Testing for Possible, Suspected, or Documented HCV Infection

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.

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 to 32 U/L 9 to 24 U/L
AST	Both sexes:	8 to 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.² 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.³

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 cause of hepatic injury or reflect the true status of hepatic function.^4,5 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.

The need to test a patient for HCV infection should be based on the patient's risk of having contracted the virus.

HCV is spread primarily by contact with 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 partners, and sexually transmitted diseases, as well as among people who use shared equipment to take cocaine intranasally.^6,7

Individuals with any risk for HCV infection should be considered for HCV testing according to the following risk categories:⁷

High risk. High-risk individuals include injection-drug users and those who received clotting factors prior to 1987. All these individuals should be tested for HCV infection.

Intermediate risk. Individuals at intermediate risk include hemodialysis patients, those with undiagnosed liver problems, and those who received blood transfusions and/or solid organs before 1992. All these individuals should be tested for HCV. Infants born to infected mothers are also at intermediate risk; testing is recommended when they reach the age of 12 to 18 months.

Low risk. Although health care and public safety workers are considered to be at low risk, testing for HCV is recommended after a possible exposure. Individuals who have sexual relations with an infected steady partner might be at low risk, but testing should still be considered.

Regardless of the test results, all at-risk patients should also be provided with counseling and continuing follow-up.^1,4,5,8-11

Antibody tests are serologic assays that are based on the immunologic characteristics of HCV.^12,13 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 2).

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. When they do occur, they do so in immunosuppressed patients (eg, organ transplant recipients and HIV-positive patients) and in patients on long-term hemodialysis.^13-16

The advantages of EIAs are that they are easy to use with automation, their variability is minimal, and they are relatively inexpensive (less than U.S. $50). The primary disadvantage of EIA testing is that detectable 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.^11-18 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).

Molecular assays such as the HCV RNA test are based on the quantification and characterization of the HCV genome.^16-19 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.¹⁶

The sensitivity of the different types of amplification varies. The TMA is the newest of the HCV RNA assays, and it is also the most sensitive.It may have the potential to detect relapsed HCV infection earlier than PCR.¹⁶ Although qualitative HCV RNA assays are more sensitive, they do not provide a quantitative value for the viral load.

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.^11,16 In the past, comparison of viral levels between assays was impossible. Adoption of standardized units of measurement (IU/mL) has eliminated this problem.^16,17,19 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 U.S. 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.

Recently it has been shown that total HCV core antigen levels correlate with HCV RNA levels. However, the utility of the HCV core antigen assay and its application to clinical practice have not yet been established.¹⁴

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 indicates that in an infected individual, 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 yet received FDA approval.

Different genotypes are more common in some areas of the world than in others (Figure 1).²⁰ 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 most common in North Africa and the Middle East.

Different tests have different capabilities in determining the diagnosis, prognosis, and treatment of HCV infection (Tables 3, 4, and 5).

EIA is the most widely used initial test for HCV infection because of both its accuracy and its low cost (Figure 2).

A positive EIA is usually followed by an HCV RNA test to document active infection. 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.