Ns4

NS4 and core

Routine lab

Yes Yes

Gen. region = genomic region of the HCV used for genotyping; Routine lab = suitable for routine diagnostic laboratory settings; 5'NC = 5' noncoding region; DM = direct method based on molecular typing; 5'UTR = 5' untranslated region; E1 = envelope 1; IM = indirect method; NS4 = nonstructural 4 region.

Routine lab

OpenGene™ DNA sequencing system (Bayer) that is combined with the automated Long-Read Tower™ for the separation of the sequenced fragments. Data are acquired with the GeneLibrarian™ module of the GeneObjects™ (Bayer) software.

Multiplex real-time polymerase chain reaction-based genotyping

The Celera HCV Genotyping assay (Celera Diagnostics, South San Francisco, CA; distributed by Abbott Laboratories, North Chicago, IL) is based on a multiplex real-time PCR technique. This assay is performed on the ABI Prism 7000, a real-time cycler (Applied Biosystems, Foster City, CA), and the analysis software used is SDS v1.01. Per sample, three reactions using three different fluorescent dyes for simultaneous detection are necessary: reaction 1 detects genotypes 1a and 1b; reaction 2 detects genotypes 2a, 2b, and 3; reaction 3 detects genotypes 4, 5, and 6.

Genotype-/subtype-specific hybridization

The Inno-LiPA HCV II (Versant; Innogenetics, Ghent, Belgium; distributed by Bayer) assay is based on the reverse-hybridization principle of RT-PCR products derived from the 5'UTR.[3] Biotinylated RT-PCR products (RT-PCR products from Cobas Amplicor™ HCV Test; Roche) are hybridized to a selection of 19 specific oligonucleotide probes immobilized on nitrocellulose membrane strips. The RT-PCR products hybridize to a probe matching the sequence of the isolate and allowing discrimination at the subtype level. HCV genotypes/

subtypes are identified by comparing the pattern of positive (colored) lines with a special interpretation chart.

Restriction fragment length polymorphism

Sequences amplified by PCR from the 5'UTR are cleaved by restriction endonucleases for identification of different HCV genotypes/subtypes. Initially, enzymes HaeIII-RsaI and HinfI-MvaI are employed for cutting the PCR fragments from the 5'UTR which is followed by cleavage using BstU1 or ScrFI. HCV genotypes/subtypes 1a, 1b, 2a, 2b, 3a, 3b, 4, 5, and 6 can be identified.[15]

DNA enzyme immunoassay

The DNA enzyme immunoassay (DEIA) (Gen-Eti-K DEIA; Sorin Biomedica, Saluggia, Italy) is based on a combination of nested PCR (core region) and DNA enzyme immunoassay.1-16-1 Amplification products are hybridized to type-specific oligonucleotides. The hybrids are detected by a standard ELISA technique which employs monoclonal antibodies reacting with double-stranded DNA.

Heteroduplex tracking assay

The heteroduplex tracking assay (HTA), also called heteroduplex mobility analysis (HMA), is based on the fact that the genetic relationship between isolates can be determined by the relative migration of the heteroduplex on gels.[17] This assay involves hybridization with probes from known HCV subtypes to RT-PCR products generated from homologous sera or from unknown samples and agarose gel electrophoresis of hybridization products.[18'19] The formation of a heteroduplex band on a gel indicates genotype and subtype.

RT-PCR with genotype-specific primers

Okamoto et al.[20] described a polymerase chain reaction (PCR)-based assay using primers for the hepatitis C core gene. This assay is just able to identify subtypes 1a, 1b, 2a, and 2b. The primary PCR with consensus primers is followed by a second-round PCR with type-specific primers. HCV subtypes are determined by the size of the amplification products generated. The original assay has been modified by Widell et al.[21] by adding another second-round PCR for identification of genotype 3.

Indirect Method

Serotyping assay

The HCV serotyping assay (SIA-3 or SIA-6 Murex HCV Serotyping assay, Murex Diagnostics Ltd, Dartford, U.K.) is based on the detection of genotype-specific antibodies directed to epitopes encoded by the NS4 region of the HCV genome. Eight (SIA-3) or 24 (SIA-6) NS4-encoded genotype-specific branched peptides are used in a competitive way to discriminate between genotypes 1-3 and 1-6, respectively.[10] With the RIBA HCV SIA (Chiron Corporation, Emeryville, CA), eight serotype-specific HCV peptide antigens, five of which are from the NS4 region of HCV subtypes 1a, 1b, 2a, 2b, and 3, and three of which are from the core region of types 1 and 2, are immobilized. Genotyping is achieved by an algorithm.[22]

Comparison of hepatitis C virus genotyping methods

The Inno-LiPA HCV II (Innogenetics), probably the most frequently used assay, has been well characterized in earlier studies.[23,24] In comparative studies, the Inno-LiPA HCV II assay (Innogenetics) and the TruGeneTM HCV 5'NC Genotyping Kit (Bayer) show concordant results on the genotype level in 100% and 99.5%, respectively.1-25-28-1 The overall accuracy for the discrimination of subtypes, however, was found to be lower for both, the Inno-LiPA HCV II assay (Innogenetics) and the TruGeneTM HCV 5'NC Genotyping Kit (Bayer) with 95.5% and 85.9%, respectively, when using the TruGeneTM HCV 5'NC Genotyping Kit (Bayer) or the Inno-LiPA HCV II assay (Innogenetics) as reference test for the alternative assay.[27] As recently reported, the

TruGeneTM HCV 5'NC Genotyping Kit (Bayer) and the Inno-LiPA HCV II assay (Innogenetics) gave concordant results on the subtype level in 90.9% of samples.[29] Accuracies for the discrimination of subtypes were 76% and 91.9% for the TruGene™ HCV 5'NC Genotyping Kit (Bayer) and 74% and 85.8% for the Inno-LiPA HCV II assay (Innogenetics), respectively, in comparison to sequence analysis of the NS5B region.[25,26] Sandres-Saune et al.[30] showed a concordance of 94%, regarding subtypes 1a or 1b, when comparing the TruGeneTM HCV 5'NC Genotyping Kit (Bayer) and a noncommercial assay using the NS5B region. The insufficient sequence polymorphism of the 5'UTR has been suggested as the major problem preventing correct discrimination of HCV subtypes. For the discrimination of subtypes 1a and 1b, it was shown that the polymorphism at position 99 of the 5'UTR may not always be linked to subtype 1a or 1b sequences detected in the core region.[23] This was also demonstrated in two comparative studies which focused on the performance of the Inno-LiPA HCV II assay (Innogenetics) versus that of NS5B sequencing with a concordance of 80% and 94%, respectively.[30,31] This limitation is common to all HCV genotyping procedures based on the analysis of the 5'UTR.[25]

Mixed HCV infections with different HCV genotypes/ subtypes may occur in patients with intravenous drug abuse, hemophilia, and those on hemodialysis. The Inno-LiPA HCV II assay (Innogenetics) shows a high sensitivity for detection of minor sequence variants[11] and may be useful for detection of mixed infections involving different HCV genotypes.[8,32] This assay was consistently able to detect as little as 1-2% of the viral population (minor genotype) compared with restriction fragment length polymorphism (RFLP) (which may detect 5-30%) in a recent study.[33]

The TruGeneTM HCV 5'NC Genotyping Kit (Bayer) or sequencing of the NS5b region is only possible if the serum HCV level exceeds 600 IU/mL.[30] To ensure accurate results, especially for low HCV levels, optimal blood collection systems containing a liquid nucleic acid stabilizer capable of stabilization nucleic acids within the blood samples even at room temperature for at least 96 hr are recommended.[34] After centrifugation, aliquots should immediately be frozen at —70°C.

In a comparative study, the TruGeneTM HCV 5'NC Genotyping Kit (Bayer) and the DEIA (Sorin Biomedica) showed concordant results on subtype level in 91%.[9] However, serological methods, while easier to perform, lack sensitivity and specificity in comparison to molecular assays.[10,13,14]

Restriction fragment length polymorphism analysis based on the 5'UTR may be more sensitive than HCV serotyping but less sensitive when compared with the Inno-LiPA HCV II assay (Innogenetics).[33,35]

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.

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