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. 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). 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". 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) . 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. 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, 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. 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.
1 Ghany MG, Strader DB, Thomas DL, Seeff LB; American Association for the Study of Liver Diseases. Diagnosis, management, and treatment of hepatitis C: an update. Hepatology. 2009;49:13PubMed Abstract
2 Gretch DR. Diagnostic tests for hepatitis C. Hepatology. 1997;26(3 Suppl 1):43S-47S.PubMed Abstract
3 Chevaliez S, Pawlotsky JM. Use of virologic assays in the diagnosis and management of hepatitis C virus infection.Clin Liver Dis. 2005;9:453-71.PubMed Abstract
4 Pawlotsky JM. Use and interpretation of virological tests for hepatitis C. Hepatology. 2002;36(5 Suppl 1):S65-73.PubMed Abstract
5 van der Poel CL, Cuypers HTM, Reesink HW, et al. Confirmation of hepatitis C virus infection by new four-antigen recombinant immunoblot assay. Lancet 1991;337:317-9.PubMed Abstract
6 Buffet C, Charnaux N, Laurent-Puig P, Chopineau S, Quichon JP, Briantais, MJ, Dussaix E. Enhanced detection of antibodies to hepatitis C virus by use of a third-generation recombinant immunoblot assay. J Med Virol 1994;43:259-61.PubMed Abstract
7 Smith BD, Teshale E, Jewett A, et al. Performance of premarket rapid hepatitis C virus antibody assays in 4 national human immunodeficiency virus behavioral surveillance system sites. Clin Infect Dis. 2011;53:780-6.PubMed Abstract
8 Centers for Disease Control and Prevention. Recommendations for prevention and control of hepatitis C virus (HCV) infection and HCV-related chronic disease. MMWR 1998;47(No. RR-19):1-39. Available at: CDC and Prevention
9 Alter MJ, Kuhnert WL, Finelli L. Guidelines for laboratory testing and result reporting of antibody to hepatitis C virus. Centers for Disease Control and Prevention. MMWR Recomm Rep 2003;52:1-16. Available at:CDC and Prevention
10 McHutchinson JG, Person JL, Govindarajan S, et al. Improved detection of hepatitis C virus antibodies in high-risk populations. Hepatology 1992;15:19-25.PubMed Abstract
11 Nakatsuji Y, Matsumoto A, Tanaka E, Ogata H, Kiyosawa K. Detection of chronic hepatitis C virus infection by four diagnostic systems: first-generation and second-generation enzyme- linked immunosorbent assay, second-generation recombinant immunoblot assay and nested polymerase chain reaction analysis. Hepatology 1992;16:300-5.PubMed Abstract
12 Chien DY, Choo QL, Tabrizi A, et al. Diagnosis of hepatitis C virus (HCV) infection using an immunodominant chimeric polyprotein to capture circulating antibodies: reevaluation of the role of HCV in liver disease. Proc Natl Acad Sci USA 1992;89:10011-5.PubMed Abstract
13 Courouce AM, Le Marrec N, Girault A, Ducamp S, Simon N. Anti-hepatitis C virus (anti-HCV) seroconversion in patients undergoing hemodialysis: Comparison of second- and third- generation anti-HCV assays. Transfusion 1994;34:790-5.PubMed Abstract
14 Vallari DS, Jett BW, Alter HJ, Mimms LT, Holzman R, Shih JW. Serological markers of posttransfusion hepatitis C viral infection. J Clin Microbiol 1992;30:552-6.PubMed Abstract
15 Cox AL, Netski DM, Mosbruger T, et al. Prospective evaluation of community-acquired acute-phase hepatitis C virus infection. Clin Infect Dis. 2005;40:951-8.PubMed Abstract
16 Stramer SL, Glynn SA, Kleinman SH, et al. Detection of HIV-1 and HCV infections among antibody-negative blood donors by nucleic acid-amplification testing. N Engl J Med 2004;351:760-8.PubMed Abstract
17 Thio CL, Nolt KR, Astemborski J, Vlahov D, Nelson KE, Thomas DL. Screening for hepatitis C virus in human immunodeficiency virus- infected individuals . J Clin Microbiol 2000;38:575-7.PubMed Abstract
18 Thomas DL, Astemborski J, Rai RM, et al. The natural history of hepatitis C virus infection: host, viral, and environmental factors. JAMA 2000;284:450-6.PubMed Abstract
19 Conry-Cantilena C, VanRaden M, Gibble J, et al. Routes of infection, viremia, and liver disease in blood donors found to have hepatitis C virus infection. N Engl J Med. 1996;334:1691-6. PubMed Abstract
20 Forman MS, Valsamakis A. Verification of an assay for quantification of hepatitis C virus RNA by use of an analyte-specific reagent and two different extraction methods. J Clin Microbiol 2004;42:3581-8.PubMed Abstract
21 Vermehren J, Kau A, Görtner BC, Göbel R, Zeuzem S, Sarrazin C. Differences between two real-time PCR-based hepatitis C virus (HCV) assays (RealTime HCV and Cobas AmpliPrep/Cobas TaqMan) and one signal amplification assay (Versant HCV RNA 3.0) for RNA detection and quantification. J Clin Microbiol. 2008;46:3880-91. PubMed Abstract
22 Schröter M, Feucht HH, Schäfer P, Zöllner B, Polywka S, Laufs R. Definition of false-positive reactions in screening for hepatitis C virus antibodies. J Clin Microbiol. 1999;37:233-4.PubMed Abstract
23 Alberti A, Chemello L, Cavalletto D, et al. Antibody to hepatitis C virus and liver disease in volunteer blood donors. Ann Intern Med. 1991;114:1010-2.PubMed Abstract
24 Lee SR, Yearwood GD, Guillon GB, et al. Evaluation of a rapid, point-of-care test device for the diagnosis of hepatitis C infection. J Clin Virol. 2010;48:15-7.PubMed Abstract
25 Lee SR, Kardos KW, Schiff E, et al. Evaluation of a new, rapid test for detecting HCV infection, suitable for use with blood or oral fluid. J Virol Methods. 2011;172:27-31. PubMed Abstract
26 Damen M, Zaaijer HL, Cuypers HTM, Vrielink H, van der Poel CL, Reesink HW, Lelie PN. Reliability of the third-generation recombinant immunoblot assay for hepatitis C virus. Transfusion 1995;35:745-749.PubMed Abstract
27 Strader DB, Wright T, Thomas DL, Seeff LB. Diagnosis, management, and treatment of hepatitis C. Hepatology 2004;39:1147-71. PubMed Abstract
28 National Institutes of Health Consensus Development Conference Statement: Management of hepatitis C: 2002--June 10-12, 2002. Hepatology 2002;36:S3-20. Available at: NIH Consensus Development Program
29 McGuinness PH, Bishop GA, Lien A, Wiley B, Parsons C, McCaughan GW. Detection of serum hepatitis C virus RNA in HCV antibody-seropositive volunteer blood donors. Hepatology 1993;18:485-90.PubMed Abstract
30 Vrielink H, van der Poel CL, Reesink HW, et al. Look-back study of infectivity of anti-HCV ELISA-positive blood components. Lancet 1995;345:95-6.PubMed Abstract
31 Sarrazin C, Teuber G, Kokka R, Rabenau H, Zeuzem S. Detection of residual hepatitis C virus RNA by transcription-mediated amplification in patients with complete virologic response according to polymerase chain reaction-based assays. Hepatology 2000;32:818-23.PubMed Abstract