Hepatitis C Workup

Updated: Oct 07, 2019
  • Author: Vinod K Dhawan, MD, FACP, FRCPC, FIDSA; Chief Editor: BS Anand, MD  more...
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Approach Considerations

Please note that guidelines for the current diagnostic workup and management of hepatitis C (HCV) infection continue to evolve rapidly. Clinicians are advised to refer frequently to HCV Guidance: Recommendations for Testing, Managing, and Treating Hepatitis C, the most recent recommendations of the American Association for the Study of Liver Diseases (AASLD) and Infectious Diseases Society of America (ISDA). Other resources can also be found at the IDSA website.

AASLD/ISDA guidelines

The AASLD/ISDA recommend the following for initial HCV testing and followup [9] :

  • Initial HCV testing: HCV-antibody test; if the result is positive, confirm current infection with a sensitive HCV-RNA test.
  • Negative HCV-antibody test but clinical suspicion of liver disease: Test for HCV RNA or followup testing for HCV antibody if HCV exposure occurred within the past 6 months; consider testing for HCV RNA in immunocompromised individuals.
  • Reinfection after previous spontaneous or treatment-related viral clearance: Obtain initial HCV-RNA testing (because an HCV-antibody test is expected to be positive).
  • Before initiation of antiviral therapy: Obtain quantitative HCV-RNA testing to document baseline viral load.
  • Selection of the most appropriate antiviral regimen: Use HCV genotype testing for guidance.
  • Positive HCV-antibody test with negative HCV RNA by polymerase chain reaction (PCR): Inform patients they do not have evidence of current (active) HCV infection.

All patients with HCV infection are recommended to have an evaluation for advanced fibrosis with the use of liver biopsy, imaging, and/or noninvasive markers to aid in decision making regarding treatment strategies and to determine whether additional measures for the management of cirrhosis should be initiated (eg, screening for hepatocellular carcinoma). [9] Patients in whom therapy is deferred should undergo repeat liver assessment on an ongoing basis.

WHO guidelines

The World Health Organization (WHO) recommends nucleic acid testing for qualitative or quantitative HCV RNA detection as well as for test of cure at 12 or 24 weeks following antiviral treatment completion. [50] In areas with limited resources, the WHO suggests using the aminotransferase/platelet ratio index (APRI) or the fibrosis-4 (FIB-4) score for evaluating hepatic fibrosis rather than other noninvasive tests that require more resources (eg, elastography, FibroTest), as follows [50] :

  • APRI = [(AST (IU/L)/AST_ULN (IU/L))×100]/platelet count (10 9 /L)
  • FIB-4= age (years) × AST (IU/L)/platelet count (10 9)/L × [ALT (IU/L)1/2]

where ALT is alanine aminotransferase, AST is aspartate aminotransferase, IU is international unit, and ULN is the upper limit of normal.

Serologic screening for HCV involves an enzyme immunoassay (EIA). These assays are 97% specific but cannot distinguish acute from chronic infection. A rapid antibody test for HCV is available. The recombinant immunoblot assay is used to confirm HCV infection.

A meta-analysis comparing point-of-care screening tests (POCTs) with rapid diagnostic tests (RDTs) indicated that POCTs are highly accurate for diagnosing hepatitis C. [51, 52] POCTs do not require special equipment or electricity and are more robust than RDTs at high temperatures; thus, they may enable expanded screening.

Healthcare personnel who sustain a needle-stick injury involving an HCV-infected patient should undergo PCR testing for HCV immediately and then every 2 months for 6 months. If HCV infection is diagnosed, therapy can be instituted.

Other baseline studies include the following [9] :

  • Complete blood cell (CBC) count with differential
  • International normalized ratio (INR)
  • Liver function tests, including levels of ALT and AST, alkaline phosphatase, albumin, and total and direct bilirubin
  • Calculated glomerular filtration rate (eGFR)
  • Thyroid function studies
  • Screening tests for coinfection with human immunodeficiency virus ( HIV) or hepatitis B virus (HBV)
  • Screening for alcohol abuse, drug abuse, and/or depression
  • Hepatitis B virus (HBV) testing with hepatitis B surface antigen (HBsAg) (to identify coinfection), as well as hepatitis B surface antibody (anti-HBs) and antibody against hepatitis B core antigen (anti-HBc) (for evidence of previous infection)
  • Serum pregnancy testing in women of childbearing age before initiating a treatment regimen that includes ribavirin or that includes direct-acting antiviral agents (DAAs) without ribavirin

The CBC demonstrates thrombocytopenia in approximately 10% of patients. Low thyroxine levels are found in approximately 10% of patients, as well. Stress testing may be necessary in appropriate patients. An ophthalmologic examination may also be necessary.

In August 2012, the Centers for Disease Control and Prevention (CDC) expanded their existing, risk-based testing guidelines to recommend a one-time blood test for HCV infection in baby boomers, the generation born between 1945 and 1965, who account for approximately three fourths of all chronic HCV infections in the United States (see Recommendations for the Identification of Chronic Hepatitis C Virus Infection Among Persons Born During 1945–1965). [53]

In June 2013, The US Preventive Services Task Force (USPSTF) also updated its 2004 HCV screening and treatment recommendations, advocating a one-time screening for all persons born between 1945 and 1965. The new recommendation arose from the fact that a lack of universal blood screening for the virus prior to 1992 placed those born between the mid-1940s and mid-1960s at an increased risk of exposure to HCV. [54, 55, 56] It was estimated that one-time HCV testing in this population could identify nearly 808,600 additional people with chronic infection. [53]

Screening for HCV in the emergency department (ED) has been found to be feasible, albeit costly. [57, 58] All individuals identified with HCV should be screened and/or managed for alcohol abuse, followed by referral to preventive and/or treatment services, as appropriate. [53]


Hepatitis C Antibody Test

Hepatitis C virus (HCV) infection is diagnosed through the detection of antibodies to recombinant HCV polypeptides. However, antibody assays do not distinguish past from current HCV infection. For this reason, follow-up testing for HCV RNA is necessary to distinguish between ongoing or prior infection in persons with HCV antibodies.

Several generations of US Food and Drug Administration (FDA)-approved enzyme immunoassays (EIAs) to measure antibodies against NS4, core, NS3, and NS5 sequences are commercially available. [59] The third-generation assay is 97% sensitive. It can detect HCV antibody at an average of 8 weeks after the onset of infection. The recombinant immunoblot assay, previously used to confirm HCV infection, is not necessary owing to the improved sensitivity of the positive EIA tests with currently recommended higher cutoff values.

False-negative results for the presence of HCV antibody can occur in persons with compromised immune systems, such as those with human immunodeficiency virus (HIV) infection, renal failure, or HCV-associated essential mixed cryoglobulinemia. False-positive EIA results can also occur; the likelihood of a false-positive result is greater in persons without risk factors and in those without signs of liver disease, such as blood donors or healthcare workers.

In 2010, the FDA approved the OraQuick HCV Rapid Antibody Test, which can be used for persons at risk for hepatitis or for those with signs or symptoms of hepatitis. The test strip can be used with a sample collected from a fingerstick or venipuncture whole blood. [60]


Qualitative and Quantitative Assays for HCV RNA

Qualitative assays

Qualitative assays can be used to test for hepatitis C virus (HCV) RNA. HCV RNA can be detected in blood using amplification techniques such as polymerase chain reaction (PCR) or transcription-mediated amplification (TMA). The following are a few of the FDA-approved PCR-based tests for qualitative HCV RNA detection [61] :

  • COBAS AmpliScreen HCV Test, version 2.0: PCR with a lower limit of detection of 50 IU/mL
  • Hepatitis C Virus Reverse-Transcriptase (RT) PCR Assay
  • UltraQual HCV-RT PCR Assay
  • Versant HCV RNA Qualitative Assay: TMA with a lower limit of detection of 9.6 IU/mL

Quantitative assays

Quantitative assays ascertain HCV RNA quantity in blood, using signal amplification (branched DNA [bDNA] assay) or target amplification techniques (PCR, TMA). RT-PCR is more sensitive than bDNA testing. The HCV RNA level in blood helps predict the likelihood of a response to treatment, and the change in HCV RNA level can also be used to monitor the therapeutic response.

The same quantitative test should be used throughout therapy to avoid confusion, and results should be reported in international units (IU) to standardize data. The Versant HCV RNA Assay, version 3.0, is based on bDNA technology and has a dynamic range of 615-7,700,000 IU/mL. Another FDA-approved HCV quantitative test is the Aptima HCV Quant Dx Assay; its limit of detection is 3.9 IU/mL in plasma and 3.4 IU/mL in serum. [62]

The following are the best laboratory evidence of acute HCV infection:

  • A positive HCV RNA test in the setting of a negative HCV antibody test (seronegative “window” period)
  • A positive HCV antibody test after a prior negative HCV antibody test (seroconversion)

It should be noted that impaired antibody production in immunosuppressed individuals may result in misleading information.


HCV Genotyping

Hepatitis C virus (HCV) genotyping is helpful for predicting the likelihood of response and duration of treatment. Genotyping can be performed by direct sequence analysis, reverse hybridization to genotype-specific oligonucleotide probes, or restriction fragment length polymorphisms (RFLPs).

In June 2013, the FDA approved the Abbott RealTime HCV Genotype II test, which, by analyzing a sample of an infected patient’s blood plasma or serum, can differentiate HCV genotypes 1, 1a, 1b, 2, 3, 4, and 5. This test is approved for use in adult, non-immunocompromised patients with known chronic HCV infection but has not been approved for diagnostic use or as a screening test for HCV genetic material. FDA approval was based partly on a comparison of the test's accuracy with that of a validated gene-sequencing method. [63, 64]

Other genotype tests are available, including the following, although none have been approved by the FDA [65] :

  • Trugene HCV 5'NC Genotyping Kit: Based on direct sequencing followed by comparison with a reference sequence database
  • Line Probe Assay (Inno LiPA HCV II): Based on reverse hybridization of PCR amplicons on a nitrocellulose strip coated with genotype-specific oligonucleotide probes
  • Versant HCV Genotyping Assay (INNO-LiPA) 2.0: Next-generation line-probe assay

In addition to HCV genotype, a growing body of research indicates that patient genetics play a role in the response to treatment. The single-nucleotide polymorphism (SNP) rs12979860, located near the IL28B gene on chromosome 19, which encodes type III interferon, is associated with more than a two-fold difference in the rate of sustained virologic response (SVR) to antiviral treatment with pegylated interferon and ribavirin. This SNP can be detected by PCR and is an independent predictor of SVR regardless of HCV genotype. [66]


Other Testing

Della Rossa et al reported that cryoglobulins are found in as many as 50% of persons with hepatitis C viral infection. [67] Hepatitis C virus (HCV) is the primary cause of essential mixed cryoglobulinemia (ie, type 2 cryoglobulinemia); as many as 90% of affected persons have HCV viremia. Cryoprecipitates usually contain large amounts of HCV antigens and antibodies. Vasculitis, arterial hypertension, purpura, lichen planus, arthralgias, and low thyroxine levels were associated with titers positive for cryoglobulin.

Other common serologic findings in patients with chronic HCV infection include one or more of the following [68] :

  • Antinuclear antibody (ANA)
  • Rheumatoid factor
  • Anticardiolipin antibody
  • Antithyroid antibody
  • Anti–smooth muscle antibody

Additional evaluations may include testing for infection with human immunodeficiency virus (HIV), screening for susceptibility to hepatitis A and hepatitis B virus infections, and for other underlying causes of liver disease (eg, autoimmune liver disease, hemochromatosis, Wilson disease, α1-antitrypsin deficiency).


Liver Biopsy

Liver biopsy is not considered mandatory before the initiation of treatment for hepatitis C, but it may be helpful for assessing the activity and severity of hepatitis C virus-related liver disease. However, some experts recommend biopsy only in the following situations:

  • The diagnosis is uncertain
  • Other coinfections or disease may be present
  • The patient being considered for treatment has normal liver enzyme levels and no extrahepatic manifestations
  • The patient is immunocompromised

Histologic Findings

Lymphocytic infiltration, moderate degrees of inflammation and necrosis, and portal or bridging fibrosis are noted in hepatitis C. Regenerative nodules are seen in patients with cirrhosis. Some patients also may have findings indicative of hepatocellular carcinoma (HCC).

Most pathologists provide separate measurements of disease activity (grade) and fibrosis (stage). Many scoring systems are used, including the Ishak (6-point scale) and the Knodell histologic activity index (18-point score); although both scoring systems are useful for assessing improvements in histologic findings in studies, they are impractical for clinical use because of interobserver disagreement.

The METAVIR score was developed by the French METAVIR Cooperative Study Group and reported by Bedossa and Poynard in 1996 [69] ; it is frequently used in European trials. This score consists of a 3-point activity scale and 4-point fibrosis score, with good agreement among pathologists. In the United States, many pathologists use a scale described by Batts and Ludwig (Batts-Ludwig score) in 1995, [70] which consists of an activity grade (0-4) and a fibrosis stage (0-4).

HCC may occur rapidly following treatment with direct-acting antiviral agents (DAAs) in HCV-related cirrhosis; thus, patients with cirrhosis should be closely monitored after DAA therapy. [71] Most of the neoplastic HCC nodules appear to have aggressive imaging features of microvascular invasion in this setting. [71]

Noninvasive methods of assessing hepatic fibrosis are in development. Current serum assays are directed at measuring breakdown products of extracellular matrix constituents (eg, glycoproteins, propeptides) and their regulatory enzymes (eg, lysyl oxidase, lysyl hydroxylase, propyl hydroxylase).


Radiologic Studies

A liver stiffness test (FibroScan) is available as a noninvasive method of staging liver disease in persons with chronic hepatitis C. Obesity, female sex, operator inexperience, and age older than 52 may give invalid results. Falsely high estimates of liver fibrosis have also been reported with acute inflammation and recent food intake.

On December 17, 2014, the FDA gave marketing approval for the Hepatiq radiologic image processing system. [72, 73] The software application uses quantitative analysis of nuclear medicine liver-spleen images to determine the severity of liver disease and to predict clinical outcomes. [73] The developer noted that Hepatiq "automates the Quantitative Liver Spleen Scan (QLSS) that has been proven to be an accurate predictor of clinical outcomes in the recently concluded HALT-C [epatitis C ntiviral ong-term reatment against irrhosis] trial." [73] The HALT-C trial was a multicenter, randomized controlled study that evaluated whether long-term interferon would suppress HCV, prevent progression to cirrhosis, prevent liver cancer, and reduce the need for liver transplantation. [74]

In a study that compared abdominal computed tomography (CT) and laboratory data from 469 HCV-infected patients with those of histopathlogic METAVIR fibrosis scores, investigators found that the use of multiparametic CT evaluation of HCV-associated liver fibrosis further improved its diagnostic performance over that of individual parameters. [75] These parameters included hepatosplenic volumetrics, texture features, liver surface nodularity (LSN) score, and linear CT measurements, as well as the fibrosis-4 (FIB-4) score and aspartate transaminase-to-platelets ratio index (APRI). The diagnostic performance of LSN plus FIB-4 scores approached that of panels with more parameters and compared favorably with elastography. [75]