Laboratory Tests and Hepatitis C
Laboratory tests for hepatitis C are generally divided into two categories including(1):
- serologic assays that detect antibodies to HCV
- assays that detect, quantify, and/or differentiate HCV RNA genomes within an infected individual. These are commonly called "hepatitis C viral load" tests and "hepatitis C genotype"
Enzyme immunoassays (EIAs)
Enzyme immunoassays (EIAs) detect the presence of antibodies in serum directed against HCV. These tests are commonly used for initial detection of hepatitis C. However, EIAs do not differentiate between acute, chronic or resolved infection.
There are three generations of the EIA, with the latest EIA being a highly sensitive test. An important issue with the EIA is that it can yield both false positive and false negative results. False negative results may occur if the test is sent during the window period before seroconversion ("serologic window" averages 8 weeks). False negative results may also occur in immunocompromised populations such as those with HIV. False positive results may occur due to cross-reactivity with other viral antigens and in immunologic disorders.
Recombinant immunoblot assay (RIBA)
Recombinant immunoblot assay (RIBA) is a highly specific test often used as a confirmatory or supplemental test in low risk populations such as blood donors.(2)
It is performed with the use of antigens from the HCV genome combined with patient's serum. It is not needed routinely to confirm the results of a positive EIA in individuals from high risk populations such as injection drug users. However, in certain clinical scenarios, such as the individual with a positive anti-HCV and negative HCV-RNA, RIBA can help confirm diagnosis.
Several different methodologies have been used to assess HCV viremia.(3) These assays may be helpful in predicting probability of treatment response and measuring treatment response. Viral load, however, does not seem to correlate with disease severity or prognosis.
HCV RNA (hepatitis C virus ribonucleic acid) tests
HCV RNA (hepatitis C virus ribonucleic acid) tests indicate presence of viremia, using target amplification techniques. These tests can detect virus within 1-2 weeks following exposure.(4) Results may not be consistent or comparable between different assays.
Qualitative tests indicate presence of virus. Qualitative tests are useful in individuals in whom EIA tests for anti-HCV are not reliable. The results are expressed as positive/negative (for the presence of HCV-RNA). These tests can detect as few as 100-1000 copies/mL. New tests such as transcription mediated amplification (TMA), have sensitivity as low as <50 copies/mL (5 IU/mL). Other tests include Amplicor HCV™ with a sensitivity of 100 copies (50 IU/mL). The specificity of qualitative tests is estimated to be >99.5%.
Quantitative tests measure the amount of virus circulating in the plasma. The quantitative measurement is less sensitive than the qualitative. Examples of quantitative tests include PCR tests such as Amplicor HCV Monitor™ and SuperQuant™. Differences exist between how test results are expressed. Thus, comparisons between assays are difficult. Results are usually expressed as as International Units/mL (IU/mL) or RNA copies/mL. Because the significance of the absolute values is unclear, clinical decisions are generally made on the categorization of grouping of "high" or "low" viral load, rather than exact levels. Generally, the cut off for high and low viral load is approximately 800,000 IU/mL.
bDNA (branched chain DNA)
bDNA (branched chain DNA) uses signal amplification technology without amplification of viral nucleic acids.(5) An example of a bDNA quantitative test includes Quantiplex(bDNA-2). Branched-DNA assays may have a lesser degree of sensitivity compared to the PCR tests. Therefore, PCR testing may be necessary to identify low-level viremia.(1)
Notes on Methods of Standardization for Quantitative HCV RNA Assays
Because of assay variability and differences in standards used, it is difficult to convert from one assay to another and compare "viral loads" over time. Most tests express units as international units (IU/mL) in order to standardize measurements. (6) It is also important to note that HCV genotype may affect the efficiency of HCV quantification. Early generation quantification assays may underestimate viral loads of genotypes 2 and 3 compared to genotype 1 strains. (7) Most current assays make adjustments for genotype.
There are at least six genotypes and over 30 subtypes of HCV. Different assays are used to determine genotype such as sequencing and hybridization. Most genotype assays use amplification of virus sequences by PCR. Assays for determining genotypes and serotypes are commonly employed in research settings. Genotypes are very useful for tailoring treatment regimens and predicting treatment response. Genotypes 2 and 3 are more likely to be cleared by interferon-based treatments than genotype 1. However, genotype alone should not be used to deny treatment to patients. For more information on genotypes and quasispecies, see HCV Genotypes and Quasispecies.
- Gretch DR. Diagnostic Tests for Hepatitis C. Hepatology 1997;26:43S-47S.
- Shakil AO, Conry-Cantilena C, Alter H, Hayashi P, Kleiner DE, Tedeschi V, Krawczynski K, Conjeevaram H, Sallie R, Di Bisceglie AM, Hepatitis C Study Group. Volunteer Blood Donors with Antibody to Hepatitis C Virus: Clinical, Biochemical, Virologic, and Histologic Features. Annals of Internal Medicine 1995;123:330-337.
- Gretch DR, dela Rosa C, Carithers RL, Willson RA, Williams B, Corey L. Assessment of hepatitis C viremia using molecular amplification technologies: correlations and clinical implications. Annals of Internal Medicine 1995;123:321-329.
- Centers for Disease Control and Prevention. Recommendations for prevention and control of hepatitis C virus (HCV) infection and HCV-related chronic disease. MMWR 1998;47(No.RR-19): 10.
- Gretch DR, dela Rosa C, Carithers RL, Willson RA, Williams B, Corey L. Assessment of hepatitis C viremia using molecular amplification technologies: correlations and clinical implications. Annals of Internal Medicine 1995;123:322.
- Ferreira-Gonzalez A, Shiffman ML. Use of diagnostic testing for managing hepatitis C virus infection. Seminars in Liver Disease 2004;24 Suppl 2:9-18.
- Martinot-Peignoux M, Boyer N, Le Breton V, Le Guludec G, Castelnau C, Akremi R,Marcellin P. A new step toward standardization of serum hepatitis C virus-RNA quantification in patients with chronic hepatitis C. Hepatology 2000;31:726-729.