Wastewater surveillance in smaller college communities may aid future public health initiatives

Laura Lee; Lescia Valmond; John Thomas; Audrey Kim; Paul Austin; Michael Foster; John Matthews; Paul Kim; Jamie Newman

doi:10.1371/journal.pone.0270385

Peer Review History

Original SubmissionJune 8, 2022
26 Jul 2022 Decision Letter - Asli Aslan, Editor PONE-D-22-16566Wastewater surveillance in smaller college communities may aid future public health initiatives PLOS ONE Dear Dr. Newman, 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 provide more detailed information on the methodology used and QA/QC protocols in your revised manuscript. Also please cite recent published papers on this topic. Please submit your revised manuscript by Sep 09 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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We will update your Data Availability statement to reflect the information you provide in your cover letter. 3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. 4. 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: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: Yes ****** 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 ****** 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 ****** 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: The authors of the manuscript PONE-D-22-16566 have conducted a nice study but there are some loopholes in the manuscript which needs to be fixed before accepted for publication. The points to be addressed are mentioned below; Please reframe the 1st sentence of the abstract. The part “since its declaration” doesn’t sound good. Maybe the sentence can be reframed to “Since the declaration of COVID-19 pandemic” or any other of your choice. In the introduction, please cite references where WBE has been imposed at school/ college/ university level. Few of the references are provided below; https://doi.org/10.1371/journal.pone.0270168 https://doi.org/10.1016/j.scitotenv.2021.146408 https://doi.org/10.1016/j.scitotenv.2021.146749 Please highlight the novelty of the study in the last paragraph of the introduction. Why was the ww sample from one WWTP heat inactivated but other one not? Was there any field blank, method blank, extraction blank? What about negative controls? What are the QA/QC measures taken into consideration? Was the LoD, LoQ determined? Was any RT-qPCR inhibition noticed? Incorporate statistical analysis. Critical scientific explanation for the obtained results are missing. Please provide better picture resolution of the figures and the fonts should be legible. Please incorporate references in support of the obtained results. The literatures provided below when cited would enhance the manuscript; https://doi.org/10.1371/journal.pone.0266407 https://doi.org/10.1128/aem.01740-21 https://doi.org/10.1016/j.scitotenv.2021.152503 https://doi.org/10.1016/j.scitotenv.2021.150264 https://doi.org/10.1371/journal.pwat.0000007 https://doi.org/10.1021/acsestwater.2c00052 Reviewer #2: This is a well-written report of SARS-CoV-2 wastewater monitoring conducted at three rural locations. The analysis is clearly reported and follows one conventional viral concentration procedure that has been commonly used despite its analytical challenges and drawbacks (e.g., <0.51% recovery of phi6 used as an indicator for SARS-CoV-2 in this paper: https://www.sciencedirect.com/science/article/pii/S0048969721058009 ) My primary concern is that the PEG precipitation method does not necessarily appear to generate concentrations that provide reliable detection or trend information. For example, the authors report low concentrations in the wastewater during the Omicron surge, lack of correlation with observed clinical case trends, and lag of wastewater data (rather than lead over clinical cases). Some of these observations could be explained by poor clinical testing or other environmental factors, but it’s also possible some of the observations result from analytical issues. The analysis appears to have been performed carefully and consistently, so I wonder how effective this particular method actually is at low concentrations (which may be most relevant in these communities). Generally, we’ve found the PEG precipitation method to perform poorly. The authors should provide more context in terms of the success (or not) of the PEG method applied in generating reliable results that can be used to capture trends in infection rates for similar contexts. If other studies have demonstrated the particular utility of this method, this would build confidence in the wastewater results reported. The normalization with PMMoV also does not appear to improve the correlations with clinical cases—is this consistent with what others have seen using this method in similar contexts? Were correlations between wastewater and clinical cases in this study better or worse with and without PMMoV normalization? I absolutely agree that rural areas and small communities can benefit from wastewater monitoring, but more sensitive and reliable methods may be needed to confidently report results (e.g., analysis of primary clarifier sludge is much more sensitive as an overall method for detection https://pubs.rsc.org/en/content/articlehtml/2022/ew/d1ew00826a ). If the wastewater measurements don’t consistently lead over clinical cases, the tool is not useful for early warning. Only analytical results (non-normalized) from WWTP plant (Figure 2C) appears to have consistent detection for N1 and N2 as well as trends that fit with expectations from the known surges in infections. Can the authors explain the poor agreement between N1 and N2 in Figure 2a? I recommend that the authors report the full suite of Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to improve the completeness of the reported results ( for further information, see Bivins et al, 2021 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8341816/ ). Especially given higher rates of non-detects, the limit of detection (LOD) in terms of genome copies / ul extract as well as genome copies/ ml wastewater sample. Standard curves are shown, but I don’t believe the LOD was reported. How non-detects were treated in triplicate qPCR analysis should also be reported (e.g., if 1 or more results of the triplicate were below the LOD, were the replicates assigned a value such as the LOD, were the results censored, or how else where they handled?). ****** 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. 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Revision 1
6 Aug 2022 Author Response 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 Style requirements have been reviewed and followed 2. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. Data has been uploaded as Supporting Information files. 3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. Data has been uploaded as Supporting Information files at the time of resubmission and reflected in the cover letter. 4. 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. Captions have been added as is appropriate to Supporting Information files. 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 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: Yes 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 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 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: The authors of the manuscript PONE-D-22-16566 have conducted a nice study but there are some loopholes in the manuscript which needs to be fixed before accepted for publication. The points to be addressed are mentioned below; Please reframe the 1st sentence of the abstract. The part “since its declaration” doesn’t sound good. Maybe the sentence can be reframed to “Since the declaration of COVID-19 pandemic” or any other of your choice. In the introduction, please cite references where WBE has been imposed at school/ college/ university level. Few of the references are provided below; https://doi.org/10.1371/journal.pone.0270168 https://doi.org/10.1016/j.scitotenv.2021.146408 https://doi.org/10.1016/j.scitotenv.2021.146749 We appreciate the suggestions for additional reference of WBE in academic institutions and the suggested citations. The abstract has been edited and relevant citations added to the introduction. Please highlight the novelty of the study in the last paragraph of the introduction. The introduction has been modified to reflect the fact that, to our knowledge, this was the first study done across two rural, primarily undergraduate university campuses that leveraged limited resources to forge partnerships and produce data that may inform public health in the future. Why was the ww sample from one WWTP heat inactivated but other one not? Many labs do not heat inactive wastewater samples and heat inactivation has been shown to have no significant impact on viral detection. As the studies reported in our manuscript were performed on two different campuses, with different safety protocols, the wastewater for one site was heat treated at the treatment facility before being taken to the university research lab in order to adhere to university safety protocol. Pecson BM et al. Environ Sci.: Water Res. Technol. 2021 Was there any field blank, method blank, extraction blank? Molecular grade water was used in the initial extraction reactions and resulted in no amplification. To conserve resources, this was not repeated with each reaction once confirmation of protocol was obtained. This has been noted in the methods. What about negative controls? No template controls were used as negative controls. This has been noted in the methods section. What are the QA/QC measures taken into consideration? As described in methods, RNA quality was first assessed in the absence of carrier RNA using an RNA IQ assay and identified 61% long, structured RNA. We also measured A260/A280 ratios in the range of 1.7 to 2.2, indicating acceptable purity of the extracted RNA, and this has been added to the methods. Was the LoD, LoQ determined? The LOD was determined using logistic regression and the method has been updated. Was any RT-qPCR inhibition noticed? No inhibition was noticed. This was previously mentioned in the supplemental information and has been moved to the methods section for clarification. Incorporate statistical analysis. We have elaborated on analysis, quality control, and technique validation in our methods section. Critical scientific explanation for the obtained results are missing. We are not sure what the reviewer is asking for with this comment. We have attempted to clearly explain all of our data, provide literature references to support our findings and conclusions, and addressed all other comments to enhance the manuscript for publication. Please provide better picture resolution of the figures and the fonts should be legible. The figures were exported at high resolution (600 dpi) and in the upload of files it was noted they will not appear high resolution in the pdf compiled for review. This should be addressed in the final proof of the paper. Please incorporate references in support of the obtained results. The literatures provided below when cited would enhance the manuscript; https://doi.org/10.1371/journal.pone.0266407 https://doi.org/10.1128/aem.01740-21 https://doi.org/10.1016/j.scitotenv.2021.152503 https://doi.org/10.1016/j.scitotenv.2021.150264 https://doi.org/10.1371/journal.pwat.0000007 https://doi.org/10.1021/acsestwater.2c00052 We appreciate this suggestion. Additional references have been added to support the results of the study. Reviewer #2: This is a well-written report of SARS-CoV-2 wastewater monitoring conducted at three rural locations. The analysis is clearly reported and follows one conventional viral concentration procedure that has been commonly used despite its analytical challenges and drawbacks (e.g., <0.51% recovery of phi6 used as an indicator for SARS-CoV-2 in this paper: https://www.sciencedirect.com/science/article/pii/S0048969721058009 ) My primary concern is that the PEG precipitation method does not necessarily appear to generate concentrations that provide reliable detection or trend information. For example, the authors report low concentrations in the wastewater during the Omicron surge, lack of correlation with observed clinical case trends, and lag of wastewater data (rather than lead over clinical cases). Some of these observations could be explained by poor clinical testing or other environmental factors, but it’s also possible some of the observations result from analytical issues. The analysis appears to have been performed carefully and consistently, so I wonder how effective this particular method actually is at low concentrations (which may be most relevant in these communities). Generally, we’ve found the PEG precipitation method to perform poorly. The authors should provide more context in terms of the success (or not) of the PEG method applied in generating reliable results that can be used to capture trends in infection rates for similar contexts. If other studies have demonstrated the particular utility of this method, this would build confidence in the wastewater results reported. The normalization with PMMoV also does not appear to improve the correlations with clinical cases—is this consistent with what others have seen using this method in similar contexts? Were correlations between wastewater and clinical cases in this study better or worse with and without PMMoV normalization? We appreciate the reviewer’s concerns over some aspects of methodology. We agree that PEG is generally not as robust as other methods. Although there are some reports of higher recoveries of enveloped viruses in wastewater using PEG than other methods (e.g. of phi6 or of CHV) (Flood MT et al. Food Envrion Virol. 2021; Barril PA et al. Sci Total Environ. 2021). Other studies indicate similar performance (e.g. 44% PEG vs 28%-65% other methods) (Ahmed W et al. Sci Total Environ. 2020) or varying reductions in recovery. Given the literature supporting the efficacy of the technique, PEG precipitation does appear to be appropriate for the circumstances presented. The use of PPMoV for normalization does help to better analyze the data at the 3 sites. This is most apparent in the non-normalized data for Ruston which does not follow the clinical case load during the peak of Omicron in January 2022 but does correlate after normalization. This normalization is critical to the study as there are variables including rainfall that may affect concentration of virus in a given sample. The greatest challenge is likely not in the methodology, but in missed sample dates and irregular sampling intervals that result from a reliance on student and city worker schedules. Despite those missing data points, the trends in viral load correlate with reported caseloads. Finally, given the nature of wastewater testing and college towns, there is a regular fluctuation in the population that can account for discrepancies in wastewater detection, timing of detection, and correlation to caseloads. We agree that this is a valid point when assessing city wastewater in college towns and so additional text has been added to the conclusion to highlight this critical aspect of the study. I absolutely agree that rural areas and small communities can benefit from wastewater monitoring, but more sensitive and reliable methods may be needed to confidently report results (e.g., analysis of primary clarifier sludge is much more sensitive as an overall method for detection https://pubs.rsc.org/en/content/articlehtml/2022/ew/d1ew00826a ). If the wastewater measurements don’t consistently lead over clinical cases, the tool is not useful for early warning. Only analytical results (non-normalized) from WWTP plant (Figure 2C) appears to have consistent detection for N1 and N2 as well as trends that fit with expectations from the known surges in infections. Can the authors explain the poor agreement between N1 and N2 in Figure 2a? We agree that as a predictive tool, wastewater testing has limitations. However, as we entered a time of at-home testing, over all less testing, and in turn less reporting, wastewater-based epidemiology provides a critical accounting of virus or other detectable pathogens in a community. This is especially useful in areas where testing is limited, where there is a large population of lower income families who may not test as often for fear of missing work, and in communities where policies have been relaxed and so again, there is less testing and reporting to public health officials. The outcome of this is illustrated in Figure 2C where the wastewater data shows high levels of virus circulating in the community despite few clinical cases being reported. This coincided with relaxed policies and at-home testing where people likely had COVID-19 and were either not testing and/or not reporting to public health officials. The problem of dashboards underreporting cases and instilling a false sense of security is increasingly recognized. This points to the necessity of this type of consistent monitoring in all communities in order to best understand disease prevalence and inform public health. As pointed out, the data reported in Figure 2A does show different levels of amplification for N1 and N2. The CDC N1 primer probe set has been reported to be more sensitive than the N2 primer probe set with the N2 more likely to result in higher Cq or non-detect in a clinical setting. An assessment of 36 laboratories testing wastewater also concluded that the N1 primer probe results in higher concentrations than N2. Whether this is inherent to the primer probe sequences or due to the stability of the target regions or both is unclear but we present both sets of data. (Pecson BM et al. Environ Sci.: Water Res Technol. 2021) I recommend that the authors report the full suite of Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to improve the completeness of the reported results ( for further information, see Bivins et al, 2021 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8341816/ ). Some of the essential details in the MIQE checklist related to reagent/buffer compositions, target/oligonucleotide sequences can be derived from our detailed description of the materials and kits used. We have explicitly added information related to nucleic acid and qPCR QA/QC such as method blank, RNA purity, result of NTC, LOD. Especially given higher rates of non-detects, the limit of detection (LOD) in terms of genome copies / ul extract as well as genome copies/ ml wastewater sample. Standard curves are shown, but I don’t believe the LOD was reported. How non-detects were treated in triplicate qPCR analysis should also be reported (e.g., if 1 or more results of the triplicate were below the LOD, were the replicates assigned a value such as the LOD, were the results censored, or how else where they handled?). We have addressed these concerns in our methods section. The LOD is reported and the handling of non-detects is described. 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 https://doi.org/10.1371/journal.pone.0270385.r002
2 Sep 2022 Decision Letter - Asli Aslan, Editor Wastewater surveillance in smaller college communities may aid future public health initiatives PONE-D-22-16566R1 Dear Dr. %Newman%, 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, Asli Aslan 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 #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: 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 #1: 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 #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: (No Response) ****** 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 ******** https://doi.org/10.1371/journal.pone.0270385.r003
Formally Accepted
7 Sep 2022 Acceptance Letter - Asli Aslan, Editor PONE-D-22-16566R1 Wastewater surveillance in smaller college communities may aid future public health initiatives Dear Dr. Newman: 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. Asli Aslan Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0270385.r004

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