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Viral Hepatitis and Liver Disease

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Laboratory Tests and Hepatitis C

for Health Care Providers

Laboratory Tests - Hepatitis C


Laboratory tests for hepatitis C are divided into four general categories:

  • Screening: Screening for hepatitis C virus (HCV) is done with a serologic test for the HCV antibody (Ab).
  • Confirmatory: Diagnosis of chronic hepatitis C requires the presence of HCV RNA, commonly called hepatitis C viral load.
  • Genotype: Once it is determined that HCV RNA is present, the specific genotype and subtype of the virus can be determined with a genotype test.
  • Drug resistance: Mutations of some proteins in HCV can allow the virus to have resistance to direct-acting antivirals (DAAs), commonly referred to as resistance-associated variants (RAVs) or resistance-associated polymorphisms (RAPs).

HCV Serologic Testing (HCV Ab)

Enzyme immunoassays for Detection of Hepatitis C Antibody

The HCV Ab test is used for initial screening for hepatitis C. The test is performed by enzyme immunoassays (EIAs), which detect the presence of hepatitis C antibodies in serum. The result of the test is reported as positive or negative. Third-generation EIAs have a sensitivity/specificity of approximately 99%. However, the presence of HCV Ab does not indicate whether the infection is acute, chronic, or resolved. A positive antibody test result should be followed up with an HCV RNA test to confirm that viremia is present.

False-Negative and False-Positive HCV Ab Results

Despite the extremely high sensitivity and specificity of the EIA test for the antibody, it is still possible to have both false-positive and false-negative results.

False-Negative HCV Ab

  1. A false-negative HCV Ab result may occur if the test is performed during the window period after acute HCV infection but before seroconversion (when the HCV Ab converts from negative to positive). The average time from infection until seroconversion is 8 weeks and is referred to as the "serologic window." If acute infection is suspected to have taken place within the past 8 weeks, it would be appropriate to order the HCV RNA test. If the HCV Ab test result is negative within the first 8 weeks after infection, it would be appropriate to retest the antibody after 8 weeks to check for seroconversion.
  2. A false-negative HCV Ab result may also occur in immunocompromised individuals such as those infected with HIV, recipients of organ transplants, and patients receiving chronic hemodialysis. If the HCV Ab result is negative in immunocompromised patients, but there is strong suspicion of HCV infection, it would be appropriate to order the HCV RNA test.

False-Positive HCV Ab

  1. A false-positive HCV Ab result may occur because of cross-reactivity with other viral antigens or the presence of immunologic disorders, such as lupus or rheumatoid arthritis.

Time for Processing HCV Ab Test Results

The turnaround time for 3rd-generation EIAs is at least 1 day. Many labs do not perform the tests on site and must send specimens to another lab for processing, which may further increase the turnaround time.

A point-of-care test is also available. The OraQuick® HCV Rapid Antibody Test is an FDA-approved test that can be performed with a fingerstick (or venous blood draw). It is also a CLIA-waived test and therefore can be used in clinic offices and outreach facilities. Results are reported as reactive or nonreactive within 20 minutes. Just as for the standard HCV Ab test done in the lab, a positive OraQuick® test must be confirmed by an HCV RNA test. The sensitivity and specificity of the test is similar to that of the laboratory-based assays.

Recombinant Immunoblot Assay for Confirmation of HCV Ab

Recombinant immunoblot assay (RIBA) is a highly specific test that in the past was used as a confirmatory test of antibody results. It still required HCV RNA testing for the diagnosis of chronic infection. The RIBA test is no longer in use or available in the United States.

HCV RNA Testing

The presence of HCV RNA is required to confirm chronic HCV infection. Therefore, a positive HCV Ab screening result must be followed by a test for the HCV RNA. The HCV RNA tests can detect virus within 1-2 weeks following exposure.

Appropriate Uses of the HCV RNA Test

There are 4 major reasons that HCV RNA tests are used:

  1. To confirm a positive HCV Ab result and make the diagnosis of current HCV infection
  2. To measure a patient's baseline viral load prior to starting HCV therapy
  3. To monitor a patient's response to therapy
  4. To determine whether a patient has achieved a sustained virologic response (SVR)

More rarely, HCV RNA is used when either very acute HCV infection is suspected or a false HCV Ab is suspected.

It would not be appropriate to repeatedly order HCV RNA viral load screening for a patient who is not on or was recently on HCV treatment, or to use the HCV viral load to determine the severity of the patient's infection or the patient's risk of developing significant liver disease.

HCV Antibody and HCV RNA Test Result Interpretations
Test OutcomeInterpretationFurther Action

HCV Ab nonreactive = negative

HCV Ab reactive = positive

HCV Ab negativeNever exposed to HCV.
  • If the antibody result is negative (nonreactive), no further action is required.
  • If acute HCV is suspected, test for HCV RNA, as the HCV Ab result may not be positive yet.
HCV Ab positiveIndicates exposure to HCV. Test for HCV RNA to determine chronic or resolved infection.
  • HCV Ab is positive in either active HCV infection or past HCV infection that has resolved. It is also possible to have a false-positive antibody result. The next step is to test for HCV RNA to determine if there is active infection.
  • Once the HCV Ab result is positive, the test does not need to be repeated in the future. The result will remain positive.
  • The antibody result also will remain positive after successful HCV treatment (achievement of an SVR).
HCV Ab positive, HCV RNA detectedActive HCV infection. If infection occurred >6 months ago, this is chronic HCV infection.
  • Provide the patient who was tested with appropriate counseling and link that person to medical care, including treatment.
HCV Ab positive, HCV RNA not detectedNo active HCV infection. Patient spontaneously cleared HCV and did not become chronically infected, or was successfully treated and achieved SVR.
  • No further action required in most cases
  • If there is concern about a false-positive antibody screen, consider a repeat HCV Ab test using a different assay.

HCV Reflex Testing

Per VA policy, an HCV RNA test is automatically performed on all positive HCV antibody specimens (reflex testing). The reflex process helps in the clinical management of the patient by avoiding the need for a patient to return for a second blood draw if the antibody result is positive.

Meaning of HCV Viral Load

The number of HCV RNA international units per milliliter of blood (i.e., the viral load) must be measured before treatment and during the course of treatment, to assess response. Before treatment, however, the HCV viral load is not related to the patient's liver disease severity or HCV prognosis. This is important for patients and providers to understand.

Note: In hepatitis B, unlike hepatitis C, a higher HBV DNA viral load does correlate with increased disease severity and increased likelihood of outcomes such as hepatocellular carcinoma.

Specific HCV RNA Assays and Range of Detectable Virus

HCV RNA tests use target amplification techniques. Several assays exist for HCV RNA testing. Methods include polymerase chain reaction (PCR), transcription mediated amplification (TMA), and branched chain DNA (bDNA) tests. Results are expressed as international units/mL (IU/mL). The different methods and different commercial assays each have a lower limit of quantification (LLOQ) and lower limit of detection (LLOD), therefore a patient's results could be reported differently depending on the assay used. HCV RNA tests must have an LLOQ of 25 IU/mL or lower when used to assess treatment response with DAAs.

LLOQ = the lowest HCV RNA level that is within the linear and analytically acceptable range of the assay.

LLOD = the lowest level of HCV RNA that is detected ≥95% of the time.

Interpreting HCV RNA Test Results

It is essential that the provider understands how to interpret HCV RNA test results, especially during the course of HCV treatment.

Result of HCV RNA TestInterpretation
A quantified viral load -- any exact numberOngoing HCV infection
"Detected"The HCV RNA is detectable but the number of international units is so low that it cannot be quantified accurately. This indicates extremely low level of virus is present.
"<12 IU/mL" or "<15 IU/mL" or "<25 IU/mL" All of these are "less than the LLOQ" HCV RNA is undetectable. No virus is detected at all in the patient's serum specimen.

HCV Genotype Testing

There are at least six HCV genotypes. These are classified as genotypes 1-6. There are also 30 subtypes of HCV, which are referred to as genotypes 1a, 1b, 2a, etc. Identifying HCV genotypes is essential for selecting treatment regimens and predicting treatment response. Within genotype 1, it is also important to determine whether the patient is subtype 1a or 1b, as this determines treatment duration and the need for ribavirin in the treatment regimen. Patients only need to be genotype tested once in their lifetime, as the genotype remains the same throughout the course of infection. Repeating a genotype test is warranted only if there is suspicion that a patient may have been reinfected with a different genotype after achieving an SVR.

Genotype testing is performed by analyzing the sequences of various regions of the HCV genome. Most genotype assays rely on the amplification of short HCV RNA regions from clinical specimens, followed by a type-specific assay, such as restriction fragment length polymorphism (RFLP) analysis, line probe reverse hybridization, or sequence analysis. Most assays target the 5' untranslated region (5' UTR), as it is the most conserved region throughout the HCV genome and is most suitable for reverse transcription polymerase chain reaction (RT-PCR) amplification.

HCV Resistance Testing (RAV testing)

DAAs are drugs that target specific steps in the life cycle of the hepatitis C virus. When these steps are disrupted, replication of HCV is stopped. DAA drug classes include NS5A inhibitors, NS5B polymerase inhibitors, and NS3/4A protease inhibitors. Resistance Associated Variants (RAVs) refer to mutations that occur in the target enzymes that confer resistance to DAAs. RAV testing is done in most patients who have failed a prior DAA-containing regimen before they initiate re-treatment with another DAA regimen. For example, genotype 3 patients are recommended to have RAV testing if they are treatment experienced before starting re-treatment with sofosbuvir/velpatasvir and to determine whether ribavirin is needed. RAV testing is occasionally done in treatment-naive patients if it may change the regimen or the duration of treatment. For example, genotype 1a patients who are treatment naive should be RAV tested before starting treatment with elbasvir/grazoprevir to determine whether ribavirin is needed or whether an extended duration of treatment is needed. Genotyping of the NS5A, NS5B, and NS3/4A genes to identify RAVs can now be accomplished by RT-PCR and population-based sequencing methods at the VA Palo Alto Public Health Reference Laboratory (PHRL) and at commercial laboratories including Monogram Biosciences (LabCorp) and Quest Diagnostics. HCV drug resistance testing should be ordered only by experienced HCV clinicians.


  1. Centers for Disease Control and Prevention. Testing for HCV infection: an update of guidance for clinicians and laboratorians. MMWR Morb Mortal Wkly Rep. 2013 May 10;62(18):362-5.
  2. Carrazin C. The importance of resistance to direct acting antiviral drugs in HCV infection in clinical practice. J Hepatol. 2016 Feb;64(2):486-504.