HCV core antigen plays an important role in the fight against HCV as an alternative to HCV‐RNA detection

Abstract Objective To discuss the clinical significance of HCV‐cAg testing in the diagnosis, activity determination, and monitoring of therapeutic effectiveness of HCV infection and its advantages compared with HCV‐RNA and anti‐HCV antibodies detection. Methods By summarizing the published literature, the advantages and significance of HCV core antigen detection were sought. Results The expression of HCV‐cAg is highly consistent with that of HCV‐RNA, but compared with HCV‐RNA, detection of HCV‐cAg is easy to operate, time saving, and low cost. HCV‐cAg can be detected within 12~15 days after infection, and the window period can be shortened by5~7 weeks. HCV‐cAg is a serological indicator of virus replication, which can distinguish previous infection of HCV or current infection. HCV‐cAg detection is more suitable for immunocompromised, hemodialysis, organ transplant patients. HCV‐cAg also can be used to monitor antiviral efficacy and predict sustained virological response (SVR). Conclusion HCV core antigen has similar clinical sensitivity to NAT and can be used as a substitute for HCV‐RNA in the diagnosis of virus infection. Combined detection of HCV‐cAg and antibody serology can help doctors detect HCV infection earlier, accurately diagnose different stages of HCV infection, and evaluate the therapeutic effect of antiviral drugs, which are beneficial in the prevention and treatment of hepatitis C.


| INTRODUC TI ON
Hepatitis C is a viral hepatitis caused by infection with the hepatitis C virus (HCV). It is often described as a "silent killer," mainly because the virus has a long incubation period and the disease is prone to become chronic. Chronic HCV infection can cause varying degrees of damage to the liver. If left untreated, it can lead to a series of complications, such as fibrosis, cirrhosis, and even liver cancer. World Health Organization (WHO) estimated that in 2015, 71 million persons were living with chronic HCV infection worldwide (global prevalence: 1%) and that 399,000 had died from cirrhosis or hepatocellular carcinoma (HCC), 1 with most of these concentrated in developing countries. 2 Due to the high missing reporting rate, the incidence of HCV infection is actually higher. HCV infection has become a serious social and public health problem because of its great harm to the health and safety of patients. 3 The WHO says that unless urgent action is taken to improve access to treatment, chronic hepatitis will cause around 10

| The deficient of current methods for detection of HCV infection
The symptoms of HCV infection are usually not easily detected. In addition, because HCV is a single positive-stranded RNA virus, it is easy to mutate and difficult to get a vaccine. There were difficulties in vaccination and fewer screening methods than for HBV. At present, commonly used detection methods include serological detection (HCV core antigen detection/anti-HCV detection) and molecular biological detection (HCV-RNA detection). Anti-HCV antibodies detection is currently the most commonly used screening tool for HCV, but it has some shortcomings: The window period of anti-HCV detection is long, acute hepatitis C patients are easy to be missed; it cannot distinguish between those who have recovered from previ- However, RNA is easy to be degraded (due to the presence of a large number of RNA enzymes in the environment, RNA is very easy to be degraded and prone to false-negative results). Test samples should not be placed at room temperature after collection. They should be stored at a low temperature immediately. Therefore, a cryogenic refrigerator should be prepared, and RNA must be extracted within 2 hours after blood is drawn. In addition, the RNA extraction process is complex and requires high requirements for operators. From what has been discussed above, HCV-RNA quantification tests are expensive, time-consuming, and require highly trained personnel. 6 The sequence of HCV core antigen (cAg) is highly conserved among all the different viral genotypes. The presence of circulating antigen is associated with complete viral particles but may also be derived from the presence of antigen/antibody complexes and a release of "RNA-free" antigen by hepatocytes that go into processes of immune-mediated lysis or apoptosis. 7 Features of HCV-cAg detection including HCV-cAg can be detected within 12~15 days after infection, and the window period can be shortened by 5~7 weeks.
HCV-cAg is a serological indicator of virus replication, which can distinguish previous infection of HCV or current infection. Compared with anti-HCV antibody test, HCV-cAg detection is more suitable for immunocompromised, hemodialysis, organ transplant patients.
HCV-cAg also can be used to monitor antiviral efficacy and predict sustained virological response (SVR).

| HCV-cAg can shorten the window period comparing with anti-HCV antibody
Anti-HCV antibody detection assays detect HCV infection after approximately 70 days, and it may be negative in the early stages of acute hepatitis. 8  A rapid diagnostic assay (HCV Ag-Ab Combo) for simultaneous detection of HCV-cAg and anti-HCV antibodies for early diagnosis of HCV infection was developed. 10 It detected HCV infection approximately 7-12 days earlier than the HCV-Ab detection assays, and its performance was not affected when testing different genotypes of HCV. It could be a useful screening assay and an alternative to HCV-RNA detection or HCV Ag-Ab ELISA when nucleic acid technologies cannot be implemented. with the lower limit of detection corresponding to the HCV-RNA load of 700-1100 IU/mL. 12 The ARCHITECT HCV Ag Assay is cheaper, faster (40 min), and simple to perform and has good sensitivity in samples with viral loads above 10,000 IU/mL. 6 Anti-HCV antibody (using ARCHITECT anti-HCV assay; Abbott Diagnostics), HCV-cAg (using ARCHITECT HCV Ag), and HCV-RNA (using Abbott Real-Time HCV assay) were detected in 298 seropositive subjects. Among them, 252 were positive for anti-HCV antibody (signal-to-cutoff ratios ≥5), 220 were positive for HCV-cAg, and 222

| HCV-cAg expression highly agreement with that of HCV-RNA
were detected with HCV-RNA. HCV-cAg significantly correlated with HCV-RNA. The accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of HCV-cAg (≥3 fmol/L) to predict HCV viremia were 99%, 99%, 100%, 100%, and 97%, respectively. 13 These results concluded that HCV-cAg was a good surrogate marker for HCV-RNA and could be used to diagnose active HCV infection in a resource-limited setting. And using HCV-cAg test to screen and identify active HCV carriers, which can reduce the cost of detection, would enable more patient access to efficacious and increasingly affordable direct-acting antivirals (DAAs) for the treatment of HCV infection. curve of HCV-cAg was 0.989. 1 pg/mL of total HCV-cAg is found to be equivalent to approximately 6607 HCV-RNA IU/mL, which implied the expression level of HCV-cAg was highly consistent with that of HCV-RNA. 15 Viral load (VL) tended to be lowest when anti-HCV and HCV-cAg were absent and to be highest when the antigen was detectable. Then, it decreased when anti-HCV appeared at a level detectable by sensitive third generation tests. 16 Using of HCV-cAg alone for patients with a very low VL (VL <3000 IU/mL) may has a false-negative result, which have enraised concerns. 17 But, the benefits of the HCV-cAg assay as a rapid, pointof-care test in population-based screening still outweigh the risk of

F I G U R E 1 Emergence of laboratory markers for HCV infection. This image describes the time point at which various markers of infection (HCV-RNA, HCV-CAG, and anti-HCV) first appeared in the body of an HCV infection
a small number of false negatives in a subset of patients since the prevalence of very low VL cohorts is <5% for now. 18 Currently, RNA-PCR tests are the gold standard. However, there is potential for the use of simpler and cheaper HCV-cAg tests to confirm HCV infection in different clinical settings. 19 The percentage HCV-RNA negative in HCV-cAg-positive results was 0.57%, while the percentage HCV-RNA positive with HCV-antigen negative was 3.52% in 24 datasets including 8136 samples. 19 Since there is strong evidence that antigen detection performs as well as RNA-based assays for HCV management, the use of HCV-cAg testing saves testing costs and increases the access of infected persons to HCV treatment. The sensitivity and specificity of HCV-cAg (ARCHITECT HCV Ag) were 76.6% and 100%, respectively, compared with HCV-RNA (Roche Diagnostics) in 204 serum samples, and the correlation between the two techniques was extremely well (Pearson coefficient = 0.951). Although the HCV-cAg assay was less sensitive than the HCV-RNA test (HCV-cAg was unable to detect viral loads below 5000 IU/mL), the correlation between both assays was excellent.
Therefore, in emergency situations (such as screening of organ donors, low-income settings, or small and low-skilled laboratories with a limited workload et al), HCV-cAg may be useful as an alternative diagnostic method for RNA detection in the diagnosis of acute or chronic HCV infection. 21 Sensitivity of HCV-cAg detection cannot be compared with that of RNA, which is especially important in samples with a low viral load. However, this drawback becomes less relevant and the correlation between both techniques is high when the VL over a specific value. Although the sensitivity for detecting low HCV-RNA VL was not optimal, the HCV-cAg showed excellent specificity and PPV. 21 The sensitivity and specificity of HCV-cAg (ARCHITECT HCV Ag) were 93.4% and 98.8% from bivariate analyses, while in quantitative studies using, HCV-cAg was highly agreement with HCV-RNA levels >3000 IU/mL. HCV-cAg assays for signal amplification have the potential to replace nucleic acid testing in settings with high HCV prevalence since it has high sensitivity, high specificity, and good correlation with HCV-RNA levels >3000 IU/mL. The positive and negative coincidence rates of HCV-cAg test (Jingda Biological Engineering Co., Ltd) and HCV-RNA were 94.00% (47/50) and 96.67% (58/60), respectively, and the total coincidence rate was 95.45% (105/110). 27 The sensitivity and accuracy of HCV-cAg test (Keshun Biological Technology Co., Ltd) were higher than those of HCV-Ab (p < 0.05). 28 The results of HCV-cAg test (Laibo Biology Science and Technology Co., Ltd) and HCV nucleic acid test were compared and analyzed. There was no significant difference between the two groups (p > 0.05). HCV core antigen detection was significantly better than anti-HCV detection (p < 0.05). Based on the analysis of various factors, it is concluded that the detection of HCV core antigen is economical and practical, and the operation is simple, and can be used as an effective means to screen HCV infection. 29 There are different platforms of conducting HCV core antigen testing; among all, Abbott ARCHITECT HCV Ag assay has the best quality and used by most studies. The HCV core antigen testing, using different platforms, showed consistent results of high sensitivity and specificity as compared to gold standard HCV-RNA.
Diagnostic value of HCV-cAg detection comparing with HCV-RNA is showed in Table 1.

| HCV-cAg detection has advantages for people with immunodeficiency or at high risk
Immunosuppressed or immunosuppressed patients may be tested negative for anti-HCV antibodies. 8 HCV is increasingly common among human immunodeficiency virus (HIV)-infected men who have sex with men. 36 Affordable and sensitive screening methods for acute HCV are necessary to successfully intervene in the current HCV epidemic among HIV-positive men having sex with men.
One study showed that HCV-cAg proved sensitive (100%) and specific (97.9%) in diagnosing acute HCV in a HIV-infected cohort when compared with HCV PCR. 31 In addition, routine use of HCV-cAg detection in screening tests instead of QRT-PCR can save potential costs. The cost per individual screen would be approximately $85 less ($108 for qRT-PCR vs $23.4 for HCV core antigen including kit, staff, and laboratory extras, although this will vary between laboratories). 31 As a quick, simple, and cost-effective test, HCV-cAg has considerable utility in screening for acute HCV. HCV-RNA in plasma, which resulted in a sensitivity and specificity of HCV-cAg of respectively 89% and 100%. Furthermore, the correlation between HCV-cAg and HCV-RNA was 0.97 (p < 0.001) upon diagnosis. 32 The data presented in this study suggest that HCV-cAg testing is a sensitive and specific method that can be used in diagnosing acute HCV in HIV-infected patients.   This pilot study demonstrated the potential for HCV-cAg testing as a reflex test to discriminate between active or past infection for seropositive individuals. 44 HCV-cAg testing has been suggested to provide a cheaper and more rapid turnaround time to issue results compared to RNA testing. 31 Use of HCV-cAg testing may be an important component for HCV screening enabling the potential for earlier diagnosis, linkage to care and commencement of treatment. 31,45 The anti-HCV antibody test had a higher false positive, with a positive predictive value of 87% compared to the repeated HCV-Ab testing in the reference laboratory. 33 However, HCV-cAg was proved to have 100% positive predictive value compared to detection of HCV-RNA. There was a strong correlation between quantitative HCV-cAg and HCV-RNA viral load (p < 0.0001), which make HCV-cAg perform well as a diagnostic test compared to HCV-RNA. 33 This result indicates that in the case of difficulty in carrying out HCV-RNA detection, HCV-cAg may be a good alternative test, particularly at higher viral loads (e.g., if HCV-RNA >104 IU/mL).
To assess the feasibility of HCV-cAg assay in community screening programmes, a study has included 2027 volunteers. The agreement between HCV-cAg and HCV-RNA was 100% in anti-HCV-positive group, and the correlation of HCV-cAg with HCV-RNA (R 2 = 0.84, p < 0.005) was good. 46 The utility of HCV-cAg testing to confirm active infection in screening programs was highlighted by the agreement between HCV-RNA and HCV-cAg.

| Prediction of Treatment Response in Patients with Chronic Hepatitis C
The high effectiveness of DAAs and its extreme safety issues requires monitoring throughout the treatment period. To evaluate the diagnostic usefulness of a HCV-cAg assay in HCV-infected patients undergoing treatment with DAAs, researchers analyzed 103 samples from 28 patients. Sensitivity and specificity were 96.2% and 100% of HCV-cAg assay comparing with RT-PCR, which shows that HCV-cAg is a reliable marker for the follow-up of HCV-infected patients undergoing treatment with DAAs. 30 The decreasing and increasing curves for RT-PCR and HCV Ag during treatment were almost the same. Therefore, both assays were excellent predictors of the success or failure of treatment.
It has been proposed that HCV-cAg assay was a more economi- To evaluate the clinical performance of HCV-cAg assay from plasma samples to monitor HCV treatment efficacy and HCV viral recurrence, HCV-cAg and HCV-RNA were detected at baseline, end of treatment response, and SVR visits. The sensitivity of the HCV-cAg assay with quantifiable HCV-RNA threshold was 94% (95% CI: 88%, 98%), 56% (21%, 86%), and 100%, respectively, while the specificity was between 98% and 100% for all time points assessed.
All six participants with viral recurrence have been detected accurately by HCV-cAg, demonstrating 100% sensitivity and specificity.
One participant with detectable (non-quantifiable) HCV-RNA and non-reactive HCV-cAg at SVR12 subsequently cleared HCV-RNA at SVR24. 50 This study indicates that confirmation of active HCV infection, including recurrent viraemia, by HCV-cAg is possible, since HCV-cAg demonstrated high sensitivity and specificity for detection of pre-treatment and posttreatment viraemia. Reduced ontreatment sensitivity of HCV-cAg may be a clinical advantage given the moves toward simplification of monitoring schedules.
It is estimated that 90% (RNA >10,000 IU/mL) of the positive HCV-RNA samples falls in the sensitivity range of HCV-cAg assays, which make the HCV-cAg test to be a cost-effective method for confirming HCV infection compared to HCV-RNA and also been proposed as a substitute for HCV-RNA in determining SVR. 51 HCV-RNA and HCV-cAg have high predictive value for sustained virological response (SVR). 52 In addition, HCV-cAg expression was highly consistent with RNA quantitative test results, and the measurement of HCV-cAg in the recent EASL guidelines is a potential alternative method for monitoring treatment response in DAA-based treatment regiments. 8

| Use of hepatitis C core antigen qualitative and quantitative tests
The qualitative test of HCV core antigen can be used for but not limited to the diagnosis of acute hepatitis C virus infection in the window period, the differential diagnosis of past infection and present infection, and the diagnosis of acute hepatitis C virus infection in immunosuppressed population and high-risk population. Since the expression of HCV core antigen is highly consistent with the viral load of HCV-RNA, HCV core antigen quantitative assay can be used as a substitute for HCV-RNA for baseline viral load analysis before antiviral therapy and response assessment during and after antiviral therapy.

| Cost-effectiveness of HCV-cAg test
Data showed that the introduction of HCV-cAg test, if compared with the standard one, would give similar effectiveness, with a lower organizational and economic impact (Economic costs for the hospital and Regional and National Health Service would be saved: A hospital could reduce the direct and indirect costs by 47.90% each and contribute to a reduction in funding at Regional or National level by 26.96%), with a good equity impact for HCV-infected patients. 53 In addition, the time needed to complete a medical report by HCV-cAg test was reduced to 90 minutes, and allowed an immediate definition of the presence or absence of HCV infection, which has a positive organizational impact. 53 Kadkhoda K pointed out that HCV core antigen used to diagnose HCV infection has a significant cost-(a minimum of 52.13% reduction in costs compared to qualitative RNA testing), labor-, and turnaround time-reducing potential. 54 Although compared with HCV-RNA, the missed detection rate of  25 WHO also signed that instead of HCV-RNA with HCV-cAg testing in acute HCV infection would reduce a few thousand dollars cost (attains the same sensitivity), and saved time effectively.
Another significant advantage of HCV Ag is that it can be measured in the same laboratory department using the same analytical system as anti-HCV. And there is no need to increase the cost of additional equipment and laboratory setup. 43 Therefore, a combination of antibody and antigen tests for screening, followed by RNA confirmation of antibody-positive Ag-negative samples, can provide equal or better diagnostic performance in a variety of situations at a lower cost.

| CON CLUS ION
On the one hand, HCV-cAg appears earlier than anti-HCV antibodies, is expressed only in active HCV infection, can differentiate between previous infection and current infection, and is not interfered by immunosuppressed or immunosuppressed patients.
On the other hand, the expression of HCV-cAg is highly consistent with that of HCV-RNA, but compared with HCV-RNA, detection of HCV-cAg is easy to operate, time saving, and low cost. Therefore, HCV core antigen has similar clinical sensitivity to NAT and can be used as a substitute for HCV-RNA in the diagnosis of virus infection. 3 Combined detection of HCV-cAg and antibody serology can help doctors detect HCV infection earlier, accurately diagnose different stages of HCV infection, and evaluate the therapeutic effect of antiviral drugs, which are beneficial in the prevention and treatment of hepatitis C.

CO N FLI C T S O F I NTE R E S T
There is no conflicts interest to report.

AUTH O R CO NTR I B UTI O N S
Wang Yuhan participated in analysis and interpretation of data.
Wang Yuhan, Wang Jie, and Jiang Ling drafted the article. Huang Yuanshuai revised it for critically important intellectual content.
Wang Yuhan, Wang Jie, Jiang Ling, and Huang Yuanshuai participated in the conception and design of the article and finally approved the version to be published.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or used during the study appear in the submitted article.