0.006/IgG p<0.04; 6 months post-therapy: IgM p<0.002/IgG p<0.004). The PR patients showed recurrent anti-HC34 antibody levels that preceded disease reactivation and detectable HCV-RNA in serum. Immune complex formation increased in some patients during treatment but did not correlate with disease activity, quantitative viraemia, antibody levels or therapy outcome.
Conclusion: Anti-HC34 antibodies, i.e. of the IgM-subtype, correlated quantitatively with viraemia and disease activity. Monitoring the antibody levels may predict the long-term therapy outcome during interferon-α treatment.

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Clinical evaluation of a single reaction, diagnostic polymerase chain reaction assay for the detection of hepatitis C virus RNA

Abstract

J Hepatol

J Hepatol

Lancet

Lancet

Lancet

Adv Clin Chem.

Gene

J Hepatol

Gastroenterelogy

Lancet

Res Virol

Chronic hepatitis C. Advances in diagnostic testing and therapy.

Arch Intern Med

Molecular biology of the hepatitis C virus: implications for diagnosis, development and control of viral disease.

Hepatology

Detection of hepatitis C viral sequences in non-A, non-B hepatitis.

Lancet

Use of aminotransferase, hepatitis C antibodies, and hepatitis C virus polymerase chain reaction RNA assays to establish the diagnosis of hepatitis C infection in diagnostic virology laboratory.

J Clin Microbiol