A direct RT-qPCR approach to test large numbers of individuals for SARS-CoV-2

Tomislav Maricic; Olaf Nickel; Ayinuer Aximu-Petri; Elena Essel; Marie Gansauge; Philipp Kanis; Dominik Macak; Julia Richter; Stephan Riesenberg; Lukas Bokelmann; Hugo Zeberg; Matthias Meyer; Stephan Borte; Svante Pääbo

doi:10.1371/journal.pone.0244824

Peer Review History

Original SubmissionJuly 28, 2020
2 Nov 2020 Decision Letter - Ronald Dijkman, Editor PONE-D-20-23519 A direct RT-qPCR approach to test large numbers of individuals for SARS-CoV-2 PLOS ONE Dear Dr. Maricic, 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 17 2020 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Ronald Dijkman, PhD Academic Editor PLOS ONE Additional Editor Comments: Dear Dr. Maricic, My apologies for the long duration of the reviewing process, however, it has been very difficult to find the appropriate number of reviewers to evaluate your work. Based on the reviewer comments (No. 2 & 3), is that the study lacks extensive validation of the direct RT-PCR protocol. After reading your manuscript, I tend agree with the reviewers that in the current manuscript version is not well addressed, and therefore I strongly encourage you to address this critical point. Best regards, Ronald Dijkman Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for stating the following in the Acknowledgments Section of your manuscript: "Funding was provided by the Max Planck Society and the NOMIS foundation." 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 funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 3. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. 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: Yes Reviewer #2: No Reviewer #3: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: N/A Reviewer #3: 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: Yes 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: 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 present a performance evaluation of different RT PCR-based detection methods for Sars-CoV-2. The main advantages of two new protocols is the use of gargles instead of nasopharyngeal swab material and poolin. They show acceptable detection performance. Major comments: none. Minor comments: Ct value, Cq value are not consistently used. élease use same wording throughout. Reviewer #2: Maricic et al. present a direct RT-qPCR approach to test large number of individuals for SARS-CoV-2. Direct RT-qPCR testing strategies are important to mitigate supply chain issues and lower cost and time for SARS-CoV-2 testing. Although the authors present an important proof of concept, several similar direct protocols have already been published and the current study lacks extensive validation of the protocol to support its major claims. Major 1. Several studies have shown that gargle lavages can be used to detect SARS-CoV-2, as well as extraction-free/direct RT-qPCR assays and therefore the originality and novelty of this study is limited. 2. The direct RT-qPCR assay has not been extensively validated. Overall, sample size for clinical testing is low, and specificity (and cross-reactivity) is only tested with 7 reference samples. The authors did test a substantial number of samples collected in the nursing home, but out of the 756 samples tested, none of those were positive. Based on these results test performance cannot be evaluated in the target population, and therefore the main claim that this method can be used for testing in nursing homes cannot be supported. 3. Sensitivity for the direct RT-qPCR assay was not determined and based on the presented data it seems like this assay can only be used for samples with a Ct value below 29 which is not sensitive enough to detect cases beyond peak viral titers. The limit of detection is an important parameter to determine the performance of a test. Minor 4. Please include primer and probe sequences in the methods section. Reviewer #3: Well performed study looking at a novel collection sample (gargle washes) with direct RT-PCR, as well as pilot study of pooling these samples in this test system. The methodology is sound and the conclusions warranted by the data, but a lack of context limits the value of the conclusions, and the discussion is overspeculative. -Describing this methodology as large-scale high-throughput is a considerable stretch. The authors' actual testing performance was 35-50 samples per weekday, 22-26 on weekends; with no positive samples. While scaling-up by pooling in theory could provide '~3,700 individuals, with ~300 Euros total reagent cost in an hour and a half', this scaling-up was not field tested. There is considerable distance between a research-ready and a clinically-ready process. -Labor is as limiting for many testing laboratories as are reagents. Please describe the setup time per run for the unpooled and pooled versions of the assay. -The operational discussion of assay use in long-term care (lines 189-197) hopelessly oversimplifies the logistics of such an approach. Recommend omitting. -The discussion in lines 203-211 is plausible but, at this time, rather contentious and by no means established science. Interruption of COVID transmission by large-scale rapid testing is conceivable, but faces formidable logistical and behavioral barriers. Most discussions of such approaches focus on home-performed testing. The authors of this study, again, fail to recognize the challenges of scaling-up centralized testing, even with simplified lab-based protocols such as they describe. A proper discussion of this topic is beyond the scope of the paper; the authors should at least soften this paragraph considerably. -Lines 212-215 speculate excessively. Please omit. -The specificity of the test appears to be roughly 98.8% (9 false-positives out of 756 samples tested). How does this impact usability and scaleability? How complex and time-consuming are the described procedures for assessing false-positives? Lines 136-138 -There is something of an imbalance between the description of this testing methodology as simple, rapid, and scaleable; which requires the gavage sample type; and the paucity of data on the clinical performance of the gavage. only ten positive samples were assessed; and the clinical sensitivity appears to be around 50%. The samples missed were high Ct value samples of (probably) modest infection-control significance; but 50% is 50% and only-ten samples are only-ten samples. I recognize that Germany during the period of this study was a low-incidence area; but the conclusions must be tempered by this major weakness. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes: Peter M. Keller Reviewer #2: No Reviewer #3: Yes:** Sheldon Campbell [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.0244824.r001
Revision 1
25 Nov 2020 Author Response Additional Editor Comments: Dear Dr. Maricic, My apologies for the long duration of the reviewing process, however, it has been very difficult to find the appropriate number of reviewers to evaluate your work. Based on the reviewer comments (No. 2 & 3), is that the study lacks extensive validation of the direct RT-PCR protocol. After reading your manuscript, I tend agree with the reviewers that in the current manuscript version is not well addressed, and therefore I strongly encourage you to address this critical point. We believe that the issue about validation is at least partially a misunderstanding. We have tested much more than the ten positive and ten negative samples to which reviewer 3 refers. We tested 756 mouthwash samples in the nursing home showing that the false positive rate is very low (Results section, line 139-140). We also tested 46 samples previously been shown to be positive and show that we detect all samples with Ct values at and below 29. See answers to the reviewers below. We may also mention that we now perform between 400 and 500 tests per week in our institute. This method is also being established at other institutions in Germany. We strongly believe that it will be useful for the manuscript to be published at this point in time. 2. Thank you for stating the following in the Acknowledgments Section of your manuscript: "Funding was provided by the Max Planck Society and the NOMIS foundation." 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. We have deleted this sentence in the Acknowledgments section. 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 funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. The sentence is OK. 3. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. A supplementary file with uncropped and unadjusted images has now been uploaded. 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. This has now been done. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: No Reviewer #3: Yes 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: N/A Reviewer #3: 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: Yes 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: 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 present a performance evaluation of different RT PCR-based detection methods for Sars-CoV-2. The main advantages of two new protocols is the use of gargles instead of nasopharyngeal swab material and pooling. They show acceptable detection performance. Major comments: none. Minor comments: Ct value, Cq value are not consistently used. élease use same wording throughout. We changed all Cq to Ct in the text and the figures. Reviewer #2: Maricic et al. present a direct RT-qPCR approach to test large number of individuals for SARS-CoV-2. Direct RT-qPCR testing strategies are important to mitigate supply chain issues and lower cost and time for SARS-CoV-2 testing. Although the authors present an important proof of concept, several similar direct protocols have already been published and the current study lacks extensive validation of the protocol to support its major claims. Similar direct RT-qPCR tests have been published but our protocol is a unique combination of using mouthwashes, direct RT-qPCR and pooling. This approach allows substantial numbers of individuals to be screened and individuals with high viral loads to be detected in a short time. We now stress in the paper that this protocol is a screening or “surveillance” approach. It is in the meantime implemented in in our institute as well as other institutions. Major 1. Several studies have shown that gargle lavages can be used to detect SARS-CoV-2, as well as extraction-free/direct RT-qPCR assays and therefore the originality and novelty of this study is limited. Our protocol is a unique combination of using gargle lavages, direct RT-qPCR, and pooling. It is a surveillance method that in our hands is currently proving valuable in the second wave of infections. We believe that it may be useful also to others. 2. The direct RT-qPCR assay has not been extensively validated. Overall, sample size for clinical testing is low, and specificity (and cross-reactivity) is only tested with 7 reference samples. The authors did test a substantial number of samples collected in the nursing home, but out of the 756 samples tested, none of those were positive. Based on these results test performance cannot be evaluated in the target population, and therefore the main claim that this method can be used for testing in nursing homes cannot be supported. The test with the 7 reference samples of which two contain another coronavirus genus (Figure 2) is a test that SARS-CoV-2 diagnostic lab in Germany has to pass. Cross-reactivity of the primers/probe combination was extensively tested in the first publication of the E-gene primers and is recommended by the WHO. We now cite this first publication the E-gene primers by Corman et al. in the Methods section. 3. Sensitivity for the direct RT-qPCR assay was not determined and based on the presented data it seems like this assay can only be used for samples with a Ct value below 29 which is not sensitive enough to detect cases beyond peak viral titers. The limit of detection is an important parameter to determine the performance of a test. As the reviewer points out, Figure 3 shows that this protocol reliably detects samples with a Ct value of 29 and lower. As we point out in Discussion section (lines 191-192), it is thus slightly less sensitive than the standard assay. However, as we now stress in the manuscript, this is a surveillance method, designed to detect individuals likely to transmit the virus. As we also point out in the manuscript (lines 192-194), there is evidence that only individuals with Ct of 30 or lower are likely to transmit the infection (NCID, 2020; Wolfel et al, 2020; Zhou et al, 2020). For example, the position statement from National Centre for Infectious Diseases Singapore (NCID, 2020) states: "when the Ct value was 30 or higher (i.e. when viral load is low), no viable virus (based on being able to culture the virus) has been found”. This is also stressed in a recent perspectives piece in NEJM (https://www.nejm.org/doi/full/10.1056/NEJMp2025631) which states: "effective surveillance regimens intended to reduce the population prevalence of a respiratory virus need to return results quickly to limit asymptomatic spread and should be sufficiently inexpensive and easy to execute to allow frequent testing”. They also say there "we need tests that can enable regimens that will capture most infections while they are still infectious." Another recent study model surveillance effectiveness considering test sensitivities, frequency, and sample-to-answer reporting time (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7325181.2/) and conclude that: “surveillance should prioritize accessibility, frequency, and sample-to-answer time; analytical limits of detection should be secondary”. We believe that our approach fulfills these needs for surveillance (daily testing, results in 5 hours, detection individuals with high viral loads (Ct <29). To make this clear, we now stress that our protocol is a surveillance strategy rather than a diagnostic procedure throughout the manuscript. Minor 4. Please include primer and probe sequences in the methods section. The probes and primers for the E-gene is now the Methods section (line 221-225). For the internal RT-qPCR control (EAV) we give the catalog number since the producer does not reveal its sequence (line 225-226. Reviewer #3: Well performed study looking at a novel collection sample (gargle washes) with direct RT-PCR, as well as pilot study of pooling these samples in this test system. The methodology is sound and the conclusions warranted by the data, but a lack of context limits the value of the conclusions, and the discussion is overspeculative. We have tried to include more context and reduce speculation in the discussion. In particular, we now stress that that what we present is a protocol that allows surveillance of personnel and residents in institutions with rapid turn-around times and detection in individuals with viral loads high enough to make them likely to be infectious. -Describing this methodology as large-scale high-throughput is a considerable stretch. The authors' actual testing performance was 35-50 samples per weekday, 22-26 on weekends; with no positive samples. While scaling-up by pooling in theory could provide '~3,700 individuals, with ~300 Euros total reagent cost in an hour and a half', this scaling-up was not field tested. There is considerable distance between a research-ready and a clinically-ready process. We agree that the protocol is not extensively field-tested. We have tempered our perhaps over- enthusiastic language throughout the manuscript. We note though, that this protocol is now used for tri-weekly testing of the ~300 employees at our institute. It is currently being implemented at other institutes in Germany. -Labor is as limiting for many testing laboratories as are reagents. Please describe the setup time per run for the unpooled and pooled versions of the assay. We now included this paragraph in the Discussion discussing this (line 196-199): “Currently, we perform tri-weekly test of an average of 150 of employees per day at our research institute. Two people do the pooling of samples and set-up of the RT-qPCR in 40 minutes. Evaluations of results and reporting follows two hours later. When individual tests need to be performed, they require another 2-3 hours.” -The operational discussion of assay use in long-term care (lines 189-197) hopelessly oversimplifies the logistics of such an approach. Recommend omitting. We have omitted this paragraph as suggested. -The discussion in lines 203-211 is plausible but, at this time, rather contentious and by no means established science. Interruption of COVID transmission by large-scale rapid testing is conceivable, but faces formidable logistical and behavioral barriers. Most discussions of such approaches focus on home-performed testing. The authors of this study, again, fail to recognize the challenges of scaling-up centralized testing, even with simplified lab-based protocols such as they describe. A proper discussion of this topic is beyond the scope of the paper; the authors should at least soften this paragraph considerably. We have now softened the paragraph by saying that we speculate that this may be possible. -Lines 212-215 speculate excessively. Please omit. We have omitted the paragraph as suggested, although we do not find it excessive to speculate that more lethal viral epidemics may appear in the future. -The specificity of the test appears to be roughly 98.8% (9 false-positives out of 756 samples tested). How does this impact usability and scaleability? How complex and time-consuming are the described procedures for assessing false-positives? Lines 136-138 We now clarify that the additional electrophoretic assay requires less than one hour. However, in the meantime, we have eliminated this issue by adding an additional primer pair and a probe (nCOV_N1, approved for SARS-CoV-2 testing by Centers for Disease Control and Prevention, USA) that target another part of the SARS-CoV-2 genome and requiring both probes to emit fluorescence. This is now mentioned in the Discussion section (line 185-190). -There is something of an imbalance between the description of this testing methodology as simple, rapid, and scaleable; which requires the gavage sample type; and the paucity of data on the clinical performance of the gavage. only ten positive samples were assessed; and the clinical sensitivity appears to be around 50%. The samples missed were high Ct value samples of (probably) modest infection-control significance; but 50% is 50% and only-ten samples are only-ten samples. I recognize that Germany during the period of this study was a low-incidence area; but the conclusions must be tempered by this major weakness. We tested more than the ten positive and ten negative samples to which the reviewer refers. We tested 46 samples previously been shown to be positive (shown in Fig. 3). We detect all samples with Ct <29. We also tested 756 mouthwash samples in the nursing home which were negative (Results, line 139-140). In the meantime, this protocol is being used for tri-weekly testing of the ~300 employees at our institute and the test is currently being implemented at other institutes in our region. We have tempered the conclusions somewhat as suggested, but remain convinced that what is described here is making a useful contribution towards limiting the spread of infections. 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes: Peter M. Keller Reviewer #2: No Reviewer #3: Yes: Sheldon Campbell https://doi.org/10.1371/journal.pone.0244824.r002
9 Dec 2020 Decision Letter - Ronald Dijkman, Editor PONE-D-20-23519R1 A direct RT-qPCR approach to test large numbers of individuals for SARS-CoV-2 PLOS ONE Dear Dr. Maricic, 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 23 2021 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Ronald Dijkman, PhD Academic Editor PLOS ONE [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 Reviewer #2: (No Response) Reviewer #3: 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 Reviewer #2: Partly Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: N/A Reviewer #3: 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: Yes Reviewer #2: Yes Reviewer #3: 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 Reviewer #2: Yes Reviewer #3: 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: The authors have addressed my comments of review round #1. The comments regarding the validation of the RT-PCR protocol raised by reviewer #2 have been addressed in the response to reviewer letter by the authors. Reviewer #2: I would like to thank the authors for revising their manuscript. The authors have partly addressed my comments, and I still have 2 remaining comments: 1. In their responses to the other reviewers and my comments, the authors state that their proposed protocol is for SARS-CoV-2 surveillance. However, I think the authors have mixed up the meaning of surveillance and screening, which refer to different types of tests in epidemiology (https://www.fda.gov/medical-devices/coronavirus-covid-19-and-medical-devices/covid-19-test-uses-faqs-testing-sars-cov-2). Surveillance is applied to the population level and not to report results back to the individual level, while screening refers to testing of individuals without symptoms. Thus, the authors should use the proper terminology throughout their manuscript. 2. In my previous review I commented on the lack of an extensive sensitivity analysis for the current protocol. I agree with the authors that screening assays may need a different level of sensitivity as compared to diagnostic assays. However, sensitivity analysis is important to enable comparisons between assays to help labs make a decision for an assay. For rapid tests and at-home test I think lower sensitivity is justified, but considering that the current assay is PCR-based and that other extraction-free (and saliva-based) PCR assays are available with a seemingly higher sensitivity I wonder what the added value of the current assay is for other labs. I urge the authors to 1) determine the limit of detection of their assay (see example guidelines for LOD here: https://www.fda.gov/media/135659/download) and 2) to add a “limitations of the study” section to their discussion to provide a rational for other labs to use this assay (as compared to other more sensitive extraction-free and non-swab based PCR assays). Reviewer #3: What is the purpose of Figure 4? It's unclear, lacks a time line, and is adequately handled in the text. Consider omitting. ****** 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: Yes: Peter M. Keller Reviewer #2: No Reviewer #3: Yes:** Sheldon Campbell [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.0244824.r003
Revision 2
14 Dec 2020 Author Response Reviewer #2: I would like to thank the authors for revising their manuscript. The authors have partly addressed my comments, and I still have 2 remaining comments: 1. In their responses to the other reviewers and my comments, the authors state that their proposed protocol is for SARS-CoV-2 surveillance. However, I think the authors have mixed up the meaning of surveillance and screening, which refer to different types of tests in epidemiology (https://www.fda.gov/medical-devices/coronavirus-covid-19-and-medical-devices/covid-19-test-uses-faqs-testing-sars-cov-2). Surveillance is applied to the population level and not to report results back to the individual level, while screening refers to testing of individuals without symptoms. Thus, the authors should use the proper terminology throughout their manuscript. We thank the reviewer for clarifying for us. We now talk exclusively about screening (and not surveillance) in the manuscript. 2. In my previous review I commented on the lack of an extensive sensitivity analysis for the current protocol. I agree with the authors that screening assays may need a different level of sensitivity as compared to diagnostic assays. However, sensitivity analysis is important to enable comparisons between assays to help labs make a decision for an assay. For rapid tests and at-home test I think lower sensitivity is justified, but considering that the current assay is PCR-based and that other extraction-free (and saliva-based) PCR assays are available with a seemingly higher sensitivity I wonder what the added value of the current assay is for other labs. I urge the authors to 1) determine the limit of detection of their assay (see example guidelines for LOD here: https://www.fda.gov/media/135659/download) and 2) to add a “limitations of the study” section to their discussion to provide a rational for other labs to use this assay (as compared to other more sensitive extraction-free and non-swab based PCR assays). 1) We have now performed the experiment suggested by the reviewer and we write the following in Results: “Next we tested the smallest number of SARS-CoV-2 RNA molecules that can be detected in the assay. We find that six RNA molecules can be detected in 19 out of 20 replicate reactions, which corresponds to limit of detection of 3,000 RNA molecules per milliliter of mouthwash (Suppl. Table 2).” Since we were able to detect only six RNA molecules the assay is very close to the theoretical limit of detection of single viral RNA molecules. The factor that limits the experimental limit of detection is the volume of the mouthwash that we can use in the assay without inhibiting the reaction. This limits also other extraction-free assays. We are not aware of any other direct assays that are substantially more sensitive. 2) We have added a last section called “Limitations and Practical Considerations” after the Discussion section (line 196 ff). It discusses the lower sensitivity and the usefulness of the assay and draws attention to these issues more than when this discussion was embedded in the Discussion. Reviewer #3: What is the purpose of Figure 4? It's unclear, lacks a time line, and is adequately handled in the text. Consider omitting. We agree that the figure is simplistic. Yet, we find it useful for illustrating a practical scheme as it implemented currently in our institute and at the retirement home. If possible, we would like to keep in the manuscript. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0244824.r004
17 Dec 2020 Decision Letter - Ronald Dijkman, Editor A direct RT-qPCR approach to test large numbers of individuals for SARS-CoV-2 PONE-D-20-23519R2 Dear Dr. Maricic, 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, Ronald Dijkman, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0244824.r005
Formally Accepted
21 Dec 2020 Acceptance Letter - Ronald Dijkman, Editor PONE-D-20-23519R2 A direct RT-qPCR approach to test large numbers of individuals for SARS-CoV-2 Dear Dr. Maricic: 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. Ronald Dijkman Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0244824.r006

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