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Screening & Diagnostic Tests
Nucleic Acid (RNA) Detection

from the
Centers For Disease Control website at:
http://www.cdc.gov/ncidod/diseases/hepatitis/c_training/

edu/2/screening-nucleic.htm

The diagnosis of HCV infection also is possible by qualitatively detecting HCV RNA using gene amplification techniques (e.g., RT-PCR ). HCV RNA can be detected in serum or plasma within 1-2 weeks after exposure to the virus and weeks before the onset of ALT elevations or the appearance of anti-HCV. Rarely, detection of HCV RNA might be the only evidence of HCV infection. Although RT-PCR assay kits for HCV RNA are available for research purposes from various manufacturers of diagnostic reagents, none have been approved by FDA. In addition, many laboratories perform RT-PCR using in-house laboratory methods and reagents.

Although not FDA-approved, RT-PCR assays for HCV infection are used commonly in clinical practice. Most RT-PCR assays have a lower limit of detection of 100-1000 viral genome copies per mL. With adequate optimization of RT-PCR assays, 75%-85% of persons who are anti-HCV-positive and > 95% of persons with acute or chronic hepatitis C will test positive for HCV RNA. Some HCV-infected persons might be only intermittently HCV RNA positive, particularly those with acute hepatitis C or with end stage liver disease caused by hepatitis C. To minimize false-negative results, serum must be separated from cellular components within 2-4 hours after collection, and preferably stored frozen at -20oC or -70oC. If shipping is required, frozen samples should be protected from thawing. Because of assay variability, rigorous quality assurance and control should be in place in clinical laboratories performing this assay, and proficiency testing is recommended.

Quantitative assays for measuring the concentration (titer) of HCV RNA have been developed and are available from commercial laboratories, including a quantitative RT-PCR (Amplicor HCV Monitor™, Roche Molecular Systems, Branchburg, New Jersey) and a branched DNA (deoxyribonucleic acid) signal amplification assay (Quantiplex™ HCV RNA Assay [bDNA], Chiron Corp., Emeryville, California) (Table 2). These assays also are not FDA-approved, and compared with qualitative RT-PCR assays, are less sensitive with lower limits of detection of 500 viral genome copies per mL for the Amplicor HCV Monitor™ to 200,000 genome equivalents per mL for the Quantiplex™ HCV RNA Assay. In addition, they each use a different standard, which precludes direct comparisons between the two assays. Quantitative assays should not be used as a primary test to confirm or exclude the diagnosis of HCV infection or to monitor the endpoint of treatment. Patients with chronic hepatitis C generally circulate virus at levels of 105 to 107 genome copies per mL. Testing for the level of HCV RNA might help predict likelihood of response to antiviral therapy, although sequential measurement of HCV RNA levels has not proven useful in managing patients with hepatitis C.

At least six different genotypes and > 90 subtypes of HCV exist. Approximately 70% of HCV-infected persons in the United States are infected with genotype 1, with frequency of subtype 1a predominating over subtype 1b. Different nucleic acid detection methods are available commercially to group isolates of HCV, based on genotypes and subtypes. Evidence is limited regarding differences in clinical features, disease outcome, or progression to cirrhosis or hepatocellular carcinoma (HCC) among persons with different genotypes. However, differences do exist in responses to antiviral therapy according to HCV genotype. Rates of response in patients infected with genotype 1 are substantially lower than in patients with other genotypes. Patients with genotype 1 may require a longer period of treatment. Thus, genotyping might be warranted among persons with chronic hepatitis C who are being considered for antiviral therapy.

http://www.cdc.gov/ncidod/diseases/hepatitis/c_training/
edu/2/screening-nucleic.htm