Hepatitis C testing today

In the United States, the incidence of what is now known as hepatitis C virus (HCV) infection was low prior to 1965, but it increased steadily over the next 25 years, primarily due to blood transfusions and to sharing of used needles and syringes.¹ HCV was identified as the main agent for post-transfusional (nonA-nonB) hepatitis in 1988, when a cDNA library derived from infectious material was constructed in phage vectors and expressed in Escherichia coli. The screen of ~106 recombinant phages resulted in identification of one positive clone expressing viral antigen.² Further analysis showed that HCV is an enveloped single-stranded positive-sense RNA virus with a remarkably heterogeneous genome of ~9.5 kb. The 5′ terminus untranslated region (UTR) and the terminal 98 nucleotides of the 3′ UTR are highly conserved; however, at the nucleotide level, there is a 31% to 33% difference between genotypes and 20% to 25% between subtypes. Genomic sequence heterogeneity leads to classification of HCV in six genotypes and numerous subtypes.³ Its heterogeneity and high mutation rate are responsible for challenges in vaccine development and for differences in treatment responses.

HCV virus identification led to the development of serologic assays in 1989 and made possible the introduction of routine HCV antibody screening of the blood supply in 1992, followed by HCV nucleic acid testing (NAT) in 1999.⁴ Safer injection practices, along with blood donor screening, resulted in a dramatic decrease in HCV incidence.

In the U.S. the National Health and Nutrition Examination Survey (NHANES) data collected from 1998 through 2008 (which did not include  the incarcerated or homeless population) estimated HCV prevalence at 3.9 million individuals (1.5%).¹ The majority of chronic HCV infections (76.5%) are in individuals born between 1945 and 1965, with an estimated antibody prevalence of 3.25%.^5,6 Recently, there have been reports of increasing HCV acute infection rates among young adults, especially in the 20 to 29 years-old age group, and the most common risk factor is intravenous drug use.^7,8

Clinical presentation and rationale for screening

Fewer than 25% of individuals with new HCV infection display acute symptoms. The Centers for Disease Control and Prevention (CDC) estimated that out of 17,000 new cases in 2010, only 2,800 patients had acute hepatitis symptoms.⁹ Without treatment, only 15% to 25% of infected people will “clear” the virus, while 75% to 85% will develop asymptomatic chronic HCV infection, which can slowly progress to chronic hepatitis, cirrhosis, and liver cancer.¹⁰ Chronic HCV infection is the most common cause of chronic liver disease and the most frequent indication for liver transplantation in the United States. Screening asymptomatic patients at risk will improve detection and provide treatment options for those individuals.¹¹ As a result of novel treatment approaches, viral clearance can be achieved in a majority of cases, thereby avoiding more than 120,000 HCV-related deaths and saving $1.5 to $7.1 billion in liver disease-related costs.^1,12

Prior to 2012, the CDC recommended HCV testing for a number of at-risk populations. In 2012, the CDC augmented its testing guidance by further recommending that all individuals born between 1945 and 1965 receive one-time testing for HCV. It is estimated that implementation of this strategy will identify 800,000 infections. In 2013, the U.S. Preventive Services Task Force (USPSTF) also updated its HCV screening recommendations to include all adults born between 1945 and 1965.¹³ Recently, New York State passed a first-in-nation law which “will ensure that all individuals born between 1945 and 1965 are offered a hepatitis C screening test or diagnostic test whenever they are a patient at a hospital, clinic or a physician’s office.”¹⁴

Diagnosing HCV

The current complete diagnosis of HCV infection requires serologic assays, which detect antibodies against HCV antigens (anti-HCV tests), and molecular assays.

HCV RNA is detected in the blood by molecular assays as early as one week after the initial infection; therefore, molecular testing is considered the gold standard as a supplemental test for the final diagnosis of acute, active, or resolved infection. Qualitative HCV RNA assays detect the presence of the HCV RNA, quantitative HCV RNA assays are able to assess the viral load, and HCV-RNA genotype tests determine the genotype of any HCV isolate. NATs have >99% specificities across all six types of HCV.¹⁵ The FDA has approved both quantitative and qualitative NAT for confirmatory diagnostics;¹¹ however, as the quantitative tests offer a broad range and a 5 IU/mL HCV RNA lower detection limit, they might gradually replace the qualitative tests.¹⁵ In the case of immunocompromised patients (i.e., HIV co-infected, transplant recipients, hemodialyzed) who are unable to mount an appropriate immune response, detection of HCV RNA is the appropriate method to accurately assess HCV infection status. NAT testing is labor-intensive and costly; therefore, novel molecular approaches have been developed as point-of-care tests: multiplex PCR detection of hepatitis B, C, and human immunodeficiency viruses 1 and 2, as well as a loop-mediated isothermal amplification (LAMP) method for HCV RNA detection.¹⁵

Serological assays are the first step in diagnosing HCV infection, and their sensitivity and specificity have been improved throughout the years. The breakthrough was represented by the first generation anti-HCV test introduced in 1990 for blood donor screening, an enzyme immunoassay (EIA) containing c100-3, a recombinant antigen derived from the NS4 peptide. Although the use of this first-generation assay decreased the incidence of transfusion-associated hepatitis C, it had low sensitivity and specificity, especially in a low-prevalence population.¹⁶ The second-generation EIA, first available in 1992, incorporated recombinant protein antigens such as c22-3 from the core and c33c and c100-3 from the non-structural proteins NS3 and NS4. Owing to the use of c22-3 antigen, which is released in the blood earlier than c100-3, this second-generation assay shortened the average “window period” from 16  to 10 weeks.¹⁷ In 1996, a third-generation EIA with improved sensitivity and specificity was approved for donor screening in the U.S. The third-generation EIA contains a reconfigured NS3 and an extra fourth antigen-NS5, shortening the window period to approximately eight weeks. Studies comparing the second- and third-generation EIA for HCV showed that the third generation EIA can better detect acute, remote nonviremic, and atypical seroconversion infections.¹⁸

Developed in the 1970s, the chemiluminescence immunoassays are widely used in clinical settings today. In 2001, the first automated enhanced chemiluminescences assay (CIA) for HCV was approved for clinical use. The enhanced chemiluminescence uses an enhancer to amplify the light signal, resulting in improved sensitivity in detecting low level analytes and better accuracy and precision. Currently, there are anti-HCV CIA assays FDA approved for screening, and their improved precision and reliability allow the initial positive results to be reported as reactive without repeat testing.¹⁹

Point-of-care assays

Despite the increased specificity and positive predictive value of the CIA assays, the likelihood of false positive results still exists, especially in a low-prevalence population; therefore, supplemental testing to confirm a reactive result is still mandatory. The need for a faster turnaround time led to the development of point-of-care rapid HCV antibody assays, based on recombinant antigens from core, NS3, NS4 and NS5. The sensitivity of these assays ranges between 86% and 99% and the specificity is greater than 99%.²⁰ These rapid assays are even more expensive than the automated immunoassays and more appropriate for low-volume laboratories and nonclinical settings.¹⁵ The FDA recently approved and granted a CLIA waiver to a rapid HCV antibody test which uses an indirect immunoassay method in a lateral flow device to detect antibodies to HCV. Immunoassays for the HCV core antigen have been developed, with sensitivities of 80% to 99% and specificities of 96% to 100% in the window period.²¹ These assays would be of most use in cases where acute HCV infection is suspected. They are not yet available in the United States.¹⁵

The updated CDC algorithm recommends that the at-risk population should initially be screened with a rapid or laboratory-conducted FDA-approved HCV antibody assay. Due to the unavailability in the United States of the recombinant immunoblot assay, used as supplemental test to confirm a reactive screening result, a positive result still must be confirmed by molecular testing for HCV RNA using an FDA-approved NAT assay.¹¹