An update on diagnostics for hepatitis C

Hepatitis C virus (HCV) is a major health concern both in the United States and abroad. Chronic infection with HCV is a leading cause of cirrhosis and, due to its association with hepatocellular carcinoma, is the most common indication for liver transplant in the United States.¹

The prevalence of HCV infection in the United States is estimated to be five to seven million cases, or 1.6 percent to 1.8 percent of the total population.² Due to a confluence of risk factors, including contaminated blood transfusions and expanded use of illicit intravenous drugs, about three-fourths of cases of chronic HCV infection in the United States belong to a single cohort of people born between 1945 and 1965.³ Thus, the U.S. Centers for Disease Control and Prevention (CDC) and United States Preventive Services Task Force recommend routine screening in that population.^4-6

HCV Ab enzyme-linked immunosorbent assay

Historically, screening for HCV infection has been performed by enzyme-linked immunosorbent assay (ELISA) for antibodies against viral proteins. Anti-HCV antibodies can be detected in infected individuals as early as four to ten weeks after exposure.⁷ Third generation ELISAs, the latest iteration available, achieve superior sensitivity over preceding versions by targeting multiple viral antigens, including the core and nonstructural (NS) proteins N3, NS4, and NS5.^8,9 Though ELISAs have high specificity when testing at-risk populations,¹⁰ they have a low positive predictive value when used for wider screening. Thus, a positive result must be confirmed by a more specific test. Other limitations of ELISA include the potential for a false-negative result in patients with deficiencies in humoral immunity—e.g., patients who are immunocompromised, who are on hemodialysis, or who are the recipients of organ transplants.

Rapid testing

Point-of-care tests (POCTs) for HCV infection have been developed to provide a presumptive diagnosis quickly and without a clinical laboratory. Rapid tests for anti-HCV antibody target the same viral antigens as do third-generation ELISAs, but can be used on fingerstick capillary whole blood as well as venipuncture-collected whole blood. Nonetheless, on evaluation by the CDC in various clinical settings, rapid POCTs were shown to perform as well as ELISA.^{11- 13}

To date, only one POCT has been approved by the FDA for the detection of anti-HCV antibody in at-risk individuals or those with suspected hepatitis.¹⁴ The approved POCT has been granted a Clinical Laboratory Improvements Amendments waiver permitting its use in physician offices, clinics, and emergency departments. Because POCTs are more costly than traditional ELISAs and cannot be batched, their utility in high volume settings is likely limited. Importantly, POCTs have not been approved for general screening.

Recombinant immunoblot assays

Recombinant immunoblot assays (RIBAs) are highly specific tests to confirm the presence of anti-HCV antibody after a positive result on ELISA. Because RIBAs require serum to react with at least two viral antigens to be called positive, patients with early infection may receive an indeterminate result if they have not yet mounted a full immune response to the virus. RIBAs are costly, slow, and somewhat impractical, in that a positive result must be followed by an assay for HCV RNA before a diagnosis of active infection can be made. Thus, RIBAs are no longer recommended for clinical use.¹⁵

Molecular testing

Nucleic acid testing (NAT) detects HCV RNA in the serum or plasma of actively infected individuals, most commonly by real-time polymerase chain reaction. In 2013, the CDC updated its guidelines to include NAT as the recommended confirmatory test for HCV infection following a positive screening ELISA.6 NAT is extremely sensitive—able to detect viral loads as low as 10-15 IU/mL¹⁶ —and highly specific, regardless of HCV genotype. Because NAT does not require circulating anti-HCV antibodies for a positive result, it may be used to assess for acute HCV infection as early as one to three weeks after exposure or in an immunocompromised host. Thus, though no NAT has been approved by regulatory agencies for the diagnosis of HCV infection, clinicians will commonly request NAT when there is suspicion for acute HCV infection despite a negative assay for anti-HCV antibody.

Once active HCV infection is confirmed, determination of the viral genotype can guide treatment and inform prognosis. The only FDA-approved and commercially available genotype assay uses real-time PCR to detect portions of the 5’untranslated region and the core/NS5B amplicon.¹⁷ In a post-market evaluation of that assay, about 2 percent of tests yielded an indeterminate result; 17 of 19 of these indeterminate cases were subsequently identified as genotype 3a.¹⁸

HCV core antigen testing

An assay for direct detection of HCV antigen from venous blood is currently available in Europe. Direct antigen testing (DAT) quantitates a viral load based on relative chemiluminescence from a bead-coated monoclonal antibody to the HCV core antigen.¹⁹ As with NAT, a positive test indicates active HCV infection and does not rely on the body’s generation of anti-HCV antibodies. Though DAT does not perform as well as NAT in the setting of low viral loads, samples submitted for DAT require less processing than do molecular assays and therefore may appeal for use in clinical laboratories. A summary of clinical diagnostics for HCV is presented in Table 1.

Figure 1. CDC algorithm for diagnosis of current HCV infection

References

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