Performance estimation of two in-house ELISA assays for COVID-19 surveillance through the combined detection of anti-SARS-CoV-2 IgA, IgM, and IgG immunoglobulin isotypes

Alfredo Ramírez-Reveco; Gerardo Velásquez; Christopher Aros; Gabriela Navarrete; Franz Villarroel-Espíndola; Maritza Navarrete; Alberto Fica; Anita Plaza; Natalia Castro; Claudio Verdugo; Gerardo Acosta-Jamett; Cristóbal C. Verdugo

doi:10.1371/journal.pone.0270388

Peer Review History

Original SubmissionJune 8, 2022
8 Aug 2022 Decision Letter - Maemu Petronella Gededzha, Editor PONE-D-22-16590Performance estimation of two in-house ELISA assays for COVID-19 surveillance through the combined detection of anti-SARS-CoV-2 IgA, IgM, and IgG immunoglobulin isotypesPLOS ONE Dear Dr. Verdugo, 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. When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf The introduction is scanty and does not give adequate background of the prevalence of SARS-COV-2 in Chile. The author must clarify how the participant number was calculated to achieve satisfactory statistical power for appropriate interpretation of results. A sample size of 94 include both cases and controls is too small to use in a disease which is pandemic. Please provide additional details regarding participant consent. Please ensure that you have specified what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. The rapid test used does not include IgA, and it is not clear how the results for the 2 in-house assay were compared with the rapid while it does not contain similar antibodies. No comparison has been performed with other formal serological assay that are either based on the ELISA or the chemiluminescent detection principle, but just a comparison with a lateral flow assay is reported which is not sufficient. The materials and methods section is poor in experimental details. I suggest, to allow a better to understanding to re-write this section. Principle and protocol of the rapid assay is not reported in the materials and methods section. Please, the authors provide it. Please list the names, sources of all reagents, software used in the methodology. Please list all the instrument used in the methodology eg. What was used to achieve a 37⁰C temperature. Line 145 should read Study design and laboratory analysis not analyses. Line 238: ‘’Figure 3B” explanation in the text does not correspond to Figure 3B/ Figure 2 B is not explained. All the abbreviations must be written in full before abbreviation. The author recommended the use of in house assay over lateral flow assay, however, several study has shown that lateral flow assay are cheap and easy to use especially in developing countries where there is a lack of infrastructure and their maintenance. Where available the infrastructure is available in university settings as mentioned in by the authors but may not be available in and as infrastructure and maintenance, that may be limited in clinics which may be remote from university laboratories. Include a table with the cost of all reagents used in the in-house to justify the use of in house as an alternative diagnostic tool. Please submit your revised manuscript by Sep 22 2022 11:59PM. 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If the need for consent was waived by the ethics committee, please include this information. 3. Thank you for stating the following in the Acknowledgments Section of your manuscript: “Pre-pandemic sampling was supported by EU-LAC Health (EULAC/FONIS T020067), through the PERITAS (Molecular epidemiological studies on pathways of transmission and long-lasting capacity building to prevent cystic echinococcosis infection) project (G.A. EULACH16/T02-0067; https://www.iss.it/en/web/iss-en/who-cc-peritas).” We note that you have provided additional information within the Acknowledgements Section that is not currently declared in your Funding Statement. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. 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Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. 5. Please amend the manuscript submission data (via Edit Submission) to include author Gerardo Velázquez, Christopher Aros, Gabriela Navarrete, Franz Villarroel-Espindola, Maritza Navarrete and Natalia Castro [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. 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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The manuscript was well written and the Authors were able to show that the two in-house ELISA assays were efficient and could be a vital tool in the surviellance studies of COVID-19, However, the study showed that using LFCI assay is still the best assay, although its is a bit expensive for poor resource limited settings, So is the Author saying that its better to use LFCI assay than the other two in-house tested assay they displayed? or what are they actually recommending? Please correct these minor findings on the manuscript listed below: Abstract Line 34, Np and RBD are abbreviated and no written in full, and it’s the first time they are being mentioned, for someone who doesn’t know COVI-19, they might not understand what they stand for. Introduction Line76, RT-qPCR mentioned for the first time, write in full then abbreviate. Materials and Methods, Line 162, Is it supposed to be 1XPBS, -Tween20 0.05% in brackets or….. ?. the same query applies for Line 168 Line 165 and line 172, was it supposed to be a reference added after as “previously described by” . Line 173, what does TMB stand for? First time mention and abbreviated. Line 473, in the text body it was receptor binding domain, now its region binding domine, is there a difference or just some typing error. Line 476, If its respectively isn’t it supposed to be written starting with the light orange and in red. Line 475, in the body text, RBD was being described as the receptor binding domain, now on the figure legend its written as the region binding domine, is it still one and the same thing or there is a difference between the two? Reviewer #2: The manuscript reports on a good advancement especially for low and middle income countries that may not have enough funds to commission population based surveillance due to cost of commercial kits. A few typographical and grammatic errors were noted. Line 31, Include free 'of' SARS-CoV-2... Line 49, replace the word 'Than' with 'to'... Line 145 and 150, replace "analyzes with 'analysis' Line 151, delete the word 'in' Line 324, replace word 'than' with "to" ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0270388.r001
Revision 1
17 Oct 2022 Author Response Response to Editor comments 2. The introduction is scanty and does not give adequate background of the prevalence of SARS-COV-2 in Chile. Introduction has been edited including a general description of the epidemic development and government response in the country lines 58-63. Despite this, we would like to highlight that a proper prevalence estimate (base on a random selection in the general population) is the outcome of a following paper from the present manuscript that will be comparing official diagnostic rates versus true prevalence estimating (base on the Se & Sp performance of the in-house ELISA test). 3. The author must clarify how the participant number was calculated to achieve satisfactory statistical power for appropriate interpretation of results. A sample size of 94 include both cases and controls is too small to use in a disease which is pandemic. Respectfully, we do not agree with the opinion of the Reviewer. Sample size calculation (method & parameters) were stated in lines 196-198 of the original submitted manuscript. In particular, we followed a sample size calculation specific for the ROC analysis (Obuchowski, NA. 2005), which is the main analysis of the paper (lines 120-122). Thus, sample size calculation was in line with the study objective, the intended use of the test, and the statistical analysis used in this research. For clarity, sample size calculation information has been moved from the “Statistical analysis” (previous manuscript version) to the “Study population and sample collection” section (lines 119-124). 4. Please provide additional details regarding participant consent. Please ensure that you have specified what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. This section has been edited (lines 144-158). 5. The rapid test used does not include IgA, and it is not clear how the results for the 2 in-house assay were compared with the rapid while it does not contain similar antibodies. As the Reviewer points out, the commercial rapid test used includes only detections of IgG and IgM and the in-house ELISA evaluated in this study we use the total detection of immunoglobulins (IgA, IgM and IgG). Despite of this, the comparison was carried out in terms of the final assessment that each tool provided. Specifically, in term of their dichotomic outcome (positive/negative). This is a very standard approach in the epidemiology, where the agreement between two tests can be assessed by a 2-by-2 table and computing Coheen’ Kappa based on the final-outcome despite having different chemistry or targets (Methods in Epidemiologic Research, 2021 ISBN-13: 978-0919013735). For clarity, we have edited the statistical section (lines 227-233). We would light to highlight that we performed a diagnostic Se & Sp estimation rather than an analytical Se & Sp estimation, which allows for the end-result comparison of two different tests, such the one conducted in the present research. Additionally, the potential effects (Se and Sp) of the presence/absence of IgA, between both tests, were discussed in lines 346-360. 6. No comparison has been performed with other formal serological assay that are either based on the ELISA or the chemiluminescent detection principle, but just a comparison with a lateral flow assay is reported which is not sufficient. We followed the STARD guidelines, which is an international effort of researchers to standardize test validation reports since 2003 (updated in 2015) (https://www.equator-network.org/reporting-guidelines/stard/). In such internationally recognized guidelines, there is no such thing as a minimum (more than one) of extra tests for comparison purposes. Independently of this, the main objective of this research (lines 110-115) was achieved through the ROC analysis. Specifically, OD cut-off, sensitivity and specificity estimates were derived directly from the ROC curves (lines 118-124 & 215-224). In this way, the comparison of the in-house ELISA tests with LFA was a complementary analysis, which was carried out with the purpose of having a reference with the most widely used serologic test in the country. Therefore, in the light of the internationally accepted standard guidelines for test validation, in addition to the study objective, we (respectfully) do not agree with the Reviewer comment that we have not performed enough analysis to address the purpose of this research. 7. The materials and methods section is poor in experimental details. I suggest, to allow a better to understanding to re-write this section Materials and methods sections has been updated 8. Principle and protocol of the rapid assay is not reported in the materials and methods section. Please, the authors provide it. The following text has been included: Serum samples for rapid test were processed according to the manufacturer's instructions (Zhuhai Livzon Diagnostics Inc.). Briefly: 10 μL of serum sample were added into the sample well of both IgM test and IgG test cassettes, then 2 drops (about 100 μL) of sample diluent were added vertically. Finally, within 1 to 15 minutes after sample addition, the results were interpreted as positive when both the control line and the test line appeared. The results were interpretated as negative when only control line appeared. 9. Please list the names, sources of all reagents, software used in the methodology. Information requested not explicitly presented in the M&M section has been included as a supplementary material (S1 Table). All software used were already informed in the statistical analysis section (lines 215-216, 227). 10. Please list all the instrument used in the methodology eg. What was used to achieve a 370C temperature. Included as a supplementary material (S1 Table). 11. Line 145 should read Study design and laboratory analysis not analyses. Updated as requested 12. Line 238: ‘’Figure 3B” explanation in the text does not correspond to Figure 3B/ Figure 2 B is not explained. The explanation in the text indicated by the reviewer corresponds to Figure 2B, and not to 3B, this was corrected in the text 13. All the abbreviations must be written in full before abbreviation. Updated as requested 14. The author recommended the use of in house assay over lateral flow assay, however, several study has shown that lateral flow assay are cheap and easy to use especially in developing countries where there is a lack of infrastructure and their maintenance. Where available the infrastructure is available in university settings as mentioned in by the authors but may not be available in and as infrastructure and maintenance, that may be limited in clinics which may be remote from university laboratories. Include a table with the cost of all reagents used in the in-house to justify the use of in house as an alternative diagnostic tool. We have edited the discussion and conclusion of the manuscript in order to provide a clearer message to the readers (lines 49-50, 308, 325, 330-331, and 381-385). In particular, we provide a more balanced conclusion in which scenario the in-house ELISA is more convenient to use. Additionally, we have included as supporting information (S2 Table) the cost of the regents used. Attachments Attachment Submitted filename: Response to Reviewer.docx https://doi.org/10.1371/journal.pone.0270388.r002
7 Dec 2022 Decision Letter - Maemu Petronella Gededzha, Editor Performance estimation of two in-house ELISA assays for COVID-19 surveillance through the combined detection of anti-SARS-CoV-2 IgA, IgM, and IgG immunoglobulin isotypes PONE-D-22-16590R1 Dear Dr. Verdugo, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Maemu Petronella Gededzha, Ph.D Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0270388.r003
Formally Accepted
27 Jan 2023 Acceptance Letter - Maemu Petronella Gededzha, Editor PONE-D-22-16590R1 Performance estimation of two in-house ELISA assays for COVID-19 surveillance through the combined detection of anti-SARS-CoV-2 IgA, IgM, and IgG immunoglobulin isotypes Dear Dr. Verdugo: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Maemu Petronella Gededzha Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0270388.r004

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