HCV core antigen is an alternative marker to HCV RNA for evaluating active HCV infection: implications for improved diagnostic option in an era of affordable DAAs

Introduction

Infection with hepatitis C virus (HCV) can lead to acute or chronic hepatitis, liver fibrosis, cirrhosis, end-stage liver disease, and hepatocellular carcinoma (Perz et al., 2006). Disease progression after HCV infection depends on factors including gender, coinfection with HIV, alcohol consumption, and duration of chronic infection (Hajarizadeh, Grebely & Dore, 2013; El-Serag, 2012). The global population seroprevalence of anti-HCV antibodies is estimated at around 1.6%, or roughly 115 million people (Gower et al., 2014). The presence of HCV antibodies can be found in spontaneous clearance, resolved infection post-treatment or persistently active disease. Early treatment for HCV infection, especially in the acute phase, can facilitate viral clearance and prevent chronic infection, thereby limiting HCV-induced liver damage and mortality (Jaeckel et al., 2001; Gerlach et al., 2003; Singal et al., 2010).

Screening for anti-HCV antibody (anti-HCV Ab) status often facilitates HCV surveillance in the community (Meffre et al., 2010; Garcia Comas et al., 2015; Morisco et al., 2016). Although simple, such an assay cannot differentiate between past and present infection and requires supplemental HCV RNA testing to confirm active HCV infection and monitor antiviral treatment. Despite its high sensitivity and reliability, an HCV RNA assay involving nucleic acid testing (NAT) and quantitative real-time RT-PCR requires skilled laboratory personnel, sophisticated equipment, and expensive reagents (Roth et al., 2012; Halfon et al., 2006). Therefore, routine screening using these tests is limited particularly for many resource-constrained setting. In contrast, testing for HCV core antigen (HCV Ag) presents a more attractive alternative owing to the lower cost and short turnaround time. HCV Ag has been shown to be an indirect marker for HCV replication comparable to the detection of HCV RNA (Schuttler et al., 2004; Bouvier-Alias et al., 2002; Ottiger, Gygli & Huber, 2013; Florea et al., 2014). In addition to serving as a reliable marker to diagnose active HCV infection, HCV Ag can also be used to evaluate the treatment response to antiviral therapy (Mederacke et al., 2009; Rockstroh et al., 2017; Alonso et al., 2017). Another advantage of the HCV Ag assay is that it can often be performed on the same instrument and simultaneously with the anti-HCV assay, an added value when determining the HCV prevalence in the community (Kuo et al., 2012; Mixson-Hayden et al., 2015).

New and effective therapeutic direct-acting antivirals (DAAs) taken orally have become widely available in recent years. DAAs have advanced HCV therapy with higher rates of sustained-virological response (SVR) post-treatment than those obtained from traditional interferon-based therapies independent of HCV genotype (Sulkowski et al., 2014; Zeuzem et al., 2014; Gane et al., 2015). Despite the availability of these highly efficient drugs, asymptomatic nature of HCV infection and expensive diagnostic screening process represent major obstacles in identifying and treating HCV-infected individuals (Cox, 2015). Therefore, a simple, cost-effective testing feasible for routine HCV screening would be ideal for low- to middle- income countries (LMIC) such as Thailand. In this study, we aimed to evaluate the diagnostic utility of HCV Ag as an alternative to HCV RNA to identify active HCV infection in a relatively high endemic area. We also assessed the cost feasibility and savings of implementing such program towards public health policy in an era of DAAs.

Materials and Methods

This follow-up study examined the prevalence and genotypes of HCV found in Petchabun and Khon Kaen province and comprised part of a previous HCV surveillance project involving 3,077 samples from high- and average-level HCV-endemic areas (Wasitthankasem et al., 2017). All HCV-seropositive individuals were informed of their status and invited to participate in confirmatory testing for HCV infection using anti-HCV, HCV Ag, and HCV RNA assays combined with liver enzyme levels and liver stiffness tests in March 2016. The study protocol was approved by the institutional review board of the Faculty of Medicine, Chulalongkorn University (IRB No. 258/58). Objectives of the study were explained to the patients and written informed consents were obtained.

Study subjects

Of the 310 eligible individuals with positive anti-HCV Ab results, 270 subjects participated in the follow-up study (Fig. 1). Another 28 subjects with anti-HCV positive status established after blood donor screening were also enrolled in this study. In all, 298 subjects (aged 34–64 years) provided demographic information and blood samples. Liver function data including aspartate aminotransferase (AST) and alanine transaminase (ALT) levels were obtained, and HCV viral loads were determined from blood plasma. The degree of liver fibrosis was assessed by transient-electrography (FibroScan, Echosens, Paris, France). Hepatitis B virus (HBV) and HIV status were previously determined using HBsAg and HIV Ag/Ab automated screening (ARCHITECT, Abbott Diagnostics, Wiesbaden, Germany).

Results

Anti-HCV, HCV Ag and HCV RNA status

A schematic diagram of anti-HCV, HCV Ag, and HCV RNA testing in this study is shown in Fig. 2. Thirty-eight out of 290 individuals with anti-HCV S/CO < 5.0 and HCV Ag < 3.0 fmol/L tested negative for HCV RNA. Of the 252 samples with anti-HCV S/CO ≥ 5, 220 samples with HCV Ag ≥ 3 fmol/L were positive for HCV RNA. In 32 samples with anti-HCV S/CO ≥ 5 and HCV Ag < 3 fmol/L, two samples were positive for HCV RNA.

Among 13 HBV reactive samples, there were six HCV RNA positive samples, five of which were positive for HCV Ag. One sample showed evidence of triple infection with HIV, HBV, and HCV.

Correlation of anti-HCV and HCV Ag with HCV RNA level

Examination of the correlations among anti-HCV, HCV Ag, and HCV RNA levels in log scale showed that anti-HCV significantly correlated with HCV RNA (log HCV RNA = −0.882 + 5.492 [log anti-HCV], R² = 0.534, p < 0.001) (Fig. 3A). Using an anti-HCV S/CO cut-off at 5, there were 30 false-positive (FP) samples with HCV RNA < 12 IU/mL (Table S1) and no false-negatives (FN). Better correlation was observed between HCV Ag and HCV RNA level (log HCV RNA = 2.67 + 0.95 [log HCV Ag], R² = 0.890, p < 0.001) (Fig. 3B). Taking an HCV Ag cut-off at 3 fmol/L, only two FN with HCV RNA levels of 501 and 1,445 IU/mL were identified (Fig. 3B and Table S1). Both samples had HCV Ag levels of 0.0 and 2.1 fmol/L and were of genotype 3a (Fig. 4B).

The mean levels of HCV RNA in the samples with genotype 1 (log 6.0 ± 0.9 IU/mL), genotype 3 (log 5.6 ± 1.1 IU/mL), and genotype 6 (log 6.1 ± 0.8 IU/mL) were determined along with the mean HCV Ag (genotype 1, log 3.5 ± 1.0 fmol/L; genotype 3, 2.9 ± 1.1 fmol/L; genotype 6, 3.8 ± 0.7 fmol/L) (Table 2). Genotypes showed significant differences in the log mean of both HCV RNA and HCV Ag (p = 0.001 and p < 0.001, respectively). Samples with HCV genotype 3 had lower levels of HCV RNA and HCV Ag than genotype 1 (p = 0.014 and p = 0.001, respectively) and 6 (p = 0.001 and p < 0.001, respectively). Based on the regression data, HCV Ag showed greatest correlation with HCV RNA in samples with genotype 1 (log HCV RNA = 2.97 + 0.87 [log HCV Ag], R² = 0.905, p < 0.001) followed by genotype 6 (log HCV RNA = 2.323 + 1.01 [log HCV Ag], R² = 0.870, p < 0.001), and genotype 3 (log HCV RNA = 2.75 + 0.97 [log HCV Ag], R² = 0.848, p < 0.001) (Fig. 4 and Table 2). Finally, the slight difference in regression data among genotypes was not statistically significant (p = 0.102).

There was no significant difference in the log mean of HCV RNA and HCV Ag between HCV mono-infection (N = 215, log 5.9 ± 1.0 IU/mL and log 3.4 ± 1.0 fmol/L) and HCV/HBV co-infection (N = 5, log 5.7 ± 1.7 IU/mL and log 3.0 ± 1.5 fmol/L) (p-value > 0.05). HCV Ag highly correlated with HCV RNA in HCV/HBV co-infection (log HCV RNA = 2.44 + 1.08 [log HCV Ag], R² = 0.968, p = 0.002), while correlation of these two markers between mono-infection and co-infection was not significantly different (p = 0.119). The one sample with triple infection (HCV/HBV/HIV) had viral load and HCV Ag at log 6.7 IU/mL and log 4.10 fmol/L, respectively.

Discussion

Timely HCV treatment, especially during the acute phase of infection, has shown promise in multiple HCV genotypes and results in improved prognosis (Jaeckel et al., 2001). Identifying actively infected individuals is therefore important in reducing HCV transmission and disease burden, especially in areas of endemicity. Although, anti-HCV screening is the primary test for diagnosis of HCV infection, supplemental testing is still needed to discriminate between resolved and viremic infection. The results of this study suggest that anti-HCV Ab tests had a fair correlation with HCV RNA and are appropriate for primary screening due to its high sensitivity. Previously, it was suggested that an appropriate cut-off could predict HCV viremia (Kuo et al., 2012). Thus, this study applied a S/CO cut-off at 5.0 for predicting a true antibody-positive result ≥95% recommended by the US CDC (https://www.cdc.gov/hepatitis/hcv/labtesting.htm). Among samples with anti-HCV S/CO ≥ 5, HCV RNA was positive in 88.1% (222 of 252), while those with S/CO < 5.0 were all negative. The latter may have resulted from a false-positive anti-HCV result or an individual with resolved disease and/or low antibody titer. Using this cut-off ratio to predict HCV infection, high diagnostic sensitivity (0.0% FN and 11.9% FP) was noted. When the cut-off ratio was adjusted to 15.0, better predictive values for HCV infection were obtained with excellent PPV (98.8%) and specificity (98.5%), albeit with poor NPV (32.8%) and sensitivity (38.3%) (Table S2). Thus, anti-HCV testing with S/CO at 5.0 was appropriate for the first-line screening.

Unlike anti-HCV Ab, HCV Ag showed excellent diagnostic validity and correlation with HCV RNA. At a cutoff of 3 fmol/L, HCV Ag was a good predictive marker of HCV viremia with 0 (0.0%) FP and 2 (2.9%) FN cases. The validity of HCV Ag testing was better than the anti-HCV marker that had 99.1% sensitivity, 100% specificity, 100% PPV, 97.1% NPV and 99.3% accuracy. It has been suggested that combining anti-HCV and HCV Ag at the appropriate cutoff point would improve the predictive value for HCV viremia, but the validity was similar to HCV Ag alone when combining those two markers (Kuo et al., 2012). In addition, HCV Ag correlates well with RNA levels and may therefore serve as a predictor for response-guided therapy and for monitoring treatment response (Nguyen et al., 2017; Alonso et al., 2017).

The 2 FN cases from the HCV Ag assay had low viral loads and were of genotype 3. These results may be related to the viral level and detection limit of the respective genotypes. Low levels of HCV RNA tended to increase the coefficient of variation and contributed to FN on HCV Ag assays (Aghemo et al., 2016; Ottiger, Gygli & Huber, 2013). Several studies suggest that the lower limit of HCV Ag detection is equivalent to HCV RNA between 1,000 and 5,000 IU/mL, therefore low RNA level in these two samples (501 IU/mL and 1,445 IU/mL) may have contributed to the FN result (Florea et al., 2014; Freiman et al., 2016; Ottiger, Gygli & Huber, 2013). In addition, adequate analytical sensitivity at 3 fmol/L of HCV Ag of genotype 3 required approximately 1,002 IU/mL of HCV RNA, which was higher than for genotype 1 (896 IU/mL) (Ross et al., 2010; Tillmann, 2014). Similar to this cohort, greater variation in genotype 3 samples with low HCV Ag but high HCV RNA was previously reported (Ottiger, Gygli & Huber, 2013; Mederacke et al., 2009). Discordant result found in this study (0.7%, 2/290) could potentially be resolved with a confirmatory RNA testing (e.g., reactive anti-HCV and non-reactive HCV Ag) (Rockstroh et al., 2017).

A meta-analysis demonstrated that HCV Ag had excellent correlation with viremia despite limitations on the effect of HCV genotypes and HBV or HIV co-infection (Freiman et al., 2016). A significant difference was observed in the log mean of viral RNA and antigen levels among genotypes, but this was not apparent in the correlation between these two markers. Previous studies also reported that HCV Ag of genotype 1, 2, 3 and 4 had good correlation with viral load (Mederacke et al., 2009; Ottiger, Gygli & Huber, 2013; Ross et al., 2010). Our study also showed good correlation between HCV Ag and RNA in HCV/HBV co-infection samples. However, poor correlation in the HBV co-infection group may sometimes occur especially with genotypes other than genotype 1 (Mederacke et al., 2012). This may be attributed to very low or very high concentration of HCV Ag relative to HCV RNA in some samples. Therefore, HCV Ag retesting should be performed in the samples with very low or very high concentrations. Excellent correlation had been reported in HCV/HIV co-infection (Mederacke et al., 2012; Thong et al., 2015), but that information was insufficient in triple infection samples. We found only one sample with HCV/HBV/HIV triple infection and elevated HCV viremia and HCV Ag. Further studies are required to elucidate the correlations in subjects with dual infections and triple infections.

Previous studies examining HCV Ag in HCV infection were conducted in industrialized countries (Mederacke et al., 2009; Mixson-Hayden et al., 2015; Rockstroh et al., 2017; Freiman et al., 2016), while data from LMIC have been scarced (Khan et al., 2017; Freiman et al., 2016). Our finding provided the evidence of the utility of HCV Ag for HCV screening in LMIC endemic for HCV due to the assay’s high diagnostic validity and lower cost than an RNA assay, which potentially could improve access to treatment and care in LMIC (Wang, Lv & Zhang, 2017; Khan et al., 2017).

Currently, the World Health Organization initiated a goal to eradicate viral hepatitis by increasing the proportion of diagnosed persons by up to 90% by the year 2030 (World Health Organization, 2017). This policy will require a substantial investment. Fortunately, affordable and highly efficacious DAA treatment is increasingly accessible (Nebehay, 2017). To enhance the likelihood of eradicating HCV, results from our study and others suggest an alternative screening and diagnostic strategy to treat HCV infection, especially in the era of DAAs (Fig. 5). When screening initially involves anti-HCV Ab and confirmatory HCV Ag assay, followed by HCV RNA testing on HCV Ag-negative samples, a cost-effective algorithm by us demonstrate a considerable reduction, roughly 48% compared to the standard expenditure (standard algorithm; anti-HCV test followed by RNA assay, File S1) and is consistent with that proposed by others (Jülicher & Galli, 2017). This strategy would effectively provide equal diagnostic performance that supports the possibility of large-scale implementation.

In conclusion, our population-based study showed high validity of HCV Ag as a reliable marker for diagnosis of active HCV infection. This marker showed excellent correlation with the viral RNA irrespective of genotypes and HBV co-infection. In addition, HCV Ag can serve as a supplemental marker after anti-HCV testing to reduce the sample number requiring further confirmatory RNA assays. A proposed cost-effective strategy would reduce the financial burden required for national screening and improve access to HCV treatment and care in the era of affordable DAAs.

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