IgG antibodies to SARS-CoV-2 in asymptomatic blood donors at two time points in Karachi

Muhammad Hasan; Bushra Moiz; Shama Qaiser; Kiran Iqbal Masood; Zara Ghous; Areeba Hussain; Natasha Ali; J. Pedro Simas; Marc Veldhoen; Paula Alves; Syed Hani Abidi; Kulsoom Ghias; Erum Khan; Zahra Hasan

doi:10.1371/journal.pone.0271259

Peer Review History

Original SubmissionSeptember 17, 2021
14 Mar 2022 Decision Letter - Joël Mossong, Editor PONE-D-21-30126Increasing IgG antibodies to SARS-CoV-2 in asymptomatic blood donors through the second COVID-19 wave in Karachi associated with exposure and immunity in the populationPLOS ONE Dear Dr. Hasan, 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. ============================== Two of the reviewers make a number of relevant constructive suggestions on how the manuscript can be improved. Please address all of these.============================== Please submit your revised manuscript by Apr 28 2022 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. 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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. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, 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. If the need for consent was waived by the ethics committee, please include this information. 3. Thank you for stating the following financial disclosure: “The study was supported by a grant from Provost’s Academic Priorities Fund, The Aga Khan University, Pakistan” Please state what role the funders took in the study. If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 4. Thank you for stating the following in the Funding Section of your manuscript: “The study was supported by a grant from Provost’s Academic Priorities Fund, The Aga Khan University.” 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: “The study was supported by a grant from Provost’s Academic Priorities Fund, The Aga Khan University, Pakistan” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 5. Please remove your figures from within your manuscript file, leaving only the individual TIFF/EPS image files, uploaded separately. These will be automatically included in the reviewers’ PDF. [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: Partly Reviewer #2: Yes Reviewer #3: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: Yes Reviewer #3: N/A ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: No Reviewer #2: Yes Reviewer #3: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? 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: Yes Reviewer #2: Yes Reviewer #3: No ******** 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: This is a review of "Increasing IgG antibodies to SARS-CoV-2 in asymptomatic blood donors through the second COVID-19 wave in Karachi associated with exposure and immunity in the population" from Hasan et al, 2022, for consideration at PLOS ONE. This paper could use numerous clarifications prior to publication, which I (hopefully) constructively suggest below. # Methods 1. Line 71 states: "Any donor with a history of COVID-19 related symptoms or confirmed infection was excluded from enrollment as a blood donor." HOWEVER, the whole premise of the paper is that you have found high seropositivity among these blood donors. And, in the discussion, you discuss the likely levels of asymptomatic infection which explain this confusion. I would suggest that, around Line 71, you explicitly note that this exclusion criterion in your methods does not prevent those who were asymptomatically or paucisymptomatically infected from donation, which is key to the present study. 2. Were the results adjusted for the sensitivity and specificity of the RBD and spike ELISAs? For that matter, what were the sensitivity and specificity of these assays? Were controls limited only to 100 known positive samples? Or were there also negative controls used? For instance, Figure 2 would be far better as seroprevalence estimates, with error bars, rather than raw sample counts which are not comparable between groups. Methods for adjustment are discussed in the context of SARS-CoV-2 here in https://elifesciences.org/articles/64206 and in general here https://www.hindawi.com/journals/eri/2011/608719/ In general, the establishment of the ELISA cutoffs should be more clearly discussed, with positive and negative controls noted or referenced. # Results 2. On Line 145, I suggest noting for the reader that 99.1% of donors were male, so the result about 99.7% of spike+ being men is not significant. Same with the 29.0±7.4 and 29.2±7.2. A reader who is skimming the paper may be surprised by these numbers. 3. Do you have any estimate of how many of the samples might be RBD+ but spike-? On Line 294, the Discussion paragraph already notes that spike ELISAs are highly specific, so would you conclude that, perhaps, your RBD ELISA is relatively insensitive? If these numbers (RBD+, spike-) are not known, then this should be noted as a limitation. 4. "Our data showed an increasing frequency of seropositivity to spike and RBD in individuals who were between their second to fourth decades of life." Typically we would expect to see statistical tests of this claim, but I do not see them. # Data Availability Data were not provided alongside submission of the manuscript. # Small Suggestions - Perhaps the numbers on Line 3 (Abstract) could be updated since it is now February 2022. - Refs 10 and 11 are the same paper, different title. - L43-50 - place the literature review in dates or waves, but not "recent" or "previous". Years from now, it may be difficult to understand this paper without specific dates. - BionaxNow spelling. (BinaxNow) - A standard curve -[of] comprising a titration... - Were tested for the presence of -[for] IgG antibodies... awkward. - Fig 1 — what do the symbols and shading mean? - "Similarly, though proportion of" -> "Similarly, the proportion of" - "Overall, only 31.2% +[of] subjects having IgG to spike" - Supplementary Fig 1 - the horizontal axis doesn't line up with the data points. What are the horizontal error bar type things? Please clarify what the colors represent, or annotate them on the plots. Reviewer #2: The author can do a follow up spike and RBD antibodies in order to assess the concept of herd immunity in those positive cases. The work is good but is not novel as already one such study has been done in Karachi. Reviewer #3: Authors attempted to assess SARS-CoV-2 seroprevalence in blood donors from Karachi, Pakistan, during the second COVID-19 pandemic wave. There are several issues regarding this manuscript: The small sample size, significant gender imbalance and the distribution of age groups. For instance they have 4 participants in the age group 51-60 years and the age range for group 1 is 17-20: 4 years. For an unbiased observation authors should have grouped participants into different age categories, for example: 17-25; 26-35; 36-45; 46-55. It is not clear from the methods section the cut off for IgG positive test results. No information regarding the sensitivity and specificity of the test was provided. The manuscript needs an English review. Some terms are inadequate and some sentences are hard to understand. Few examples: Page 10: The current study performed December 2020 until February 2021 at the time of the second COVID-19 wave shows a further rise (to 53%) in seropositivity in Karachi. Page 11: the secondary questionnaire was administered later… ******** 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: Yes:** SAMRA WAHEED Reviewer #3: 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/. 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Revision 1
3 May 2022 Author Response PONE-D-21-30126 Increasing IgG antibodies to SARS-CoV-2 in asymptomatic blood donors through the pandemic in Karachi reflects exposure and immunity in the population PLOS ONE 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 -Thank you. We have made the required style changes in the manuscript. 2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, 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. If the need for consent was waived by the ethics committee, please include this information. -Written consent from each study participant. Only adults aged 18 years and over were included in the study. 3. Thank you for stating the following financial disclosure: “The study was supported by a grant from Provost’s Academic Priorities Fund, The Aga Khan University, Pakistan” Please state what role the funders took in the study. If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. -Thank you for pointing this out. This has been added to the cover letter. 4. Thank you for stating the following in the Funding Section of your manuscript: “The study was supported by a grant from Provost’s Academic Priorities Fund, The Aga Khan University.” 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: “The study was supported by a grant from Provost’s Academic Priorities Fund, The Aga Khan University, Pakistan” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. - Thank you. This has been added to the cover letter. 5. Please remove your figures from within your manuscript file, leaving only the individual TIFF/EPS image files, uploaded separately. These will be automatically included in the -reviewers’ PDF. -Thank you. We will upload the image files separately. Reviewers' comments: ________________________________________ 5. Review Comments to the Author Reviewer #1: This is a review of "Increasing IgG antibodies to SARS-CoV-2 in asymptomatic blood donors through the second COVID-19 wave in Karachi associated with exposure and immunity in the population" from Hasan et al, 2022, for consideration at PLOS ONE. This paper could use numerous clarifications prior to publication, which I (hopefully) constructively suggest below. # Methods 1. Line 71 states: "Any donor with a history of COVID-19 related symptoms or confirmed infection was excluded from enrollment as a blood donor." HOWEVER, the whole premise of the paper is that you have found high seropositivity among these blood donors. And, in the discussion, you discuss the likely levels of asymptomatic infection which explain this confusion. I would suggest that, around Line 71, you explicitly note that this exclusion criterion in your methods does not prevent those who were asymptomatically or paucisymptomatically infected from donation, which is key to the present study. - We thank the reviewer for this comment, the exclusion criteria has now been revised (line#80-81) 2. Were the results adjusted for the sensitivity and specificity of the RBD and spike ELISAs? For that matter, what were the sensitivity and specificity of these assays? Were controls limited only to 100 known positive samples? Or were there also negative controls used? For instance, Figure 2 would be far better as seroprevalence estimates, with error bars, rather than raw sample counts which are not comparable between groups. Methods for adjustment are discussed in the context of SARS-CoV-2 here in https://elifesciences.org/articles/64206 and in general here https://www.hindawi.com/journals/eri/2011/608719/ In general, the establishment of the ELISA cutoffs should be more clearly discussed, with positive and negative controls noted or referenced. - thank you for pointing this out. We have now included the calculation for sensitivity and specificity of the RBD and spike ELISA assays in the manuscript. The assay validation is explained more clearly. We used 45 COVID-19 convalescent sera as positive controls and 55 healthy sera from pre-pandemic period as negative controls. - We have added confidence intervals (95%) to the results to explain the range of antibody responses investigated in each condition tested. # Results 2. On Line 145, I suggest noting for the reader that 99.1% of donors were male, so the result about 99.7% of spike+ being men is not significant. Same with the 29.0±7.4 and 29.2±7.2. A reader who is skimming the paper may be surprised by these numbers. _ Thank you for pointing this out. We have re-written this line to avoid any confusion. 3. Do you have any estimate of how many of the samples might be RBD+ but spike-? On Line 294, the Discussion paragraph already notes that spike ELISAs are highly specific, so would you conclude that, perhaps, your RBD ELISA is relatively insensitive? If these numbers (RBD+, spike-) are not known, then this should be noted as a limitation. - None of the samples were spike negative but RBD positive. We have expanded our discussion of the results to describe our observation. The sensitivity and specificity of spike and RBD ELISAs are discussed. IgG to both these proteins were significantly correlated with each other. - Further, it is noted that the dynamics of IgG to each of these proteins may differ in COVID-19. 4. "Our data showed an increasing frequency of seropositivity to spike and RBD in individuals who were between their second to fourth decades of life." Typically we would expect to see statistical tests of this claim, but I do not see them. - Thank you for this suggestion. We have re-analysed the data according to the age groups suggested by the reviewer. After doing so, we did not find any difference between IgG seropositivity to spike or RBD across the age groups. This has been corrected in the manuscript. # Data Availability -Data availability statement is corrected. The data has been depicted in tables and figures used for the manuscript. The full set will be available upon acceptance of the publication. Additional material can be provided upon request. # Small Suggestions - Perhaps the numbers on Line 3 (Abstract) could be updated since it is now February 2022. - Refs 10 and 11 are the same paper, different title. - Thank you – this correction has been made. - L43-50 - place the literature review in dates or waves, but not "recent" or "previous". Years from now, it may be difficult to understand this paper without specific dates. - BionaxNow spelling. (BinaxNow) This correction has been made - A standard curve -[of] comprising a titration... Correction made - Were tested for the presence of -[for] IgG antibodies... awkward. correction made - Fig 1 — what do the symbols and shading mean? - "Similarly, though proportion of" -> "Similarly, the proportion of" - "Overall, only 31.2% +[of] subjects having IgG to spike" - Supplementary Fig 1 - the horizontal axis doesn't line up with the data points. What are the horizontal error bar type things? Please clarify what the colors represent, or annotate them on the plots. - Thank you we have made this correction. Reviewer #2: The author can do a follow up spike and RBD antibodies in order to assess the concept of herd immunity in those positive cases. The work is good but is not novel as already one such study has been done in Karachi. -Thank you for this comment. We appreciate that there have been previous studies which have investigated the seroprevalence of antibodies to SARS-CoV-2. However, we believe it is of value to add to this body of literature in the context of different study populations and also locations across the country. Importantly, we discuss the value of using different antibody assays for identification of SARS-CoV-2 infections. Also, the possible significance of IgG to spike and RBD. Given that whilst spike is associated with protection to the virus, it is IgG to RBD which is directly correlated with neutralizing activity against SARS-CoV-2. Reviewer #3: Authors attempted to assess SARS-CoV-2 seroprevalence in blood donors from Karachi, Pakistan, during the second COVID-19 pandemic wave. There are several issues regarding this manuscript: 1. The small sample size, significant gender imbalance and the distribution of age groups. For instance they have 4 participants in the age group 51-60 years and the age range for group 1 is 17-20: 4 years. For an unbiased observation authors should have grouped participants into different age categories, for example: 17-25; 26-35; 36-45; 46-55. -Thank you for this suggestion, we have re-analysed the data in the age categories as suggestion by the reviewer. 2. It is not clear from the methods section the cut off for IgG positive test results. No information regarding the sensitivity and specificity of the test was provided. - We have added details regarding assay sensitivity and specificity and also cut-offs for IgG in the manuscript 3. The manuscript needs an English review. Some terms are inadequate and some sentences are hard to understand. Few examples: - A review of the English in the manuscript as been conducted and corrections made. 4. Page 10: The current study performed December 2020 until February 2021 at the time of the second COVID-19 wave shows a further rise (to 53%) in seropositivity in Karachi. - correction made 5. Page 11: the secondary questionnaire was administered later… - correction made.________________________________________ Attachments Attachment Submitted filename: PONE-reviewers_v2.docx https://doi.org/10.1371/journal.pone.0271259.r002
20 Jun 2022 Decision Letter - Joël Mossong, Editor PONE-D-21-30126R1Increasing IgG antibodies to SARS-CoV-2 in asymptomatic blood donors through the pandemic in Karachi reflects exposure and immunity in the populationPLOS ONE Dear Dr. Hasan, 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. ============================== ACADEMIC EDITOR:One of the reviewers has some minor comments. Please address these before resubmitting ============================== Please submit your revised manuscript by Aug 04 2022 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. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Joël Mossong, PhD Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: No ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? 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: Yes ****** 6. 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: # Review 2 I have only a few more suggestions, which are small in nature. Overall, the manuscript is improved, and I find that the authors have addressed my comments, as well as the comments of the other reviewer. I thank the authors for their attentiveness, and recommend publication after some small adjustments are made: - My biggest suggestion is to consider a change in title. Between the two time points, the authors do not show increasing seroprevalence. This fact makes the title potentially misleading. One alternative title might be: "IgG antibodies to SARS-CoV-2 in asymptomatic blood donors at two time points in Karachi" or something straightforward and clear like that. - I suggest stating in the first part of the abstract that individuals with known COVID-19 or symptoms in the prior 4 weeks were excluded. - L168. "Hence" almost implies that the prior two percentages naturally lead to the third percentage. I suggest simply deleting "hence" to avoid this impression. - The figures appear low-resolution and blurry to me. Please check before final submission. ****** 7. 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 ******** [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.0271259.r003
Revision 2
27 Jun 2022 Author Response Joël Mossong, PhD Academic Editor PLOS ONE Dear Dr. Mossong, Thank you for the Editorial and Reviewer feedback. We have addressed the concerns raised and hope that the manuscript will now be acceptable for publication in PLoSONE Thank you Best wishes, Zahra Hasan >>> PONE-D-21-30126R1 Increasing IgG antibodies to SARS-CoV-2 in asymptomatic blood donors through the pandemic in Karachi reflects exposure and immunity in the population PLOS ONE Dear Dr. Hasan, 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. ============================== ACADEMIC EDITOR: One of the reviewers has some minor comments. Please address these before resubmitting ============================== Reviewer 1 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: No >> Response: We thank the reviewer for pointing this out. We have now included all supporting data used for the manuscript as S1 Appendix. ________________________________________ 6. 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: # Review 2 I have only a few more suggestions, which are small in nature. Overall, the manuscript is improved, and I find that the authors have addressed my comments, as well as the comments of the other reviewer. I thank the authors for their attentiveness, and recommend publication after some small adjustments are made: - My biggest suggestion is to consider a change in title. Between the two time points, the authors do not show increasing seroprevalence. This fact makes the title potentially misleading. One alternative title might be: "IgG antibodies to SARS-CoV-2 in asymptomatic blood donors at two time points in Karachi" or something straightforward and clear like that. >>> We thank the reviewer for their comments and have modified the title of the paper as suggested. - I suggest stating in the first part of the abstract that individuals with known COVID-19 or symptoms in the prior 4 weeks were excluded. >> We thank the reviewer for their comments and have added this information in the Abstract - L168. "Hence" almost implies that the prior two percentages naturally lead to the third percentage. I suggest simply deleting "hence" to avoid this impression. >> We have removed the work ‘Hence’ from L168 - The figures appear low-resolution and blurry to me. Please check before final submission. >> We thank the reviewer for their comments and have redone the TIF conversions for the files so that they are of better resolution Attachments Attachment Submitted filename: Response to reviewers1.docx https://doi.org/10.1371/journal.pone.0271259.r004
28 Jun 2022 Decision Letter - Joël Mossong, Editor IgG antibodies to SARS-CoV-2 in asymptomatic blood donors at two time points in Karachi PONE-D-21-30126R2 Dear Dr. Hasan, 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, Joël Mossong, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0271259.r005
Formally Accepted
18 Jul 2022 Acceptance Letter - Joël Mossong, Editor PONE-D-21-30126R2 IgG antibodies to SARS-CoV-2 in asymptomatic blood donors at two time points in Karachi Dear Dr. Hasan: 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. Joël Mossong Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0271259.r006

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