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HCV testing: Gold standard vs. HCV core antigen testing

HCV testing provides valuable information that aids in disease prevention and in the initiation of care.

Currently, the diagnosis of HCV is at minimum a two-step process that involves screening for exposure to HCV using an indirect serological assay to detect HCV antibodies and confirmation of active infection using a nucleic acid test (NAT) to detect viral RNA in the plasma or serum (i.e., viral load).

While these two tests are able to identify and diagnose patients with active HCV infection, they’re only as successful as their availability allows. The HCV antibody test is relatively inexpensive with a quick turnaround time (TAT) and can be performed by most clinical laboratories, but a large number of patients who test positive for HCV antibodies are lost to follow-up and never receive a confirmatory HCV RNA test. The two-step diagnostic process is an onerous one for the patient, and the resultant loss of patients to follow-up is a significant obstacle in the continuum of care.

NATs: expensive and time-consuming

In addition to the time burden experienced by the patient, other challenges also factor into the low rate of confirmatory testing.

• NATs are performed by highly trained staff at specialized laboratories that have the capacity to run complex assays; an estimated 84% of people do not have access to these specialized laboratory facilities.
• For those patients who do have access, the cost of the test itself is often prohibitive: an HCV RNA assay may cost up to 120 USD.
• The effect of these limitations is marked in low-income, resource-poor settings.

What is the HCV core antigen?

More affordable, less complex diagnostic testing has the potential to greatly improve access to care for people with chronic HCV in low- to middle-income (LMIC) areas and to help forge the way toward the elimination of HCV. A promising alternative to HCV viral load testing is HCV core antigen (HCVcAg) testing.

The HCVcAg is a highly conserved and antigenic protein across different strains of HCV that forms part of the HCV viral particle; in fact, the target protein is more stable than HCV RNA. The protein is released into the plasma and is easily quantified as an indirect measure of HCV replication. The HCVcAg is measurable at about the same time as HCV RNA, appearing in the serum one to two days after HCV RNA during the course of infection.

HCVcAg is quantified using an immunoassay. Immunoassays are both precise and reliable, and they have several advantages over NATs. Compared with NATs, most immunoassays

• are fully automated and relatively simple to perform
• have a shorter TAT; the TAT for an HCVcAg assay is about an hour, while the TAT may be up to 7 hours for an HCV RNA test
• are considerably less expensive at about 1 to 9 USD per test
• require smaller sample volumes

Clinical utility: Can core antigen testing replace the gold standard?

But how does the HCVcAg test compare to HCV RNA testing from a clinical standpoint? The cost and availability of the test don’t mean much if the test is inferior. The first-generation HCVcAg tests were understandably unpopular due to their low sensitivity compared to RNA testing; the new chemiluminescent microparticle immunoassay (CMIA) tests, however, are far superior to these first-generation tests, with a sensitivity 16 to 25 times greater.

The suggested utility of the HCVcAg test is two-fold: as a confirmatory diagnostic test and as treatment monitoring – that is, for the assessment of sustained virologic response (SVR). Several studies comparing the clinical performance of HCVcAg versus HCV RNA assays have found a high correlation between the two testing methods in both settings.

Commercially available HCVcAg assays have demonstrated highly sensitive and specific diagnoses of active HCV infection in a range of populations. According to most studies comparing the diagnostic accuracy of the core antigen and HCV RNA assays, the main diagnostic limitation of HCVcAg testing is lower sensitivity at viral loads between 1,000 and 3,000 IU/mL. Since the overwhelming majority of patients with chronic HCV infection have HCV RNA measurements greater than 1,000 IU/mL, core antigen assays may be suitable for screening and diagnosis of chronic HCV, particularly in at-risk populations estimated to have a high prevalence of HCV infection.

With respect to treatment monitoring, how do the kinetics of HCVcAg compare to those of HCV RNA in infected patients on direct-acting antiviral (DAA) medication? A study assessing the utility of HCVcAg testing in identification of SVR found that, initially, HCVcAg fell faster than HCV RNA. At week 2 of treatment, concordance between the two testing methods was 40%; this increased to a 95% concordance rate by the end of treatment. Even with the initial discordance (due to the rapid effect of treatment on HCVcAg), the results of this study showed that HCVcAg testing was as informative and accurate as HCV RNA for predicting treatment success. Furthermore, previously mentioned lower sensitivity of the HCVcAg assay compared to HCV RNA assay shouldn’t hinder the ability to detect treatment failures, either, as the increased viral load following treatment failure leads to values much higher than the 3,000 IU/mL sensitivity threshold.

Is there still a place for treatment monitoring in the DAA era?

Speaking of DAA treatment, the superior efficacy and greatly improved safety profile of these medications compared to interferon (IFN) and ribavirin (RBV)-containing regimens begs the question: is regular treatment monitoring even necessary?

Historically, establishment of baseline viremia and an early decrease in viral load were the strongest predictors of treatment response. Monthly on-treatment measurement of viral RNA was used to determine the duration of treatment: so-called “response-guided therapy.”

Today, the extremely high on-treatment response rates and cure rates associated with DAA treatment have diminished the importance of using HCV RNA levels to predict response to treatment.

Additionally, residual HCV RNA detected at the end of DAA treatment is not an uncommon occurrence and has no association with viral relapse. This may be an argument in support of HCVcAg testing, as the decreased sensitivity of the assay may not detect this clinically insignificant residual RNA, avoiding any unnecessary worry for the patient. Strengthening that argument is the fact that HCVcAg is more readily undetectable than HCV RNA, which may help to improve patients’ motivation and adherence to treatment, as they will see treatment results more quickly.

The outcome of HCV treatment is still based on HCV RNA testing, but due to the superior efficacy of DAAs, future HCV testing guidelines may focus on the inclusion of—or even replacement by—HCVcAg testing in certain settings; LMIC settings, in particular.

• The most recent update to the European Association for the Study of the Liver (EASL) guidelines includes the addition of HCVcAg testing as an alternate to HCV RNA when RNA testing is not affordable or available.
• The 2017 World Health Organization (WHO) guidelines also state that an HCVcAg assay with similar sensitivity to HCV RNA testing may be considered as a more accessible and affordable alternative.

A consensus, of course, has not been reached regarding core antigen testing. The most recent guidelines from the American Association for the Study of Liver Diseases (AASLD) still recommend quantitative HCV viral load testing after 4 weeks of treatment, meaning that patients are expected to undergo testing at four different times: baseline, week 4 of treatment, end of treatment, and end of post-treatment follow-up; assessments of genotype and fibrosis stage are also required to determine DAA treatment eligibility.

A look to the future

In the future, using only HCVcAg as a diagnostic assay in high-prevalence populations, such as injection drug users and prison populations, may also reduce the loss of patients to follow-up. The use of HCVcAg as a diagnostic test may also allow for same-day initiation of treatment due to the short TAT of immunoassays compared to RNA testing.

Point-of-care (POC) testing methods are also being investigated for their utility in high-prevalence, underserved populations. In particular, dried blood spot (DBS) testing using capillary whole blood from heel or finger sticks may be useful in settings that lack the ability to collect venous blood samples and in those individuals with poor venous access. At present, DBS testing is poorly correlated to RNA testing, though it is still useful as a qualitative diagnostic test. The affordability and simplicity of DBS testing makes it a viable alternative in LMIC populations, where a sensitive qualitative assay that can readily identify those with active infection will go a long way toward containing the spread of HCV and initiating care for those who need it.

Since the first detection of the HCV core antigen in 1996, HCVcAg testing has come a long way.

• Current well-performing assays boast a diagnostic accuracy similar to HCV RNA testing at viral loads over 3,000 IU/mL.
• Core antigen immunoassays are more convenient in that they don’t require specially trained laboratory personnel or special facilities; in fact, the HCVcAg test can be performed on the same analyzer and at the same time as the HCV antibody test.
• They are cost-effective, require smaller samples, have quicker TAT compared to RNA tests, and the test samples are more stable.
• Core antigen testing has been highly correlated to RNA testing regardless of genotype, even in patients coinfected with hepatitis B virus (HBV) and human immunodeficiency virus (HIV).

HCVcAg testing may not replace HCV RNA testing altogether, but its utility in high-prevalence, low-income populations is clear.

References

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