The hepatitis C virus (HCV) is a member of the Flaviviridae family of viruses, which are associated with both human and animal diseases. Hepatitis caused by HCV is the most common chronic bloodborne infection in the United States. Over 4 million Americans are believed to be infected. HCV can also be transmitted through blood transfusion. HCV causes inflammation of the liver, and up to 80% of those exposed to the virus develop a chronic infection, which can lead to liver inflammation, cirrhosis, cancer, and death. Eventually, up to 20% of people with HCV may develop cirrhosis of the liver or other severe liver diseases. As in other forms of hepatitis, individuals may be infected with the virus but may not realize they are carriers because they do not have any symptoms. Because of the risk of serious illness, people with HCV need to be followed closely by a physician with experience evaluating this infection. Since the full length HCV cDNA was first cloned in 1989, significant progress has been made in characterizing its molecular biology (Lamballerie, 1996). But, the natural history of HCV infection is still largely unclear and current treatment options for patients are limited. There is no vaccine for HCV, and the only available treatment, a combination of alphainterferon and ribavirin, is efficacious in only a minority of patients (Wang and Heinz, 2001). The life cycle of the HCV is poorly understood due to the lack of an efficient cell culture system (Cohen, 1999). There is an urgent need to develop a highly sensitive detection method for studying possible extrahepatic sites for the replication of hepatitis C virus. We recently established a cell culture system for the replication of HCV by using human T and B leukemia cell lines. (Hu et al., 2003) This model should represent a valuable tool for the detailed study of the initial steps of the HCV replication cycle and for the evaluation of antiviral molecules. Currently, appropriate therapeutic and vaccine strategies for HCV have not been developed. Early detection and prevention of HCV infection are most important for blood safety.
It is a formidable task to design primers and probes for sensitive nucleic acid level diagnostic assays throughout the open reading frame of the HCV genome because of a high mutation rate in this genomic region. However, the untranslated region of about 341 nucleotides contains highly conserved domains, which allows for stable primer design and sensitive diagnostic tests, both qualitative and quantitative, which have equivalent sensitivity against the known six various genotypes of HCV.
Antibodies to the Hepatitis C Virus (Anti-HCV)
In 1990, the first specific test for HCV, the major cause of "non-A, non-B" hepatitis was introduced. Now, a third-generation ELISA kit is available to detect antibodies to HCV, and screening blood for HCV antibodies is recommended. These assays are based on detection of serum antibody to various HCV antigens because these antibodies are nearly universally present in patients who are chronically infected with HCV (Major et al., 2001). The HCV screening tests are known to have significant limitations, and positive samples should be further tested by HCV confirmatory tests.
Guidelines provided by the CDC recommend that HCV antibody screening test positive samples should be confirmed with serologic or nucleic acid supplemental testing. HCV confirmatory tests include the recombinant immunoblot assay in which several recombinant peptide antigens are applied on a strip that is then probed with the patient's serum. In this way, the response to individual antigens can be recognized, and some false-positive ELISA results can be eliminated
(e.g., RIBA, Chiron HCV 3.0 and PCR assay) (e.g., Roche COBAS AMPLICOR HCV Test, version 2.0). Laboratories can choose to perform this testing on all positive specimens or based on screening test positive (signal to cutoff) ratios. The positive predictive values (s/co) can vary depending on the prevalence of infection in the population being screened.
HCV antibodies are not generally detectable for at least 6 weeks and may not appear for several months. Acute HCV infections are relatively rare among blood donors, but the antibody tests often fail to detect these patients in the window period between the time of infection and the time of appearance of antibody detectable by the above assays. High-sensitivity detection of HCV during the window period is a long-term technical challenge in the field. Tests for HCV RNA genome detection based on the PCR or other highly sensitive RNA detection systems have been used for the diagnosis of acute hepatitis (Major et al., 2001). Sensitive detection of HCV RNA based on RT-PCR or other nucleic acid amplification techniques can be readily accomplished with kits that are now available commercially. For example, in 1999 the FDA approved Roche's Amplicor HIV-1 Monitor Ultra Sensitive quantitative assay. It can measure HIV levels at as few as 50 copies/mL and another commercial kit, the LCx HIV RNA Quantitative Assay from Abbott Laboratories, also has a detection limit at 50 copies/mL. Some research papers even showed a sensitivity limit at 1 copy (Palmer et al., 2003). In fact, a qualitative assay should be much more sensitive than a quantitative assay for HIV/HCV screening. A sensitive qualitative HCV molecular detection assay will possibly interdict and virtually prevent all transfusion-associated HIV/HCV. The current sensitivity standard for clinical diagnostics is 100 copies per mL, but since there has been an improvement in technology, this would be the time to change sensitivity standard to 50 copies per mL.
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