Comparison of anti-HCV combined with HCVcAg (Elecsys HCV Duo immunoassay) and anti-HCV rapid test followed by HCV RNA analysis using qRT-PCR to identify active infection for treatment

Sitthichai Kanokudom; Kittiyod Poovorawan; Pornjarim Nilyanimit; Nungruthai Suntronwong; Ratchadawan Aeemjinda; Sittisak Honsawek; Yong Poovorawan

doi:10.1371/journal.pone.0313771

. 2024 Nov 18;19(11):e0313771. doi: 10.1371/journal.pone.0313771

Comparison of anti-HCV combined with HCVcAg (Elecsys HCV Duo immunoassay) and anti-HCV rapid test followed by HCV RNA analysis using qRT-PCR to identify active infection for treatment

Sitthichai Kanokudom ^1,², Kittiyod Poovorawan ³, Pornjarim Nilyanimit ¹, Nungruthai Suntronwong ¹, Ratchadawan Aeemjinda ¹, Sittisak Honsawek ^2,^*, Yong Poovorawan ^1,^4,^*

Editor: Gheyath K Nasrallah⁵

PMCID: PMC11573151 PMID: 39556526

Abstract

Hepatitis C virus (HCV) infection can cause acute and chronic hepatitis, leading to liver cirrhosis and hepatocellular carcinoma. The World Health Organization aims to eliminate viral hepatitis by 2030 through extensive screening and treatment. To achieve this goal, comprehensive and widespread screening is essential for diagnosis and treatment. This study aims to evaluate the diagnostic sensitivity and specificity of the Elecsys^® HCV Duo immunoassay (Duo-assay), which simultaneously detects anti-HCV antibodies (Duo/anti-HCV) and HCV core antigen (Duo/HCVcAg) in a single sample, compared with initially antibody testing followed by quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, this study aimed to evaluate a relationship between Duo/HCVcAg and qRT-PCR assay in different genotypes. A total of 769 plasma samples were tested using the Duo-assay to further evaluate the test’s performance and conduct Duo/HCVcAg correlation analysis using qRT-PCR for each genotype. Among the active infection group (anti-HCV+/RNA+; n = 473), the Duo-assay showed 100% sensitivity for detecting Duo/anti-HCV and 70.6% for Duo/HCVcAg. In the resolved infection group (anti-HCV+/RNA–; n = 176), the assay showed 100% sensitivity for Duo/anti-HCV and 100% specificity for Duo/HCVcAg. In the non-infected group (anti-HCV–/RNA–; n = 120), the assay showed 100% specificity for both Duo/anti-HCV and Duo/HCVcAg. Moreover, no correlation was observed between the Duo/HCVcAg and HCV RNA tests, irrespective of genotype. These findings indicate that the Duo-assay is highly sensitive for detecting anti-HCV and specifically identifies patients with active infection. Nevertheless, cases with anti-HCV+/HCVcAg–results should undergo additional confirmation with western blot/immunoblot and qRT-PCR to ensure diagnostic accuracy, especially in Blood donation facilities.

Introduction

Hepatitis C virus (HCV) infection can cause acute and chronic liver diseases, potentially leading to liver cirrhosis and eventually the possible development of liver cancer [1]. The World Health Organization (WHO) recently estimated that 58 million people worldwide are infected with HCV, an 18% decrease from 71.1 million people reported in 2015 [2, 3]. Currently, there is no effective vaccine against hepatitis C. In 2016, the WHO announced a global HCV elimination strategy with the ambitious aim of screening at least 90% of the world’s population, with >80% of those testing positive for HCV expected to be treated, hopefully resulting in a 65% reduction in liver-related mortality by 2030 [2, 4].

Beginning in 2018, several countries pledged to implement measures and campaigns to reach the WHO goal of HCV elimination by 2030, including Thailand [5]. This pledge included a test-to-treat strategy involving a rapid diagnostic test (RDT) as the first step, followed by verification of active infection using a qualitative real-time polymerase chain reaction (qRT-PCR) assay for HCV RNA. Our previous study was conducted in 11 districts of Phetchabun Province, Thailand, between 2019 and 2022 and initially identified 323,672 individuals using a RDT. The predominant genotype among the patients in Phetchabun Province was 6f, followed by 3a and 1a [6, 7]. A total of 11,676 (5.8%) individuals tested positive for anti-HCV. Among these subjects, 75% (4,157/5,527) were positive for HCV RNA, confirming an active HCV infection. Approximately 300 of these patients are currently undergoing treatment [6].

To eliminate HCV in accordance with the WHO’s policy, screening and treatment of individuals are necessary. This approach would reduce the transmission of HCV to uninfected individuals and reduce the number of cases that progress to HCV-related liver disease. Antibody testing is the standard diagnostic procedure for HCV infection in blood donors, individuals with acute or chronic infection, and those who have a resolved infection. The standard two-step process for detecting active HCV infection includes an initial anti-HCV test followed by a confirmatory HCV RNA test, which can be both time-consuming and costly [3]. The WHO has subsequently approved the use of HCV core antigen (HCVcAg) tests alone as an alternative method for the diagnosis of active infection, reserving the use of HCV RNA testing for treatment decisions with direct-acting antivirals (DAAs). HCVcAg testing using Architect showed high concordance with HCV RNA detection in several studies, ranging from 89.7% to 95% [8]. Following this, the Thailand’s National Health Security Office (NHSO), Ministry of Public Health (MOPH) of Thailand and other partners implemented WHO-based guidelines for diagnosis and treatment for HCV patients [9–11]. The approach follows a test-to-treat strategy using a two-step process [6, 9]. However, only the isolated HCVcAg test from Architect allow to use in Thailand. Therefore, it is essential to evaluate alternative tests to determine their effectiveness for HCV diagnosis.

In 2022, the newly developed commercial Elecsys^® HCV Duo immunoassay (hereafter referred to as Duo-assay) (Roche Diagnostics GmbH, Mannheim, Germany) is designed to detect early infection (window phase) in individuals who are negative for antibodies against HCV and to confirm active infection. The assay consists of two test modules: one designed to detect HCVcAg using monoclonal antibodies targeting the cAg, and another module designed to detect anti-HCV using synthetic peptides and a recombinant protein representing the core, NS3, and NS4 antigens to identify anti-HCV. The Duo-assay enables the simultaneous detection of Duo/anti-HCV and Duo/HCVcAg in a single specimen [12, 13]. Previously, the Duo-assay was evaluated in a multicenter study in Germany with regard to detecting HCV infection in blood donors (n = 20,634) and routine samples (n = 2,531). The results were compared with those of eight commercially available anti-HCV tests, followed by confirmation using nucleic acid testing (NAT), immunoblotting, or qRT-PCR. The Duo-assay demonstrated a specificity of >99.9% among blood donors and routine samples and a sensitivity of 99.6% among positive samples [13]. However, the efficacy for evaluating active/resolved infection in independent clinical samples is limited.

Additionally, our previous study demonstrated that genotypes 3a and 6 reduced the sensitivity of HCVcAg detection using the Architect HCVcAg assay compared to gold-standard qRT-PCR [14]. Another study also observed a positive correlation between HCVcAg and qRT-PCR in genotype 1b [15]. Currently, there are no reports elsewhere evaluating the performance of the Duo assay for HCVcAg of different genotypes.

The primary objective of this study was to evaluate the diagnostic sensitivity and specificity of the Duo-assay with regard to Duo/anti-HCV and Duo/HCVcAg compared standard anti-HCV testing followed by qRT-PCR confirmation among actively infected or resolved infected samples, and blood donor samples with HCV-negative. The study also examined whether detection of Duo/HCVcAg using the Duo-assay was suitable as an alternative to qRT-PCR. We also performed the correlation between Duo/HCVcAg and HCV RNA viral load and also deeply investigated whether different genotypes of HCV affect the performance of Duo/HCVcAg detection.

Materials and methods

Study design and sample selection

The study was carried out between February and April 2024 at the Center for Excellence in Clinical Virology, Chulalongkorn University, Bangkok. The study protocol was approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University (IRB Number 693/66) and conducted in accordance with the principles of the Declaration of Helsinki. All data used in the final analysis of this study were fully anonymized to protect participant privacy. As a result, no individual informed consent was required. The anonymized data can be made available upon reasonable request to the corresponding author, ensuring compliance with ethical standards.

We obtained the leftover plasma samples from a previous study [9], “Elimination of Hepatitis Virus in Thailand (Micro-elimination Model) Phetchabun Province Model Aiming for clearance Hepatitis virus by 2030” (IRB 023/63), to evaluate the performance of the Duo-assay in this study. In the previous study, these samples were collected from patients aged 35–64 years and were subjected to HCV antibody screening using the Bioline^™ HCV RDT (Abbott Korea Inc., Gyeonggi-do, Korea) (Fig 1). Samples with negative anti-HCV RDT (non-reactive) were excluded, whereas positive anti-HCV RDT samples (reactive) were subsequently confirmed as positive for HCV RNA by qRT-PCR using the COBAS AmpliPrep/COBAS TaqMan HCV Test, v2.0 (Roche Molecular Systems, Pleasanton, CA). Consequently, samples were classified into two groups according to the results of anti-HCV RDT and HCV RNA including samples tested positive for anti-HCV RDT and HCV RNA were classified as the active infection group (n = 478), while those that tested positive anti-HCV RDT and negative for HCV RNA were categorized as the resolved infection group (n = 176). Additionally, leftover donated plasma from another previous study [16], which tested negative for anti-HCV antibody using an enzyme-linked immunosorbent assay (ELISA) and HCV RNA negative by qRT-PCR using the COBAS, was obtained as a control group, referred to as the non-infected group (n = 120). All samples were done nucleic acid testing at sampling sites, and sent the left to us at 4°C. Those samples were storage at −20°C and underwent only one freeze-thaw (FT) cycle for qRT-PCR and no more than three FT cycles for antigen and antibody testing.

Fig 1 — Five samples from the infected group were excluded because two samples had clotted and could not be analyzed using the COBAS analyzer, and three samples had no complete sequence data. A total of 769 samples were included in this study for further analysis.

In the current study, a total of 774 plasma samples from previous studies were reassessed for HCV antibody using the Bioline^™ HCV RDT. All samples were subjected to analyze using the Duo-assay on the Cobas^® 8000 e801 analyzer (Roche Diagnostics GmbH) according to the manufacturer’s instructions. Individual Duo, Duo/anti-HCV and Duo/HCVcAg results were also accessible. However, five samples were excluded due to the clotted samples and incomplete sequencing data. Thus, there were totally 769 samples included for further analysis, comprising 473 in the active infection group, 176 in the resolved infection group, and 120 in the non-infected group.

Laboratory assessment

Serological assay

The Duo-assay is an electrochemiluminescence immunoassay (ECLIA) used to screen for HCV infection in serum and plasma samples. Samples were processed using the Cobas^® 8000 e801 machine following the manufacturer’s instructions (Roche Diagnostics GmbH). The results were reported as cutoff index (COI) values in three categories: the main result (Duo) and two modules consisting of Duo/anti-HCV and Duo/HCVcAg. The Duo result was automatically calculated from the Duo/anti-HCV and Duo/HCVcAg values, with a COI of less than 1.0 indicating a non-reactive result, and a COI of 1.0 or greater indicating reactivity. Similarly, for both Duo/anti-HCV and Duo/HCVcAg, COI values below 1.0 indicate non-reactive results, while values of 1.0 or higher indicate reactivity for anti-HCV and HCVcAg, respectively.

Furthermore, four samples from the resolved infection group showed a discrepancy between the anti-HCV RDT, which had positive results, and the Duo/anti-HCV module, which had negative results. These samples were reanalyzed using the chemiluminescent microparticle immunoassay (CMIA) of the Architect anti-HCV assay with an Architect PLUS i1000SR analyzer (Abbott GmbH, Wiesbaden, Germany) according to the manufacturer’s instructions. The results were interpreted as reactive for Architect HCVcAg when equal to or greater than 1.0 signal-to-cutoff ratio (S/C), whereas results below 1.0 S/C were considered to indicate non-reactive samples.

Quantitative HCV RNA test

Duo/anti-HCV positive and HCV RNA positive plasma samples were subjected to qRT-PCR analysis using the Cobas^® 4800 system (Roche Diagnostics) according to the manufacturer’s instructions, as previously described [14]. This platform enables quantification of HCV RNA viral load for genotypes 1–6, with a lower detection limit of 15 to 10⁸ IU/mL.

HCV genotyping

Briefly, HCV RNA was extracted from Duo/anti-HCV positive and HCV RNA positive plasma samples using a QIAamp Viral RNA Mini kit (Qiagen, Hilden, Germany), and cDNA was subsequently synthesized using a cDNA using ImProm-II^™ Reverse Transcription system (Promega, Madison, WI). PCR of the partial core gene was conducted in two groups utilizing two primer sets, following a previously published protocol [14]. The resulting PCR products were subjected to Sanger sequencing (1st Base Laboratory, Selangor, Malaysia) and subsequently genotyped based on BLASTN search results (http://www.ncbi.nlm.nih.gov).

Data analysis

The results of diagnostic testing using the Duo-assay and qRT-PCR (as a reference standard) were compared and used to evaluate assay performance in terms of sensitivity and specificity, considering positive samples and non-infected samples, respectively. In this study, performance was evaluated using the MedCal Software Ltd. Diagnostic Test Evaluation Calculator, version 22.026 (https://www.medcalc.org/calc/diagnostic_test.php).

Linear regression analyses, correlations (R²), and statistical analyses of differences between Duo/HCVcAg in terms of COI and logarithmic HCV RNA in terms of IU/mL were performed using GraphPad Prism 10 software (GraphPad Software, Boston, MA). The normality of the distribution of log HCV RNA values (in IU/mL) between reactive and non-reactive HCVcAg samples was assessed using the Kolmogorov-Smirnov test. Because the data were not normally distributed, the non-parametric Mann-Whitney U test was used to compare logarithmic levels of HCV RNA between HCV Ag reactive and non-reactive samples. For normally distributed samples, the parametric Welch t-test was used instead. A p-value <0.05 was considered statistically significant.

Results

Reactivity of plasma samples for Duo/anti-HCV and Duo/HCVcAg

A total of 769 plasma samples were analyzed using the Duo-assay. The results indicated that 473 samples from the active infection group were reactive in the Duo and Duo/anti-HCV (100% sensitivity). Of these 473 samples, 334 (70.6% sensitivity) were reactive for Duo/HCVcAg, whereas 139 (29.4%) were non-reactive.

In the resolved infection group (n = 176), 172 samples were reactive in the Duo/anti-HCV, whereas 4 samples were non-reactive. Additionally, all samples in this group were non-reactive in the Duo/HCVcAg. To additionally validate the sensitivity of the Duo/anti-HCV assays in the resolved infection group, 4 non-reactive samples were retested using the Architect anti-HCV assay. The results ultimately confirmed that these 4 samples were either non-reactive for Duo/anti-HCV or false positives by RDT.

Concurrently, all 120 samples in the non-infected group were non-reactive in the Duo, Duo/anti-HCV, and Duo/HCVcAg tests (100% specificity) (Fig 2).

These findings underscore that the Duo-assay achieves 100% sensitivity for detecting anti-HCV in individuals with active infections. It has approximately 70% sensitivity for detecting HCVcAg, suggesting that nearly 30% of active infection cases were underestimated. Moreover, the Duo-assay showed 100% sensitivity in both the Duo and Duo/anti-HCV and demonstrated 100% specificity for Duo/HCVcAg in individuals with resolved infections.

No correlation observed between Duo/HCVcAg and quantitative HCV RNA

To identify that the detection of Duo/HCVcAg was able to quantitate an amount of HCV. A total of 473 samples from active infected individuals (anti-HCV+, HCV RNA+) were subjected to HCV genotyping, and the results are described in Table 1. The four most common genotypes in the Phetchabun cohort in this study were 6f, accounting for 41.6%, followed by 3a at 31.7%, 1a at 14.0%, and 1b at 7.2%. By contrast, there was no available data regarding genotypes of the group negative for HCV RNA (n = 296).

Table 1. Proportion of HCV genotyping of the study samples.

Total number of samples: 769	Number (%)
Active infection group (anti-HCV+, HCV RNA+), n = 473
• Genotype 1a	66 (14.0)
• Genotype 1b	34 (7.2)
• Genotype 3a	150 (31.7)
• Genotype 3b	10 (2.1)
• Genotype 6f	197 (41.6)
• Genotype 6i	8 (1.7)
• Genotype 6n	7 (1.5)
• Genotype 6v	1 (0.2)
Resolved infection group (anti-HCV+, HCV RNA−), n = 176	N/A
Non-infected group (anti-HCV−, HCV RNA−), n = 120	N/A

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N/A, no data available due to the absence of HCV RNA.

No significant correlation was observed between Duo/HCVcAg results and HCV RNA viral load among genotypes 1a, 1b, 3a, and 6f (R² ranging from 0.067 to 0.212) (Fig 3A–3D). In terms of test sensitivity, the Duo/HCVcAg was able to detect genotypes 1a (80.3%), 1b (76.5%), 6f (71.6%), and 3a (62.0%) (Table 2). We also compared the HCV RNA viral load between Duo/HCVcAg reactive and non-reactive samples. Duo/HCVcAg reactive samples had significantly higher titers than non-reactive samples across genotypes 1a, 1b, 3a, and 6f (p<0.05) (Table 2). These data suggest that the sensitivity of the Duo/HCVcAg assay is affected by the level of HCV RNA present in plasma samples. Our results suggest that the level of Duo/HCVcAg detection was not significantly correlated with the amount of HCV RNA. This indicates that the Duo/HCVcAg module is not suitable for the quantitative determination of HCV RNA.

Fig 3 — Scatter plot illustrates the relationship between Duo/HCVcAg module, monitored based on the automated Duo/HCVcAg in COI, and log HCV RNA level in IU/mL across different genotypes, including 1a (A), 1b (B), 3a (C), and 6f (D). Correlations (R²) were calculated from trendlines. Statistical significance was set at p<0.05.

Table 2. Comparison between Duo/HCVcAg and viral load for 447 samples from the active infection group, including genotypes 1a, 1b, 3a, and 6f.

Genotype	Module	Interpretation	Number	Log median [IQR] (min-max) in IU/mL	p-value^*
1a	Duo/HCVcAg	Reactive	53	6.63 [6.35–6.9] (5.20–7.18)	0.0141
1a	Duo/HCVcAg	Non-reactive	13	5.88 [4.69–6.37] (3.52–7.02)	0.0141
1b	Duo/HCVcAg	Reactive	26	6.50 [5.98–6.71] (4.63–7.47)	0.0068
1b	Duo/HCVcAg	Non-reactive	8	4.59 [3.44–5.84] (2.38–6.36)	0.0068
3a	Duo/HCVcAg	Reactive	93	6.52 [6.21–6.99] (4.69–7.43)	<0.0001
3a	Duo/HCVcAg	Non-reactive	57	5.30 [4.81–5.92] (2.88–7.33)	<0.0001
6f	Duo/HCVcAg	Reactive	141	7.00 [6.61–7.24] (5.02–7.66)	<0.0001
6f	Duo/HCVcAg	Non-reactive	56	6.18 [5.41–6.99] (3.66–7.58)	<0.0001

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*p-value comparison of HCV RNA viral load between Duo/HCVcAg reactive and non-reactive samples.

Discussion

The main purpose of this study was to evaluate the performance of one-step antibody and antigen tests for the screening and diagnosis of HCV infection in unidentified samples. The Duo-assay, which includes an anti-HCV module, exhibited 100% sensitivity for samples from the active infection group (RDT+/PCR+). Of these, 70.6% were reactive in the Duo/HCVcAg module, in contrast to previous studies reporting a sensitivity of 95.7% for the automated Architect HCVcAg assay, designed specifically for HCVcAg detection in chronic hepatitis C patients, including those co-infected with HIV or hepatitis B virus (HBV) [17].

In the resolved infection group (RDT+/PCR−), 172 of 176 samples showed concordance with the sensitivity of the two-step reference standard, which combines RDT followed by qRT-PCR analysis. However, the results for four samples were inconsistent. These samples were then re-analyzed using a different automated diagnostic test, the Architect Anti-HCV assay. The results indicated that the RDT had produced false-positive results for anti-HCV. Among 172 resolved infection samples, there was 100% agreement with the Duo-assay: 100% were positive for anti-HCV, and 100% were negative for Duo/HCVcAg, corresponding to the two-step RDT+/PCR− results. The automated analysis results were more accurate than those interpreted visually using RDTs [18]. This incidence was supported by previous studies showing that seven manufactured anti-HCV RDTs (Alere Trueline, Benesphera, AccuTest, Abon, Blue Cross, Healgen, and SD Bioline) exhibited lower sensitivity, ranging from 65.3% to 83.0%, when compared with automated CMIA using the Architect anti-HCV assay [18, 19].

All resolved infection samples tested negative in the Duo/HCVcAg module. In the blood donor group (RDT−/RNA−), all samples were negative in the Duo, Duo/anti-HCV, and Duo/HCVcAg modules (100% specificity). Based on these collective results, we concluded that the Duo and Duo/anti-HCV modules exhibited 100% sensitivity throughout the cohort for anti-HCV+ samples, the active infection and resolved infection groups, and 100% specificity with HCV confirmed-negative samples. These results were consistent with those of a previous study conducted in Germany, which demonstrated a sensitivity of up to 99.9% among known HCV confirmed-positive samples and specificity of 99.6% among blood donor samples [12, 13]. Moreover, our data suggest that the German study calculated the Duo-assay results based solely on the anti-HCV module [12, 13]. However, our data indicates that approximately 30% of active infection group may be missed and incorrectly classified as resolved infection group (i.e., lower accuracy). Despite this limitation, to ensure accurate monitoring of treatment response in some patients, samples that test positive for anti-HCV but negative for HCVcAg should undergo follow-up an alternative HCV RNA testing to confirm the infection status.

We were the first to report the influence of genotypes on the performance of the Duo/HCVcAg assay and its relationship with viral load. This study revealed found no correlation between Duo/HCVcAg and HCV viral load corresponding to genotypes 1a, 1b, 3a and 6f when using ECLIA, although we monitored the correlation between cAg levels in COI and HCV viral load (with a median of >4.5 log IU/mL) among actively infected individuals. This is because the Duo-assay is designed for in vitro qualitative interpretation. Therefore, we suggest that the COI scale determined using the Duo-assay should not be used to calculate the correlation with HCV viral load. Unlike previous studies demonstrating a correlation between cAg level and HCV RNA viral load, this discrepancy arises from the design of the CMIA of the Architect HCVcAg test specifically for quantitative analysis, with the result reported in fmol/mL [14]. Other recent studies have suggested that detecting HCVcAg using the Architect assay provides high sensitivity for samples with a viral load >10,000 IU/mL [20] and that cAg can be detected within 12–15 days after infection [21]. Moreover, another study observed that 32 of 145 actively infected patients tested positive for a viral load ≥1,000 IU/mL (high titer), whereas 113 of 145 tested positives for a viral load <1,000 IU/mL (low titer). When comparing the positivity between viral load and Architect HCVcAg results showing a high correlation (r = 0.890; p<0.001), this study demonstrated a sensitivity of 93.9% in the high-titer group and 50.0% in the low-titer group, with a specificity of 99.1% [22].

These results were consistent with those of the current study, which showed that samples seropositive in the Duo/HCVcAg test had significantly higher levels of HCV RNA compared with non-reactive samples among patients with active infection. However, no correlation was observed between HCV RNA level and Duo/HCVcAg result, considering both reactive and non-reactive samples. Furthermore, our previous study revealed that Architect HCVcAg determined was significantly correlated with HCV RNA, regardless of HCV genotype and/or co-infection with HBV, with accuracy, sensitivity, and specificity values of 99%, 99%, and 100%, respectively [23]. Furthermore, another previous study demonstrated a strong correlation between Architect HCVcAg and HCV RNA results, with an R² value of 0.889 and p-value of <0.001 [19]. The Duo-assay combines two modules, including total anti-HCV antibodies that target the core, NS3, NS4A, and NS5A proteins [12], which might interfere with binding to cAg during the reaction, whereas the Architect assay was specifically designed for detecting the cAg alone.

We also explored the sensitivity of the test among different genotypes. The Duo-assay was designed using recombinant proteins representing HCV genotype 1. This was consistent with previous finding revealing that Architect HCVcAg quantification is highly correlated with genotype 1, whereas genotypes 3 and 6 exhibit lower correlations with HCV RNA levels [14]. This was consequently supported by previous studies showing that genotype 1 is commonly prevalent worldwide, accounting for 49.1%, followed by genotype 3 at 17.9%. By contrast, genotypes 5 and 6 collectively are the least common, constituting <5% of cases [24–26]. Previous studies reported that genotype 3 (particularly 3a) is highly prevalent in Southeast Asia, including Thailand, which was associated with the significant increase in narcotics use during the Vietnam War in the 1960s-1970s and unawareness regarding asymptomatic/resolved hepatitis cases associated with medical transfusions thereafter [27–29]. The current study identified genotype 6 as predominant in Phetchabun Province, which is located in the southern part of northern Thailand. Our data was concordant with those of a previous study conducted in 2018 involving a sample size of 4,769 subjects [7]. Understanding the variety of genotypes is crucial for development of effective screening processes and the use of DAAs in therapeutics, which could facilitate achievement of the goal of eliminating hepatitis C by 2030.

The results of this study support development of guidelines for identifying new patients and shorten the HCV screening process, potentially leading to more cost-effective surveillance. This can influence clinical decision-making and treatment timing, particularly in resource-limited regions where HCV RNA testing may be limited or unavailable. The results obtained from the two modules using the Elecsys^® HCV Duo immunoassay (Duo-assay) can be categorized into four categories, as illustrated in Fig 4. First, if the results are anti-HCV−/cAg−, the patient is categorized as free of HCV infection; consequently, no further follow-up is needed. For the second category, if infection is diagnosed in the early stages (anti-HCV−/cAg+), known as acute hepatitis, treatment may not be immediately necessary. Instead, the patient can undergo another blood test after a few months to confirm natural clearance of the virus by the immune system. In early HCV infection, 15–45% of individuals experience spontaneous viral clearance, leading to the presence of anti-HCV antibodies and the absence of detectable HCV RNA within a few months [3, 29, 30]. Approximately 55–85% of infected individuals progress to chronic infection, characterized by the presence of anti-HCV antibodies and detectable HCV RNA [3, 29–32]. For the third category, patients with a result of anti-HCV+/cAg− (approximately 30% misclassified due to a false negative for cAg) require additional nucleic acid testing (RNA+) to determine if the infection is active, and if so, guide the appropriate course of treatment. Conversely, patients with an RNA− result do not require further follow-up. In the fourth category, patients with a result of anti-HCV+/cAg+ are deemed as needing treatment.

Fig 4 — Duo-assay results can be interpreted according to four patterns, consisting of three sets of one-step (anti-HCV−/cAg−, anti-HCV−/cAg+, and anti-HCV+/cAg+) and one set of two-step (anti-HCV+/cAg−) processes. Samples with a result of anti-HCV+/cAg− must be confirmed using nucleic acid testing as the second step.

Some limitations were observed in this study. First, demographic characteristics were not provided due to the anonymity of the sample sites. The reference standards for evaluating a performance agreement of Duo/anti-HCV antibodies were unavailable. However, our study focused specifically on assessing the Duo-assay’s ability to differentiate between individuals with active infection (anti-HCV+ and Duo/HCVcAg +) and those with resolved infection (anti-HCV+ and Duo/HCVcAg–). Another limitation is that we did not obtain samples showing RDT−/PCR+ results, which represent early infection/window-phase cases. This was because the criteria used in the previous study did not include additional confirmation by PCR in the case of a negative RDT result, as this is rare [11]. Anti-HCV antibodies are typically detected after several weeks of infection.

Conclusion

The Elecsys^® HCV Duo immunoassay, which allows determination of antibodies and antigens in a single sample, is beneficial for determining HCV status. The results of this study suggest that the Duo-assay provides high sensitivity for detecting chronic HCV infection with high specificity for the diagnosis of active HCV infection. However, samples with anti-HCV+/cAg− results must be confirmed using HCV qRT-PCR or RNA detection. This tool could significantly reduce the cost of HCV RNA viral load testing, reduce turn-around time for screening and diagnostic and expedite treatment for patients. Additionally, the Elecsys^® HCV Duo immunoassay could assist in achieving the goal of eliminating hepatitis C globally by 2030.

Acknowledgments

We would like to express our gratitude to Bangpakok International Hospital for providing us with the equipment and facility for sample testing, and to all participants for their contributions and support of this project.

Data Availability

All relevant data are within the manuscript.

Funding Statement

This work was partially supported by the Roche Diagnostics (Thailand) Ltd., the Center of Excellence in Clinical Virology, Chulalongkorn University, the King Chulalongkorn Memorial Hospital and the Second Century Fund (C2F) of Sitthichai Kanokudom, Chulalongkorn University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0313771.r001

Decision Letter 0

Gheyath K Nasrallah

24 Sep 2024

PONE-D-24-35203Comparison of anti-HCV combined with HCV cAg (Elecsys® HCV Duo immunoassay) and anti-HCV rapid test followed by HCV RNA analysis using qRT-PCR to identify active infection for treatmentPLOS ONE

Dear Dr. Poovorawan,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Your manuscript were reviewed by two experts in the field. Although both mentioned that you manuscript is very interesting, yet many comments were generated and need to be fixed.

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[This work was partially supported by the Roche Diagnostics (Thailand) Ltd., the Center of Excellence in Clinical Virology, Chulalongkorn University, the King Chulalongkorn Memorial Hospital and the Second Century Fund (C2F) of Sitthichai Kanokudom, Chulalongkorn University.].

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Comments to the Author

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Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript presents a comprehensive evaluation of the Elecsys® HCV Duo immunoassay, highlighting its potential as a diagnostic tool for Hepatitis C virus (HCV) infection. The study offers valuable insights into the assay's performance, particularly in its ability to detect both HCV antibodies and core antigen. However, there are several critical aspects that need to be addressed to enhance the manuscript's clarity, the robustness of the findings, and the practical implications of the assay. Below are specific comments and concerns that should be addressed before the manuscript can be considered for publication.

Minor Comments:

1. Ensure consistent tense usage. For example, "This study aimed to evaluate" could be revised to "This study aims to evaluate" to match the present tense used throughout the abstract.

2. There are a few instances where commas are either missing or incorrectly placed. For example, in the sentence "The results were interpreted as reactive for Duo, as calculated from the highest score among anti-HCV and HCV cAg results," a comma after "Duo" seems unnecessary.

3. The manuscript might benefit from ensuring consistency in the use of italics and bold for emphasis. For instance, "HCV" is sometimes italicized and other times not. Consistency is key.

4. Ensure that all acronyms are defined at their first occurrence. For example, "ECLIA" and "COI" are used without prior definition.

5. The resolution of the figures is very poor. Consider using color coding to differentiate between the different groups in figure 1. This would help visually separate different parts of the flow and make the figure more engaging.

Major comments

1. One of the primary concerns with this manuscript is that it does not substantially add to the body of knowledge already established by the 2022 study on the Elecsys® HCV Duo immunoassay. The earlier study provided a comprehensive multicenter evaluation of the assay's specificity and sensitivity across diverse populations and settings. While your manuscript offers localized data from a Thai cohort, the overall findings align closely with those of the 2022 paper, without introducing significant new insights or advancements. To strengthen the manuscript, the authors should highlight the novelty of their study and what their study add to the exiting literature.

2. The manuscript briefly mentions the lack of correlation between HCV core antigen (cAg) levels and HCV RNA. This is an important point, but it needs a more in-depth analysis. The discussion should explore potential reasons for this lack of correlation and its implications for the use of the Elecsys® HCV Duo immunoassay as a standalone diagnostic tool. Additionally, compare these findings with the 2022 study, which did not emphasize this issue, and discuss whether this observation is novel or aligns with other emerging data in the field.

3. The manuscript explicitly states that there was no overall correlation found between HCV core antigen (cAg) levels and HCV RNA. Given this finding, the rationale for proceeding with genotyping and sequencing is unclear. The authors need to explain why they chose to perform genotyping after determining that there was no significant correlation between cAg and RNA. Was the genotyping intended to investigate whether specific HCV genotypes might influence this lack of correlation, or was there another hypothesis being tested? Without this explanation, the inclusion of genotyping and sequencing appears disconnected from the primary findings, raising questions about the necessity and relevance of these procedures within the study. This clarification is crucial for understanding the study's design and the implications of the results.

4. The manuscript should reference any existing literature that suggests genotyping might influence the correlation between cAg and RNA levels. If such references exist, they should be cited to justify the decision to include genotyping in the study. If no such references are available, the authors should clarify that their approach was exploratory and explain the reasoning behind this exploration. This clarification is crucial for understanding the study's design and the implications of the results.

5. The manuscript reports that a significant number of samples in the active infection group tested positive for anti-HCV but negative for HCV cAg. This finding is critical, as it impacts the reliability of using cAg as a standalone marker for active infection. The authors should discuss potential reasons for this discrepancy, such as assay sensitivity, low viral loads, or technical factors. They should also address the clinical implications of these findings, particularly the importance of confirming anti-HCV+/cAg- results with HCV RNA testing to avoid false negatives.

Reviewer #2: Reviewer Comments to Authors: Manuscript No.: PONE-D-24-35203

Comparison of Elecsys® HCV Duo assay versus Anti-HCV RDT followed by qRT-PCR to Identify active infection

I went through the manuscript, and after thoroughly reviewing and evaluating the scientific attributes and

level of importance of the manuscript, I am hereby sending the following comments to the Authors:

The authors’ aim was to compare the Elecsys® HCV Duo immunoassay and the traditional two-step

process, which involves initial anti-HCV antibody testing followed by quantitative real-time polymerase

chain reaction (qRT-PCR) for detecting active hepatitis C infections. Additionally, the study aimed to

evaluate the relationship between HCVcAg and qRT-PCR assay in different genotypes.

Comments to Authors:

1. While the study holds significant value, especially within the context of the global effort to

eliminate HCV, the authors should consider the following points:

A. Introduction:

i. In line 69: include a reference on the adoption of HCV core antigen testing by WHO

recommendation. This will provide credibility regarding the global significance of core antigen

testing.

ii. Provide a brief mention of the national guidelines for HCV testing in Thailand and whether

HCV core antigen testing has been adopted in these guidelines. This will help ground the study

in the context of local diagnostic practices and highlight the relevance of your findings for

Thailand’s national guidelines.

iii. Elaborate on the advantages of using the assay of anti-HCV combined with HCV cAg in real

world settings, particularly its implications for reducing costs and turnaround time in HCV

screening and diagnostics.

B. Materials and Methods:

i. Flow and Clarity: Improving the flow of the methodology would significantly enhance its

clarity and make it easier for readers to follow. A more logical structure would ensure that

each step of the study is clearly outlined, facilitating comprehension. For example, as noted

below, you could improve the transition between the previous and current study methods for

better readability.

ii. Serology assay in the Laboratory assessment Section: Reorganize this section by starting with

information on how the samples were categorized into active infection and resolved infection

groups, referencing the use of Bioline™ HCV RDT from the previous study (Figure 1). Then, follow this by explaining how the current study employed the Elecsys® Duo assay (anti-HCV & HCV cAg) to further assess these groups. This approach will make it easier for readers to understand how the two testing methods are related and integrated into the study.

iii. Sample Processing: More detail should be mentioned on how samples were processed, in

particular in regard to the handling of frozen samples and any potential effects on antigen or

RNA stability.

iv. Line 169 - Reference Standard: In line 169, it is mentioned that qRT-PCR was used as the

reference standard to evaluate the Duo-assay's performance in terms of sensitivity and

specificity. While this is correct for the HCV core antigen (HCV cAg) component of the assay,

it is not correct for the anti-HCV component. For anti-HCV testing, a more specific assay is

required for confirmation, such as the Line Immunoassay (LIA). Typically, tests like the INNO

LIA HCV Score are used to confirm the presence of HCV antibodies with greater specificity.

Therefore, it is important to clarify what reference standard was used to evaluate the

performance of the Duo anti-HCV component. If a Line Immunoassay or a similar confirmatory

test was utilized, this should be explicitly mentioned. Doing so will enhance the accuracy of

the methodology and ensure that the reported sensitivity and specificity values for the anti

HCV assay are appropriately validated against the correct reference standard.

C. Results:

i. The result part needs improvement in terms of clarity and interpretation. By Embedding brief

interpretations within the results to highlight the clinical or practical significance of the

findings, without waiting for the discussion section.

ii. Line 191 and 194: In lines 191 and 194, you refer to "the Duo and anti-HCV modules." For

clarity and consistency, I suggest changing this to "the Duo/anti-HCV modules" throughout

the manuscript wherever the Duo and anti-HCV are mentioned. This will provide a clearer and

more concise reference to the different components of the assay, making it easier for readers

to follow.

iii. Line 197: In line 197, you mention "To validate the sensitivity of the Duo and Elecsys® anti

HCV assays." If the study only used the Elecsys® Duo assay (which includes both anti-HCV and

HCV cAg components), and not a separate Elecsys® anti-HCV assay, please revise this

sentence for accuracy. Instead, it should read: "To validate the sensitivity of the Elecsys® Duo

(anti-HCV) assay." This will ensure that readers are not confused about whether multiple

assays were used, and it accurately reflects the methodology. If both the Duo and separate

anti-HCV assays were used, make sure this distinction is clearly outlined.

D. Discussion:

i. While the Elecsys® Duo assay demonstrates 100% sensitivity for detecting anti-HCV

antibodies, its 70% sensitivity for detecting HCV core antigen (cAg) indicates that 30% of

active infections might be missed if relying solely on cAg detection. This limitation should be

explicitly acknowledged in the manuscript, particularly in the context of resource-limited

settings where access to HCV RNA testing may be restricted or unavailable. In these settings,

the inability to detect all active infections could impact clinical decision-making, delaying

treatment initiation for some patients. Discussing the importance of complementary testing

(e.g., follow-up with RNA testing) where possible, or alternative strategies to manage false

negatives, will make the discussion more balanced and practical.

ii. Detailed discussions on clinical applicability will enhance the quality of the manuscript.

E. Informed consent statement:

i. The ethical considerations concerning anonymized data usage should be emphasized more.

F. Funding:

i. To further reduce any potential conflict of interest (COI) concerns, it is recommended that

you include the following statement in the Funding section: "The funders had no role in study

design, data collection and analysis, decision to publish, or preparation of the manuscript."

This statement will reassure readers and reviewers that the research was conducted

independently and that the funding sources did not influence any aspect of the study's design,

execution, or reporting. This addition will enhance the transparency of the manuscript and

ensure that any concerns regarding objectivity are adequately addressed.

G. Figures and Tables:

i. Provide a legend for each figure and table to guide the reader in interpreting the data,

especially for less familiar readers

ii. The tables should be revised to be clearer and more informative.

H. Minor Comments:

i. Review the manuscript for minor grammatical issues. Shorter, clearer sentences would

improve readability.

ii. Consistency in terminology (e.g., using "Elecsys® Duo assay" versus "Duo-assay") should

be maintained throughout the text.

**********

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Reviewer #1: No

Reviewer #2: No

**********

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Attachment

Submitted filename: Reviewer Comments to Authors PONE-D-24-35203 (Ahmed Ismail).pdf

pone.0313771.s001.pdf^{(129.5KB, pdf)}

PLoS One. 2024 Nov 18;19(11):e0313771. doi: 10.1371/journal.pone.0313771.r002

Author response to Decision Letter 0

29 Oct 2024

Response to editor (R1)

Journal Requirements:

Response: We ensured that the first draft MS and revision version are provided according to the PLOS ONE's style requirements.

2. Thank you for stating the following financial disclosure:

If this statement is not correct you must amend it as needed.

Response: This statement has been added accordingly.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

Response: Ethics statement has been appeared only in the Method section (Lines 112-118).

____________________________________________________________________________________________________________________________________________________________________

Response to Reviewer1 (R1)

Minor Comments:

1. Ensure consistent tense usage. For example, "This study aimed to evaluate" could be revised to "This study aims to evaluate" to match the present tense used throughout the abstract.

Response: This has been revised accordingly. (line 28)

Response: This Sentence has been rewritten in the revied version.

3. The manuscript might benefit from ensuring consistency in the use of italics and bold for emphasis. For instance, "HCV" is sometimes italicized and other times not. Consistency is key.

Response: All terms used in this version have been corrected for consistency.

4. Ensure that all acronyms are defined at their first occurrence. For example, "ECLIA" and "COI" are used without prior definition.

Response: We have rechecked all acronyms that have been mentioned at the first met.

Response: All figures have been reconstructed for consistency and readability, and we have implemented the color coding as per your recommendation. All figures have been converted by the PACE generator software to correct format before resubmission.

Major comments

Response: Some additional paragraphs were included to address the study's gap (Line 74-79), and we are continuing the investigation of DUO/cAg testing by using the Duo-assay in clinical samples. The 2022 study focused on blood donors and routine clinical samples, calculating sensitivity and specificity based on 31 out of 32 positive samples (Table 3, DOI: 10.1016/j.jcv.2022.105293). In contrast, our study expands upon this by calculating these metrics from a larger cohort of actively infected and resolved cases.

Moreover, we extend to investigate the performance using samples from individuals with active phase and those who recovered from HCV infection to monitor the recurrent HCV infection. We also performed the correlation between Duo/HCVcAg and HCV RNA and also deeply investigated whether different genotypes of HCV affect the performance of Duo/HCVcAg detection. (lines 105-107)

Response 2.1 Notably, we found no correlation between the DUO/HCVcAg assay and RT-PCR, as discussed in detail (Lines Line 308-318). Previous studies predominantly examined this correlation using the Architect HCVcAg assay (Abbott)

Our previous study also found the correlation between cAg (Architect) and RT-PCR

“Hansoongnern P, Pratedrat P, Nilyanimit P, Wasitthankasem R, Posuwan N, Wanlapakorn N, et al. An amino acid substitution in HCV core antigen limits its use as a reliable measure of HCV infection compared with HCV RNA. PLOS ONE. 2023;18:e0287694. “

Additionally, compare these findings with the 2022 study, which did not emphasize this issue, and discuss whether this observation is novel or aligns with other emerging data in the field.

Response 2.2: We ensured that our manuscript presents novel findings. The 2022 study primarily involved blood donors and routine samples, most of which were from healthy (non-infected) individuals. In contrast, our current study evaluates the performance of the DUO-assay (Elecsys, Roche) in samples from infected, resolved infection, and non-infected individuals.

Additionally, we are the first to report the influence of genotypes on this assay (Elecsys HCV Duo immunoassay, Roche). (Lines 308-310)

Response:

Firstly, we tried to study the correlation from overall samples between cAg and HCV RNA levels (among 1a, 1b, 3a and 6f). The result showed the no significant and negligible positive correlation (R-squared = 0.06730, p<0.0001). Then, to identify deeply if we analyze upon different genotype, this will appear that are there any relationship among cAg and HCV or not. However, the result showed it still has no correlation between cAg and HCV RNA for respective genotypes.

we expected that the genotype might influence to cAg detection due to the previous study in Hansoongnern et. al, 2022 showed the relation between specific genotype when used the architect cAg assay. However, our studies showed that the Elecsys DUO/cAg has no correlated with RT-PCR result. The reason had been described in discussion (lines 308-318)

Response: We appreciated on your comment. However, do same change in the revised version. Although HCV RNA detection was a gold standard for diagnosis of HCV infection, the performance of HCV RNA comparing with Architect HCVcAg (Abbott GmbH) might be affected by the genotype diversity (a). In addition, the study of correlation between HCV RNA and Architect HCVcAg antigen (identified using CMIA (Abbott)) in different HCV genotype demonstrated a positive correlation between HCV RNA and HCV cAg was observed in only genotype 1b (b). Therefore, we decided to analyze the correlation between HCV cAg and HCV RNA in our study. The review literature for genotyping might influence the correlation between cAg and RNA levels have been cited in lines 95-99. (introduction)

(a) “Hansoongnern P, Pratedrat P, Nilyanimit P, Wasitthankasem R, Posuwan N, Wanlapakorn N, et al. An amino acid substitution in HCV core antigen limits its use as a reliable measure of HCV infection compared with HCV RNA. PLOS ONE. 2023;18:e0287694. “

(b) “Xiang Y, Lai XF, Chen P, Yang Y. The correlation of HCV RNA and HCV core antigen in different genotypes of HCV. J Clin Lab Anal. 2018;33:e22632.” https://pmc.ncbi.nlm.nih.gov/articles/PMC6430366/

Response: I agree with your comment regarding the lower sensitivity of the Duo/HCVcAg assay. However, WHO guidelines do permit the use of cAg as a marker for evaluating infection and monitoring treatment response in active cases. Evidence from previous studies using the Architect assay has demonstrated its successful application in clinical samples with high viral loads, while showing lower sensitivity in samples with low viral loads. In many high-income countries with advanced healthcare systems, RT-PCR is still regarded as the gold standard for all samples. This suggests that RT-PCR remains necessary to confirm results in anti-HCV+/cAg- cases (This has been mentioned in discussion Lines 370-373 and conclusion line 395-403).

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Response to Reviewer2 (R1)