Chemistry, toxicology & urinalysis

• Hepatitis C testing involves measurement of hepatitis C virus (HCV) specific antibodies and HCV RNA, which can be used to screen, diagnose and monitor HCV infections, as well as guide direct acting HCV antiviral therapy
• A combination of HCV serology and nucleic acid test (NAT) results can help determine whether a patient has current or past HCV infection
  • In addition, quantitative NAT, genotyping assays and sequencing based drug resistance tests are important for predicting treatment response and tailoring therapy

• ICD-10
  • B17.1 - acute hepatitis C
  • B18.2 - chronic viral hepatitis C
  • B19.2 - unspecified viral hepatitis C

• ICD-11
  • 1E50.2 - acute hepatitis C
  • 1E51.1 - chronic hepatitis C

• Methodology
  • Chemiluminescent immunoassay (CLIA) or electrochemiluminescent immunoassay (ECLIA) are the most common methods for hepatitis C serology testing performed on automation analyzers (Clarke: Contemporary Practice in Clinical Chemistry, 4th Edition, 2020)
  • Polymerase chain reaction (PCR) and reverse transcriptase PCR (RT-PCR) are the most common molecular methods to detect HCV RNA, including qualitative and quantitative NAT assays
  • Direct sequencing of a specific PCR amplified portion of the viral genome is the most definitive method for HCV genotyping
  • Sequencing of the NS proteins using PCR or RT-PCR method has been used to detect HCV viral drug resistance mutations

• HCV serology test
  • There is only 1 HCV serology test clinically offered in the U.S., anti-HCV, which detects total HCV antibodies
    • Presence of HCV antibody indicates HCV infection but cannot differentiate current infection versus resolved past infection
  • HCV core antigen test, currently available only outside of the U.S., is another HCV serology test that can be used as a more cost effective alternative to HCV RNA NAT
    • Clinical utility of HCV core antigen for screening or diagnosis of HCV infection is limited because it is less sensitive compared to HCV RNA NAT assays and may become undetectable after seroconversion in some patients with current HCV infection (J Hepatol 2005;42:S108)
  • Current generation of HCV serology testing is highly sensitive and specific
    • However, when used in patient populations with low disease prevalence, the positive predictive value may be compromised
    • This is especially true with near cut off test results
    • Because immunoassay design varies significantly (e.g., antibody selection, cutoff setup, reagent formulation), testing with a different assay can be considered if distinction between true positive and biologic false positive anti-HCV result is desired
  • A point of care, CLIA waived, rapid HCV antibody test has been made available using the lateral flow immunoassay technology, which provides comparable performance to enzyme immunoassays
  • Rarely, HCV serology test results may be misleading in immunocompromised patients who failed to produce sufficient antibodies
• HCV molecular test
  • Various HCV molecular tests are available for clinical use, including qualitative NAT assays for detection of virus presence, quantitative NAT assays for measurement of viremia (viral load), sequencing assays for HCV RNA genotyping and detection of drug resistance mutations
  • Presence of HCV RNA alone does not distinguish between acute and chronic infection
    • Transiently negative HCV RNA can be seen in acute infection, while low or fluctuating viral load is indicative of acute infection
  • Limit of detection (LoD) and lower limit of quantitation (LLoQ) are important for NAT assays as the definition of cure relies on a not detected result at the end of the treatment cycle
    • Current recommendation considers an HCV NAT assay as appropriate with an LLoQ of 25 IU/mL and LoD of 15 IU/mL, at the least (Hepatology 2020;71:686)
  • HCV genotyping is recommended before therapy initiation for patients with a prior treatment failure or for those ineligible for simplified HCV treatment, to guide DAA regimen selection and to determine therapy duration (Ann Intern Med 2020;173:ITC33)
  • Because the DAA regimens target HCV NS proteins, including NS3, NS4A, NS5A and NS5B, HCV drug resistance mutation tests based on sequencing of NS proteins are important for the identification of resistance associated substitutions (RASs) (Ann Intern Med 2020;173:ITC33)
    • When tailored DAA regimen is required, a combination of genotype and RASs information together with host factors are used to determine treatment plan

Hepatitis C testing results interpretation (adapted from MMWR Morb Mortal Wkly Rep 2013;62:362)

Anti-HCV	HCV RNA	Result interpretation
Nonreactive	Not indicated (unless recent exposure suspected)	Report nonreactive HCV antibody
Reactive	Not performed	Presumptive HCV infection Consistent with current infection Or past infection that has resolved Or biologic false positivity for HCV antibody
Reactive	Detected	Current HCV infection
Reactive	Not detected	No current HCV infection If distinction between true positive and biologic false positive anti-HCV result is desired, consider testing with another HCV antibody assay if repeat positive with the initial test Repeat HCV RNA testing can be considered if there is strong clinical indication or if there is a concern regarding specimen quality

Which of the following is the most appropriate test for the diagnosis of current hepatitis C virus (HCV) infection?

1. Anti-HCV antibody
2. Anti-HCV antibody with reflex to HCV RNA NAT
3. HCV core antigen
4. HCV genotyping
5. HCV RNA NAT

B. Anti-HCV antibody and HCV RNA NAT. The diagnosis of current HCV infection requires both antibody and RNA tests. Answers A and E are incorrect because neither HCV antibody nor HCV RNA alone is sufficient for diagnosis of current HCV infection. Also, it is clinically very uncommon to have positive results with the NAT test alone without subsequent RNA detection, even in cases with acute HCV infection. Answer C is incorrect because HCV core antigen is less sensitive than HCV RNA and may become undetectable after seroconversion in some patients with current HCV infection. Currently, there is no FDA approved HCV core antigen assay offered in the U.S. Answer D is incorrect because a genotyping test is used for pretreatment assessment in certain patients diagnosed with a current HCV infection.

Comment Here

Reference: Hepatitis C testing

In a patient that does not have recent exposure or a known risk for hepatitis C virus (HCV) infection, what is the most likely interpretation of the hepatitis C testing results below and what is the appropriate action to take?

• Anti-HCV antibody: reactive
• HCV RNA: not detected

1. Acute HCV infection; link patient to medical care and treatment
2. Chronic HCV infection; link patient to medical care and treatment
3. False negative HCV RNA result; repeat HCV RNA testing
4. No current HCV infection; no further action is required

D. No current HCV infection; no further action is required. A negative RNA result indicates no current HCV infection. A reactive HCV antibody result could be due to past infection (true positive) or biological false positive. Answers A and C are incorrect because the clinical indication is low for a transiently negative HCV RNA during acute infection or a false negative HCV RNA result. Answer B is incorrect because HCV RNA would be positive in chronic HCV infection.

Comment Here

Reference: Hepatitis C testing