MM=molecular method; IM=immunological method; IU=WHO international HCV RNA standard; DL = detection limit. aTest only available through the National Genetics Institute. bA total of 5.2 HCV RNA copies/IU.

MM=molecular method; IM=immunological method; IU=WHO international HCV RNA standard; DL = detection limit. aTest only available through the National Genetics Institute. bA total of 5.2 HCV RNA copies/IU.

products (Amplicor HCV Monitor 2.0) have been semi-automated with the Cobas Amplicor (Roche Diagnostic Systems).[8'9] AmpErase® (uracil-N-glycosylase, or UNG) is used, which destroys dUTP-containing ampli-cons from previous amplifications and provides safety from contamination of amplicons of previously amplified samples. In contrast to signal amplification techniques, the amplified target is quantified.

The Superquant Assay (NGI)

The HCV Superquant assay (NGI), which is based on a multicycle RT-PCR method with an internal control, followed by Southern blot detection for the quantification of HCV RNA, shows a relatively large dynamic range from approximately 40 x 101 to 2.0 x 106 IU/mL. A cDNA is produced from extracted HCV RNA. This cDNA is then amplified in four separate PCRs with inclining cycles. The PCR products are visualized by Southern blotting, and subsequent quantification is accomplished by densitome-try, comparing the product band intensity of the wild-type HCV to the internal control product band intensity.[10] The Superquant assay utilizes robotic instruments for agarose gel electrophoresis, vacuum transfer Southern blot hybridization, and immunostaining.[11]

The NASBA-QT Test (Organon Teknika)

Like RT-PCR, in the NASBA-QT test (Organon Teknika), the target nucleic acid is first amplified and then the amplification products are quantified in comparison with three different synthetic internal standard RNA calibrators.[12] This isothermal amplification technique uses RNA, not DNA. Instead of a precipitation of RNA by alcohol used in RT-PCR, the extraction is performed by adherence of RNA to silica particles. The amplification of target RNA involves the coordinated activities of three enzymes—reverse transcriptase, RNase H, and T7 RNA polymerase—in four steps: extension, degradation, DNA synthesis, and cyclic RNA amplification. RNA calibrators are added in three concentrations to control efficiency in extraction and enzymatic amplification. Quantification results from the ratio of signals of the specimen HCV RNA divided by the signal of the appropriate internal calibrator.

The RTD-PCR Test (5'-Nuclease PCR or TaqMan PCR)

Morris et al.[13] described the application of a fluorogenic probe-based (TaqMan probes) PCR assay (TaqMan; Applied Biosystems) for the detection of HCV RNA in serum and plasma. This assay allows the direct detection of specific PCR products by monitoring the increase in fluorescence of a dye-labeled oligonucleotide probe. TaqMan PCR uses the 5'-3' endonuclease activity of Taq DNA polymerase to digest a probe labeled with a fluorescent reporter and quencher dye, and inclining PCR cycles result in exponential amplification of PCR products and fluorescence activity. Takeuchi et al.[14] described the establishment of a real-time detection system for real-time quantitative HCV PCR—the ABI PRISM 7000 instrument (Applied Biosystems). This instrument combines thermal cycling, fluorescent detection, and application-specific software in a single instrument.

Now, the Cobas TaqMan HCV test, a real-time homogenous quantitative PCR using TaqMan technology, and the Cobas TaqMan 48 or 96 analyzer are introduced for routine diagnostic laboratory. In this quantitative assay, the recombinant Thermus specie Z05 polymerase is used, which offers reverse transcriptase, polymerase, and endo-nuclease activity. Roche Diagnostic Systems states a detection limit of 10 IU/mL and a high dynamic range between 30 x 101 and 2 x 108 IU/mL.

The bDNA Test

Chiron Corporation has developed a bDNA signal amplification for viral load detection of hepatitis C.[15] The pelleted virus is lysed with proteolytic agents and detergents to release RNA and then is added to wells of a microwell plate. RNA is captured by hybridization with a set of specific synthetic oligonucleotide capture probes immobilized to the plate. A set of target probes hybridizes to both the viral RNA and the preamplifier probes (the backbone of the branched complex). The capture probes and the target probes bind to the 5' UTR and core region of the HCV genome. The amplifier probe subsequently hybridizes to the preamplifier, forming a bDNA complex. By a series of hybridizations, multiple bDNA amplifier molecules and alkaline phosphatase-labeled probes are then hybridized to this immobilized complex. Detection is achieved by a chemiluminescent substrate. The emitted signal directly correlates to the amount of HCV RNA present.

The HCV Core Antigen ELISA—trak-C Test

Recently, as alternative to molecular quantitative detection of HCV load, a quantitative immunoassay for the measurement of total hepatitis C nucleocapsid core antigen—the trak-C test (Ortho-Clinical Diagnostics, Inc.)—in the presence or absence of antibodies to HCV in human serum or plasma was introduced.1-16-1

Comparison of Assays

In comparison with the HCV Superquant assay, only available through NGI and not accessible to other laboratories, quantification with the Cobas Amplicor HVC Monitor 2.0 test (Roche Diagnostic Systems), suitable for routine laboratory settings, has been shown to be linear up to viral loads of 8.5 x 105 IU/mL by appropriate dilution.[17] Results of both tests correlated significantly. Moreover, Konnick et al.[18] demonstrated a good agreement between the Cobas Amplicor HVC Monitor 2.0 (Roche Diagnostic Systems) and the Superquant (NGI) assay results.

Through some technical improvements in probe design and background reduction, bDNA Version 3.0, the Versant HCV RNA 3.0 test (Bayer Diagnostics), now shows a broader range of 5.2 x 102 to 8.3 x 106 IU/mL for quantification of HCV RNA[19] compared with the previous range of bDNA 2.0 (Chiron Corporation) of 2 x 105 to 5 x 107 copies/mL.[15] Later, the reportable range was further improved (3.2 x 103 to 40 x 106 copies/mL; conversion factor: 5.2 HCV RNA copies/IU). Beld et al.[19] found a good correlation for viral load (expressed in international units per milliliter) obtained from the Amplicor HCV Monitor 2.0 test (Roche Diagnostic Systems) and the Versant HCV RNA 3.0 test (Bayer Diagnostics).

Zanetti et al.[20] demonstrated a good correlation of the trak-C test (Ortho) with the Cobas Amplicor HCV Monitor 2.0 test (Roche Diagnostic Systems) except for HCV RNA levels <5 x 104 IU/mL, where the trak-C test was negative due to a lower sensitivity compared to the Cobas Amplicor HCV Monitor 2.0 test (Roche Diagnostic Systems). An additional new quantitative assay based on PCR (the LC x HCV Quantitative test; Abbott, Chicago, IL) is being evaluated.

Sample preparation is currently thought to be the major weakness in the molecular detection of HCV RNA. Conventional sample preparation protocols are usually time consuming, labor intensive, and sensitive to contamination producing false-positive results. Therefore, besides ready-to-use sample preparation kits, automated specimen preparation systems such as the Cobas Ampli-prep (Roche Diagnostic Systems) designed for Cobas Amplicor PCR and, in the future, for Cobas TaqMan 48 and 96 analyzer were developed and brought to the market. It could be demonstrated that quantitative test results were comparable between a combination of the Cobas Ampliprep with the Cobas Amplicor HCV Monitor 2.0 test (Roche Diagnostic Systems) and the Cobas Amplicor HCV Monitor 2.0 test (Roche Diagnostic Systems) alone within ±0.5 log10.[21] Standardization for any commercially available assay or method is essential for providing reliable information for appropriate patient management, especially if, following the recommendations of EASL Consensus Panel,[22] a clinically relevant threshold of 2 x 106 copies/mL is used as a guideline for determining the outcome of treatment with interferon plus ribavirin in patients infected with genotypes 1, 4, and 5, or defining the duration of treatment. The NIH Consensus Statement[23] defined early viral response (EVR) as a minimum of 2 log10 decrease in viral load during the first

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

Get My Free Ebook

Post a comment