Serum HCV RNA levels assessed by quantitative NASBA®: stability of viral load over time, and lack of correlation with liver disease

Hollingsworth, Rosalind; Sillekens, Peter; Deursen, Peter van; Neal, Keith; Irving, William L.

doi:10.1016/s0168-8278(96)80115-2

Cited by 64 publications

(54 citation statements)

References 21 publications

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“…26 Several studies have evaluated methods for the quantitation of HCV RNA, 27 such as quantitative RT-PCR 7,28,29 and, more recently, bDNA, 8,9,30 the semiquantitative Amplicor assay, 31 and the NASBA method. 32 Ours is the first comparison of the sensitivity, linearity, and reproducibility of three quantitative HCV-RNA assays based on the EUROHEP panel, which contains dilutions of titrated plasma samples, 11 a titrated (CID/mL) chimp serially diluted plasma pool, samples from anti-HCV-positive patients, and samples from patients with chronic HCV infection treated with IFN, tested before, during, and after therapy.…”

Section: Discussionmentioning

confidence: 99%

Comparative evaluation of hepatitis C virus RNA quantitation by branched DNA, NASBA, and monitor assays

et al. 1999

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Since its discovery in 1989, 1 it has been demonstrated that hepatitis C virus (HCV) is a major cause of chronic hepatitis throughout the world. The routine virological diagnosis is based on serological tests detecting antibodies to HCV. However, in the absence of an efficient culture system for HCV, or viral antigen assays, direct detection of HCV is based on nucleic acid amplification and hybridization methods. HCV RNA was principally detected with in-house nested polymerase chain reaction (PCR) methods, but new standardized assays such as Amplicor (Roche Diagnostics Systems, Basel, Switzerland) and nucleic acid sequence-based amplification (NASBA) (Organon Teknika, Boxtel, the Netherlands) have been developed to detect HCV RNA on a routine basis 2,3 or for research purposes. 4,5 Several studies have shown a relation between pretreatment viral load and the response to interferon (IFN) therapy, 6-10 creating a need for standardized HCV-RNA quantification assays. Currently, the most frequently used kit is the branched DNA (bDNA) assay (Quantiplex HCV RNA, Chiron Diagnostics, Emeryville, CA), a well-standardized signalamplification method. Other assays are based on HCV-RNA amplification (Amplicor HCV Monitor, Roche Diagnostics Systems, and NASBA HCV). The aim of this study was to compare the NASBA, bDNA, and Monitor HCV assays in terms of reproducibility, sensitivity, and linearity in various clinical settings and using standardized panels. Two qualitative methods (in-house nested PCR and Amplicor) were used as standards for sensitivity. Specificity was not a focus of this study, because specificity studies were performed by the manufacturers during the test registration procedure, and because we routinely obtain very good specificity in our lab. Moreover, negative samples were included in each series of tests (anti-HCV-and PCR-negative blood donors). MATERIALS AND METHODS Pelicheck HCV-RNA EUROHEP PanelThis panel was used to evaluate the sensitivity and linearity of each assay. It contains two dilution panels of the EUROHEP genotype 1 and 3 plasma standards. The plasma standards have been characterized in two collaborative EUROHEP studies. 11,12 The EUROHEP HCV-RNA genotype 1b and 3a plasma standard dilutions were prepared from a pool of plasma units negative for hepatitis B surface antigen, anti-hepatitis B core antibodies (Ab), anti-HCV Ab, anti-human immunodeficiency virus-1 or -2 Ab, anti-human T-lymphotropic virus type 1 Ab, syphilis, HCV RNA, hepatitis B virus DNA, and human immunodeficiency virus RNA. The undiluted plasma samples are not available. According to the EUROHEP group, the undiluted plasma corresponding to the genotype 1 panel should contain 38 Ϯ 8 ϫ 10 5 genome equivalents per milliliter (eq/mL) in the bDNA 2.0 assay, and 2.8 ϫ 10 5 copies per milliliter (cps/mL) in the HCV RNA Monitor assay. The potency of the

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Section: Discussionmentioning

confidence: 99%

Comparative evaluation of hepatitis C virus RNA quantitation by branched DNA, NASBA, and monitor assays

et al. 1999

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“…Either trivial or consistent differences of viral load over time have been described after observation periods ranging from days to months. [8][9][10][11][12] To assess the spontaneous behavior of serum HCV-RNA levels over a reasonable length of time upon close observation, we monitored chronic HCV carriers who had been followed longitudinally without receiving any antiviral therapy and then analyzed their patterns of virus replication in relation to biochemical and histological activity of liver disease. …”

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confidence: 99%

Hepatitis C virus RNA profiles in chronically infected individuals: Do they relate to disease activity?

et al. 1999

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Fluctuations of hepatitis C virus (HCV)-RNA serum levels were monitored in a multicenter study in 76 chronic HCV carriers who had been followed longitudinally without receiving antiviral therapy to assess their relation with the course of liver disease activity. Forty-four patients had normal transaminases over more than 2 years, while 32 additional patients had fluctuating levels. Viral load was measured in serial serum samples prospectively collected for 10 to 12 months in 54 patients and in sera stored yearly up to 8 years in an additional 22 patients. In patients tested monthly, a lesser extent of fluctuations was detected in cases with constantly normal transaminases as compared with those with fluctuating transaminases. In the former group, the mean difference between maximum and minimum values observed in each individual patient was 0.7 Log, while in the latter group, it was 1.3 Log (P ‫؍‬ .0004). Most of these patients experienced, on average, three peaks of viremia over 1 year. The range of variation observed upon yearly testing was between 0.2 and 2.2 Log and did not reach statistical significance between the two groups. In conclusion, a careful viral replication profile can be achieved only by monthly testing, because longer time intervals could miss viremia fluctuations. HCV-RNA levels are more stable in asymptomatic HCV carriers than in patients with biochemical activity of liver disease. (HEPATOLOGY 1999;29: 585-589.)Chronic infection with hepatitis C virus (HCV) is characterized by persistent viremia. HCV RNA is usually detected in serum by sensitive polymerase chain reaction (PCR)-based techniques and has become a useful tool for diagnosis and monitoring. Besides methods for qualitative detection of viremia, a number of procedures to quantify serum HCV RNA have been developed, including end-point dilution PCR, 1 competitive PCR, 2,3 isothermal nucleic acid amplification, 4 and signal-amplification branched DNA. 5 Routine use of these techniques in a wide clinical setting is hampered by problems of specificity and sensitivity, lack of reproducibility, poor standardization, and high cost. 6 Recently, methods for quantitative assessment of HCV-RNA levels have become commercially available and are being extensively evaluated in clinical studies, particularly in patients treated with interferons and antivirals. 7 Little is known about viral kinetics in untreated patients, because most evaluations of the level of viremia are based on single determinations. If wide spontaneous fluctuations occur, they could mimic treatment-induced effects or lead to under-or overestimate baseline values. In a few published studies, different time intervals have been evaluated and discordant conclusions have been reported. Either trivial or consistent differences of viral load over time have been described after observation periods ranging from days to months. [8][9][10][11][12] To assess the spontaneous behavior of serum HCV-RNA levels over a reasonable length of time upon close observation, we monitored chronic HCV car...

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“…Whereas all other quantification methods are based on the assumption that the amplification efficiencies are identical between two individual reactions, the internal standard approach is not dependent on this. The method has been applied successfully for PCR (3), RT-PCR (4,5) and NASBA (6,7).…”

Section: Introductionmentioning

confidence: 99%

“…NASBA has been applied for the detection of viral genomes (6,7,10), viroids (11), rRNAs (12) and mRNAs (13)(14)(15)(16)(17). The NASBA reaction mixture contains oligonucleotide primers and three enzymes: AMV-RT, RNase H and T7 RNA polymerase for target specific amplification.…”

Section: Introductionmentioning

confidence: 99%

“…This makes the method useful for specific mRNA detection in a background of genomic DNA even for intronless genes (13). NASBA proved to be a successful tool in the detection of mRNAs (13)(14)(15)(16)(17) and in the quantification of viral RNAs in combination with the internal standard approach (6,7). Combining these two applications makes NASBA a potentially very suitable approach for the quantification of mRNAs in DNA backgrounds.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A novel quantitative multiplex NASBA method: application to measuring tissue factor and CD14 mRNA levels in human monocytes

Deursen¹,

Gunther²,

Riel

et al. 1999

Nucleic Acids Research

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A new method to quantify two individual mRNAs in a single NASBA reaction is described. In this study, tissue factor and CD14 mRNAs were used as a model system. RNA ratios of -4 to +4 log units were determined with good precision (within 0.3 log) and accuracy (within 0.2 log). By measuring both mRNAs in human monocytes that were stimulated with LPS, the multiplex Q-NASBA proved to be a successful tool to monitor the expression levels of two individual mRNAs in a single-tube amplification system. The method has potential in all fields in which quantitative information is needed on two individual RNAs.

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Serum HCV RNA levels assessed by quantitative NASBA®: stability of viral load over time, and lack of correlation with liver disease

Cited by 64 publications

References 21 publications

Comparative evaluation of hepatitis C virus RNA quantitation by branched DNA, NASBA, and monitor assays

Comparative evaluation of hepatitis C virus RNA quantitation by branched DNA, NASBA, and monitor assays

Hepatitis C virus RNA profiles in chronically infected individuals: Do they relate to disease activity?

A novel quantitative multiplex NASBA method: application to measuring tissue factor and CD14 mRNA levels in human monocytes

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