Discussion

Overview of HCV Diagnostic Tests

The laboratory tests used to diagnose hepatitis C virus (HCV) infection consist of serologic assays that detect human antibodies against HCV (anti-HCV) and molecular assays that detect HCV nucleic acid[1,2,3,4]. The serologic tests used to diagnose HCV include three different types of assays that can detect antibodies to hepatitis C virus: enzyme immunoassay (EIA), chemiluminescent assay (CIA), and recombinant immunoblot assay (RIBA). The 3rd generation EIA test is now the dominant HCV screening test used in clinical practice and this assay detects antibodies against epitopes derived from the HCV core, nonstructural 3, nonstructural 4, and nonstructural 5 regions. The RIBA originally was developed as a high-specificity confirmatory test for patients with a positive EIA result, but the importance of the RIBA has diminished with the marked improvement in specificity of the 3rd generation EIA tests and with the more widespread use of molecular assays. The recombinant immunoblot assay (RIBA) identifies the specific antigens to which antibodies are reacting in the EIA, and the results are interpreted as positive (2 or more antigens), indeterminate (1 antigen), or negative (0 antigens) [5,6]. More recently, a point of care "rapid" HCV immunoassay antibody test has been approved for diagnostic purposes, providing another type of initial antibody screening test; this rapid test is an immunoassay that contains proteins from the HCV core, NS3, and NS4 regions[7]. The molecular assays used detect HCV RNA are typically categorized as qualitative or quantitative assays. The US FDA has approved multiple qualitative and quantitative HCV RNA assays; the molecular HCV RNA assays are also referred to as nucleic acid tests (NAT) or nucleic acid amplification tests (NAAT).

Initial Screening Tests for HCV Infection

Although the optimal strategy for diagnosing chronic hepatitis C virus (HCV) infection can vary, all testing algorithms begin with the use of a serologic assay (EIA or CIA) that detect antibodies to HCV. The 3rd generation HCV EIA is currently widely used as the initial screening test to diagnose chronic HCV infection and it has an estimated 98% sensitivity for detecting antibody to HCV[8,9,10,11,12,13,14]. Indeed, the sensitivity is almost 100% in healthy adults with chronic HCV infection. The HCV EIA test can represent a false-negative result in patients with acute (or recent) HCV infection[13,14,15], patients on long-term hemodialysis, and immunosuppressed persons (including those with advanced HIV infection and organ transplant recipients)[17]. In all other cases, the HCV EIA serves as an outstanding screening test for HCV infection because of its high sensitivity and relatively low cost. The 3rd generation HCV EIA tests has good specificity, but the predictive value of a positive result varies substantially based on the pre-test probability of HCV infection. For example, in injection-drug users, a group at greater risk for HCV infection, all positive HCV EIA results likely represent "true positives"[18]. False-positive results can occur in some patients who have autoimmune liver disease or hypergammaglobulinemia. With the EIA test, a signal-to-cutoff ratio can be calculated and used as a predictor of true positive tests. The signal-to-cutoff ratio is calculated by dividing the optical density value of the patient sample by the optical density of the assay cutoff for that specific run. It is important to understand that all positive EIA results should be verified with an independent supplemental test of high specificity (HCV RNA testing or RIBA) [19]. The serologic tests can establish that a person has been infected with HCV at some point in time, but they do not distinguish active from past (resolved) infection.

Supplemental (Confirmatory) Tests for HCV Infection

All positive HCV screening serologic tests require confirmation. The major tests used for supplemental (confirmatory) testing include HCV RNA tests and HCV RIBA tests. The RIBA test has very high specificity and can determine whether a positive EIA test represents true HCV infection. A positive RIBA, however, does not distinguish whether the patient has active or resolved HCV infection. An indeterminate RIBA can represent a false-positive result, especially if the individual tested has low risk for HCV infection. An indeterminate result can also occur transiently in individuals with acute HCV infection who are in the process of seroconversion and occasionally in chronically infected individuals. In such cases, a repeat RIBA at least 1 month later or HCV RNA testing may clarify the situation. The EIA-positive, RIBA-indeterminate sera in a true positive situation will typically contain patterns that show reactivity to core (c22-3) or NS3 antigens (c33-c bands), which are less prone to cross-reactivity than NS5 antigens.

The molecular HCV RNA tests provide an advantage over the RIBA, since a positive HCV RNA test both verifies the HCV EIA and establishes the presence of chronic HCV infection. The HCV RNA tests used for diagnostic purposes include the qualitative and quantitative assays. Previously, the qualitative HCV RNA test was the most common molecular test used for HCV diagnosis, primarily because of the very high sensitivity. The qualitative assay, however, only determines the presence or absence of HCV RNA. In contrast, a quantitative HCV RNA test can determine whether chronic HCV infection is present and generate an HCV RNA level, which can provide prognostic information for treatment. Further, in recent years, real-time PCR technology for quantitative HCV RNA testing has become widely available and real-time PCR has been reported to be as sensitive as qualitative assays, providing a very low limit of detection and a broad dynamic quantitative range across the different HCV gentoypes[20,21]. Most experts now recommend using quantitative HCV RNA assays (typically real time PCR assays) as the preferred supplemental test for persons with a positive HCV antibody screening test.

Approach to Diagnosing Chronic HCV Infection

General Approach: Different testing algorithms have been utilized to evaluate patients for chronic HCV infection and some experts have suggested the optimal approach depends on the patient's underlying risk profile, the goals of testing, and the setting in which future care will be provided. The most common contemporary strategy (Figure 1) to diagnose chronic HCV infection consists of initial screening with an HCV EIA antibody test (to determine whether the patient has ever been infected with HCV), followed by supplemental testing of positive screening tests with a quantitative HCV RNA assay (to confirm the positive EIA and to determine whether they have active or resolved hepatitis C infection). In patients with a positive EIA and negative HCV RNA, either the RIBA or the EIA signal-to-cut-off ratio can be used to sort out whether the patient has resolved HCV infection or a false-positive EIA.

EIA Negative: If the initial EIA test is negative, the patient is considered not infected with HCV, and, in general, no further testing for HCV is needed. The exception to this approach occurs in patients with an increased likelihood of having a false-negative EIA test (acute HCV infection, hemodialysis, and immunocompromising diseases); in these situations, a negative EIA should be followed by a HCV RNA test.

EIA Positive: All patients with a positive EIA screening test should undergo supplemental testing, preferably with an HCV RNA test. Patients with a positive EIA and a positive HCV RNA have confirmed active HCV infection and should undergo further medical evaluation for hepatitis C. A positive EIA screening test followed by a negative HCV RNA test most commonly occurs in patients who were infected with HCV but spontaneously cleared the virus, or in those with a false-positive EIA screening test. The strategy of using HCV RNA testing to follow up on a positive EIA tells a provider what they need to care for the patient but, if negative, it does not differentiate a false positive EIA from resolved HCV infection. In this situation (positive EIA and negative HCV RNA), a RIBA can be performed to sort out these two possible scenarios. If the RIBA is negative (positive EIA, negative HCV RNA, negative RIBA), the initial EIA is considered as a false-positive result and the patient is considered not infected with HCV. If the RIBA is positive (positive EIA, negative HCV RNA, positive RIBA) the patient most likely has resolved HCV infection. Alternatively, the EIA signal-to-cut-off ratio can be used in this situation, with a high signal-to-cut-off ratio indicating resolved HCV infection and a low signal-to-cut-off ratio indicating a false reactive EIA. Another option is to repeat the HCV RNA test to make sure that the first HCV RNA test was not falsely negative due to a technical laboratory error or because there are rare instances in which HCV RNA is intermittently undetectable in persons with chronic HCV infection. Any patient who has evidence of either false positive EIA or resolved infection should undergo repeat screening with HCV RNA if they have an exposure to HCV in the future.

Strategies with Low Pretest Probability of Infection

Some experts have recommended a different approach to the HCV testing process for patients who have no risk factors for HCV and thus have a low pretest probability of having a true positive HCV test. This scenario most often occurs when testing for HCV in healthy blood donors or persons undergoing a medical evaluation for a life insurance policy. In this situation, a positive HCV EIA result would be unexpected and some experts would recommend using the RIBA (not the HCV RNA) for supplemental testing. The rationale for this approach is that RIBA is better (and more cost-effective) than HCV RNA for sorting out possible false-positive EIA tests in persons unlikely to have chronic HCV. If the RIBA is negative (EIA-positive and RIBA-negative) the EIA test should be considered a false-positive test result and the patient considered not infected with HCV. Several studies have demonstrated that HCV RNA is not detected in sera from these patients, and these individuals demonstrate no clinical signs of hepatitis on follow-up[22,23]. Thus, patients with EIA-positive, RIBA-negative sera do not require further testing. If the RIBA is positive (EIA-positive and RIBA-positive), the patient has either resolved or active infection and HCV RNA testing should be performed to determine to distinguish the two.

Use of Rapid HCV Screening Tests

In 2010, the FDA issued approval for the first rapid HCV test—the OraQuick HCV Rapid Antibody Test[24]. As of March 2012, this test was approved for use in whole blood samples collected by either fingerstick or venipuncture in individuals 15 and older. This test also can be performed with oral swab samples[25], but the oral swab test is not yet FDA approved. The OraQuick HCV Rapid Antibody test is a point-of-care test that that provides results in 20 minutes. The rapid HCV antibody test provides increased feasibility of testing in outreach settings, such as syringe program sites and methadone clinics, where persons who inject drugs or who may not regularly access health care receive services. In addition, the point-of-care testing allows for prompt delivery of on-site test results without requiring clients to return on another day. All reactive OraQuick HCV Rapid Antibody tests are considered a preliminary positive result and require supplemental testing. Confirmation is particularly important given some reported variability in sensitivity (79 to 99%) and specificity (80 to 100%) depending on sites and tests[7]. Two additional rapid tests have been developed but do not have FDA approval: Chembio PPP HCV test and the Multiplo Rapid HIV/HCV Antibody Test.

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    Figure 1. CV Testing Algorithm

    Abbreviations: EIA = enzyme immunoassay; RIBA=recombinant immunoblot assay

    Figure 1