Antiviral Therapy 2012; 17:1431–1435 (doi: 10.3851/IMP2463)

Review The potential role of HCV core antigen testing in diagnosing HCV infection George J Dawson1* Infectious Disease Research and Development, Abbott Laboratories, Abbott Park, IL, USA

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*Corresponding author e-mail: [email protected]

The potential uses of serological tests that detect HCV core antigens in biological fluids are highlighted. The most common serological tests utilized to detect exposure to HCV rely on the detection of antibodies to HCV. However, these tests cannot distinguish between individuals who have resolved their infection and those who remain actively infected with HCV. By contrast, the HCV core antigen test detects circulating HCV core antigen and identifies individuals who are actively infected with HCV.

There is increasing interest in using the HCV core antigen test as a reflex test for seropositive individuals to identify individuals who are actively infected with HCV. In addition, the HCV core antigen test can be utilized to detect the early phase of HCV infection prior to the development of antibodies, both in the blood bank setting and in the diagnostic laboratory. Lastly, quantitative versions of the HCV core antigen test can be used to monitor the effectiveness of antiviral therapy.

Introduction Shortly after the realization that many cases of both community-acquired and transfusion-associated hepatitis were not attributable to hepatitis A virus (HAV) or HBV, efforts were initiated to discover the aetiological agent(s) responsible for these cases of non-A, non-B hepatitis. Classical viral discovery methods were initially applied to identify the agent(s), followed by efforts to discover the agent(s) using molecular-based technologies. In 1988, one of the agents was identified utilizing a molecular-based immunoscreening technique; the discovered agent was renamed HCV. In the process of its discovery, epitopes were identified that were useful both in blood donor screening tests and diagnostic tests. The first-generation antibody test for HCV utilized a recombinant antigen encoded by a non-structural protein (NS4) of the HCV genome. The next generation of tests improved detection of HCV-infected individuals by supplementing the NS4 recombinant protein with other HCV recombinant proteins, including the NS3 protein, the core protein and, in some cases, the NS5 protein, all encoded by the single open reading frame of the HCV RNA genome. The progression of events that led to the successful commercialization of serological tests for HAV, HBV or for HIV was quite different from the events leading to the successful introduction of serological tests for HCV. For the former viruses, the initial antibody tests utilized native viral proteins, followed by the generation of suitable ©2012 International Medical Press 1359-6535 (print) 2040-2058 (online)

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recombinant antigens that would replace the native viral proteins. For HCV, there was no access to native viral proteins during the early stage of assay development, and serological tests were confined to the use of synthetic peptides and recombinant proteins. Furthermore, for HBV, and to some extent for HIV, serological tests are utilized to detect circulating viral antigens indicating early active infection with these viruses (for example, p24 antigen detection for HIV) or to diagnose active infection (for example, hepatitis B surface antigen for HBV infection). Thus, after the discovery of HCV, considerable efforts were undertaken to continue to pursue classical viral studies, with attempts to explore the use of native viral proteins in antibody tests, to adapt the virus to cell culture, and to determine the utility of serological tests that detect circulating antigen in serum of HCV-infected individuals. This review focuses on the studies that detect circulating HCV core antigens in serum. The first published results that identified HCV viral antigen in the serum of HCV-infected individuals was reported in 1992 [1]. This pioneering study indicated that circulating HCV core antigen could be detected via an enzyme-linked immunosorbent assay (ELISA) sandwich antigen test. The method required that large volumes of HCV-seropositive plasma be subjected to buoyant density centrifugation followed by treatment with detergents to expose viral proteins, prior to performing the ELISA. In  1431

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Table 1. Detection of HCV genotyped samples: the ARCHITECT HCV Core Antigen Test compared with the Amplicor HCV Monitor 2 Assay Genotype Specimens, n

Detection, % (n/total n) ARCHITECT HCV Core Antigen Test Amplicor HCV Monitor 2 Assay

1 1a 1a/1b 1b 2 2a 2a/2c 2b 3a 3k 4 4a 4a/4c 4c/4d 4c/4d/43 5/5a 6a 6i Total

100 (3/3) 100 (53/53) 100 (10/10) 100 (42/42) 100 (1/1) 100 (2/2) 100 (4/4) 100 (20/20) 100 (24/24) 100 (2/2) 100 (1/1) 100 (16/16) 67 (2/3) 100 (3/3) 100 (1/1) 100 (9/9) 100 2/2) 100 (1/1) 99.5 (196/197)

3 53 10 42 1 2 4 20 24 2 1 16 3 3 1 9 2 1 197

100 (3/3) 100 (53/53) 100 (10/10) 100 (42/42) 100 (1/1) 100 (2/2) 100 (4/4) 100 (20/20) 88 (21/24) 100 (2/2) 100 (1/1) 100 (16/16) 67 (2/3) 100 (3/3) 100 (1/1) 100 (9/9) 100 (2/2) 100 (1/1) 98.0 (193/197)

Reprinted from [3] with permission from Elsevier. A total of 197 samples positive for HCV RNA via the Amplicor HCV 2.0 Qualitative Assay (Roche Diagnostics, Indianapolis, IN, USA) were tested with the ARCHITECT HCV Core Antigen Test (Abbott Laboratories, Abbott Park, IL, USA) and the Amplicor HCV Monitor 2 Assay (Roche Diagnostics).

1995, it was demonstrated that HCV core antigen could be detected and quantified in serum via a simple fluorescent enzyme immunoassay [2]. This procedure required a precipitation step followed by solubilization of the pellet both to reveal the HCV core antigen and to destroy antibodies that may prevent detection of the HCV core protein. The processed sample could then be tested for HCV core antigen. Since these early reports, methodologies have evolved and become more simplified, and at least one assay (the ARCHITECT HCV Core Antigen Test; Abbott Laboratories, Abbott Park, IL, USA) is fully automated [3]. There have been numerous studies indicating the potential utility of HCV core antigen testing in three different types of situations: to distinguish actively infected HCV-­ seropositive individuals from those who have resolved HCV infection; to identify HCV infection in the pre-seroconversion window period; and to monitor the efficacy of antiviral therapy using a quantitative HCV core antigen assay. This review provides a brief overview of key features in development of these three areas.

Identification of active HCV infection in seropositive individuals Approximately 30% of HCV-infected individuals resolve their infection, while 70% of infected 1432 

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individuals develop chronic infection characterized by the continuous detection of both antibodies to HCV and HCV RNA [4]. There have been numerous reports documenting the ability of the HCV core antigen test to confirm HCV infection in seropositive individuals, with studies conducted during the early days of HCV core antigen testing [1,2,5] to more recent studies using more sensitive methodologies [3,6–9]. In all these studies, the detection of HCV core antigens in seropositive individuals requires that specimens be treated with agents in order to inactivate antibodies and disrupt immune complexes. As reported previously [8], a commercialized version of the HCV core antigen enzyme immunoassay detected approximately 1.5 pg/ml of HCV core antigen (corresponding roughly to 27,000–30,000 IU/ml HCV RNA). A more recently commercialized test (the ARCHITECT HCV Core Antigen Test) had a sensitivity of approximately 0.06 pg/ml or (3 fmol/l), resulting in a significant increase in sensitivity over the previous assay and a stronger correlation with HCV RNA testing. The ARCHITECT HCV Core Antigen Test detected most or all of the samples from a collection of HCV genotyped samples, and was comparable to the Amplicor HCV RNA Test (Roche Diagnostics, Indianapolis, IN, USA) in identifying HCV-infected individuals [3] (Table 1). In a recent study, the ARCHITECT HCV Core Antigen Test detected 112 of 118 HCV-RNA-positive ©2012 International Medical Press

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HCV core antigen testing

Figure 1. Possible algorithm for HCV diagnostics

Possible algorithm Anti-HCV

Positive

Negative

Active replication?

Highly suspicious for HCV infection (for example, elevated ALT in patients at risk for hepatitis C) despite negative anti-HCV

HCV core antigen

Negative

Positive

Previous infection

Active infection

Highly suspicious for HCV infection (for example, elevated ALT) despite negative HCV core antigen

HCV core antigen

Ongoing monitoring

Positive

Negative

HCV core antigen

Active infection

Highly suspicious for HCV

HCV RNA

HCV RNA

Reprinted from [7] with permission from Elsevier. ALT, alanine aminotransferase; anti-HCV, antibody against HCV core antigen.

samples, with 5 of the 6 negative samples having HCV RNA levels 3,000 IU/ml, and detected approximately 20% of samples with HCV RNA levels