When should Hepatitis C Virus (HCV) Ribonucleic Acid (RNA) testing be performed?

When Should HCV RNA Testing Be Performed

HCV RNA testing should be performed immediately after any positive anti-HCV antibody test to confirm active infection, and directly (without antibody testing first) in suspected acute hepatitis C, immunocompromised patients with unexplained liver disease, and persons at risk for reinfection. 1, 2

Primary Testing Algorithm

Initial Diagnosis of HCV Infection

• Start with anti-HCV antibody testing followed by reflex HCV RNA testing for all patients being screened for hepatitis C infection, using a single blood collection to eliminate the need for return visits 2, 3
• Perform HCV RNA testing on all anti-HCV positive samples automatically to avoid incomplete testing, which historically occurs in approximately one-third of patients 3
• Use a sensitive molecular method with a lower limit of detection <50 IU/mL (ideally ≤15 IU/mL) for HCV RNA detection 1, 4

When to Bypass Antibody Testing and Go Directly to HCV RNA

Acute hepatitis C: HCV RNA testing is required as the initial test because HCV RNA appears 1-2 weeks after infection, while anti-HCV antibodies may not be detectable for 8-12 weeks 1

Immunocompromised patients: Test HCV RNA directly when hepatitis is present but anti-HCV antibodies may be undetectable or delayed, including HIV-positive patients, transplant recipients, and those on immunosuppressive therapy 1

Recent exposure (within 6 months): Perform HCV RNA testing 4-6 weeks after exposure for early diagnosis in persons with occupational needlestick injuries or other known exposures 1

Suspected reinfection: Use HCV RNA testing (not antibody testing) in patients with prior HCV clearance who have ongoing risk factors, as antibodies remain positive indefinitely after clearance 2, 5, 4

Specific Clinical Scenarios

Post-Exposure Testing Protocol

• Immediately after exposure: Test baseline anti-HCV and ALT 1
• At 4-6 weeks: Perform HCV RNA testing if early diagnosis is desired and baseline anti-HCV was negative 1
• At 4-6 months: Repeat anti-HCV and ALT testing for definitive diagnosis if earlier tests were negative 1

Monitoring for Reinfection

• Annual HCV RNA testing is required for people who inject drugs and men with HIV who have unprotected sex with men 2
• At least every 12 months: Test HCV RNA in patients with history of spontaneously cleared HCV and ongoing risk factors 5
• With any unexplained ALT elevation: Perform immediate HCV RNA testing in patients with prior HCV infection 5

Confirming Treatment Success

• At 12 weeks post-treatment (SVR12): Perform quantitative HCV RNA testing with undetectable result (<15 IU/mL) defining cure 4
• Use the same laboratory and assay type for pre-treatment and post-treatment testing to ensure consistency 4

Interpretation of Results

Anti-HCV Positive, HCV RNA Positive

• Indicates current active HCV infection requiring evaluation for treatment 2
• Proceed with quantitative HCV RNA and genotyping prior to initiating antiviral therapy 1

Anti-HCV Positive, HCV RNA Negative

• Suggests past resolved infection or false-positive antibody test 2
• Retest HCV RNA a few weeks later if acute hepatitis is present, as intermittent viremia can occur 1
• If persistently HCV RNA negative, patient does not have current infection but is not protected from reinfection 2

Anti-HCV Negative, Clinical Suspicion Remains

• Perform HCV RNA testing if acute infection suspected (within window period), patient is immunocompromised, or unexplained hepatitis is present 1

Critical Pitfalls to Avoid

Never rely on antibody testing alone to determine active infection status, as antibodies persist after viral clearance and cannot distinguish current from past infection 2, 4

Do not miss the window period in acute infection—HCV RNA becomes positive 1-2 weeks after exposure while antibodies may take 8-12 weeks to develop 1

Avoid incomplete testing by ensuring automatic reflex HCV RNA testing on all positive antibody results rather than requiring separate physician orders 3

Do not use antibody testing to monitor for reinfection in previously infected patients—only HCV RNA testing can detect new infection 2, 5

Never use ALT levels alone to determine infection status or treatment success, as liver enzymes can normalize despite persistent viremia or remain elevated from other causes 4