Clinical trial and detection of SARS-CoV-2 by a commercial breath analysis test based on Terahertz technology

Meila Bastos De Almeida; Regina Aharonov-Nadborny; Eran Gabbai; Ana Paula Palka; Leticia Schiavo; Elis Esmanhoto; Irina Riediger; Jaime Rocha; Ariel Margulis; Marcelo Loureiro; Christina Pettan-Brewer; Louise Bach Kmetiuk; Ivan Roque De Barros-Filho; Alexander Welker Biondo

doi:10.1371/journal.pone.0273506

Peer Review History

Original SubmissionMarch 24, 2022
26 Apr 2022 Decision Letter - Davor Plavec, 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-22-07738Clinical trial and detection of SARS-CoV-2 by a commercial breath analysis test based on Terahertz technologyPLOS ONE Dear Dr. Biondo, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== ACADEMIC EDITOR:There are some minor comments presented by reviewers. Please revise according to the suggestions of the reviewers or write a detailed rebuttal on a point-by-point basis. ============================== Please submit your revised manuscript by Jun 10 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Davor Plavec, MD, MSc, PhD, Prof. 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. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information. 3. Thank you for stating the following financial disclosure: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. At this time, please address the following queries: a) Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution. b) State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” c) If any authors received a salary from any of your funders, please state which authors and which funders. d) If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 4. 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. 5. 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. 6. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments: Please revise according to the suggestions of the reviewers or write a detailed rebuttal on a point-by-point basis. [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: Yes Reviewer #3: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: Yes 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: Interesting results that should be tested in the broader population and different circumstances but this work is pointing in the direction that this system can be used in Covid 19 pandemic and maybe for the further possible epidemics. The test for discrimination between the respiratory infection and specific respiratory infection is warranted. Reviewer #2: In the paper authors presented results of clinical trial in which evaluate a commercial breath analysis test (TERA.Bio®) for detecting the SARS-CoV-2 present in exhaled air samples of suspicious persons. All components of the manuscript are correctly presented, including introduction, results, statistical analysis, discussion and conclusions. However, the study conducted has certain limitations; 1. the potential bias of symptomatic cases which may have partially compromised the assessment of breath analysis thest (BAT) for asymptomatic patients 2. the BAT did not tested in asymptomatic patients and patients in the first days of infection or after 7 days of the onset of symptoms 3. the small sample size of infected persons (70 confirmed infected persons) 4.relatively limited detection of occurrence of other respiratory viruses, or other pathogens (including bacterial), for evidence of co-infection in patinets with COVID-19 . All these limitations were anticipated by the authors and suggested how to improve them. Reviewer #3: This is a well-written paper with innovations in SARS-COV2 diagnostics. In the Materials and Methods section, it would be good to explain the detailed procedure as it relates to the collection of exhaled air samples to prevent the spread of SARS-CoV2. It is visible that it is done in disposable air collection kit, but can it be sampled next to the patient (since these are all outpatient samples, not from hospialized patients). What about safety to the enviroment during the sampling? Does the time between sampling and analysis influences the results? ****** 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 Reviewer #3: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. 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.0273506.r001
Revision 1
3 Jun 2022 Author Response Manuscript #PONE-D-22-07738 Title “Clinical trial and detection of SARS-CoV-2 by a commercial breath analysis test based on Terahertz technology” 1. Thank you for stating the following Funding Information in your manuscript: "Brazilian National Council for Scientific and Technological Development -CNPq (material support) (2020-1/402341- AWB and CPB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." We note that you have provided funding information that is currently declared in your Fund-ing Statement. However, funding information should not appear in any areas of your manu-script. 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. Answer: Funding information was removed from the manuscript. 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 ex-ceeds 20,000 characters) Reviewer #1: Interesting results that should be tested in the broader population and differ-ent circumstances, but this work is pointing in the direction that this system can be used in Covid 19 pandemic and maybe for the further possible epidemics. The test for discrimina-tion between the respiratory infection and specific respiratory infection is warranted. Answer: Authors are thankful to reviewer for the positive comment. Reviewer #2: In the paper authors presented results of clinical trial in which evaluate a commercial breath analysis test (TERA.Bio®) for detecting the SARS-CoV-2 present in exhaled air samples of suspicious persons. All components of the manuscript are correctly presented, including introduction, results, statistical analysis, discussion and conclusions. Answer: Authors are thankful to reviewer for the positive comment. However, the study conducted has certain limitations; 1. the potential bias of symptomatic cases which may have partially compromised the as-sessment of breath analysis thest (BAT) for asymptomatic patients 2. the BAT did not tested in asymptomatic patients and patients in the first days of infection or after 7 days of the onset of symptoms 3. the small sample size of infected persons (70 confirmed infected persons) 4.relatively limited detection of occurrence of other respiratory viruses, or other pathogens (including bacterial), for evidence of co-infection in patients with COVID-19 . All these limitations were anticipated by the authors and suggested how to improve them. Answer: Authors are thankful to reviewer for the positive comment, since all above limita-tions have already been reported and discussed intext. Reviewer #3 (green intext): This is a well-written paper with innovations in SARS-COV2 diagnostics. In the Materials and Methods section, it would be good to explain the detailed procedure as it relates to the collection of exhaled air samples to prevent the spread of SARS-CoV2. Changed: Reviewer is right, and information was added. Now you read: “The study protocol has included non-hospitalized patients who were re-ferred by the Curitiba city health professionals after suspicious RT-qPCR results. Thus, exhaled air samples and nasopharyngeal swab samples were concomitantly collected and tested by the official city service. Each patient sampled by nasopharyngeal swab was also given a dischargeable kit of exhaled air analyzer containing an identified sampling device, which was uncovered in both sides with opened capsule and the melamine membrane in-ternally coupled. Patients were then asked to keep a 2-meter distance from the operator, swallow all saliva in the mouth and deeply blow the device for 5 times. After blown, the pa-tient was asked to close both device sides with the correspondent lids (within the kit) until hear a click noise of sealed lid, capturing the exhaled breath constituents inside the device. The operator received the device, inspected the complete lid closure, and disinfected the external parts with 70% alcohol. After taken to the laboratory, device was opened and cap-sule with internal membrane removed and inserted into the equipment for analysis. Sam-ples were collected up to 6 hours maximum after sampling, which insured best readings due to membrane integrity, ideal absorption of electromagnetic wave and wave propagation into the exhaled air sample. After analysis, device was discharged as hospital contaminated garbage.” (Page 9-10, Lines 238-258). It is visible that it is done in disposable air collection kit, but can it be sampled next to the patient (since these are all outpatient samples, not from hospitalized patients). What about safety to the environment during the sampling? Does the time between sampling and anal-ysis influences the results? Changed: Reviewer is right, and information was added. Now you read: “The study protocol has included non-hospitalized patients who were re-ferred by the Curitiba city health professionals after suspicious RT-qPCR results. Thus, exhaled air samples and nasopharyngeal swab samples were concomitantly collected and tested by the official city service. Each patient sampled by nasopharyngeal swab was also given a dischargeable kit of exhaled air analyzer containing an identified sampling device, which was uncovered in both sides with opened capsule and the melamine membrane in-ternally coupled. Patients were then asked to keep a 2-meter distance from the operator, swallow all saliva in the mouth and deeply blow the device for 5 times. After blown, the pa-tient was asked to close both device sides with the correspondent lids (within the kit) until hear a click noise of sealed lid, capturing the exhaled breath constituents inside the device. The operator received the device, inspected the complete lid closure, and disinfected the external parts with 70% alcohol. After taken to the laboratory, device was opened and cap-sule with internal membrane removed and inserted into the equipment for analysis. Sam-ples were collected up to 6 hours maximum after sampling, which insured best readings due to membrane integrity, ideal absorption of electromagnetic wave and wave propagation into the exhaled air sample. After analysis, device was discharged as hospital contaminated garbage.” (Page 9-10, Lines 238-258). Attachments Attachment Submitted filename: Rebuttal letter_BreathTerahertz _03rdJun2022.docx https://doi.org/10.1371/journal.pone.0273506.r002
16 Jun 2022 Decision Letter - Davor Plavec, Editor PONE-D-22-07738R1Clinical trial and detection of SARS-CoV-2 by a commercial breath analysis test based on Terahertz technologyPLOS ONE Dear Dr. Biondo, 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 revise as suggested by the reviewer. Please submit your revised manuscript by Jul 31 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Davor Plavec, MD, MSc, PhD, Prof. Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments: Please revise as suggested by the reviewer. [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 #4: (No Response) ******** 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 #4: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #4: 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 #4: 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 #4: 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 #4: A clinical trial was conducted which aimed to evaluate the commercial breath analysis test TERA.Bio (r) and a deterministic algorithm for detecting SARS-CoV-2 compounds exhaled in air samples. Results were compared to RT-qPCR as the gold standard. The sensitivity and specificity of the test was 92.6% and 96.0%, respectively. Minor revisions: 1- State and justify the study’s target sample size with a pre-study statistical power calculation. 2- In the statistical analysis section indicate the methods used to compare the features listed in table 1. Provide more precise p-values rather than p < 0.05. 3- Lines 364-370 and subsequent sections: In the statistical analysis section, state the method used to calculate the confidence intervals. 4- Table 2: In addition to frequencies, provide the corresponding percentages. ****** 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 #4: 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.0273506.r003
Revision 2
1 Jul 2022 Author Response Re: PLOS ONE Decision: Revision required [PONE-D-22-07738R1] Dear Dr Plavec, Please find attached our reviewed manuscript entitled “Clinical trial and detection of SARS-CoV-2 by a commercial breath analysis test based on Terahertz technology” [PONE-D-22-07738R1]. All the corrections and suggestions indicated by the reviewers were fully ad-dressed, accepted, and rewritten. A response letter has been added to pinpoint the editor and reviewers’ requests, with a detailed list of corrections and a description of changes made within the manuscript. To better identify the corrections of each reviewer, changes were highlighted in different colors in the revised manuscript. Thank you for the opportunity and please do not hesitate to con-tact us with any further question. Sincerely, Alexander Welker Biondo, DMV, MSc, PhD. Professor, Department of Veterinary Medicine, UFPR, Brazil Visiting Professor, Purdue University, IN, USA E-mail: abiondo@ufpr.br / Phone: +55 (41) 3350-5812 Manuscript #PONE-D-22-07738R1 Title “Clinical trial and detection of SARS-CoV-2 by a commercial breath analysis test based on Terahertz technology” Reviewer #4: A clinical trial was conducted which aimed to evaluate the commercial breath analysis test TERA.Bio (r) and a deterministic algorithm for detecting SARS-CoV-2 com-pounds exhaled in air samples. Results were compared to RT-qPCR as the gold standard. The sensitivity and specificity of the test was 92.6% and 96.0%, respectively. Minor revisions: 1- State and justify the study’s target sample size with a pre-study statistical power calcula-tion. Changed: Reviewer is right, that information was missing. Explanation was included in the Study Sample Size and Data Validation Section of the manuscript. The correspondent ref-erence was cited in the sentence. Now you read: “The assumed positivity of RT-qPCR for sampling calculation in the clinical trial herein was 25%, based on epidemiological COVID-19 reports of Curitiba city, which had an estimated population of approximately 1.9 million habitants at the time. Aiming at a type I error (α) of 0.05, and an estimated precision of 0.05, a minimum sample size of 400 subjects was obtained to statistically evaluate the BAT test, considering a 90% sensitivity as minimum for a useful diagnostic test (30). Representing a consecutive, casuistic, ran-dom, and homogeneous sampling, collected from all subjects who presented themselves at the hospital in the period, a total of 570 successful samplings were included in the present study.” (Page 7-8, Lines 173-183). 2- In the statistical analysis section indicate the methods used to compare the features listed in table 1. Provide more precise p-values rather than p < 0.05. Changed: The sentences were included in the Statistical Analysis section of the manu-script. P values were included, whenever higher than 0.0001. P values lower than 0.0001 are not expressed by the software used for statistical analysis (SPSS). Now you read: “Sociodemographic, epidemiological, and clinical characteristics were pre-sented as percentages, alongside their 95% proportion confidence intervals, and by arith-metic mean and standard deviation. The results were presented in contingency tables, al-lowing the calculation of sensitivity, specificity, and predictive values for the BAT. Assess-ment of the association between categorical variables; and the BATs and RT-qPCR tests was performed by Fisher’s exact test. Continuous variables were compared using Student’s unpaired t-test. The precision of the BAT was assessed using ROC curve analysis. Statisti-cal significance was assumed for P<0.05. In addition, machine learning was based on “training data” that had been collected prior to the study herein. Over 4,000 subjects were tested using the BATs at several loca-tions worldwide including Brazil. The THz spectrum features for healthy and infected (nega-tive and positive) subjects were processed using ML techniques, to establish a mathemati-cal algorithm for COVID-19 classification. The statistical analyses were performed using the SPSS version 17 software.” (Page 11-12, Lines 290-306). 3- Lines 364-370 and subsequent sections: In the statistical analysis section, state the method used to calculate the confidence intervals. Changed: The information was included in the Statistical analysis Section of the manu-script. Now you read: “The results from the RT-qPCR test and SARS-CoV-2 BAT for all subjects was presented (Table 2). Using RT-qPCR method as the gold standard, the commercial BAT method was found to have 92.7% sensitivity (CI 84.1-97.6%) and 96.0% specificity (CI 93.9-97.5%). The positive predictive value (PPV) and negative predictive value (NPV) were determined as 76.5% (CI 66.3-85.0%) and 99.0% (CI 97.6-99.7%), respectively. Fisher's exact test showed a statistically significant association between results (P < 0.0001). The area under the ROC curve of the total sampling was 0.94 (SD 0.19; CI 0.91-0.98) (Figure 4). Figure 4. ROC curve for the entire sample. Considering only the symptomatic patients and using RT-qPCR as the gold standard, BAT presented 92.4% sensitivity (CI 83.2-97.5%) and 95.9% specificity (CI 93.1-97.7%). The PPV was determined as 81.3% (CI 70.7-89.4%) and the NPV as 98.5% (CI 96.5-99.5%). Fisher's exact test showed a statistically significant association between results (p<0.0001). The area under the ROC curve calculated only with symptomatic patients was 0.94 (standard error 0.20; CI 0.90-0.98). Given that the optimum sampling window of opportunity for RT-qPCR has been reported to comprise the first seven days with symptoms, the data were stratified for analysis including the symptomatic patients who had shown symptoms for up to seven days. In such scenario, the results from SARS-CoV-2 BAT, compared with the RT-qPCR method as the gold standard, showed 90.2% sensitivity (CI 76.0-97.3%) and 95.5% specificity (CI 90.9-98.2%). The PPV was determined as 84.1% (CI 69.9-93.4%) and the NPV as 97.4% (CI 93.4-99.3%). Again, Fisher's exact test showed a statistically significant association between the results obtained through the two methods (P < 0.0001). The area under the ROC curve calculated only for symptomatic patients who had shown symptoms up to seven days was 0.93 (standard error 0.03; CI 0.87-0.98). Considering only the asymptomatic patients, the results showed 100.0% sensitivity (CI 39.7-100.0%) and 96.3% specificity (CI 92.1- 98.6%), with PPV of 40.0% (CI 12.2-73.8%) and NPV of 100.0% (CI 97.7-100.0%). Fisher's exact test showed a statistically significant association between results (P < 0.0001). The area under the ROC curve calculated only with asymptomatic patients was 0.98 (standard error 0.01; CI 0.96-1.00). The limitation of this method was a false positive rate of 23.5%, indicating that the positive predictive value of the test may be compromised in this sampling.” (Page 15-17, Lines 358-401). 4- Table 2: In addition to frequencies, provide the corresponding percentages. Changed: The percentages were included in Table 2. Attachments Attachment Submitted filename: Response letter_BreathTerahertz _01stJul2022 final.docx https://doi.org/10.1371/journal.pone.0273506.r004
11 Jul 2022 Decision Letter - Davor Plavec, Editor PONE-D-22-07738R2Clinical trial and detection of SARS-CoV-2 by a commercial breath analysis test based on Terahertz technologyPLOS ONE Dear Dr. Biondo, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== ACADEMIC EDITOR: Please make suggested corrections. ============================== Please submit your revised manuscript by Aug 25 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Davor Plavec, MD, MSc, PhD, Prof. Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments : Dear Authors, please make corrections suggested by the reviewer. [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 #4: (No Response) ******** 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 #4: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #4: 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 #4: 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 #4: 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 #4: Minor revisions: (Note that line numbers refer to those in the track changes version of the manuscript.) 1- Line 183: In the sample size justification section, state the statistical power that was attained. 2- Table 1: Some of the p-values display a comma in place of a decimal. Perhaps these errors resulted when the document was formatted by the journal. 3- In the "Statistical analysis" section, list and describe the statistical methods used to estimate the p-values shown in Table 1. Also indicate the statistical methods used to generate 95% confidence intervals. ****** 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 #4: 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.0273506.r005
Revision 3
16 Jul 2022 Author Response 1- Line 183: In the sample size justification section, state the statistical power that was attained. Changed: The statistical power was included. Now you read: “Aiming at a type I error (α) of 0.05, and an estimated precision of 0.05, a minimum sample size of 400 subjects was obtained to statistically evaluate the BAT test, considering a 90% sensitivity as minimum for a useful diagnostic test (30). Representing a consecutive, casuistic, random, and homogeneous sampling, collected from all subjects who presented themselves at the hospital in the period, a total of 570 successful samplings were included in the present study, with statistical power of 0.819.” (Page 8, Lines 193). 2- Table 1: Some of the p-values display a comma in place of a deci-mal. Perhaps these errors resulted when the document was formatted by the journal. Changed: Number have been corrected at table 1. 3- In the "Statistical analysis" section, list and describe the statistical methods used to estimate the p-values shown in Table 1. Also indicate the statistical methods used to generate 95% confidence intervals. Changed: Statistical methods were inserted. Now you read: “Student's t-test was used to determine estimate the p-values and chi-square test to generate 95% confidence intervals.” (Page 11, Lines 299-301). Attachments Attachment Submitted filename: Response to reviewers_BreathTerahertz _16thJun2022.docx https://doi.org/10.1371/journal.pone.0273506.r006
20 Jul 2022 Decision Letter - Davor Plavec, Editor PONE-D-22-07738R3Clinical trial and detection of SARS-CoV-2 by a commercial breath analysis test based on Terahertz technologyPLOS ONE Dear Dr. Biondo, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== ACADEMIC EDITOR: Dear Authors, please consult with the statistician to improve the text of your Statistical analysis as this is obviously the problem for you and the root of corrections. ============================== Please submit your revised manuscript by Sep 03 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Davor Plavec, MD, MSc, PhD, Prof. Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments: Dear Authors, please consult with the statistician to improve the text of your Statistical analysis as this is obviously the problem for you and the root of corrections. [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 #4: (No Response) ******** 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 #4: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #4: 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 #4: 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 #4: 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 #4: Minor Revisions: The recently added sentence at line 299 is not clear: "Student's t-test was used to determine estimate the p-values and chi-square test to generate 95% confidence intervals." A student's t-test is used for comparing the mean differences in two groups. A chi-square tests is used for comparing differences in proportions. Table 1 summarizes both means and proportions. Furthermore, the student's unpaired t-test is mentioned earlier in this paragraph. Improve the clarity of this first paragraph in the "Statistical analysis" section. ****** 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 #4: 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.0273506.r007
Revision 4
24 Jul 2022 Author Response Re: PLOS ONE Decision: Revision required [PONE-D-22-07738R3] Dear Dr Plavec, Please find attached our reviewed manuscript entitled “Clinical trial and detection of SARS-CoV-2 by a commercial breath analysis test based on Terahertz technology” [PONE-D-22-07738R3]. All the corrections and suggestions indicated by the reviewers were fully ad-dressed, accepted, and rewritten. A response letter has been added to pinpoint the editor and reviewers’ requests, with a detailed list of corrections and a description of changes made within the manuscript. To better identify the corrections of each reviewer, changes were highlighted in different colors in the revised manuscript. Thank you for the opportunity and please do not hesitate to con-tact us with any further question. Manuscript #PONE-D-22-07738R3 Title “Clinical trial and detection of SARS-CoV-2 by a commercial breath analysis test based on Terahertz technology” Reviewer #4: Minor Revisions: The recently added sentence at line 299 is not clear: "Student's t-test was used to determine estimate the p-values and chi-square test to generate 95% confidence intervals." A student's t-test is used for com-paring the mean differences in two groups. A chi-square tests is used for comparing differences in proportions. Table 1 summarizes both means and proportions. Furthermore, the student's unpaired t-test is mentioned earlier in this paragraph. Improve the clarity of this first par-agraph in the "Statistical analysis" section. Changed: Sentence was rewritten to clarify. Now you read: “Sociodemographic, epidemiological, and clinical char-acteristics were presented as percentages, arithmetic means and standard deviations. Differences in proportions were compared by chi-square test with calculated 95% confidence intervals. The results were presented in contingency tables, allowing the calculation of sensitivity, specificity, and predictive values for the BAT. Assessment of associa-tion between categorical variables, and BAT and RT-qPCR tests was performed by Fisher’s exact test. Mean differences in variables were compared by p-values of Student’s unpaired t-test, assuming statistical significance when p<0.05. Precision of the BAT was assessed using ROC curve analysis.” (Page 11, Lines 290-300). https://doi.org/10.1371/journal.pone.0273506.r008
10 Aug 2022 Decision Letter - Davor Plavec, Editor Clinical trial and detection of SARS-CoV-2 by a commercial breath analysis test based on Terahertz technology PONE-D-22-07738R4 Dear Dr. Biondo, 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, Davor Plavec, MD, MSc, PhD, Prof. 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 #4: 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 #4: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #4: (No Response) ****** 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 #4: (No Response) ****** 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 #4: (No Response) ****** 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 #4: (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 #4: No ******** https://doi.org/10.1371/journal.pone.0273506.r009
Formally Accepted
12 Sep 2022 Acceptance Letter - Davor Plavec, Editor PONE-D-22-07738R4 Clinical trial and detection of SARS-CoV-2 by a commercial breath analysis test based on Terahertz technology Dear Dr. Biondo: 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. Davor Plavec Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0273506.r010

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 .