Rapid genomic surveillance of SARS-CoV-2 in a dense urban community of Kathmandu Valley using sewage samples

Rajindra Napit; Prajwol Manandhar; Ashok Chaudhary; Bishwo Shrestha; Ajit Poudel; Roji Raut; Saman Pradhan; Samita Raut; Pragun G. Rajbhandari; Anupama Gurung; Rajesh M. Rajbhandari; Sameer M. Dixit; Jessica S. Schwind; Christine K. Johnson; Jonna K. Mazet; Dibesh B. Karmacharya

doi:10.1371/journal.pone.0283664

Peer Review History

Original SubmissionMay 11, 2022
11 May 2022 Author Response Attachments Attachment Submitted filename: ES Paper Reviewers comments.docx https://doi.org/10.1371/journal.pone.0283664.r001
26 Oct 2022 Decision Letter - Nadim Sharif, Editor PONE-D-22-13706Rapid genomic surveillance of SARS-CoV-2 in a dense urban community using environmental (sewage) samplesPLOS ONE Dear Dr. Karmacharya, 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 Dec 08 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. 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We would also like to express our gratitude to the One Health Institute of the University of California-Davis and the USAID-funded PREDICT project for providing us with laboratory resources. Whole-genome sequencing was done at the Intrepid Nepal Genomic Center. Some of our work was partially funded by the Australian Development Agency and PSI grant (the Netherlands). We would like to thank all these agencies for their support. And finally, we would like to thank everyone at Intrepid Nepal Pvt Ltd, Center for Molecular Dynamics Nepal, and BIOVAC Nepal for their tireless work even during the nationally enforced lockdown in Nepal.” 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. 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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: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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). 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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: In the present manuscript PONE-D-22-13706 "Rapid genomic surveillance of SARS-CoV-2 in a dense urban community using environmental (sewage) samples", the authors have studied/presented the genomic surveillance of SARS-CoV-2 in wastewater samples Kathmandu, Nepal, by two different procedures, MiSeq (Illumina) and MinION (ONT). This is a well-written manuscript, the scope of which is really important in the current pandemic, and more important in countries like Nepal, as stated by the authors. As I can see, 4 reviewers have also evaluated the manuscript, and this is a revised or resubmitted manuscript. Although, I have not seen the initial submission, the authors have tried to address the comments/critishisms of the reviewers to some extent. Major comments 1. The results of the study are not well presented, not really validated and moreover not novel for the readers. 2. In this regard, the fig. 1-3 does not include any results but describing the sample sites/plan and the basic analytical procedures. 3. Fig. 6, including the mutations/SNPs detected is poor, given the fact that WGS was performed. 4. The authors must present the %percentage of the mutations/SNPs detected and moreover to translate them to specific SARS-CoV-2 variants. 5. Several discrepancies between MiSeq and MinION are presented. This is very important, and the authors should discuss it in detail. 6. The authors should describe in detail the quality control of the NGS libraries and the final sequencing output. 7. The quantification of viral load in WW samples and the "persons infected" formula are not clearly presented and described. 8. The authors should pre 9. And most importantly, I cannot draw any specific and major conclusions from the results and discussion presented. Reviewer #2: Napit et al. studied the genomic surveillance of SARS-CoV-2 in sewage from Kathmandu, Nepal. The authors aimed to establish a genomic-based environmental surveillance system by monitoring SARS-CoV-2 in sewage samples via PCR and WGS approaches. Although the experiments in the manuscript are well-organized and the manuscript itself is also written in a good way, this reviewer can see right away the shortages of this study. The following are listed some major issues for the authors' consideration. 1) One of the main issues is the data quality of the sequencing. I looked into some of the raw reads the authors deposited on GitHub. Since those data are directly related to the conclusions, it will not stand if such bad quality data was used. The author should consider toning down some of the conclusions from Fig. 6. 2) Considering the environmental variants which might cause damage to lots of biological samples, the mutations the authors found has simply after the virus particles expose to sewage. It is essential to show some correlation to some clinical results to see if the mutations were presented in society. 3) This reviewer really appreciates this kind of excellent effort in studying SARS-CoV-2 in developing countries like Nepal. The authors can discuss the significance of this study extensively since the novelty of this study is not apparent, especially in the methodology and results. One possible direction is to mention that sewage might become one of the possible infection routes for SARS-CoV-2, and we should be cautious since some mutations were found in environmental samples, such as sewage, in this study. ****** 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.0283664.r002
Revision 1
1 Dec 2022 Author Response Reviewer 1 In the present manuscript PONE-D-22-13706 "Rapid genomic surveillance of SARS-CoV-2 in a dense urban community using environmental (sewage) samples", the authors have studied/presented the genomic surveillance of SARS-CoV-2 in wastewater samples Kathmandu, Nepal, by two different procedures, MiSeq (Illumina) and MinION (ONT). This is a well-written manuscript, the scope of which is really important in the current pandemic, and more important in countries like Nepal, as stated by the authors. SNo Comment Response 1 The results of the study are not well presented, not really validated and moreover not novel for the readers. The result section has been updated with more elaborate figure from supplementary data (figure 6) and table no. 3 has been added to make it more effective. 2 In this regard, the fig. 1-3 does not include any results but describing the sample sites/plan and the basic analytical procedures. These figures are intended to elaborate the procedures/methodology and not the results- they are in methodology section. 3 Fig. 6, including the mutations/SNPs detected is poor, given the fact that WGS was performed. The figure has been removed with the supplementary figure S1 which is more elaborate and depicts all the SNP with its relative abundance in y-axis while SNP position in x-axis. 4 The authors must present the % percentage of the mutations/SNPs detected and moreover to translate them to specific SARS-CoV-2 variants. The mutation % has been added in supplementary table S3 and S9, Major circulating variant has been added in line 266-269 and table 3. The result is more specifically highlighted in the result section as well (line 301-304, 359-364 and 296-300). At the time of this study, variant of concerns was not classified and tools like Frejya that uses similar SNPs frequency-based algorithm and database containing all variant of concerns can actually predict circulation variant fractions (https://github.com/andersen-lab/Freyja) was not available. 5 Several discrepancies between MiSeq and MinION are presented. This is very important, and the authors should discuss it in detail. The discrepancies has been highlighted both in result section line 292-293 Supplementary table S7. 6 The authors should describe in detail the quality control of the NGS libraries and the final sequencing output The quality control has been added in line 190-193 and 202-206. 7 The quantification of viral load in WW samples and the "persons infected" formula are not clearly presented and described. The formula was taken from the previous similar research paper; all the procedure was already described in those literature so was not mentioned earlier. The explanation has been added in line 135-137. 8 And most importantly, I cannot draw any specific and major conclusions from the results and discussion presented. The discussion has been updated to add some clarity, we intended to demonstrate and highlight importance of genomic surveillance for disease like SARS-CoV-2. However, it is not feasible always for country like Nepal where fundings are not readily available, in such condition using low-cost sequencing to assess the situation and then if something important comes up then go for Miseq or clinical genomic surveillance of the community would be better option. Described in line 354-370, 380-392. Reviewer 2 Napit et al. studied the genomic surveillance of SARS-CoV-2 in sewage from Kathmandu, Nepal. The authors aimed to establish a genomic-based environmental surveillance system by monitoring SARS-CoV-2 in sewage samples via PCR and WGS approaches. Although the experiments in the manuscript are well-organized and the manuscript itself is also written in a good way, this reviewer can see right away the shortages of this study. The following are listed some major issues for the authors' consideration. SNo Comment Response 1 One of the main issues is the data quality of the sequencing. I looked into some of the raw reads the authors deposited on GitHub. Since those data are directly related to the conclusions, it will not stand if such bad quality data was used. The author should consider toning down some of the conclusions from Fig. 6. All the data was not of bad quality, few had low sequencing coverage, which is common in environmental samples as they do not have high viral load to that of clinical samples that has been acknowledged with all the table on coverage and depths in supplementary table and also in the manuscripts. The figure 6 has also been replaced with the Supplementary figure S1. Those low coverage samples had failed to distinguish lineage to high resolution (table no 3 has been added) for clarity. A line has been added in discussion highlighting the same (line 370-371) 2 Considering the environmental variants which might cause damage to lots of biological samples, the mutations the authors found has simply after the virus particles expose to sewage. It is essential to show some correlation to some clinical results to see if the mutations were presented in society. The comparison with clinical samples were in discussion section but result was updated to contain variant calling data from consensus sequences in result section and table no. 3. 3 This reviewer really appreciates this kind of excellent effort in studying SARS-CoV-2 in developing countries like Nepal. The authors can discuss the significance of this study extensively since the novelty of this study is not apparent, especially in the methodology and results. One possible direction is to mention that sewage might become one of the possible infection routes for SARS-CoV-2, and we should be cautious since some mutations were found in environmental samples, such as sewage, in this study. The discussion (line 385-390 and 392-395) has been updated to highlight the effort needed to track pandemic in country like Nepal cheaply and quickly in time. Attachments Attachment Submitted filename: rebutal_letter.docx https://doi.org/10.1371/journal.pone.0283664.r003
4 Dec 2022 Decision Letter - Nadim Sharif, Editor PONE-D-22-13706R1Rapid genomic surveillance of SARS-CoV-2 in a dense urban community using environmental (sewage) samplesPLOS ONE Dear Dr. Karmacharya, 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 Jan 18 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. 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, Nadim Sharif, M.Sc. 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. Additional Editor Comments: Figure 1, 2 and 5 are not necessary for the main documents. Actually these figures reduced the quality of the manuscript. For the map figures, the authors should use high resolution and detailed maps of the study regions. It seems the maps are copied from google map or similar application. It is totally unacceptable. Use your originally created figures to use in the manuscript. Figure 2 is not necessary in this manuscript. Remove figure 2 and improve figures 1 and 5. Specify the reference genome sequence accession number against which you aligned your sequences and compared form mutational analysis. The discussion section requires major improvement. Compare your findings with similar studies in your country and other countries and describe the similarities and differences with appropriate reasoning. [Note: HTML markup is below. Please do not edit.] [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.0283664.r004
Revision 2
9 Dec 2022 Author Response Reviewer 1 In the present manuscript PONE-D-22-13706 "Rapid genomic surveillance of SARS-CoV-2 in a dense urban community using environmental (sewage) samples", the authors have studied/presented the genomic surveillance of SARS-CoV-2 in wastewater samples Kathmandu, Nepal, by two different procedures, MiSeq (Illumina) and MinION (ONT). This is a well-written manuscript, the scope of which is really important in the current pandemic, and more important in countries like Nepal, as stated by the authors. SNo Comment Response 1 The results of the study are not well presented, not really validated and moreover not novel for the readers. The result section has been updated with more elaborate figure from supplementary data (figure 6- now figure 4) and table no. 3 has been added to make it more effective. 2 In this regard, the fig. 1-3 does not include any results but describing the sample sites/plan and the basic analytical procedures. These figures are intended to elaborate the procedures/methodology and not the results- they are in methodology section. Still figure 1 and 2 has been removed. 3 Fig. 6, including the mutations/SNPs detected is poor, given the fact that WGS was performed. The figure has been removed with the supplementary figure S1 which is more elaborate and depicts all the SNP with its relative abundance in y-axis while SNP position in x-axis. 4 The authors must present the % percentage of the mutations/SNPs detected and moreover to translate them to specific SARS-CoV-2 variants. The mutation % has been added in supplementary table S3 and S9, Major circulating variant has been added in line 266-269 and table 3. The result is more specifically highlighted in the result section as well (line 301-304, 359-364 and 296-300). At the time of this study, variant of concerns was not classified and tools like Frejya that uses similar SNPs frequency-based algorithm and database containing all variant of concerns can actually predict circulation variant fractions (https://github.com/andersen-lab/Freyja) was not available. 5 Several discrepancies between MiSeq and MinION are presented. This is very important, and the authors should discuss it in detail. The discrepancies has been highlighted both in result section line 292-293 Supplementary table S7. 6 The authors should describe in detail the quality control of the NGS libraries and the final sequencing output The quality control has been added in line 190-193 and 202-206. 7 The quantification of viral load in WW samples and the "persons infected" formula are not clearly presented and described. The formula was taken from the previous similar research paper; all the procedure was already described in those literature so was not mentioned earlier. The explanation has been added in line 135-137. 8 And most importantly, I cannot draw any specific and major conclusions from the results and discussion presented. The discussion has been updated to add some clarity, we intended to demonstrate and highlight importance of genomic surveillance for disease like SARS-CoV-2. However, it is not feasible always for country like Nepal where fundings are not readily available, in such condition using low-cost sequencing to assess the situation and then if something important comes up then go for Miseq or clinical genomic surveillance of the community would be better option. Reviewer 2 Napit et al. studied the genomic surveillance of SARS-CoV-2 in sewage from Kathmandu, Nepal. The authors aimed to establish a genomic-based environmental surveillance system by monitoring SARS-CoV-2 in sewage samples via PCR and WGS approaches. Although the experiments in the manuscript are well-organized and the manuscript itself is also written in a good way, this reviewer can see right away the shortages of this study. The following are listed some major issues for the authors' consideration. SNo Comment Response 1 One of the main issues is the data quality of the sequencing. I looked into some of the raw reads the authors deposited on GitHub. Since those data are directly related to the conclusions, it will not stand if such bad quality data was used. The author should consider toning down some of the conclusions from Fig. 6. All the data was not of bad quality, few had low sequencing coverage, which is common in environmental samples as they do not have high viral load to that of clinical samples that has been acknowledged with all the table on coverage and depths in supplementary table and also in the manuscripts. The figure 6 (now figure 4) has also been replaced with the Supplementary figure S1. Those low coverage samples had failed to distinguish lineage to high resolution (table no 3 has been added) for clarity. A line has been added in discussion highlighting the same (line 370-371) 2 Considering the environmental variants which might cause damage to lots of biological samples, the mutations the authors found has simply after the virus particles expose to sewage. It is essential to show some correlation to some clinical results to see if the mutations were presented in society. The comparison with clinical samples were in discussion section but result was updated to contain variant calling data from consensus sequences in result section and table no. 3. 3 This reviewer really appreciates this kind of excellent effort in studying SARS-CoV-2 in developing countries like Nepal. The authors can discuss the significance of this study extensively since the novelty of this study is not apparent, especially in the methodology and results. One possible direction is to mention that sewage might become one of the possible infection routes for SARS-CoV-2, and we should be cautious since some mutations were found in environmental samples, such as sewage, in this study. The discussion (line 400-409) has been updated to highlight the effort needed to track pandemic in country like Nepal cheaply and quickly in time. Attachments Attachment Submitted filename: rebutal_letter.docx https://doi.org/10.1371/journal.pone.0283664.r005
14 Mar 2023 Decision Letter - Basant Giri, Editor Rapid genomic surveillance of SARS-CoV-2 in a dense urban community using environmental (sewage) samples PONE-D-22-13706R2 Dear Dr. Karmacharya, 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. However, I suggest to re-phrase the title as follows: Rapid genomic surveillance of SARS-CoV-2 in a dense urban community of Kathmandu valley using sewage samples. 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, Basant Giri, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): 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 #2: 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 #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: N/A ****** 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 #2: Yes ****** 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 #2: 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 #2: I have no further comments on this revised manuscript and the updated version should be acceptable for Plos ONE. ****** 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 #2: No ******** https://doi.org/10.1371/journal.pone.0283664.r006
Formally Accepted
21 Mar 2023 Acceptance Letter - Basant Giri, Editor PONE-D-22-13706R2 Rapid genomic surveillance of SARS-CoV-2 in a dense urban community of Kathmandu Valley using sewage samples Dear Dr. Karmacharya: 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. Basant Giri Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0283664.r007

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