The hepatitis C virus (HCV) infection remains one of the most prevalent blood-borne viral infections worldwide and is a leading cause of life-threatening liver diseases including cirrhosis and hepatocellular cancer (HCC) [1]. In 2020, the global prevalence of viremic HCV infection was 0.7%, corresponding to 56.8 million infections [2]. Based on nationwide studies in Korea, the current prevalence of HCV infection in the Korean population ranges from 0.71% to 0.78%, with prevalence increasing with age [3,4].

In 2016, the World Health Organization introduced a global goal of eliminating viral hepatitis as a major public health threat by 2030 by reducing the incidence of new chronic infections by 90% and the mortality rate by 65% [5]. The availability of highly effective direct-acting antiviral (DAA) treatment for HCV infection has made the global elimination of HCV theoretically feasible. However, the majority of people infected with HCV are not diagnosed and remain untreated [6,7], which hinders the achievement of this global goal. Consequently, there has been a change in the HCV infection management system, including scaling up screening and simplification of care and treatment services [6–8].

The diagnosis of HCV infection is classically made based on the detection of antibodies to HCV (anti-HCV) at the first step, with confirmation on HCV RNA or HCV core antigen tests. In other words, anti-HCV is usually used as a screening test for HCV infection, and anti-HCV reactive samples require further supplemental testing for HCV RNA or HCV core antigen to definitively confirm whether there is an HCV infection with viremia. Although anti-HCV assays are highly sensitive and specific for detecting chronic HCV infection in patients [9], this test has a high false-positive (FP) rate of 15–60% (mean, 35%), especially in low-risk populations with an anti-HCV prevalence of < 10% [10–12]. This results in unnecessary additional tests and monitoring of the individuals who test anti-HCV positive. To overcome this problem, several studies have been conducted to reduce FP results by adjusting the signal-to-cutoff (S/CO) ratio or conducting additional tests [13–19].

The current guidelines recommend performing reflex HCV RNA testing for all positive anti-HCV samples, but the availability of HCV RNA testing in clinical laboratories and blood banks may be limited because of its high cost and the requirement for qualified personnel and specialized equipment. Considering the easy accessibility to medical facilities and low prevalence of HCV infection, Korea is an excellent setting to evaluate whether reflex HCV RNA testing for all positive anti-HCV samples is a suitable diagnostic strategy for HCV infection.

This study aimed to determine the correct anti-HCV S/CO ratio to distinguish true-positive (TP) from FP HCV infection, estimate the true prevalence of HCV infection using the optimal anti-HCV S/CO ratio, and finally propose a suitable strategy for the diagnosis of HCV infection in low prevalence areas such as Korea.

The anti-HCV assay with high sensitivity and specificity has been widely used as a screening test for HCV infection. However, this test has a relatively high FP rate, especially in low-risk populations [10–12]. Our study found that 34.7% of patients had FP anti-HCV results, out of all patients in whom TP or FP HCV infection could be ascertained. A previous Korean study showed that about 50% of patients had FP anti-HCV results [23]. In our study, when we applied an anti-HCV S/CO ratio of 5 as the definition for anti-HCV positivity, the prevalence of HCV infection decreased from 1.22% to 0.65%. This indicates that the FP anti-HCV rate was about 45% in our study, which was comparable with the result of a previous Korean study [23]. This indicates that FPs of anti-HCV assays may occur with a high frequency of 45–50% in countries like Korea with low HCV prevalence rate of less than 1%. This high rate should be considered at the time of diagnosis as well as in the estimation of HCV infection prevalence. FPs result in the performance of unnecessary additional tests, such as HCV RNA testing and imaging techniques such as ultrasonography. In addition, HCV infection requires continuous monitoring due to the possibility of progression to cirrhosis and HCC. Thus, false positivity may exert various health, economic, and psychological impacts on both patients and providers.

A positive anti-HCV result may indicate a current active infection, a past resolved infection, or an FP reaction. RIBA is commonly used as a supplementary serologic anti-HCV assay to differentiate false positivity from true HCV exposure because of its robust specificity [10]. A positive RIBA result indicates current active or past resolved infection. However, the US Centers for Disease Control and Prevention (CDC) issued an update on the HCV testing approach, including sequential testing of anti-HCV screening-positive samples with the HCV RNA test alone without RIBA due to the unavailability of RIBA and a strategic change in the management of HCV infection from diagnosis to treatment [24]. Despite the CDC’s decision to remove RIBA from the diagnostic algorithm for HCV infection, the present study demonstrated that RIBA may still be a useful tool in specific circumstances, such as low prevalence countries like Korea, considering the high FP anti-HCV results and difficulty distinguishing between past resolved and FP HCV infection without RIBA.

In the current study, we were able to differentiate TP from FP HCV infection using RIBA and HCV RNA testing, and we analyzed anti-HCV titers using ROC curves in order to determine an optimal S/CO ratio for the prediction of HCV viremia and TP HCV infection. These determined cutoff values of anti-HCV S/CO ratio for HCV viremia (anti-HCV S/CO ratio of 8) and TP HCV infection (anti-HCV S/CO ratio of 5) could provide clinical significance in the diagnosis of HCV infection, especially in circumstances of unavailability of the RIBA test. In terms of the levels of anti-HCV S/CO ratio for predicting HCV viremia, an anti-HCV S/CO ratio of 8 showed a high diagnostic accuracy, with a sensitivity, specificity, PPV, and NPV of 99.7%, 87.5%, 87.4%, and 99.7%, respectively. This value is comparable with those of previous studies [10,25,26]. Regarding the levels of anti-HCV S/CO ratio for predicting TP HCV infection, we set an anti-HCV S/CO ratio of 5 as an optimal cutoff value, which is applicable to estimate the true prevalence of HCV infection.

Various commercially available anti-HCV assay kits show high agreement in detecting anti-HCV [27]. However, they use different methods and molecular targets for generating and detecting signals and, as a result, may have different levels of diagnostic performance. Some studies reported discrepancies among assays and great variability in cutoff points for predicting TP HCV infection, such as 200 for the Elecsys assay, 19 for the Elecsys II assay, 3–5 for the ARCHITECT assay, 7–8 for the Vitros assay, 11 for the ADVIA Centaur, and three for the Access assay [27–30]. Although anti-HCV assays are highly sensitive and specific, their diagnostic accuracy requires standardization and improvement.

After introduction of highly effective DAA treatment for HCV infection, the HCV management strategy has evolved to focus on the diminution of undiagnosed or untreated patients with HCV infection, which includes scaling up the screening process, reflex HCV RNA testing, and simplification of management. Although these strategies improve HCV infection diagnosis and linkage to care, as well as support earlier treatment initiation, increased screening for HCV infection in a population with a low prevalence rate leads to a higher number of FP results. It is well known that prevalence affects PPV. Therefore, the decline of HCV prevalence after the DAA era further increases FP rates in anti-HCV results. At that time, our study results, an anti-HCV S/CO ratio of 5 for predicting TP HCV infection, may require revision.

The Korean Association for the Study of the Liver recently suggested implementing HCV screening test in the National Health Screening Program (NHSP) as part of an effort toward global elimination of viral hepatitis as a public health problem by 2030. When HCV screening tests are introduced in the NHSP, the high FP rate of anti-HCV assays in Korea should be considered and our study results could provide guidance.

The anti-HCV prevalence of our study (1.22%) was higher than that of previous Korean reports (0.71–0.78%), which might be caused by differences in the study population. For example, the participants in our study were usually inpatients, whereas previous Korean studies were conducted on health check-up examinees or the general population. Thus, our study could have included higher-risk populations for HCV infection. Considering the variability of cutoff points of the S/CO ratio for predicting HCV infection according to the types of anti-HCV assays, our results using only the ARCHITECT assay are not applicable to all anti-HCV positive samples. This study also focused on anti-HCV positive results. Therefore, we did not analyze false negativity in anti-HCV assays because we did not evaluate HCV RNA in anti-HCV negative results. This retrospective study could not ascertain TP or FP for HCV infection in all anti-HCV positive patients because not all anti-HCV positive patients were tested for HCV RNA and RIBA. Regardless of these limitations, our study could provide significant clinical information for the diagnosis of TP HCV infection.

In conclusion, because the FP anti-HCV result are unacceptably high in low prevalence areas like Korea, false positivity should be considered and different diagnostic approaches may be required when diagnosing chronic HCV infection. Revision of the anti-HCV S/CO ratio could be useful and applicable to these conditions because it showed high diagnostic accuracy for predicting HCV viremia and TP HCV infection.