Antigen concentration, viral load, and test performance for SARS-CoV-2 in multiple specimen types

Allison Golden; Michelle Oliveira-Silva; Hannah Slater; Alexia Martines Vieira; Pooja Bansil; Emily Gerth-Guyette; Brandon T. Leader; Stephanie Zobrist; Alan Kennedy Braga Ferreira; Erika Crhistina Santos de Araujo; Catherine Duran de Lucena Cruz; Eduardo Garbin; Greg T. Bizilj; Sean J. Carlson; Mariana Sagalovsky; Sampa Pal; Vin Gupta; Leo Wolansky; David S. Boyle; Deusilene Souza Vieira Dall’Acqua; Felipe Gomes Naveca; Valdinete Alves do Nascimento; Juan Miguel Villalobos Salcedo; Paul K. Drain; Alexandre Dias Tavares Costa; Dhélio Pereira; Gonzalo J. Domingo

doi:10.1371/journal.pone.0287814

Peer Review History

Original SubmissionJanuary 13, 2023
20 Feb 2023 Decision Letter - Md Maruf Ahmed Molla, Editor PONE-D-23-01146Antigen concentration, viral load, and test performance for SARS-CoV-2 in multiple specimen typesPLOS ONE Dear Dr. Golden, 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. Please submit your revised manuscript by Apr 06 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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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 2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 3. Thank you for stating the following financial disclosure: "This work was supported by grants from The Rockefeller Foundation [2020 HTH 039] and Amazon.com [2D-04020007] to GJD. Rockefeller and Amazon's contributions to the study and publication are represented by authors VG and LW. FGN and VAN were supported by the National Council for Scientific and Technological Development [grant 403276/2020-9] and Inova Fiocruz / Fundação Oswaldo Cruz [grant VPPCB-007-FIO-18-2-30 - Knowledge generation]. FGN is a National Council for Scientific and Technological Development (CNPq) fellow. Benchmarking work cited and that was used in analysis was funded by the Bill & Melinda Gates Foundation (https://www.gatesfoundation.org/) via grant INV-016821. Other than the contributions by authors VG and LW, funders did not have any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript." We note that one or more of the authors is affiliated with the funding organization, indicating the funder may have had some role in the design, data collection, analysis or preparation of your manuscript for publication; Rockefeller Foundation, Amazon, Inova Fiocruz (Fundação Oswaldo Cruz) In other words, the funder played an indirect role through the participation of the co-authors. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please do the following: (1) Review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. These amendments should be made in the online form. (2) Confirm in your cover letter that you agree with the following statement, and we will change the online submission form on your behalf: “The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.” 4. Thank you for stating the following in the Acknowledgments Section of your manuscript: "This work was supported by grants from The Rockefeller Foundation [2020 HTH 039] and Amazon.com [2D-04020007] to Gonzalo J Domingo. Felipe Gomes Naveca and Valdinete Alves do Nascimento were supported by the National Council for Scientific and Technological Development [grant 403276/2020-9] and Inova Fiocruz / Fundação Oswaldo Cruz [grant VPPCB-007-FIO-18-2-30 - Knowledge generation]. Felipe Gomes Naveca is a National Council for Scientific and Technological Development (CNPq) fellow. Benchmarking work cited and that was used in analysis was funded by the Bill & Melinda Gates Foundation(https://www.gatesfoundation.org/) via grant INV-016821. The Bill & Melinda Gates Foundation did not have any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript." We note that you have provided funding information that is not currently declared in your Funding Statement. However, 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. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "This work was supported by grants from The Rockefeller Foundation [2020 HTH 039] and Amazon.com [2D-04020007] to GJD. Rockefeller and Amazon's contributions to the study and publication are represented by authors VG and LW. FGN and VAN were supported by the National Council for Scientific and Technological Development [grant 403276/2020-9] and Inova Fiocruz / Fundação Oswaldo Cruz [grant VPPCB-007-FIO-18-2-30 - Knowledge generation]. FGN is a National Council for Scientific and Technological Development (CNPq) fellow. Benchmarking work cited and that was used in analysis was funded by the Bill & Melinda Gates Foundation (https://www.gatesfoundation.org/) via grant INV-016821. Other than the contributions by authors VG and LW, funders did not have any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. [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: Partly ******** 2. 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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No Reviewer #2: Yes ****** 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: Comments to the Author In this manuscript Number PONE-D-23-01146 entitled"Antigen concentration, viral load, and test performance for SARS-CoV-2 in multiple specimen types", the authors provided an overview of the SARS-CoV-2 diagnostic techniques within different specimens in Brazil with their clinical manifestations. This paper presents an interesting topic with a broad appeal to readers and presents a balanced view. However, it can be further improved and specific concerns need to be addressed before publication: 1. The title can be better modified to “The correlation between SARS-CoV-2 Antigen concentration and viral load using different techniques and specimens”. 2. 31-32 no need to repeat “specimen”. 3. 33 no need for “cases”. 4. 66 “The performance” correction. 5. 69,71 repeated word “understanding” please rephrase. 6. 86 “The design” correction. 7. 35, 88, 200, 201 can you please make it clear about the 214 cases and 50 cases in these positions, also in the supplementary table A? shouldn’t you have 214 cases and 65 positive cases, so what is this 50? 8. In table 1, can you address the symptomatic, asymptomatic/oligosymptomatic cases, as you mentioned in 274, 275 9. In Supplementary A, there are more than 65 positive cases of close contacts and in 202-203 “Among all the close contacts, 65 tested as SARS-CoV-2 positive by the reference assay at least once “, please make it clear. Reviewer #2: The article titled 'Antigen concentration, viral load and test performance for SARS-CoV-2 in multiple specimen types' emphasizes on the comparison between the RT-PCR and RDT method. However, the importance of Nucleocapsid Protein o 'N Antigen' as the standard for viral load and antigen concentration measurement was not explained as there are other antigens such as 'ORF1b, S' that are also used in SARS-CoV-2 diagnostic methods. RT-PCR was not conducted on ANS sample and RDT was not conducted on NPS sample. As it was mentioned as a limitation, so how does this impact the findings of this study? In page 8, the heading 'Antigen testing' is kind of misleading. it may be addressed as 'Rapid diagnostic testing/RDT of N antigen'. Also in page 10, the paragraph 'Antigen concentration determination' was not well versed. Table 1 needs to be rewritten and explained precisely. The grammatical and typographical errors needs to be corrected. ****** 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: Yes: Ahmed H. Mousa 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.0287814.r001
Revision 1
24 May 2023 Author Response Dear Editors, Thank you for the opportunity to revise the manuscript and we are grateful to the reviewers for their thoughtful review and hope we have addressed their comments. Please find below the detailed replies in bold text below each comment. Kind Regards, Allison Golden Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. 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 Thank you, figure references and legend placement have been updated. 2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 3. Thank you for stating the following financial disclosure: "This work was supported by grants from The Rockefeller Foundation [2020 HTH 039] and Amazon.com [2D-04020007] to GJD. Rockefeller and Amazon's contributions to the study and publication are represented by authors VG and LW. FGN and VAN were supported by the National Council for Scientific and Technological Development [grant 403276/2020-9] and Inova Fiocruz / Fundação Oswaldo Cruz [grant VPPCB-007-FIO-18-2-30 - Knowledge generation]. FGN is a National Council for Scientific and Technological Development (CNPq) fellow. Benchmarking work cited and that was used in analysis was funded by the Bill & Melinda Gates Foundation (https://www.gatesfoundation.org/) via grant INV-016821. Other than the contributions by authors VG and LW, funders did not have any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript." We note that one or more of the authors is affiliated with the funding organization, indicating the funder may have had some role in the design, data collection, analysis or preparation of your manuscript for publication; Rockefeller Foundation, Amazon, Inova Fiocruz (Fundação Oswaldo Cruz) In other words, the funder played an indirect role through the participation of the co-authors. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please do the following: (1) Review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. These amendments should be made in the online form. (2) Confirm in your cover letter that you agree with the following statement, and we will change the online submission form on your behalf: “The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.” Thank you for this, we have updated the Financial Disclosure to the following: "This work was supported by grants from The Rockefeller Foundation [2020 HTH 039] and Amazon.com [2D-04020007] to GJD. Rockefeller and Amazon's contributions to the study and publication are represented by authors VG and LW. FGN and VAN were supported by the National Council for Scientific and Technological Development [grant 403276/2020-9] and Inova Fiocruz / Fundação Oswaldo Cruz [grant VPPCB-007-FIO-18-2-30 - Knowledge generation]. FGN is a National Council for Scientific and Technological Development (CNPq) fellow. Benchmarking work cited and that was used in analysis was funded by the Bill & Melinda Gates Foundation (https://www.gatesfoundation.org/) via grant INV-016821. The Bill & Melinda Gates Foundation did not have any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors VG and LW, contributed to the study design, decision to publish, and review of the manuscript." 4. Thank you for stating the following in the Acknowledgments Section of your manuscript: "This work was supported by grants from The Rockefeller Foundation [2020 HTH 039] and Amazon.com [2D-04020007] to Gonzalo J Domingo. Felipe Gomes Naveca and Valdinete Alves do Nascimento were supported by the National Council for Scientific and Technological Development [grant 403276/2020-9] and Inova Fiocruz / Fundação Oswaldo Cruz [grant VPPCB-007-FIO-18-2-30 - Knowledge generation]. Felipe Gomes Naveca is a National Council for Scientific and Technological Development (CNPq) fellow. Benchmarking work cited and that was used in analysis was funded by the Bill & Melinda Gates Foundation(https://www.gatesfoundation.org/) via grant INV-016821. The Bill & Melinda Gates Foundation did not have any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript." We note that you have provided funding information that is not currently declared in your Funding Statement. However, 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. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. We have removed this language and the updated Financial Disclosure has been noted in the cover letter. Please include your amended statements within your cover letter; we will change the online submission form on your behalf. Please see above in our revised financial disclosure. 5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. This has been done. Reviewers' comments: Reviewer #1: Comments to the Author In this manuscript Number PONE-D-23-01146 entitled"Antigen concentration, viral load, and test performance for SARS-CoV-2 in multiple specimen types", the authors provided an overview of the SARS-CoV-2 diagnostic techniques within different specimens in Brazil with their clinical manifestations. This paper presents an interesting topic with a broad appeal to readers and presents a balanced view. However, it can be further improved and specific concerns need to be addressed before publication: 1. The title can be better modified to “The correlation between SARS-CoV-2 Antigen concentration and viral load using different techniques and specimens”. Thank you for this suggestion, but after further internal consultation it was felt that the current title is most appropriate. 2. 31-32 no need to repeat “specimen”. Thank you for this suggestion. We have updated the sentence to state the following: The relationship between N-antigen concentration and viral load within and across different specimens guides the clinical performance of rapid diagnostic tests (RDT) in different uses. 3. 33 no need for “cases”. Addressed above. 4. 66 “The performance” correction. The word “the” has been incorporated. 5. 69,71 repeated word “understanding” please rephrase. Thank you. Removed the second “understanding” 6. 86 “The design” correction. Changed “This” to “the”. 7. 35, 88, 200, 201 can you please make it clear about the 214 cases and 50 cases in these positions, also in the supplementary table A? shouldn’t you have 214 cases and 65 positive cases, so what is this 50? This has been clarified in lines 201-207 and additionally in the Supporting information in Table S2. 8. In table 1, can you address the symptomatic, asymptomatic/oligosymptomatic cases, as you mentioned in 274, 275 Thank you for this suggestion. Because of the low numbers of cases, we have opted to present the performance across all close contacts with a confirmed RT-PCR result since separating these from the asymptomatic/oligosymptomatic cases results in broader confidence intervals. Breakdown of numbers of each category of patient disposition for each category are now listed in Table S2. Because asymptomatic cases overall showed lower levels antigen (Figure 1), it is expected performance would be lower in asymptomatic and oligosymptomatic cases given the correlations between viral load and N antigen concentrations in cognate specimens presented in this study. 9. In Supplementary A, there are more than 65 positive cases of close contacts and in 202-203 “Among all the close contacts, 65 tested as SARS-CoV-2 positive by the reference assay at least once “, please make it clear. Thank you for pointing this out. We hope the new Table S2 and additional wording in lines 201-207 clarifies the distinction between all the numbers raised by the reviewer. Household close contact study participants may have multiple specimens associated. Reviewer #2: 1.The article titled 'Antigen concentration, viral load and test performance for SARS-CoV-2 in multiple specimen types' emphasizes on the comparison between the RT-PCR and RDT method. However, the importance of Nucleocapsid Protein o 'N Antigen' as the standard for viral load and antigen concentration measurement was not explained as there are other antigens such as 'ORF1b, S' that are also used in SARS-CoV-2 diagnostic methods. Thank you for this. We have inserted the following statement in the introduction: ” The majority of RDT used to screen for COVID-19 infection do so by detecting the nucleocapsid or N-antigen.” 2.RT-PCR was not conducted on ANS sample and RDT was not conducted on NPS sample. As it was mentioned as a limitation, so how does this impact the findings of this study? Thank you for this, • we have added the following limitation in line 335, “(b) RDTs were not conducted on NPS samples,” • we added the following statement line 344: “While strong antigen to viral load relationships were observed in the NPS and saliva samples, this study did not directly measure the same relationship in ANS samples.” 3. In page 8, the heading 'Antigen testing' is kind of misleading. it may be addressed as 'Rapid diagnostic testing/RDT of N antigen'. Thank you for this. This section actually refers to determination of antigen concentration. So we have updated the section subheading to read: “Measuring antigen concentration” 4. Also in page 10, the paragraph 'Antigen concentration determination' was not well versed. We have changed this to measurement of “Antigen concentration in clinical samples” 5. Table 1 needs to be rewritten and explained precisely. Thank you, we have updated Table 1. 6. The grammatical and typographical errors needs to be corrected. We have reviewed the article again and corrected any errors found. Have the authors made all data underlying the findings in their manuscript fully available? Reviewer #1: Yes Reviewer #2: No Note to editors: All the study data minus the antigen concentration data is already available in dataverse as cited. On acceptance of the manuscript by the journal the spreadsheet will be updated to include the antigen concentration data. Attachments Attachment Submitted filename: PONE-D-23-01146 response to review.docx https://doi.org/10.1371/journal.pone.0287814.r002
13 Jun 2023 Decision Letter - Md Maruf Ahmed Molla, Editor Antigen concentration, viral load, and test performance for SARS-CoV-2 in multiple specimen types PONE-D-23-01146R1 Dear Dr. Golden, 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, Md Maruf Ahmed Molla Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0287814.r003
Formally Accepted
3 Jul 2023 Acceptance Letter - Md Maruf Ahmed Molla, Editor PONE-D-23-01146R1 Antigen concentration, viral load, and test performance for SARS-CoV-2 in multiple specimen types Dear Dr. Golden: 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. Md Maruf Ahmed Molla Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0287814.r004

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