Sunday, August 14, 2011

Laboratory Diagnosis of Hepatitis C virus (HCV)

• Serologic Assays
Components of previous and current commercial anti-HCV antibody test systems licensed in the United States. Location on the HCV polyproteins of recombinant antigens and peptides used in ELISA and recombinant immunoblot assay (RIBA) antibody test systems. Recombinant antigens are expressed in yeast as fusion proteins with superoxide dismutase. The first clone identified, 5-1-1, was used in hybridization assays to identify three overlapping clones that together constituted a 363–amino acid peptide termed C100-3, which was then expressed at high levels in yeast and used in the first-generation ELISA (Kuo et al., 1998).
Other antigens were added later to make the second-generation commercial ELISA. The new approved system, the Ortho HCV version 3.0 ELISA test system includes three recombinant antigens, c22-3, c200, and NS5. At present, there are two licensed ELISA systems available in the United States, and several other systems are available in other countries. The version 3.0 may shorten the window period. The most commonly used supplemental assay is RIBA to help exclude false-positive ELISA results. In this assay, several recombinant peptide antigens are applied on a strip, which is then probed with the patient's serum. In this way, some false-positive ELISA results can be eliminated (Gretch, 1997).
• Antigen detection
Immunoassays using MAbs for the detection of the core antigen of HCV (HCVcAg) have been developed. In this assay, the viral envelope and the preexisting anti-core antibodies are stripped off the HCVcAg by detergents; the core antigen is then detected by standard immunoassay. Although the HCVcAg was detected in 94.5% of 59 patients who had detectable HCV RNA, it was not shown that the assay actually had similar sensitivity to detect virus at a very low level (Aoyagi et al., 1999).
• Molecular detection
RT-PCR techniques are currently the most sensitive methods for detecting HCV RNA. Qualitative methods detect presence of circulating HCV RNA; for monitoring patients on antiviral therapy and quantitative methods determine concentration of HCV RNA, may be useful for assessing the likelihood of response to antiviral therapy. Quantitative assays are less sensitive than qualitative assays and should not be used as primary test to confirm or exclude the diagnosis of HCV infection. Currently, testing for HCV RNA is available on a research basis and no tests have been approved by the U.S. Food and Drug Administration. Because of assay variability, results of HCV RNA testing should be interpreted cautiously (Beltrami et al., 2000).
• HCV Genotype
Group isolates of HCV based on genetic differences into six genotypes and > 90 subtypes; with new therapies, length of treatment may vary based on genotype. Genotyping of HCV has become important in the treatment of infection and for epidemiological studies. It is based on analysis of an amplified segment of the genome (such as 5' UTR) commonly used for virus detection. The sequence of 5' UTR is highly conserved between genotypes, but genotype specific differences compromising one or more substitutions can detected by probe hyperdization, by changes in restriction endonuclease sites, or by direct sequencing of the 5' UTR ( Ross et al., 2000).

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