Performance of Elecsys^® HCV Duo Immunoassay for Diagnosis and Assessment of Treatment Response in HCV Patients with or without HIV Infection

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this study, authors investigated the performance of Elecsys® HCV Duo immunoassay for diagnosis and assessment of treatment response in HCV patients. There are some minor issues to be dealt with.

1. How did authors confirm the HCV genotypes? The methodology of it should be mentioned.

2. What is the LOD of the HCV RNA quantification method used in this study? Can the negative results represent that HCV RNA level <12 IU/mL?

3. Did authors compare whether HIV coinfection could affect the performance of Elecsys® HCV Duo immunoassay?

Author Response

In this study, authors investigated the performance of Elecsys@ HCV Duo immunoassay for diagnosis and assessment of treatment response in HCV patients. There are some minor issues to be dealt with.

Comments 1: How did authors confirm the HCV genotypes? The methodology of it should bementioned.

Response 1: Thank you for pointing this out. We added details of HCV genotype identification in the method section line 97-101 as below.   “HCV genotypes were identified through nucleotide sequencing of the core and NS5B regions, followed by phylogenetic analysis. The lower limit and upper limit of the HCV RNA detection were < 12 IU/mL and 100,000,000 IU/mL, respectively. Details of HCV genotype identification and HCV RNA quantification were described in a previous study.(1)”

Comments 2: What is the LOD of the HCV RNA quantification method used in this study? Can the negative results represent that HCV RNA level <12 IU/mL?

Response 2: Thank you for pointing this out. Our study's lower limit of detection (LOD) of HCV RNA detection was < 12 IU/mL. We stated the LOD in line 99 of the method section as below. Therefore, the negative result represented HCV RNA < 12 IU/mL.   “HCV genotypes were identified through nucleotide sequencing of the core and NS5B regions, followed by phylogenetic analysis. The lower limit and upper limit of the HCV RNA detection were < 12 IU/mL and 100,000,000 IU/mL, respectively. Details of HCV genotype identification and HCV RNA quantification were described in a previous study.(1)”     Comment 3: Did authors compare whether HIV coinfection could affect the performance of Elecsys@ HCV Duo immunoassay   Response 3: Thank you for your suggestion. The subgroup analysis of the Elecsys HCV Duo test for HCV diagnosis and SVR assessment in patients with HCV mono-infection and HCV-HIV coinfection is presented in Supplementary Tables 1, 2, and 3. We compared result of both subgroup analysis in line 154-157 and line 168-172 of result section as below.   “In subgroup analysis, the combination of anti-HCV and HCV-Ag showed similar sensitivity and specificity for diagnosing HCV infection in patients, with 100% sensitivity and 100% specificity in mono-infection and 99.3% sensitivity and 100% specificity in co-infection. (Supplementary table1).”   “The subgroup analyses of patients with mono-infection and coinfection are shown in Supplementary tables 2 and 3, respectively. HCV-Ag showed an excellent diagnostic performance in HCV mono-infected patients with a sensitivity and specificity of 100%. However, the sensitivity declined in co-infected individuals to 50%, while maintaining a comparable specificity of 96.15%.”

Reviewer 2 Report

Comments and Suggestions for Authors

This study evaluates the performance of the Elecsys® HCV Duo immunoassay in diagnosing chronic HCV infection and assessing sustained virological response (SVR) after treatment with direct-acting antivirals (DAAs) in patients with or without HIV infection. The assay’s combined detection of both HCV antibodies (anti-HCV) and HCV core antigen (HCV-Ag) showed excellent diagnostic sensitivity and specificity, making it a potentially effective tool for managing HCV infection in resource-limited settings. However, the performance for assessing SVR post-DAA therapy was less consistent, particularly in terms of sensitivity. The study demonstrates that the Elecsys® HCV Duo immunoassay offers near-perfect sensitivity and specificity for HCV diagnosis. The combination of anti-HCV and HCV-Ag significantly improves diagnostic performance, and it is particularly suitable for diagnosing both mono- and coinfected patients. The authors highlight the Elecsys® assay as a cost-effective option that simplifies the testing process, which is critical for high-risk populations like those with HIV coinfection. The inclusion of 200 patients, many with HIV coinfection, strengthens the study’s external validity and provides important data on the assay’s robustness in more complex cases of HCV diagnosis.

On the other hand, the sensitivity for detecting SVR using HCV-Ag alone was relatively low (57.14%). This needs to be discussed more thoroughly. For instance, why the assay’s accuracy drops in this context and how it compares to other available methods should be examined. This limitation needs a stronger emphasis, especially for clinicians considering the assay for post-treatment monitoring. Moreover, the subgroup analyses of HCV mono-infected vs. co-infected patients are limited by small sample sizes. Increasing the cohort size for both groups would likely lead to more reliable results and possibly provide a clearer understanding of the assay's performance for SVR.

It should be clear that the study is predominantly focused on HCV genotype 1 patients, which limits generalizability to other genotypes. A more detailed comparison between different HCV genotypes and their impact on the assay's performance should be included. Testing on a wider range of genotypes or referencing literature on this aspect could improve this section.

Regarding the results, some results, particularly those regarding SVR evaluation, are presented without sufficient context. For instance, a comparative analysis with other diagnostic methods for SVR, such as HCV RNA testing, would add value. This comparison would help highlight whether the trade-off in sensitivity is acceptable given the assay’s affordability and ease of use.

While the conclusion focuses on the assay's diagnostic performance, it would benefit from a more nuanced discussion of its limitations, particularly in post-treatment settings. The limitations regarding SVR sensitivity need clearer emphasis, and recommendations for future research should include investigating how to improve sensitivity in this context.

I will suggest:

1.       Expand Discussion on Limitations: More focus is needed on the limitations, particularly the low sensitivity for SVR assessment. This could include potential solutions, such as combining HCV-Ag with HCV RNA testing or using other biomarkers.

2.       Enhance Statistical Analysis: Subgroup analyses should be improved with a larger cohort. Consider statistical adjustments for small sample sizes or applying more rigorous statistical methods to account for this limitation.

3.       Clarify Genotype-Specific Findings: Include additional data or literature on how the assay performs across other HCV genotypes, especially those prevalent in other regions.

4.       Improve Data Presentation: Strengthen the clarity and depth of the SVR-related data by comparing it to alternative methods or diagnostic tools, offering readers a clearer understanding of how the Elecsys® HCV Duo immunoassay stacks up in this area.

 

Author Response

This study evaluates the performance of the Elecsys@ HCV Duo immunoassay in diagnosing chronic HCV infection and assessingsustained virological response (SVR) after treatment with direct-acting antivirals (DAAs) in patients with or without HIV infection. The assay' combined detection of both HCV antibodies (anti-HCV) and HCV core antigen (HCV-Ag) showed excellent diagnostic sensitivity and specificity, making it a potentially effective tool for managing HCV infection in resource-limited settings. However, the performance for assessing SVR post-DAA therapy was less consistent, particularly in terms of sensitivity. The study demonstrates that the Elecsys@ HCV Duo immunoassay offers near-perfect sensitivity and specificity for HCV diagnosis. The combination of anti-HCV and HCV-Ag significantly improves diagnostic performance, and it is particularly suitable for diagnosing both mono- and coinfected patients. The authors highlight the Elecsys@ assay as a cost-effective option that simplifies the testing process, which is critical for high-risk population like those with HIV coinfection. The inclusion of 200 patients, many with HIV coinfection, strengthens the study's external validity and provides important data on the assay's robustness in more complex cases of HCV diagnosis.

 

On the other hand, the sensitivity for detecting SVR using HCV-Ag alone was relatively low (57.14%). This needs to be discussed more thoroughly. For instance, why the assay's accuracy drops in this context and how it compares to other available methods should be examined. This limitation needs a stronger emphasis, especially for clinicians considering the assay for post-treatment monitoring. Moreover, the subgroup analyses of HCV mono-infected vs.co- infected patients are limited by small sample sizes. Increasing the cohort size for both groups would likely lead to more reliable results and possibly provide a clearer understanding of the assay's performance for SVR.

 

It should be clear that the study is predominantly focused on HCV genotype 1 pationts, which limits generalizability to other genotypes. Amore detailed comparison between different HCV genotypes and their impact on the assay's performance should be included. Testing on awider range of genotypes or referencing literature on this aspect could improve this section.

 

Regarding the results, some results, particularly those regarding SVR evaluation, are presented without sufficient context. For instance, a comparative analysis with other diagnostic methods for SVR, such as HCV RNA testing, would add value. This comparison would help highlight whether the fade-off in sensitivity is acceptable given the assay's affordability and ease of use.

 

While the conclusion focuses on the assay's diagnostic performance, it would benefit from a more nuanced discussion of its limitations, particularly in post-treatment settings. The limitations regarding SVR sensitivity need clearer emphasis, and recommendations for future research should include investigating how to improve sensitivity in this context.

 

Response : We appreciate your suggestion and have edited the data to address the suggestions provided in point-by-point responses.

 

Comment 1: Expand Discussion on Limitations: More focus is needed on the limitations, particularly the low sensitivity for SVR assessment. This could include potential solutions, such as combining HCV-Ag with HCV RNA testing or using other biomarkers.

Response 1: Thank you for your suggestion. Due to the low sensitivity of HCV-Ag for SVR assessment, we proposed that patients with positive HCV-Ag after DAA treatment should confirm treatment failure with HCV RNA testing. We also edited Figure 1 to clarify the clinical application.

We re-edited this limitation in discussion section lines 212-220, as below

 

“According to the decreased sensitivity of HCV-Ag for SVR assessment, we propose that patients with post-treatment positive HCV-Ag should undergo additional HCV RNA testing to confirm the presence of detectable HCV RNA, as outlined in Figure 1. Nonetheless, the HCV-Ag sub-result of the Elecsys@ HCV Duo test demonstrated high specificity and NPV across the entire cohort, with an acceptable low false negative rate of 1.57%. In this regard, our data might indicate that HCV-Ag could be useful for assessing SVR after DAA therapy. As over 95% of patients achieved SVR in the DAA era, a negative HCV-Ag result could strongly indicate SVR and reduce the need for HCV RNA confirmatory tests.”

 

Comment 2: Enhance Statistical Analysis: Subgroup analyses should be improved with a larger cohort. Consider statistical adjustments for small sample sizes or applying more rigorous statistical methods to account for this limitation.

 

Response 2: Thank you for your feedback. To address the discordance between the HCV-Ag and HCV RNA results, we reviewed the details of non-SVR patients and found that 2 out of 3 patients with HCV-HIV co-infection had HCV RNA levels of less than 3,000 IU/mL. This could potentially lead to a false negative result and reduced sensitivity of HCV-Ag performance. We have included this information in the discussion section, lines 231-234, and supplementary table 4. However, we also emphasize the importance of validating the performance of HCV-Ag for assessing SVR in cohorts that compare patients with HCV-mono and HIV co-infection in the limitation section.

 

Line 231-234 “Upon reviewing the non-SVR patients in supplementary table 4, two out of three patients in a subgroup of HIV-HCV co-infection had HCV RNA below 3,000 IU/mL, which could result in false negative HCV-Ag findings.”

 

Comment 3: Clarify Genotype-Specific Findings: Include additional data or

literature on how the assay performs across other HCV genotypes, especially those prevalent in other regions.

 

Response 3: Thank you for your suggestion. Only one previous study using the Elecsys@ HCV Duo test included patients with HCV genotype 1-6 and showed excellent diagnostic performance. (Line 226-227 in the manuscript) Therefore, we believed that the Elecsys@ HCV Duo test can be utilized across all HCV genotypes. Our data support its use in genotype 1, with further studies needed for validation in other genotypes.

 

Comment 4: Improve Data Presentation: Strengthen the clarity and depth of the SVR-related data by comparing it to alternative methods or diagnostic tools, offering readers a clearer understanding of how the Elecsys@ HCV Duo immunoassay stacks up in this area.

 

Response 4: Thank you for your feedback. We have reviewed previous studies that used HCV-Ag for SVR assessment. Most of these studies utilized the Architect core antigen assay by Abbott Diagnostics and demonstrated good sensitivity and specificity, as described in lines 208-211. Therefore, in comparison with the reduced sensitivity of the HCV-Ag sub-result of our study, we propose further HCV RNA testing for patients who test positive for HCV-Ag after treatment.

(Line 208-211) “Previous studies utilizing the Architect core antigen assay by Abbott Diagnostics for SVR assessment have reported reduced accuracy during treatment but good accuracy at 12 weeks post-treatment, with a sensitivity ranging from 88.9% to 100% and excellent specificity of 97.7% to 100%. ”

Reviewer 3 Report

Comments and Suggestions for Authors

 In the submitted manuscript, the authors present the results of the Elecsys® HCV Duo test, regarding the diagnosis of HCV infection in monoinfected and coinfected with HIV. The authors show the effectiveness of the study in both groups, presenting also the limitations of the test that relate to the evaluation of DDA treatment. I consider that the study design is appropriate.

The obtained results are adequately analyzed and discussed in comparison with the results of other tests. I would recommend the authors to look at the bibliography and look for more literary sources from the last 5 years.

Author Response

In the submitted manuscript, the authors present the results of the Elecsys@ HCV Duo test, regarding the diagnosis of HCV infection in mono- and coinfected with HIV. The authors show the effectiveness of the study in both groups, also presenting the limitations of the test that relate to the evaluation of DDA treatment. I consider that the study design is appropriate. The obtained results are adequately analyzed and discussed in comparison with the results of other tests. I would recommend the authors to look at the bibliography and look for more literary sources from the last 5 years.

 

Response : Thank you for your suggestion. We have included literature published in the last 5 years, including works by Feld et al., Page et al., and Cartwright et al in line 240-243. ^(1-3) Most of the recent literature on the diagnosis of HCV emphasizes a simplified algorithm, which aligns with our study. However, none of the studies have validated the performance of the Elecsys HCV Duo test for HCV diagnosis or evaluated its performance for SVR assessment.