Detection of HCV-RNA

Qualitative assays (RT-PCR, TMA).
After detection of HCV in 1989 the first test systems to confirm ongoing, replicating hepatitis C were based on RT-PCR. Due to a high conservation of the 5/ nontranslated region (NTR) of the HCV genome throughout the different geno- and subtypes, primers complementary to this region were chosen for reliable diagnostics (Christoph, 2004).
Due to their lower detection limits in comparison with quantitative HCV-RNA assays, qualitative HCV-RNA tests clinically are used for diagnosis of acute hepatitis C in which HCV-RNA concentrations are fluctuating and may be very low and for confirmation of virologic response during, at the end and after antiviral therapy (Christoph, 2004).
By the end of 1993 a first standardized RT-PCR based assay for detection of HCV-RNA was introduced (AmplicorTM HCV, Roche Molecular Systems, Pleasanton, CA, USA). The Amplicor™ HCV system is a combined single tube-, single enzyme-, single primer set RT-PCR assay (Christoph, 2004).
More recently, a second test system based on transcription mediated amplification (TMA) was approved for qualitative detection of HCV-RNA (VersantTM Qualitative HCV, Bayer Diagnostic, Emeryville, CA, USA). HCV-RNA detection by this technique consists of threesteps which are performed in a single tube: (i) target capture; (ii) target amplification and; (iii) specific detectionof target amplicons by hybridization protection assay.
Due to its extreme high sensitivity the TMA-based assay (lower detection 5–10 IU/mL) is able to detectresidual HCV-RNA amounts not observed by standard RT-PCR-based tests (lower detection limit 50 IU/mL). In 7–33% of patients treated with (pegylated) interferon-alfa and ribavirin with negative results by RT-PCR at the end of treatment residual HCV-RNA concentrations were detectable by TMA (Sarrazin et al., 2000; Comanor et al., 2001 and Sarrazin et al., 2001).
Similar results were obtained during therapy at week 24 for decisions of treatment discontinuation in HCV-RNA positive patients. Whether patients with low HCV-RNA levels (TMA positive/RT-PCR negative) will benefit from prolongation of therapy is still unknown (Mihm et al., 2004).
2.2.10.2.2.2. Quantitative assays (RT-PCR, real time RT-PCR bDNA)
Measurement of HCV-RNA concentration in serum/blood is an important parameter for management of chronic hepatitis C. While no correlation was observed between the HCV-RNA viral load and the severity or the progression of liver disease HCV-RNA concentration is an important predictor for response to antiviral therapy. In multiple studies an inverse correlation of lower pretreatment HCV-RNA blood levels with higher rates of sustained virologic response to (pegylated) interferon alfa combination therapy with ribavirin was observed. Furthermore, after initiating interferon-based therapy the initial decline of HCV-RNA levels is used for determination of virologic non-response at week 12 and week 24 of treatment. Patients without an initial decline of HCV-RNA of at least 2 log steps and/or absolute values above 30,000 IU/mL have shown to become virologic non-responders in 98–100% of cases (Poynard et al., 2000; Fried et al., 2002; Berg et al., 2003; Davis et al., 2003 and Mihm et al., 2004).
Thus, according to current recommendations treatment should be discontinued on the basis of this week 12 HCV-RNA decline rule. Highly sensitive HCV-RNA detection assays (lower detection limits ≤50 IU/mL) are used for determination of virologic response at week 24 of therapy. As patients with detectable HCV-RNA at this time point will not achieve a sustained virologic response in 98–100% of cases again treatment can be discontinued at week 24 on the basis of a positive highly sensitive HCV- RNA test (Fig. 4) (Poynard et al., 2000; Fried et al., 2002; Berg et al., 2003; Davis et al., 2003 and Mihm et al., 2004).
Five different quantitative HCV-RNA detection assays are approved by the legal authorities and are commercially available. Three are based on standard RT-PCR (SuperquantTM, National Genetics Institute, Los Angeles, CA, USA; Cobas AmplicorTM HCV Monitor version 2, Roche Molecular Systems, Pleasanton, CA, USA; LCxTM HCV-RNA Quantitative, Abbott Laboratories, North Chicago, IL, USA) and one is based on branched DNA technique (VersantTM Quantitative HCV-RNA, bDNA 3.0, Bayer Diagnostics, Emeryville, CA, USA). Recently, the first real-time PCR based assay for HCV-RNA quantification was approved (COBAS TaqManTM , Roche Molecular Systems, Pleasanton, CA, USA) and in the future additional real-time PCR based HCV quantification assays may follow as this technique has the potential of highly sensitive, linear quantification of RNA and DNA targets (Cristoph, 2004).

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