Direct detection of hepatitis C virus (HCV) RNA from whole blood, and comparison with HCV RNA in plasma and peripheral blood mononuclear cells

Schmidt, Warren N.; Klinzman, Donna; LaBrecque, Douglas R.; Macfarlane, Donald E.; Stapleton, Jack T.

doi:10.1002/jmv.1890470208

Cited by 46 publications

(63 citation statements)

References 21 publications

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“…12 Our data demonstrating increased analytical and clinical sensitivity of HCV RNA detection in plasma using the ultracolumn cationic detergent modification are not in agreement with previously published data suggesting that treatment of plasma with catrimox-14 does not enhance the clinical sensitivity of HCV RNA detection. 13 One difference between the two studies is the method of HCV RNA detection.…”

Section: Discussioncontrasting

confidence: 91%

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Increased Sensitivity of the Roche COBAS AMPLICOR HCV Test, Version 2.0, Using Modified Extraction Techniques

Forman

Valsamakis

2004

The Journal of Molecular Diagnostics

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Processing modifications were made to the COBAS AMPLICOR HCV version 2.0 assay to enhance sensitivity. Two methods of specimen concentration, centrifugation ("ultraspin") and cationic detergent plus silica membrane ("ultracolumn"), were compared to the standard method. The effect of these changes on assay sensitivity and specificity was examined using commercial hepatitis C virus (HCV) preparations. The limits of detection (LOD, defined as detection of HCV RNA in > 95% of replicates) of genotype 1a were 50, 12, and 6 by standard method, ultraspin and ultracolumn, respectively. For genotype 1b, the LOD was 25 IU/ml, 12 IU/ml, and 3 IU/ml; for 2b, it was 50, 12, and 3; for 3a, it was 25, 12, and 1.5; for 4 it was 18, 4, and 2; for 5a, it was 38, 9, and 2; and for 6a it was 47, 6, and 3. No false positives were detected after ultraspin when controls containing high or low HCV concentrations were alternated with normal human plasma. Plasmas in which HCV RNA was not detected by the standard assay were re-tested with modified methods to assess the effect of altered processing in clinical specimens. Three of 152 specimens with no detectable HCV RNA by the standard method were positive by ultraspin and 2 of 109 were positive by ultracolumn, suggesting that these methods may increase assay sensitivity in clinical specimens. Accurate, sensitive detection of hepatitis C virus (HCV) in serum or plasma is essential for diagnosis of HCV infections and assessing response to anti-HCV therapy. The standard, FDA approved COBAS AMPLICOR HCV version 2.0 assay has a reported LOD of 50 IU/ml for HCV genotype 1b. 1 This assay detects HCV RNA during active HCV infection. 2 It has been used to diagnose viremia 3,4 and to assess treatment response. 5 Therapy for chronic HCV infection has evolved from interferon alfa (IFN-alfa) alone, to second generation combination therapy with IFN-alfa plus ribavirin for either 24 or 48 weeks, to the latest generation of pegylatedinterferon ␣ with ribavirin (pegIFN-alfa/riba) for either 24 or 48 weeks. The lowest rates of response and highest rates of relapse were observed with IFN-␣ monotherapy. 6 Treatment with pegIFN-␣/riba resulted in the highest rates of response and the lowest rates of relapse. 5,7 Variables that potentially affect rates of virologic relapse include treatment regimen, genetic variability of the virus, and HCV RNA detection assay sensitivity. The ability to detect very low levels of virus may prove useful in identifying patients who may relapse at end of treatment. Transcription-mediated amplification (TMA) has been used to assess the presence of residual HCV RNA in plasma from patients treated with pegINF-alfa-2a and INF-alfa-2a. 8 In patients who relapsed after therapy, residual HCV RNA was detected in 7% of plasma samples at end of treatment with pegINF-alfa-2a and 33% of samples following therapy with INF-alfa-2a.The aim of this study was to increase the sensitivity of COBAS AMPLICOR HCV version 2.0 through alteration of extraction methods. Two modifications were used to extra...

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Section: Discussioncontrasting

confidence: 91%

“…To test whether this was true, three dilutions (12,6, and 3 IU/ml) of an HCV 1b panel member were centrifuged and supernatants (800 l) were extracted using the ultracolumn method. Ultraspin pellets and ultracolumn eluates were then tested by COBAS AMPLICOR HCV version 2.0.…”

Section: Lod Studiesmentioning

confidence: 99%

Increased Sensitivity of the Roche COBAS AMPLICOR HCV Test, Version 2.0, Using Modified Extraction Techniques

Forman

Valsamakis

2004

The Journal of Molecular Diagnostics

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“…Samples of WB (200 µL), freshly prepared Pl (200 µL), and PBMCs (1 ϫ 10 6 cells) were pipetted into 1.0 mL of Catrimox for RNA extraction as previously described. 25 PBMCs were prepared by Ficoll-Hypaque from aliquots of WB and washed three times with balanced salt solution before use. Final washes of PBMCs from HCV-positive patients were previously shown to be negative for HCV RNA sequences.…”

Section: Methodsmentioning

confidence: 99%

“…Final washes of PBMCs from HCV-positive patients were previously shown to be negative for HCV RNA sequences. 25 After purification of RNA, nested RT-PCR was performed using primers from the highly conserved HCV 5Ј-nontranslated region. 25 Positive and negative HCV RNA control reactions were run with each batch of samples.…”

Section: Methodsmentioning

confidence: 99%

“…25 After purification of RNA, nested RT-PCR was performed using primers from the highly conserved HCV 5Ј-nontranslated region. 25 Positive and negative HCV RNA control reactions were run with each batch of samples. DNA products (250 base pairs) were analyzed on 1.6% agarose gels using a 1-kilobase ladder as standard and stained with ethidium bromide.…”

Section: Methodsmentioning

confidence: 99%

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Effect of interferon therapy on hepatitis C virus RNA in whole blood, plasma, and peripheral blood mononuclear cells

Schmidt¹,

Wu²,

Brashear³

et al. 1998

Hepatology

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Fifty-two patients with chronic hepatitis C virus (HCV)infection were treated with standard doses of interferon alfa-2b. During treatment, HCV RNA detection was studied in samples of whole blood (WB), plasma (Pl), and peripheral blood mononuclear cells (PBMCs). Individuals were classified as sustained responders (SRs), complete responders with relapse (CRs), partial responders (PRs), or nonresponders (NRs) according to normalization of serum alanine transaminase (ALT) during treatment and follow-up. Before treatment, 100% of WB samples and more than 95% of Pl and PBMC samples were positive for HCV RNA. During treatment, there was progressive clearance of HCV RNA from Pl and PBMCs in SRs and CRs, but CRs had significantly more positive WB samples during and following treatment (P Ͻ.0001). At 6 months, only 10% of CR patients were positive by Pl assay, but 50% were positive by WB assay (P Ͻ.01). In the PR group, all WB samples remained positive throughout treatment, although 25% to 40% of PBMC and Pl samples became negative for HCV RNA during the first 2 months of therapy (WB Ͼ Pl or PBMC; P Ͻ .001). However, at later times during treatment most Pl and PBMC samples in the PR group were positive. Samples from the NR group showed no clearance of HCV RNA from WB, Pl, or PBMC fractions. These data document the increased sensitivity of WB assays for detecting HCV RNA in the peripheral blood of patients during interferon therapy. Furthermore, our findings suggest that WB analysis of HCV RNA may be a useful parameter to monitor in determining the end point of interferon therapy.(HEPATOL-OGY 1998;28:1110-1116.)Hepatitis C virus (HCV) is a small, enveloped RNA virus of approximately 9,500 nucleotides that is responsible for more than 90% of parenterally transmitted cases of non-A, non-B hepatitis. 1-3 Infection with HCV usually results in chronic active hepatitis, which may progress to cirrhosis. Currently, end-stage liver disease resulting from chronic HCV infection is a major reason for referral of patients for orthotopic liver transplantation in the United States. 4,5 Interferon therapy is the only approved treatment for chronic hepatitis C. Approximately 50% of patients who are treated respond with normalization of serum aminotransferase levels, although more than half of these responders will relapse after discontinuation of the drug. Thus, approximately 20% of treated patients have a durable, sustained response. 6,7 Although the reasons responsible for relapse are unclear, variables such as length of treatment, HCV genotype, and histological evidence of inflammation and fibrosis at the start of therapy appear to correlate with treatment outcomes. 3,[8][9][10][11] Reverse-transcription (RT) polymerase chain reaction (PCR) techniques to detect HCV RNA during interferon therapy have been widely employed and are considered to be important for optimum management. 12,13 A high level of HCV RNA in serum before treatment is a poor prognostic sign for sustained response; unfortunately, clearance of virus from serum or plasma ...

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Size‐ and Distance‐Dependent Nanoparticle Surface‐Energy Transfer (NSET) Method for Selective Sensing of Hepatitis C Virus RNA

Griffin

Singh

Senapati

et al. 2008

Chemistry A European J

195

255

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We report size- and distance-dependent surface-energy transfer (SET) properties of gold nanoparticles for recognizing hepatitis C virus (HCV) RNA sequence sensitively and selectively (single-base mutations) in a homogeneous format. We have demonstrated that quenching efficiency increases by three orders of magnitude, as the particle size increases from 5 to 70 nm. Due to this extraordinarily high K(SV), nanoparticle SET (NSET) detection limit can be as low as 300 fM concentration of RNA, depending on the size of gold nanoparticle. We have shown that the distance-dependent quenching efficiency is highly dependent on the particle size and the distance at which the energy-transfer efficiency is 50 %, ranges all the way from 8 nm, which is very close to the accessible distance of conventional Förster resonance energy transfer (FRET), to about 40 nm by choosing gold nanoparticles of different diameters. Our result points out that dipole-to-metal-particle energy transfer and NSET models provide a better description of the distance dependence of the quenching efficiencies for 8 nm gold nanoparticle, but agreement is poor for 40 and 70 nm gold nanoparticles, for which the measured values were always larger than the predicted ones.

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Direct detection of hepatitis C virus (HCV) RNA from whole blood, and comparison with HCV RNA in plasma and peripheral blood mononuclear cells

Cited by 46 publications

References 21 publications

Increased Sensitivity of the Roche COBAS AMPLICOR HCV Test, Version 2.0, Using Modified Extraction Techniques

Increased Sensitivity of the Roche COBAS AMPLICOR HCV Test, Version 2.0, Using Modified Extraction Techniques

Effect of interferon therapy on hepatitis C virus RNA in whole blood, plasma, and peripheral blood mononuclear cells

Size‐ and Distance‐Dependent Nanoparticle Surface‐Energy Transfer (NSET) Method for Selective Sensing of Hepatitis C Virus RNA

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