Comprehensive Evaluation of the Elecsys® HCV Duo Immunoassay for Active Hepatitis C Detection: Insights from the DEPCDUO Study

Comprehensive Evaluation of the Elecsys® HCV Duo Immunoassay for Active Hepatitis C Detection: Insights from the DEPCDUO Study | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 14 August 2025 V1 Latest version Share on Comprehensive Evaluation of the Elecsys® HCV Duo Immunoassay for Active Hepatitis C Detection: Insights from the DEPCDUO Study Authors : Paul-Elie Gosset , Célia Sentis 0009-0001-4303-0686 , Christophe Ramiere , Fabien Zoulim , and Caroline Scholtes 0000-0003-0691-8480 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175515688.88437052/v1 529 views 235 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Early diagnosis of Hepatitis C virus (HCV) infection is a cornerstone of elimination strategies. Fourth-generation antigen/antibody combination assays, such as Elecsys® HCV Duo, promise to bridge the diagnostic gap left by antibody-only testing. In this real-world study, we assessed Elecsys® HCV Duo performance using seroconversion panels and routine clinical samples, focusing on sensitivity, specificity, and positive predictive value (PPV) for active HCV detection, including challenging scenarios with low viral loads and HIV co-infections. The assay shortened the serological window by up to 203 days. When both antigen and antibody were detected, PPV reached 100%, enabling confident diagnosis without immediate RNA confirmation. The antigen component alone achieved a PPV of 97.6%, even in a low-prevalence setting (0.33%), better than previously reported assays. However, sensitivity for low viral loads (≤ 5 log IU/mL) was limited, with 95.5% of such cases missed. Elecsys® HCV Duo offers a powerful tool for earlier detection and streamlined care, particularly in high-risk and resource-limited contexts. Yet, confirmatory RNA testing remains essential for low viral load cases. Broader validation is needed to confirm its utility in diverse populations, reinfection surveillance, and cost-effective screening strategies. TITLE PAGE Title Comprehensive Evaluation of the Elecsys® HCV Duo Immunoassay for Active Hepatitis C Detection: Insights from the DEPCDUO Study Running title Elecsys® HCV Duo Performance Authors Paul-Elie Gosset, Célia Margueron-Sentis, Christophe Ramière, Fabien Zoulim, Caroline Scholtès Dr. Paul-Elie GOSSET [email protected] Hospices Civils de Lyon, Department of Virology; Université de Lyon, Université Claude Bernard Lyon 1, F-69000 LYON, France Dr. Célia MARGUERON-SENTIS [email protected] Hospices Civils de Lyon, Department of Virology; Lyon Hepatology Institute, IHU EVEREST; F-69000 LYON, France Dr. Christophe RAMIERE [email protected] OrchID 0000-0002-1981-7155 Hospices Civils de Lyon, Department of Virology; INSERM Unit 1111, CIRI; Université de Lyon, Université Claude Bernard Lyon 1; Lyon Hepatology Institute, IHU EVEREST; F-69000 LYON, France Pr. Fabien Zoulim [email protected] Hospices Civils de Lyon, Department of Hepatology; INSERM Unit 1350 PathLiv; Université de Lyon, Université Claude Bernard Lyon 1; Lyon Hepatology Institute, IHU EVEREST; F-69000 LYON, France Corresponding author: Dr. Caroline SCHOLTES Virologie department, Hôpital de la Croix Rousse, Hospices Civils de Lyon 103 Grande-Rue de la Croix-Rousse 69317 LYON CEDEX 04 Telephone: +33472003763 OrchID 0000-0003-0691-8480 [email protected] Hospices Civils de Lyon, Department of Virology; INSERM Unit 1350 PathLiv; Université de Lyon, Université Claude Bernard Lyon 1; Lyon Hepatology Institute, IHU EVEREST; F-69000 LYON, France No funding was received for this study. The authors declare no conflicts of interest. The study was approved by the local ethics committee (approval no. 24-5089). By default, patients provide general consent for the use of their data upon hospital admission and are informed of the study through a transparency portal. Abstract Early diagnosis of Hepatitis C virus (HCV) infection is a cornerstone of elimination strategies. Fourth-generation antigen/antibody combination assays, such as Elecsys® HCV Duo, promise to bridge the diagnostic gap left by antibody-only testing. In this real-world study, we assessed Elecsys® HCV Duo performance using seroconversion panels and routine clinical samples, focusing on sensitivity, specificity, and positive predictive value (PPV) for active HCV detection, including challenging scenarios with low viral loads and HIV co-infections. The assay shortened the serological window by up to 203 days. When both antigen and antibody were detected, PPV reached 100%, enabling confident diagnosis without immediate RNA confirmation. The antigen component alone achieved a PPV of 97.6%, even in a low-prevalence setting (0.33%), better than previously reported assays. However, sensitivity for low viral loads (≤ 5 log IU/mL) was limited, with 95.5% of such cases missed. Elecsys® HCV Duo offers a powerful tool for earlier detection and streamlined care, particularly in high-risk and resource-limited contexts. Yet, confirmatory RNA testing remains essential for low viral load cases. Broader validation is needed to confirm its utility in diverse populations, reinfection surveillance, and cost-effective screening strategies. Keywords: Hepatitis C diagnosis, HCV core antigen, Fourth-generation assay, Elecsys® HCV Duo, early detection, HCV elimination strategy. Introduction Hepatitis C virus (HCV) infection remains a significant global health concern. In 2022, the World Health Organization (WHO) estimated that approximately 50 million people were living with HCV, with an annual incidence of 1 million cases (1). Among those infected, about 70% develop chronic infection, which progresses to cirrhosis in 10–20% of cases and increases the annual risk of hepatocellular carcinoma (HCC) by 1–4% if left untreated. These complications contribute to over 242,000 deaths globally each year (2). HCV diagnosis in France follows a two-step process: initial detection of anti-HCV antibodies (HCVAb) to determine exposure, followed by nucleic acid amplification tests (NAATs) to confirm active infection (3). Antibody detection is commonly performed using third-generation serological tests, which identify total HCVAb (IgM and IgG) (4). However, these tests face several limitations. The antibody detection window is long, typically 6–12 weeks post-infection, and may extend beyond 12 months in immunocompromised individuals, including people living with HIV (PLWHIV) (5,6). Moreover, serological tests cannot distinguish between active and resolved infections. NAATs, while reliable for detecting active infections, are costly, time-intensive, and require specialized expertise and equipment. Early and accessible HCV detection is critical for curbing the epidemic, particularly among high-risk populations such as PLWHIV and people who inject drugs. In these groups, delayed seroconversion and barriers to healthcare access pose diagnostic challenges (7). The WHO has prioritized eliminating hepatitis C as a public health threat by 2030, emphasizing improved screening strategies to identify individuals with chronic HCV infection. This is particularly pressing as only 36% of HCV-infected individuals worldwide are estimated to be aware of their serological status (1). Given that HCV is largely asymptomatic until advanced stages, there is an urgent need for simple, cost-effective, and reproducible methods to identify active infections. HCV core antigen (HCVcAg) detection has emerged as a promising alternative to NAATs (8). HCVcAg can be detected in peripheral blood within 12–15 days of infection, approximately 1–2 days after HCV RNA, significantly shortening the diagnostic window compared to antibody testing alone (9). Recognized as a reliable marker of viral replication, HCVcAg detection is endorsed by the European Association for the Study of the Liver (EASL) as a viable substitute for NAATs (grade A1) (8). Recent studies, including a meta-analysis by Sepulveda-Crespo et al., have demonstrated the strong analytical performance of the Abbott® Architect HCV Antigen test for diagnosing active HCV infections (10). However, simultaneous testing with the HCVAb assay on the same Architect can result in false positives, highlighting the need for careful evaluation of combined diagnostic approaches. Fourth-generation serological tests that detect both HCVcAg and HCVAb in parallel offer an opportunity to address these challenges. Such tests not only reduce the diagnostic window but also minimize reliance on molecular testing, which may be inaccessible or prohibitively expensive in resource-limited settings. The Elecsys® HCV Duo test (Roche Diagnostics GmbH, Mannheim, Germany) is an electrochemiluminescence immunoassay (ECLIA) designed to simultaneously detect HCVcAg and HCVAb in separate, parallel reactions within 27 minutes. Compatible with serum, heparinized plasma, or EDTA, this CE-marked test provides results expressed as an index, with the ability to identify which marker is reactive in positive cases. Previous study by Majchrzak et al. showed that this test had a specificity of 99,9% and a sensitivity of 99.6% similar or better than competitors third or fourth generation assays for HCV screening. In the present study the primary aim was to evaluate the performance of the Elecsys® HCV Duo test under real-world conditions, with a particular focus on the “antigen” module’s ability to identify active HCV infections. Additionally, the assay was retrospectively assessed on plasma samples with low viral loads to further investigate its diagnostic sensitivity. II. Materials and Methods Study Design This study evaluated the results obtained with the Elecsys® HCV Duo test during routine implementation at the Hospices Civils de Lyon (HCL) virology laboratory over a six-month period (May 2, 2023, to November 2, 2023). A total of 24,656 tests were conducted, of which 22,731 unique results were included after excluding multiple concordant screenings from the same patient. The study protocol was designed and reported in accordance with the STARD (Standards for Reporting Diagnostic Accuracy Studies) guidelines, approved by the local ethics committee (approval no. 24-5089) and conducted in accordance with the Declaration of Helsinki and applicable national regulations. All antigen-positive samples were subjected to quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for HCV RNA detection (cobas® HCV, Roche Diagnostics GmbH, Mannheim, Germany), which served as the reference method for confirming viral replication. Samples with newly detected HCVAb alone were also tested for HCV RNA. When feasible, HCV genotyping was performed using the ViroKey® SQ FLEX system (VELA Diagnostics GmbH, Hamburg, Germany). Additional clinical and biological data, including patient age, hepatitis type (acute or chronic), alanine aminotransferase (ALT) levels, symptomatology, risk factors, HIV co-infection status, treatment with direct-acting antivirals (DAAs), HCV genotype, hepatic fibrosis or cirrhosis, hepatocellular carcinoma (HCC), hepatic transplantation (HT), and the prescribing service, were collected for positive cases. During the study period, a total of 1340 viral loads (VLs) were performed, including 749 samples with associated serology. For viral loads performed without accompanying serology, 41 positive VL samples were identified and subsequently tested using the Elecsys® HCV Duo assay. Due to insufficient sample volume, 3 samples could not be tested. Additionally, the Elecsys® HCV Duo test’s reactivity for its two components (HCVcAg and HCVAb) was evaluated using samples collected between April 2022 and May 2023 with a viral load (VL) ≤ 5 log IU/mL (n=21). To compare screening performance with the third-generation HCVAb test previously used in the laboratory (Atellica aHCV, Siemens Healthcare Diagnostics, Munich, Germany), sera from the laboratory’s seroconversion panel (n=19) were tested using the Elecsys® HCV Duo assay. These sera corresponded to the last samples testing negative for total anti-HCV antibodies using the third-generation method, followed by subsequent detection of an active infection. All sera were collected within one year prior to confirmed seroconversion. Sera that were negative with the 3rd-generation test and positive with the 4th-generation test were subsequently tested by RT-PCR to confirm the presence of active infection. Statistical Analysis Statistical analyses were performed using GraphPad Prism 10 software. Descriptive statistics were expressed as counts and percentages. Differences in means for antigenic indexes or age across subpopulations were assessed using Student’s t -test. A p -value < 0.05 was considered statistically significant. The sensitivity and specificity of the Elecsys® HCV Duo test for detecting active HCV infection were evaluated against viral load results, with true positives defined as samples with HCVcAg levels Characteristic (ROC) curve analysis was performed to determine the optimal antigenic index threshold for interpreting results in patients with anti-HCV antibodies. III. Results Over a six-month period, 24,656 Elecsys® HCV Duo tests were conducted at the Hospices Civils de Lyon, a tertiary hospital encompassing all clinical specialties. After excluding 1,925 duplicate tests, 22,731 unique analyses were included. Approximately 30% of test prescriptions originated from screening centers such as CeGIDD (free screening center for sexually transmitted illnesses) or infectious disease departments, with smaller contributions from hepato-gastroenterology (4.2%), addictology/psychiatry (3.9%), and hematology/oncology departments (9%). The median age of tested individuals was 37 years (mean: 42.3 years, range: 0–101 years). Among the unique tests, 297 (1.3%) were positive, with the majority (85.9%, n=255) showing antibody positivity alone. Antigen-only positivity was rare (1.0%, n=3), while 13.1% (n=39) were positive for both antigen (Ag) and antibody (Ab) (Figure 1). Figure 1 : Overview of Elecsys® HCV Duo screening and confirmatory tests results + Positive, - Negative, Ag Core Antigen; Ab HCV antibodies, VL viral load Biological characteristics of the tested samples are summarized in Table 1 . Patients with active HCV infection were significantly older (mean: 48.3 years, median: 49.5 years) than those with negative screening results (mean: 42.2 years, median: 37 years, p =0.0088). Table 1 : Biological characteristics of samples tested with the Elecsys(r) HCV Duo test NA = not available, Ag = antigen, Ab = antibody.*in the case of active infection, mean antigenic index: 0.65 (p-value <0.0001) (n=34). Mean antigenic index (min-max) 88.5 (2.82-258) 9.9 (1.1-62.8) 0.56* (0.48-0.97) 0.54 (0.49-0.99) Median antigenic index 4.8 4.1 0.54 0.53 Mean HCV VL log UI/mL 7.0 (n=2) 6.3 (n=39) 5.04 (n=34) NA Age (mean) 40.7 48.9 54.7 42.1 HIV co-infection (%) 2 (66.6%) 3 (7.7%) 19 (7.4%) 1102 (4.9%) Genotype 1a 1b 2 3a 4 Not performed 1 0 0 0 1 0 17 7 3 5 3 4 3 1 1 1 1 24 NA Antigen and Antibody Results In this study, 9 samples were initially found positive for HCV Ag alone. However, after reanalysis following the manufacturer’s recommendations, only 3 samples retained an index value above the positivity threshold of 1, with values ranging from 2.82 to 258. The mean index value for the initial false positives was 3.45 (range: 1.02–8.1). Two of these 3 showed a positive viral load (VL). Notably, none of these patients exhibited hepatic cytolysis. All 39 samples with dual Ag+/Ab+ results corresponded to active infections (confirmed HCV RNA-positive), with mean and median viral loads of 6.3 log IU/mL (range: 5.0–7.4 log IU/mL). Interestingly, only 53.8% (n=21) of these patients exhibited elevated ALT levels (mean ALT: 9.1 times the upper normal limit). Among antibody-only positive samples (n=255), 13.3% (n=34) had active infections, as evidenced by positive HCV RNA. Mean HCV RNA level of these samples with negative HCV Ag was 5.04 log IU/mL (median 5.19 log IU/mL, [1.48-7.23]). Although negative according to the manufacturer’s threshold, these samples exhibited a higher mean antigenic index (0.65) compared to cured or resolved cases (0.54, p <0.0001). Overall, for patients with antibody-positive results (n=294), there was a significant difference between the antigenic index of HCV-positive and HCV-negative viral load ( p <0.0001, Figure 2a). Moreover, in order to determine an antigenic index threshold for detecting active infections in antibody-positive individuals, a ROC analysis identified a threshold of 0.625 with a sensitivity of 77% and a specificity of 98.6% (Figure 2B). In total, 1126 of the patients who were subjected to serological testing (n=22731) were identified as being infected with HIV. Of these, 24 exhibited positive-HCV serology (2.1%). Figure 2 : Antigenic Index Analysis in Antibody-Positive Patients A) Comparison of antigenic index values (log) in antibody-positive patients according to viral load results. Dotted line corresponds to the Elecsys® threshold of positivity. B) ROC curve: determination of an antigenic index threshold in antibody-positive patients to identify active infection. All samples with a positive viral load (n=41) without serology initially requested, were also screened as positive by Elecsys® HCV Duo. Among these, 48.7% had a positive antigenic valence (n=20), with a mean antigenic index of 14.93 (median: 5.15 ) and negative (n=18) with a mean antigenic index of 0.69 (median: 0.65 , range: 0.53–0.96 ). The overall mean VL for these samples was 5.44 log IU/mL (median: 5.9 log IU/mL ). Overall, The Elecsys® HCV Duo assay demonstrated 99.9% specificity and 100% sensitivity for detecting HCV infection. However, focusing on the ability of the antigenic-valence to detect active HCV infection, sensitivity dropped to 54.6% but increased to 70.9% for viral loads above 5 log IU/mL. Low viral load Between April 2022 and May 2023, 12% of HCV-positive VLs were below 5 log IU/mL (mean 2.98 log IU/mL). Low VL were associated to factors such as ongoing DAA therapy, acute hepatitis profiles, or underlying conditions like cirrhosis or dialysis. All samples were Ab-positive but only one HCV Ag-positive. Consequently, the sensitivity of the Elecsys® HCV Duo assay for detecting HCV infection in cases with low viral loads (≤5 log IU/mL) was 100% but only 4.5% to identify active HCV infection. Additionally, the study demonstrated a moderate correlation between viral load and the antigenic index (r² = 0.43). Notably, capsid antigen was more frequently (68%) positive when viral loads exceeded 5 log IU/mL (Figure 3). Figure 3: Correlation between HCV viral load (log UI/mL) and antigenic index (log) of Elecsys® HCV Duo assay. Characteristics of HCV infections A total of 66 cases of chronic HCV infection were identified, with half of them displaying Ag-/Ab+ profiles. Most chronic cases were asymptomatic and often linked to intravenous drug use (Table 2b). Table 2: Clinico-Biological Characteristics of Patients with Acute (a) and Chronic (b) Active HCV Infections Ag+/Ab- (n=2) Ag+/Ab+ (n=8) Ag-/Ab+ (n=4) Mean antigenic index 131.4 14.7 0.73 Median antigenic index 131.4 3.55 0.73 Mean HCV VL log IU/mL 7 6.02 4.35 Median HCV VL log IU/mL 7 5.4 3.9 Age mean (years) 48.5 34.5 34.5 ALTN>N 2 (100%)0 08 (100%) 1 (25%)3 (75%) Clinical featuresNo symptomsJaundice, abdominal painCirrhosis, fibrosisOthers 2 (100%)000 3 (37.5%)3 (37.5%)02 (25%) 1 (25%)2 (50%)00 Genotype1a4d 1 (50%)1 (50%) 6 (75%)2 (25%) 4 (100%)0 Ag + /Ab + (n=32) Ag - /Ab + (n=34) Mean antigenic index 8.48 0.65 Median antigenic index 4.05 0.62 Mean HCV VL log IU/mL 6.34 5.04 Median HCV VL log IU/mL 6.30 5.23 Sex-ratio (M/F) 2.5 2.7 Age mean (years) 52,4 47,8 ALT N > N Not available 15 (46.8%) 14 (43.8%) 3 (9.4%) 20 (58.8%) 14 (41.2%) 0 Clinical features No symptoms Jaundice, abdominal pain Cirrhosis, fibrosis Others (including HCC) Not available 28 (87.5%) 2 (6.25%) 0 2 (6.25%) 0 24 (70.6%) 1 (2.9%) 5 (14.7%) 1 (2.9%) 3 (8.8%) Genotype 1a 1b 2 3a 4d Co-infection 1a,3a Not realised 12 (37.5%) 7 (21.9%) 3 (9.4%) 5 (15.6%) 1 (3.1%) 0 4 (12.5%) 5 (14.7%) 1 (2.9%) 2 (5.9%) 4 (11.8%) 2 (5.9%) 1 (2.9%) 19 (55.9%) Overall, 14 cases of acute HCV infection (defined as a positive HCV serology and/or positive HCV VL in patients with a negative history less than 6 months old) were identified during the study period (Table 2a). All of these patients were male (mean age: 39 years) and exhibited high-risk factors for HCV infection such as high-risk behaviors (men who had unprotected sex with men or chemsex. 43% were coinfected with HIV. Seven cases were identified with dual Ag+/Ab+ profiles, while two were Ag+ only. In five cases, acute infection was detected via HCV RNA direct testing due to suggestive clinical symptoms following high-risk behaviour. Subsequent serology on these plasmas was Ab-positive in all 5 cases, but only one for the Ag valence with index value ranging from 0.53 to 0.93. These patients had significantly lower HCV VLs than antigen-positive patients (4.35 log IU/mL versus 6.22 log IU/mL, p=0.002, but the majority was infected with genotype 1a as for HCV Ag-positive patients. Importantly, genotypic analysis revealed that the Elecsys® HCV Duo test effectively detected genotypes 1 through 4, without differences in serological profiles between antigen-positive and “false” antigen-negative results. This suggests that the antigenic detection component of the Elecsys® HCV Duo test is capable of reliably identifying various HCV genotypes circulating in France. Seroconversion panel Retrospective analysis of previously identified acute infections showed that the Elecsys® HCV Duo would have been negative on its Ab-valence in all samples such as the third generation test used at the time of screening, but positive for the Ag valence in 12 of the 19 samples, with an antigenic index ranging from 1.7 to 468. Among these 12 samples, 9 were retrospectively confirmed with a positive VL for HCV, confirming the true positivity of the Elecsys® HCV test. Six out of the 12 patients had normal ALAT. Use of the Elecsys® HCV Duo test therefore would have enabled a reduction in screening time in 63.2% of cases with a mean of 93.5 days (min = 0 days; max = 253 days). Discussion Early diagnosis of HCV infection is essential to limit transmission and improve patient outcomes. Traditional HCV antibody-based assays suffer from a prolonged serological window, limiting their ability to detect infections at an early stage. Fourth-generation combined antigen/antibody assays, such as Elecsys® HCV Duo, address this gap by simultaneously detecting HCVcAg and HCVAb. In our real-life evaluation, Elecsys® HCV Duo shortened the serological window by up to 253 days in one case, a delay that could lead to significant secondary transmission, particularly in high-risk groups such as people who inject drugs or immunocompromised patients, including those with HIV. These findings confirm previous reports and highlight the test’s potential for earlier diagnosis and linkage to care. Dual Ag/Ab positivity demonstrated excellent reliability for active infection (PPV = 100%), supporting direct initiation of DAA therapy without systematic confirmatory RNA testing, as also observed by Bui et al. (11). In contrast, Ag-only results required confirmation due to occasional false positives, consistent with manufacturer recommendations. Our false-positive rate (n = 1/22,731) was lower than that reported by Majchrzak et al. (13/20,634), underlining the assay’s high specificity (12). The lower false-positive rate observed in the present study may be attributable to better control of the pre-analytical phase in a level 3 hospital compared with the multicenter study. Similarly, a meta-analysis by Sepulveda-Crespo et al. showed that HCV Ag assays may have a low PPV (<59%) in settings where the prevalence of active infection is below 1%, necessitating confirmatory HCV RNA testing to ensure diagnostic accuracy (10). On the other hand, it exceeded 80% in high-prevalence populations (≥5%). Prevalence of active hepatitis C in the study was about 0.33%. According to the meta-analysis, with this prevalence of active hepatitis C, the probability of the test being a true positive should be between 12 and 42%. A PPV of 97.6% was observed for the detection of antigen valence, which is not consistent with their results but might be explained by our lower sensitivity. EASL (8) and WHO guidelines support HCV-Ag as a screening alternative to RNA in resource-limited settings, but emphasize confirmation where prevalence is low or clinical suspicion persists. However, this recommendation is based on the high negative predictive value of the HCV Ag test alone (10). The use of combined techniques to replace NAATs should be approached with caution. Moreover, a recent study investigated the role of HCVcAg results in assessing SVR instead of HCV viral load. They obtained a good specificity (97.4%) and a low false positivity rate (1.57%) but a relatively low sensitivity (57.1%) which might be explained by the difference of sensitivities in detecting viremia. Thus, HCV-Ag testing could be helpful for assessing SVR after DAA therapy, with a HCV viral load performed when HCV-Ag is still positive (13). Indeed, the present study revealed a non-negligible number of false-negative antigen results associated with positive antibodies, which could lead to genuine active infection being ignored (n=34). Adjusting the Ag cut-off to 0.625 improved PPV, in line with Bui et al. that suggested 0.7 threshold. In acute HCV infections, capsid antigen was positive in 71.4% of cases, either alone or in combination with antibody-positivity. Thus, detection of capsid antigen associated with clinical (jaundice, vomiting) and biological (increased ALT) criteria would suggest an acute hepatitis C infection. However, false negatives of antigen valence were observed in almost a third of cases. These false negatives were all associated with a low viral load (≤ 5 log IU/mL) and, in ¾ of cases, with HIV co-infection. As a reminder, the natural history of chronic HCV infection includes a zenith of VL secondary to infection, followed either by a plateau persistence of VL or by a nadir linked to the establishment of the immune response before a re-ascension of VL during chronicization of the infection (14,15). A sample taken at the time of the ramp-up phase of the VL or at its nadir may explain the presence of these false negatives. The main limitation of the Elecsys® HCV Duo was reduced sensitivity for low HCV VL (≤ 5 log IU/mL): only 4.5% in this subgroup, with most false negatives linked to ongoing DAA therapy, HIV-coinfection, cirrhosis, or dialysis. This aligns with Bertisch et al., who demonstrated that only one-third of patients with VLs ≤ 3.5 log IU/mL were capsid antigen-positive (16). Consequently, it reinforces that Ag-negative/Ab-positive results should not exclude active infection. This highlights the importance of confirmatory HCV RNA testing, particularly in cases with a low VL or where is suspected. Interestingly, the assay identified reinfections in patients with previous HCV cure, with HCVcAg detected in 2 of the 3 observed cases, supporting its potential role in surveillance of high-risk populations. Although further validation in larger cohorts is required, our results suggest that combined Ag/Ab testing could play a role in identifying reinfection in high-risk individuals. The widespread application of the Elecsys® HCV Duo test in various clinical departments underscores its versatility and adaptability across healthcare settings. Its use in high-risk populations, particularly those served by CeGIDD centers and infectious disease departments, aligns with WHO’s elimination targets by 2030, especially in settings with limited molecular capacity. Elecsys® HCV Duo is a valuable tool for early HCV detection and streamlined patient management, particularly when dual Ag/Ab positivity is present. However, confirmatory RNA remains essential for Ag-only positives and Ab-positive/Ag-negative cases in high-risk contexts. Future work should focus on improving sensitivity for low VLs, validating adjusted cut-offs, and assessing cost-effectiveness in diverse and decentralised screening programmes. REFERENCES 1. World Health Organization, Global hepatitis report 2024: action for access in low- and middler-income countries [Internet]. [accessed 18 déc 2024]. https://www.who.int/publications/i/item/97892400916722. Axley P, Ahmed Z, Ravi S, Singal AK. Hepatitis C Virus and Hepatocellular Carcinoma: A Narrative Review. J Clin Transl Hepatol. 2018; 6. 3. HAS. Recommandations de prise en charge des personnes infectées par le virus de l’hépatite C. 2024. 4. HAS. Stratégies de dépistage biologique des hépatites virales B et C. 2011. 5. 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PLOS ONE. 2015; 10(4):e0122232. 15. Martinello M, Hajarizadeh B, Grebely J, Dore GJ, Matthews GV. Management of acute HCV infection in the era of direct-acting antiviral therapy. Nat Rev Gastroenterol Hepatol. 2018; 15(7):412‑24. 16. Bertisch B, Brezzi M, Negro F, Müllhaupt B, Ottiger C, Künzler-Heule P, et al. Very Low Hepatitis C Viral Loads in Treatment-naive Persons: Do They Compromise Hepatitis C Virus Antigen Testing? Clin Infect Dis. 2020; 70(4):653-659. Information & Authors Information Version history V1 Version 1 14 August 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords disease control hepatitis c virus research and analysis methods virus classification Authors Affiliations Paul-Elie Gosset Hospices Civils de Lyon View all articles by this author Célia Sentis 0009-0001-4303-0686 Hospices Civils de Lyon View all articles by this author Christophe Ramiere Hospices Civils de Lyon View all articles by this author Fabien Zoulim Hospices Civils de Lyon View all articles by this author Caroline Scholtes 0000-0003-0691-8480 [email protected] Hospices Civils de Lyon View all articles by this author Metrics & Citations Metrics Article Usage 529 views 235 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Paul-Elie Gosset, Célia Sentis, Christophe Ramiere, et al. Comprehensive Evaluation of the Elecsys® HCV Duo Immunoassay for Active Hepatitis C Detection: Insights from the DEPCDUO Study. Authorea . 14 August 2025. 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