Swab pooling: A new method for large-scale RT-qPCR screening of SARS-CoV-2 avoiding sample dilution

Ana Paula Christoff; Giuliano Netto Flores Cruz; Aline Fernanda Rodrigues Sereia; Dellyana Rodrigues Boberg; Daniela Carolina de Bastiani; Laís Eiko Yamanaka; Gislaine Fongaro; Patrícia Hermes Stoco; Maria Luiza Bazzo; Edmundo Carlos Grisard; Camila Hernandes; Luiz Felipe Valter de Oliveira

doi:10.1371/journal.pone.0246544

Peer Review History

Original SubmissionOctober 14, 2020
15 Dec 2020 Decision Letter - Jean-Luc EPH Darlix, Editor Transfer Alert This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present. PONE-D-20-32256 Swab pooling: a new method for large-scale RT-qPCR screening of SARS-CoV-2 avoiding sample dilution PLOS ONE Dear Dr. de Oliveira, 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. In agreement with the referee ii have major critiques and concerns on this manuscript: 1- the time line of the overall process from swab collection, swab pooling, analysis by RTPCR and availability of the data; at the same time there is no parallel comparison between the method proposed and the standard one or that using antigen detection. 2- Ct values of 40 or above seems much to high; details should be given regarding the COV2 ODNs used in this study, and why Ct's of 40 have been used. Please submit your revised manuscript by Jan 28 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, Jean-Luc EPH Darlix, MG, Ph.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. In your revised cover letter, please address the following prompts: a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially identifying or sensitive patient information) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. Please see http://www.bmj.com/content/340/bmj.c181.long for guidelines on how to de-identify and prepare clinical data for publication. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. We will update your Data Availability statement on your behalf to reflect the information you provide. 3. Thank you for stating the following in the Financial Disclosure section: 'This work was supported by BiomeHub Biotechnologies.' We note that one or more of the authors have an affiliation to the commercial funders of this research study, BiomeHub Biotechnologies. a. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form. Please also include the following statement within your amended Funding Statement. “The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.” If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement. b. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc. Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. c. Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf. Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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: In the current paper, Christoff et al. report a large-scale swab-pooling method for SARS-CoV-2 detection. The overall idea is that this method avoids sample dilution and is based on pooling nasopharyngeal swabs from multiple patients thus decreasing laboratory manipulation. The manuscript is well written and experiments seem to be correctly performed. However, since the authors argue on the high reliability of the assay, decreased lab manipulation, and an overall reduction of 77% of the PCR tests, I have the following concerns about its implementation in practice which need to be addressed: 1. The sampling time and sample handling are increased compared to the “classical” test since 1 tube will be systematically collected apart of the “pool tube” and 2 swabs will be needed per patient instead of 1. Moreover, in practice it is not always possible to perform sampling from both nostrils especially in children. 2. Although the swab pooling could decrease the number qRT-PCR reactions, this would not be valid for populations where the infectivity rate is high and consequently, the percentage of positive PCR samples (10-30% or even more). In fact, in the current study, the positivity rate of the tests is 1,26% which is quite low compared to the actual situation in many countries where high numbers such as 40% of test positivity rates could be observed. In such cases, the number of qRT-PCR reactions will even increase. 3. One of the main challenges of SARS-CoV-2 diagnostics is its rapidity in order to isolate/quarantine the positive patients. The proposed method can only discard at first the negative ones, but further analyses are needed to confirm the true positive cases which may take 2 more days from sampling, a period during which the patients are highly contagious. 4. No comparison of the proposed method with the SARS-CoV-2 antigen tests is made. The latter are currently becoming quite popular especially for large scale screens of potentially infected populations and asymptomatic patients and can reveal infection in less than 30 min. 5. I have reserves about considering qPCR cut off values of 40 qC and above as positive. What is the PCR program and the primers that were used? This section needs to be more detailed (not only references) since the whole study is based on qPCR. It would be worthy to include as a supplementary figure a sample image of PCR plots for the different SARS-CoV-2 genes. 6. Figure 5 could be included in Figure 4. ****** 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 [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.0246544.r001
Revision 1
22 Dec 2020 Author Response Response to reviewers PONE-D-20-32256 Swab pooling: a new method for large-scale RT-qPCR screening of SARS-CoV-2 avoiding sample dilution PLOS ONE Dear Dr. de Oliveira, 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. In agreement with the referee ii have major critiques and concerns on this manuscript: 1- the time line of the overall process from swab collection, swab pooling, analysis by RTPCR and availability of the data; at the same time there is no parallel comparison between the method proposed and the standard one or that using antigen detection. 2- Ct values of 40 or above seems much to high; details should be given regarding the COV2 ODNs used in this study, and why Ct's of 40 have been used. Response: All editor's questions were also addressed within the responses to the reviewer's comments. We also included in the manuscript the timeline of sample processing considering negative and positive pools. After sample arrival at the laboratory, patients from positive pools have results released within 48-72 hours; patients from negative pools have results released within 24 hours. In this manuscript, the swab pooling method was compared to a standard method, the individual nasopharyngeal RT-qPCR assay, performed as recommended by WHO and regulatory agencies in most countries. Thus, our pooling method was compared to the standard RT-qPCR diagnostic method. Some Cq values above 40 were included (very few cases that appeared along our study) just to show all the data we had, since this manuscript is a demonstration of a new approach for SARS-CoV-2 screening. CDC and Charité-Berlin protocols, the ones used worldwide, perform 45 amplification cycles with a cutoff of 40 Cq, but as we deal with clinical samples and biological data, these extreme thresholds should be carefully analyzed. We do not intend to state that all Cqs of 40 and above should be considered positive, but we included these few cases to highlight that such things happen with real biological samples and should be evaluated along with the laboratory expertise. Several SARS-CoV-2 papers (as the links below) showed Cqs of 40, which is a very common practice for viral RNA detection in clinical samples, given the low viral load in the samples and the inherent PCR stochasticity in that range. https://www.eurosurveillance.org/content/10.2807/1560-7917.ES.2020.25.32.2001483 https://www.nature.com/articles/s41564-020-0761-6 https://jcm.asm.org/content/58/5/e00310-20 https://jamanetwork.com/journals/jamainternalmedicine/fullarticle/2769235 https://www.nature.com/articles/s41467-020-18611-5 https://jamanetwork.com/journals/jama/fullarticle/2765837 https://academic.oup.com/cid/article/71/16/2073/5828059 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7267454/ https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)30154-9/fulltext Journal Requirements: A revised cover letter version was submitted including the statements required. All the data used in the study was made available. The financial disclosure statement was adjusted as requested. Reviewer #1: 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. Response: Data and code to reproduce all the analyses included in this manuscript are provided in Supplementary information. Reviewer #1: In the current paper, Christoff et al. report a large-scale swab-pooling method for SARS-CoV-2 detection. The overall idea is that this method avoids sample dilution and is based on pooling nasopharyngeal swabs from multiple patients thus decreasing laboratory manipulation. The manuscript is well written and experiments seem to be correctly performed. However, since the authors argue on the high reliability of the assay, decreased lab manipulation, and an overall reduction of 77% of the PCR tests, I have the following concerns about its implementation in practice which need to be addressed: 1. The sampling time and sample handling are increased compared to the “classical” test since 1 tube will be systematically collected apart of the “pool tube” and 2 swabs will be needed per patient instead of 1. Moreover, in practice it is not always possible to perform sampling from both nostrils especially in children. Response: We thank the reviewer for the important consideration. In fact, there can be an increase in sampling time compared to workflows that perform the collection of only one swab. However, several countries guidelines already suggest two swabs in the collection procedure, from both nostrils, from nasopharynx and oropharynx, among several other variations around the world. This minimal difference for sample collection in the swab polling method is of negligible magnitude, especially when compared to the overall decrease in average processing time per patient, i.e., screening over 20 thousand individuals would be far more time consuming with no pool testing at all. With the swab pooling method implemented here, the collection procedure last less than 2 minutes. Within the laboratory, sample handling from swab pooling is faster and safer compared to the classical sample pooling procedure, once steps of making new identifications, aliquots, and sample manipulation to make the pools are substantially laborious. These steps also require more people involved, which is a critical operational limitation. Additional sample manipulation could also lead to increased chance of contamination or even sample/pool misidentifications. In the swab pooling method, we aimed to reduce and simplify such laborious and time-consuming requirements. These considerations are detailed in lines 236-250. In the rare cases when two swabs cannot be sampled, individual testing is performed without major operational harm and processed as a single diagnostic test. 2. Although the swab pooling could decrease the number qRT-PCR reactions, this would not be valid for populations where the infectivity rate is high and consequently, the percentage of positive PCR samples (10-30% or even more). In fact, in the current study, the positivity rate of the tests is 1,26% which is quite low compared to the actual situation in many countries where high numbers such as 40% of test positivity rates could be observed. In such cases, the number of qRT-PCR reactions will even increase. Response: We agree with the reviewer that the cost-effectiveness of swab pooling relies on limited positivity rate. The infection rate of 1,26% reflects our local scenario from May-July 2020, when the samples were collected. Noteworthy, this is a limitation inherent to the idea of pool testing in general - the scale gains are inversely proportional to the number of positive individuals in the sampled population. In fact, our methodology is flexible to accommodate varying pool sizes effortlessly with fixed dilution factor, which can be used to customize pool testing to scenarios of higher expected prevalence. In traditional pool testing, varying pool size means varying dilution factors and laboratory protocols, which may demand additional validation. These considerations are discussed in the paper (lines 259 to 264). 3. One of the main challenges of SARS-CoV-2 diagnostics is its rapidity in order to isolate/quarantine the positive patients. The proposed method can only discard at first the negative ones, but further analyses are needed to confirm the true positive cases which may take 2 more days from sampling, a period during which the patients are highly contagious. Response: We share the concern about waiting time for testing, a major issue almost a year after the first COVID-19 outbreak. We do not claim that swab pooling supersedes massive individual testing if this is a viable option. Yet, the demand for SARS-CoV-2 testing has reached levels far beyond the testing capacity of most countries, and massive screenings are considerably rare. Also, by saving a substantial number of RT-qPCR assays, swab pooling reduces the demand for laboratory supplies, optimizing the currently overloaded supply chain. In this scenario, swab pooling boosts testing capacity when the target population is expected to yield a low positivity rate - e.g. screening of asymptomatic individuals, in which case the people being tested are not choosing between pool testing and a faster alternative, but between pool testing and no testing at all. In this sense, the strategic advantage of pool testing is discarding negative individuals, as is the case with any screening strategy. Finally, it’s important to notice that waiting time rarely exceeds 48 hours independently of test positivity, and a negative result may be released even sooner. 4. No comparison of the proposed method with the SARS-CoV-2 antigen tests is made. The latter are currently becoming quite popular especially for large scale screens of potentially infected populations and asymptomatic patients and can reveal infection in less than 30 min. Response: Antigen testing for SARS-CoV-2 is an important strategy that has become quite popular. However, the Diagnostic flow diagram for the detection of acute SARS-CoV-2 infection in individuals with clinical suspicion for COVID-19, recommended by the World Health Organization, is still based on nucleic acid amplification methods (Fig 1). Here, we have compared swab pooling with the standard of care (individual RT-qPCR tests) and present it as a reliable and efficient alternative to complement available strategies. Our method has allowed the screening of over 20 thousand individuals who would have been tested by neither individual RT-qPCR or antigen testing. 5. I have reserves about considering qPCR cut off values of 40 qC and above as positive. What is the PCR program and the primers that were used? This section needs to be more detailed (not only references) since the whole study is based on qPCR. It would be worthy to include as a supplementary figure a sample image of PCR plots for the different SARS-CoV-2 genes. Response: This specific threshold value of 40 has been used before and is stated in the CDC interim guidelines (https://www.fda.gov/media/134922/download). Also, the protocol was used as described in the Charité-Berlin publication (https://doi.org/10.2807/1560-7917.ES.2020.25.3.2000045). Recently, assessment of analytical sensitivity and efficiency of SARS-CoV-2 primer–probe sets also employed a cutoff of 40 Cq for clinical samples (https://www.nature.com/articles/s41564-020-0761-6). Other studies also related the COVID-19 infectiousness with RT-PCR cycle thresholds and showed several Cq values above 40 (https://www.eurosurveillance.org/content/10.2807/1560-7917.ES.2020.25.32.2001483). In our study, we performed 45 cycles in the RT-qPCR reaction, and for conservativeness, we opened pools with 40 Cq or little higher for any of the tested genes given that lower viral loads have higher Cq values and a higher rate of false-negatives due to the RT-qPCR methodology limitations. We only had 3 pools with Cqs higher than 40 for the E gene (41.06, 40.58, 40.58) and 3 individuals (41.93, 41.61, 40.99), and one exceptional pool with Cq 44.79 for the RdRp gene as its E gene Cq was much lower than 40, which could indicate some PCR deviation. These 4 pools and 3 individuals is a really small fraction of all the data we had (163 pools and 246 individuals) and is explained in lines 186-193. We only included these data to demonstrate the full range of results obtained for the method experimental validation. The PCR program and the primers used were described in the methodology section according to their original references. The Charité-Berlin protocol is one of the most well-known and recommended protocols to be followed by WHO (lines 113-120). These experiments were performed in several RT-qPCR plates, it will be a little impractical and also uninformative to concatenate all the RFU values (fluorescence at each PCR cycle) to construct the PCR plots for all samples. All Cq values were already demonstrated in the new figure 4C comparing the Cq of individuals and their respective pools. The raw data are available in the supplementary information. 6. Figure 5 could be included in Figure 4. Response: We thank the reviewer for the suggestion and have moved Figure 5 to Figure 4C. https://doi.org/10.1371/journal.pone.0246544.r002
14 Jan 2021 Decision Letter - Jean-Luc EPH Darlix, Editor PONE-D-20-32256R1 Swab pooling: a new method for large-scale RT-qPCR screening of SARS-CoV-2 avoiding sample dilution PLOS ONE Dear Dr. de Oliveira, 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. I still have a number of critiques: (i) the reason of using amplification cycles (Ct) of 40 or above is not clear; (ii) there is no comparison using COV2 antigen detection; (iii) several statements do not seem appropriate ie beginning of the discussion section should include the fact that the extensive COV2 testing is essential for monitoring viral infection in the human population and a prerequisite for investigating how the virus spreads. Please submit your revised manuscript by Feb 28 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, Jean-Luc EPH Darlix, MG, Ph.D. Academic Editor PLOS ONE [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.0246544.r003
Revision 2
18 Jan 2021 Author Response Swab pooling: a new method for large-scale RT-qPCR screening of SARS-CoV-2 avoiding sample dilution (PONE-D-20-32256R1) Rebuttal letter: 1. The reason for using amplification cycles (Ct) of 40 or above is not clear. We have modified the text accordingly to clarify this point. We included more detailed information to consider a sample positive for SARS-CoV-2, which states as follows: “For pool samples, detecting at least one gene with Cq value lower than 40 was enough for pool opening and subsequent individual testing; for individual samples, detecting both genes with Cq lower than 40 was required to consider it positive for SARS-CoV-2". This was added to the methods section in lines 121-127. Three individual samples had divergent results (one gene with Cq lower than 40 and another with Cq slightly higher than 40), but it was not possible to collect new samples. For precaution, the patients were treated as positive and, hence, their results were kept in the analysis. This was detailed in the rewritten lines 203-211. 2. There is no comparison using COV2 antigen detection. We understand the interest in antigen tests due to the benefits of point-of-care diagnosis. However, comparisons involving antigen testing go beyond the scope of this work for several reasons: A. Antigen testing has lower sensitivity than RT-qPCR: while antigen testing has become popular, it is recommended that negative results should be confirmed by RT-qPCR due to inferior sensitivity (1). Even though it is suggested that antigen tests are sensitive enough to detect active infection only, there is substantial uncertainty regarding its actual sensitivity, with reported values ranging from 80% to 98% under varying validation conditions (2-4). Most works assessing antigen tests used RT-qPCR as a unique reference, as we do here. Future works comparing multiple methods should be useful, but beyond our current operational capacity and scope. B. Antigen testing and swab pooling target different populations: while swab pooling, as any other pooling strategy, targets low prevalence populations, antigen testing has been recommended to target symptomatic individuals (1,4). In fact, a recent assessment comparing antigen testing to standard RT-qPCR found a sensitivity of 80% (32 of 40) among symptomatic individuals but of only 41% (7 of 17) for asymptomatic individuals (3). Even though this matter certainly requires further investigation, there is not enough evidence to support the widespread adoption of rapid antigen tests as a substitute of molecular testing for the screening of asymptomatic people and other low prevalence populations. Both the FDA and Brazil’s National Health Surveillance Agency (ANVISA) recommend that negative antigen test results should be subsequently confirmed by RT-qPCR, which may further delay the process of ruling out infection (1,4). The use of antigen tests for screening is suggested only if molecular testing is not feasible or if turnaround times are too long. C. Our aim is solely to propose a reliable and efficient alternative for large-scale screening in low prevalence populations: massive molecular testing for SARS-CoV-2 places logistic, operational, and financial challenges. The more alternatives available the higher our ability to address issues due to insufficient testing. Here, our proposal improves on sample pooling, a strategy successfully implemented worldwide (5-6). Finally, works addressing different variations of pool testing continue to be published without any comparison to antigen testing (5-6). The value of our contribution lies in the improvement over the two main drawbacks from sample pooling: sample dilution and increased laboratory workload. Our method has allowed reliable screening of over 20 thousand individuals who would not have been tested by either individual RT-qPCR or antigen testing. 3. Several statements do not seem appropriate i.e. the beginning of the discussion section should include the fact that the extensive COV2 testing is essential for monitoring viral infection in the human population and a prerequisite for investigating how the virus spreads. While we are not sure which statements the editor refers to, we have reviewed our discussion section and included the suggested observations (lines 205-206), in addition to minor modifications along the manuscript. REFERENCES 1. FDA. 2020. A Closer Look at Coronavirus Disease 2019 (COVID-19) Diagnostic Testing. Available at: https://www.fda.gov/media/143737/download. Accessed: 15/01/2021. 2. https://doi.org/10.1016/S0140-6736(20)32635-0 3. http://dx.doi.org/10.15585/mmwr.mm695152a3 4. ANVISA. 2021. NOTA TÉCNICA Nº 7/2021. Available at: https://www.gov.br/anvisa/pt-br/centraisdeconteudo/publicacoes/servicosdesaude/notas-tecnicas/nota-tecnica-no-7-de-2021.pdf. Accessed: 15/01/2021. 5. https://www.thelancet.com/journals/laninf/article/PIIS1473-3099(20)30362-5/fulltext https://www.nature.com/articles/s41586-020-2885-5 Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0246544.r004
21 Jan 2021 Decision Letter - Jean-Luc EPH Darlix, Editor Swab pooling: a new method for large-scale RT-qPCR screening of SARS-CoV-2 avoiding sample dilution PONE-D-20-32256R2 Dear Dr. de Oliveira, 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, Jean-Luc EPH Darlix, MG, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0246544.r005
Formally Accepted
26 Jan 2021 Acceptance Letter - Jean-Luc EPH Darlix, Editor PONE-D-20-32256R2 Swab pooling: a new method for large-scale RT-qPCR screening of SARS-CoV-2 avoiding sample dilution Dear Dr. Oliveira: 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 Professor Jean-Luc EPH Darlix Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0246544.r006

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .