Serologic Antibody Assays
Initial testing for the diagnosis of hepatitis C infection uses serologic assays that detect human antibodies generated as a response to hepatitis C virus (HCV) infection. A positive HCV antibody test indicates HCV infection at some point in time, but it does not differentiate whether the person has resolved or current HCV infection.
- Enzyme Immunoassay (EIA): In the United States, the 3rd generation EIA test is the assay most frequently used for initial HCV antibody testing. The 3rd generation EIA detects antibodies that bind to recombinant antigens derived from four viral regions: core, nonstructural 3, nonstructural 4, and nonstructural 5 (Figure 1). The EIA test is reported as positive or negative based on an absorbance signal compared with a cut-off value. The 3rd generation HCV EIA has a sensitivity of approximately 98%. Rare circumstances associated with a false-negative EIA include patients with major immunosuppression (advanced HIV infection or organ transplantation recipients), patients with chronic renal failure on long-term hemodialysis, and patients with acute or early HCV infection. The HCV EIA has a reported specificity greater than 99%, with false-positive tests occurring more frequently when performing testing in populations that have a very low hepatitis C prevalence.
- Chemiluminescence Immunoassay (CIA): The CIA test is an antibody test similar to the EIA, but for HCV testing, the CIA is used less frequently than the EIA test. For the diagnosis of HCV, the CIA has similar sensitivity and specificity as the 3rd generation EIA.
- Point-of-Care Rapid Immunoassays: The OraQuick HCV Rapid Antibody Test was FDA approved in 2010 as a point-of-care test for use with whole blood samples obtained either by venipuncture or fingerstick. This OraQuick Rapid Antibody Test can be used as an alternative to the 3rd generation EIA for initial HCV antibody testing. The OraQuick test is read between 20 to 40 minutes after the test device is inserted into the buffer (Figure 2) and the result is either reactive or nonreactive (Figure 3). A patient with a reactive test should be considered to have a preliminary positive test and should undergo supplemental HCV testing. In 2011, the FDA granted a Clinical Laboratory Improvement Amendments (CLIA) waiver for the OraQuick HCV Rapid Antibody Test. Two additional point-of-care rapid HCV antibody tests have been developed but are not FDA approved.
- Recombinant Immunoblot Assay (RIBA): The HCV RIBA is no longer available for use in the United States. The RIBA test identifies specific antibodies generated in response to HCV antigens (Figure 4) and the test is interpreted as positive (2 or more antigens), indeterminate (1 antigen), or negative (0 antigens). The RIBA antibody test, when it was available, provided useful information in persons who had a positive HCV EIA and a negative HCV RNA test. In this circumstance, the RIBA could differentiate whether the patient had resolved HCV infection or had a biologic false-positive EIA.
Molecular HCV RNA Tests
Molecular diagnostic tests for hepatitis C specifically detect HCV RNA and the process is commonly referred to as a Nucleic Acid Test (NAT) or Nucleic Acid Amplification Test (NAAT). The HCV NAT becomes positive approximately 1 to 2 weeks after initial HCV infection. The NAT test has become the gold standard supplemental test for patients who have a positive HCV EIA screening test. The NAT can determine whether a patient with a positive HCV antibody test has current (active) or resolved HCV infection. In addition, the NAT can be used to diagnose individuals with acute HCV infection. The results for the commercially available quantitative HCV RNA assay are given in International Units (IUs).
- Qualitative HCV RNA: The qualitative HCV RNA tests provide a yes or no answer to whether detectable HCV RNA is present in the sample. The qualitative HCV RNA assays are FDA approved for HCV diagnostic purposes. These tests, however, do not provide a quantitative level of HCV and are not used for baseline HCV RNA levels or for monitoring response to therapy.
- Quantitative HCV RNA: The quantitative HCV RNA tests are not FDA-approved for HCV diagnostic purposes. More recently, however, with the introduction of ultrasensitive HCV quantitative RNA assays (that detect as few as 5 copies/mL), the quantitative HCV RNA has achieved a similar level of diagnostic sensitivity as the qualitative assay. In addition, quantitative HCV RNA assays generate an actual HCV RNA level that may provide useful information as a baseline HCV RNA. Because the sensitivity of the quantitative HCV RNA assays has dramatically improved, many clinicians have utilized the quantitative HCV RNA for diagnostic purposes.