Medicine Today Internal Medicine Webcast Series

Hepatitis C Management:

Testing for Possible,Suspected, or
Documented HCV Infection

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Key Points

  • A positive enzyme immunoassay (EIA) is usually followed by HCV RNA testing to confirm antibody specificity and to document active infection.

  • A negative HCV RNA assay in a patient with a positive EIA indicates that either the infection has resolved or the initial EIA was a false-positive. The distinction can be made by a recombinant immunoblot assay.

  • Although EIA is generally regarded as the initial test for HCV, in some special circumstances HCV RNA testing should be performed regardless of EIA results.

Since the hepatitis C virus was cloned in 1989, technological advances in molecular biology have led to the development of several serologic and molecular tests to determine the presence of HCV. Clinicians and clinical investigators now have the ability to detect the virus and identify its subtypes, which facilitates the management of patients with chronic HCV infection.

Four categories of hepatitis C laboratory tests are available: (1) liver enzyme tests, (2) tests to detect antibodies to HCV, (3) tests to detect the virus, and (4) HCV genotyping.

Liver Enzyme Tests

The two liver enzymes that are measured in the evaluation of patients with HCV infection are alanine aminotransferase (ALT), also known as serum glutamate pyruvate transaminase (SGPT), and aspartate aminotransferase (AST), also known as serum glutamic oxaloacetic transaminase (SGOT) (Table 1).

Table 1: Range of normal ALT and AST values
ALT Men
Women
10-32 U/L
9-24 U/L
AST Both sexes
8-20 U/L

The reference values for normal AST and ALT levels can vary among laboratories. In general, most laboratories have used asymptomatic "normal" individuals for these determinations. It has become increasingly clear that the presence of obesity, obesity-related nonalcoholic fatty liver disease, and female gender can affect the level of ALT.4 Furthermore, liver enzyme levels can fluctuate over time and the presence of one normal value is not sufficient to determine ALT levels. Finally, liver histology may not always correlate with ALT values. Compared with patients who have elevated ALT levels, HCV-infected patients with normal ALT values appear to have liver disease that is at an earlier histologic stage and less active. However, 25% to 30% of such patients have significant histologic fibrosis, with 5% to 10% having bridging fibrosis or cirrhosis.5

Simultaneous elevations of aminotransferase levels indicate some degree of hepatocellular injury. However, the absence of any elevation does not rule out significant injury or hepatic fibrosis. Liver enzyme tests do not reveal the extent of hepatic injury or reflect the true status of hepatic function.6 In patients with risk factors for HCV infection and abnormal liver enzyme levels, HCV infection is probable but not certain. Thus, liver enzymes are neither sensitive nor specific for the diagnosis of HCV infection.

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HCV Transmission and Candidates for HCV-Specific Testing

HCV is spread primarily by contact with infected blood and blood products. Blood transfusions and the use of shared or unsterilized needles and syringes have been the primary means of HCV transmission in the United States. With the advent of routine blood screening for HCV antibody in the United States in 1991, transfusion-related transmission has almost disappeared, leaving injection-drug use as the most common risk factor for contracting HCV. Nevertheless, many patients acquire HCV without any known exposure to blood or any drug use. There appears to be a slightly increased risk of HCV infection among people with high-risk sexual behavior, multiple sexual partners, and sexually transmitted diseases as well as among people who share equipment to take cocaine intranasally.

Individuals with any risk for HCV infection should be considered for HCV testing according to Table 2.

Table 2
Risk Factor Risk of Infection Testing Recommended?
History of injecting drug use High Yes
Received clotting factors made before 1987 High Yes
Hemodialysis patients Intermediate Yes
Received blood and/or solid organs before 1992 Intermediate Yes
Evidence of undiagnosed liver disease Intermediate Yes
Infants born to infected mothers Intermediate After 12-18 mos. old
Sexual relations with multiple partners Low No*
Sexual relations with an infected partner Low No*
Healthcare/public safety workers Low Only after known exposure

*Regardless of the test results, all at-risk patients should also be provided with counseling and follow-up.7

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HCV Antibody Tests

Antibody tests are serologic assays that are based on the immunologic characteristics of HCV.8 The two types are (1) the enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA), and (2) the recombinant immunoblot assay (RIBA).

EIA. EIA is the initial serologic test used for HCV screening. Its sensitivity and specificity are excellent, and its positive predictive value in high-risk patients is quite high. A patient with a positive EIA is presumed to have HCV infection until proven otherwise; EIAs cannot distinguish between resolved and active infection. HCV antibodies usually become detectable 8 weeks following exposure. Several EIAs are available (Table 3).

Table 3: EIAs for specific HCV antigens
Assay Antigen
Abbott HCV EIA 2.0 Core, NS3, NS4
Abbott HCV EIA 3.0 Core, NS3, NS4, NS5
Abbott IMx HCV 3.0 Core, NS3, NS4, NS5
Abbott AxSYN HCV 3.0 Core, NS3, NS4, NS5
Bio-Rad Monolisa Anti-HCV Core, NS3, NS4
Bio-Rad Access HCV Ab Plus Core, NS3, NS4, NS5
Innogenetics Innotest HCV Ab IV Core, NS3, NS4, NS5
Ortho HCV 3.0 ELISA Core, NS3, NS4, NS5
Ortho Vitro Anti-HCV Core, NS3, NS4, NS5

False positives are rare now, but they were common with earlier generations of these assays. When false positives do occur, they usually do so in patients with autoimmune liver disease or hypergammaglobulinemia who have normal liver enzyme levels and no risk factors for HCV infection. False negatives are also uncommon and usually occur only in immunosuppressed patients (eg, organ transplant recipients and HIV-positive patients) and in patients on long-term hemodialysis.

The advantages of EIAs are that they are easy to use with automation, their variability is minimal, and they are relatively inexpensive (less than US $50). The primary disadvantage of EIA testing is that antibodies may not be detectable in immunosuppressed patients or early in the course of infection.

RIBA. Because the first generations of EIA tests were plagued by false positives, researchers developed the RIBA as a supplemental semiquantitative assay to refine the specificity of positive anti-HCV EIAs. RIBA can identify false-positive EIA results that are sometimes seen in patients with no apparent risk factors for HCV infection and in patients with other immune system-mediated diseases, such as rheumatoid arthritis. However, RIBAs are becoming obsolete because their function can be performed better by HCV RNA testing.9 Currently, the primary purpose of RIBA testing is to distinguish between resolved HCV infection (EIA positive, HCV RNA negative, RIBA positive) and a false-positive EIA (EIA positive, HCV RNA negative, RIBA negative).

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Virus Detection Tests

Molecular assays such as the HCV RNA test are based on the quantification and characterization of the HCV genome.9 The HCV RNA test determines the presence of the virus itself rather than its antibodies. The HCV RNA test measures the amount of HCV RNA in the blood via target amplification with reverse transcriptase polymerase chain reaction (PCR), transcription-mediated amplification (TMA), or a signal amplification technique such as a branched DNA (b-DNA) assay. Amplification is necessary because the amount of virus in serum is generally very low. Regardless of the method of amplification, HCV RNA detection represents definitive proof that an infection exists.

The sensitivity of the different types of amplification varies. TMA assays appear to be the most sensitive for detecting HCV RNA. The newest testing is done with the Fast PCR. This type of testing is shortens the typical time for PCR from 1.5 hours to 35 minutes. It may have the potential to detect relapsed HCV infection earlier than PCR. Although qualitative HCV RNA assays are more sensitive, they do not provide a quantitative value for the viral load. In fact, many laboratories do not offer qualitative testing any more.

Another testing technique integrates a PCR-based assay with laser scanning technology to excite fluorescent dyes present in the specially designed TaqMan® probes. It is a fully integrated system for real-time detection of PCR. The cycle-by-cycle detection of the increase in the amount of PCR product is quantified in real time by the special probes. TaqMan® is highly reproducibility and reliability.10

HCV RNA is customarily done at 12 and 24 weeks during the treatment course, at the end of treatment, and 6 months after treatment has been completed. However, recent information shows that HCV RNA testing at 4 weeks identifies patients with strong opportunity towards sustained virological response.

In the past, comparison of viral levels between assays was impossible. The adoption of standardized units of measurement (IU/mL) has eliminated this problem.11 It should be borne in mind that differences in HCV viral load of 0.5 log or less are within the range of testing variability and may not have clinical significance.

Several PCR-, TMA-, and b-DNA-based commercial assays are currently available. The US Food and Drug Administration (FDA) has approved two qualitative HCV RNA assays: the manual Amplicor™ version 2.0 assay and the semi-automated Cobas Amplicor™ version 2.0 assay, both of which are marketed by Roche Molecular Systems. The only quantitative HCV RNA assay that has been approved by the FDA is the Versant™ HCV RNA version 3.0 assay, marketed by Bayer Diagnostics.

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HCV Genotyping

The ability of HCV to undergo high rates of mutation allows it to escape the effects of the immune system and to resist the impact of antiviral therapy. High rates of mutation coupled with the absence of an efficient repair mechanism have resulted in a great deal of genetic heterogeneity among HCV strains. The genetic heterogeneity of HCV is reflected in a variety of genotypes (30% to 50%), subtypes (15% to 30%), isolates (5% to 15%), and HCV quasispecies (1% to 5%). In the spectrum of this genetic heterogeneity, quasispecies indicate that HCV circulates as a population of viruses that are very similar (1% to 5% differences in base pair).

HCV is classified according to different genotypes (genotypes 1 through 6) based on differences in genomic sequences. Identification of a particular HCV genotype does not predict the natural history of the disease but does have important ramifications for the likelihood of response to therapy and therapy duration. For example, patients with genotypes 2 and 3 generally respond better to treatment and do not need as long a course of therapy. On the other hand, patients with HCV genotype 1 have lower rates of response and require a longer duration of therapy (this is discussed in more detail in the section on Treatment of Uncomplicated Chronic HCV Infection).

HCV genotypes are determined by restriction fragment length polymorphism (RFLP), by direct sequence analysis, or by reverse hybridization to genotype-specific oligonucleotide probes. Once the HCV genotype has been identified, there is no need to repeat the test. HCV genotyping assays have not received FDA approval.

Different genotypes are more common in some areas of the world than in others (Figure 1).12 For example, genotype 1 is most common in the United States (accounting for 70% to 75% of all cases) followed by genotype 2 (10% to 15%). Genotypes 2 and 3 are more common in Europe than in the United States, and genotype 4 is the most common in North Africa and the Middle East.

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Clinical Utility of HCV Tests

Different tests have different capabilities for determining the diagnosis, prognosis, and treatment of HCV infection (Table 4, Table 5, and Table 6). EIA is the most widely used initial test for HCV infection because of both its accuracy and its low cost.

Table 4: HCV Tests
Diagnosis Labs
Acute HCV HCV RNA
Chronic HCV EIA; if EIA is (+), then HCV RNA
Vertical transmission HCV RNA
Occupational exposure HCV RNA, EIA
Prognosis None
Antiviral Treatment None
Decision to treat EIA, HCV RNA (+), HCV genotyping
Response evaluation HCV RNA by sensitive assay
Sustained eradication HCV RNA by sensitive assay



Table 5: HCV Tests
Diagnosis Treatment
Tests Screen Confirmation Assessing
Response
Predicting
Response & Logic
EIA X      
RIBA   X (?)    
HCV RNA qualitative   X X  
HCV RNA quantitative   X X X
HCV genotype       X
AST/ALT     X (?)  



Table 6: Interpretation of Hepatitis C Testing
Anti-HCV HCV RNA (PCR) Interpretation
Negative Negative No infection
Positive Positive Acute or chronic infection
Negative Positive Early infection
Chronic infection in immunosuppressed
Positive Negative Resolved infection
Chronic infection with low-level viremia
False-positive-antibody
Passively acquired antibody

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Testing Algorithm

A positive EIA is usually followed by an HCV RNA test to document active infection (Figure 2 and Figure 3). Since HCV RNA levels in patients with chronic HCV infection are within the range of the quantitative assays, many experts evaluate EIA-positive patients with a quantitative HCV RNA assay. In unusual cases, the HCV RNA quantitative test may be negative, but the (more sensitive) HCV RNA qualitative assay will be positive.

A negative qualitative HCV RNA assay in a patient with a positive EIA indicates that either the infection has resolved or the initial EIA was a false positive. The distinction can be made by RIBA. A positive RIBA generally indicates that an infection has cleared spontaneously. A negative RIBA indicates that the initial EIA was a false positive.

Exceptions. EIA is generally regarded as the initial test for HCV. In some cases, HCV RNA testing should be performed following a negative EIA. As mentioned earlier, the presence of conditions associated with diminished antibody production—such as immunosuppression, HIV infection, or the long-term hemodialysis—can lead to a false-negative EIA result.

Another exception to the testing algorithm concerns patients in the early stage of acute HCV infection. At the time of testing, some of these patients may not yet have developed an antibody response, which can take approximately 8 weeks to manifest. In such a case (eg, in a patient who has been recently exposed), the negative EIA may be followed by HCV RNA testing for verification.

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