Diagnosis of Clostridioides difficile infection by analysis of volatile organic compounds in breath, plasma, and stool: A cross-sectional proof-of-concept study

Teny M. John; Nabin K. Shrestha; Gary W. Procop; David Grove; Sixto M. Leal Jr; Ceena N. Jacob; Robert Butler; Raed Dweik

doi:10.1371/journal.pone.0256259

Peer Review History

Original SubmissionJuly 29, 2020
4 Nov 2020 Decision Letter - Timothy J Garrett, Editor PONE-D-20-23689 Diagnosis of Clostridioides difficile infection by analysis of volatile organic compounds in breath, plasma, and stool – a cross-sectional proof-of-concept study. PLOS ONE Dear Dr. John, 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. We had comments from 3 reviewers who all believed the work is exciting. They each have provided comments and I request that you prepare a response addressing each of the comments. Please submit your revised manuscript by Dec 19 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Timothy J Garrett, PhD Academic Editor PLOS ONE Additional Editor Comments: Thank you for your submission. I have carefully reviewed all the comments from the reviewers and agree that a major revision is needed. Please carefully consider and appropriately respond to each comment. Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for including the following ethics statement on the submission details page: 'Approved by Cleveland Clinic institutional review board, IRB# 18-030. Written informed consent obtained from all participants' Please also include this information in the ethics statement in the Methods section of your manuscript. 3. We noted in your submission details that a portion of your manuscript may have been presented or published elsewhere. "Findings were presented at ID week 2018 as a poster and abstract was published in Open Forum of Infectious Diseases." Please clarify whether this publication was peer-reviewed and formally published. If this work was previously peer-reviewed and published, in the cover letter please provide the reason that this work does not constitute dual publication and should be included in the current manuscript. [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: No Reviewer #2: Partly 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: No 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: Notable point on the article is the use of paired controls to reduce if not eliminate any bias due to other factors unrelated to the disease. Some typographical errors found but not in a way that can influence the article. Line 125 Capitalize C on Celsius Fig 1 “Stautus” to “status” However, a major issue that is detrimental to the study is the use of breath metabolites as metrics to differentiate or classify stool and plasma samples. It is not surprising that there is low accuracy in these samples, and therefore invalid comparison. The authors have even stated that there is a characteristic odor of infected stool, which also suggests to the readers that there may be challenges in using SIFT MS for detection of these characteristic compounds. A helpful suggestion is to reclassify the stool and plasma samples using their intrinsic metabolites. Reviewer #2: The authors provide a very brief manuscript describing the use of SIFT-MS to analyze molecules released from different samples from patients and controls for the detection of C. difficile. The paper has some interesting data, but needs to be expanded on a number of different points to enable clearer understanding of the need for the research, reproducibility of the methods for data analysis, more thorough evaluation of the results, and confirmation that the results support the conclusions that are presented in the manuscript. Major critiques include: 1. Please provide the individual data for the patients including clinical status and values for each analyte observed in SIFT-MS. 2. Example mass spectra for each sample type would be helpful to the reader. 3. Representation of the data using principal component analysis, hierarchical clustering with a heat map, or some similar approach would enable the authors’ claims of separation of groups (C. diff and control) to be evaluated more effectively. At the current time, the claims for separation of the groups can’t be evaluated by the way the data are presented. The draft is well written, but minor editing for spelling and grammar is needed. Additional specific requests for revisions are included below. Abstract: Please add detail about why C. difficile is important and what methods are currently available for its detection to describe the need for the research presented here. Introduction: In the previous GC-MS work on C. difficile, which compounds could be used to differentiate infected people from controls? Are they detected with SIFT-MS? Table 1: Do the authors expect that the confounding variables from other conditions (e.g. cancer) will impact the differences between the groups? Data may need to be presented in the figures with color codes to indicate patients these other chronic diseases. Why was the K nearest neighbors strategy selected? Please explain the rationale. Do any of the molecules detected in SIFT-MS have potential as individual biomarkers? This point is addressed in part by the comparisons and p values in the supplemental tables, but it should also be presented in the results section of the manuscript. Supplemental Figures and Tables need captions on the page with the data or image. Supplemental Tables need headings to explain the data, which are presented as average and standard deviation but not labeled. Reviewer #3: Here the authors present a clinical study which shows that breath samples could be used to diagnose if someone has a CDI. In addition to breath, they also analyzed the headspace of both plasma and stool. Both of the other diagnostic mediums had poor predictive power which could have been cause by the loss in VOCs during the sample handling process. I find the scientific work interesting and generally well done. With that said, this paper would benefit if the authors would expand the introduction and addressed concerns below. Introduction: This section is very short. It almost reads like an abstract. The authors should do a better job introducing breath analysis, its prior use for CDI, and the various analytical techniques. The section could end on the rationale used for the selection of SIFT-MS. This section should be at least two paragraphs longer. Materials and Methods: Why were weekend samples excluded? Please do a better job explaining the rationale. Why were samples incubated before analysis? Is this common practice? Table 1: Does not significantly contribute to the paper. I would recommend moving this to the supplemental section or shortening it for the main text. Supplemental Tables 1-3. Given that these numbers are at the heart of the study, I would move supplemental table 1 into the main text. For all supplemental tables, what are the units? What is pos and neg? CDI pos, CDI neg? Please label more clearly. Supplemental Figure 1 and 2 need figure legends. Results: I understand that the entire 22 VOC panel was used for the ROC curves. However, was there a subset of VOCs in the panel that was MORE predictive than the full panel for each type of sample? If so what? Other general comments: Why was blood plasma used? Why not whole blood? The sample processing of whole blood to plasma would significantly change and or remove important VOCs. ****** 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/. 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Revision 1
2 Apr 2021 Author Response Thank you for reviewing our manuscript and providing valuable suggestions. We hereby respond to reviewer and editors comments in a question and response format. 1. 'Approved by Cleveland Clinic institutional review board, IRB# 18-030. Written informed consent obtained from all participants. Please also include this information in the ethics statement in the Methods section Response from authors: Thank you for your suggestion. We included these key sentences in the methods section. 2. We noted in your submission details that a portion of your manuscript may have been presented or published elsewhere. "Findings were presented at ID week 2018 as a poster and abstract was published in Open Forum of Infectious Diseases." Please clarify whether this publication was peer-reviewed and formally published. If this work was previously peer reviewed and published, in the cover letter please provide the reason that this work does not constitute dual publication and should be included in the current manuscript. Response from authors: Findings were presented as a poster at ID week 2018, the abstract of it was published in a supplement of OFID as a conference abstract. While conference abstracts are reviewed by the scientific committee, they are not considered peer-reviewed. Reviewer # 1 comments: 1. Some typographical errors found but not in a way that can influence the article. Line 125 Capitalize C on Celsius and Fig 1 “Stautus” to “status” Response from authors: Thank you; both these typographical errors are corrected. 2. However, a major issue that is detrimental to the study is the use of breath metabolites as metrics to differentiate or classify stool and plasma samples. It is not surprising that there is low accuracy in these samples, and therefore invalid comparison. The authors have even stated that there is a characteristic odor of infected stool, which also suggests to the readers that there may be challenges in using SIFT MS for detection of these characteristic compounds. A helpful suggestion is to reclassify the stool and plasma samples using their intrinsic metabolites. Response from authors: Thank you for your suggestion. The characteristic odor of stool in patients with Clostridioides difficile infection (CDI) is well known, and it is thought to be secondary to metabolites in the stool. Studies have shown that dogs can scent stool odor and correctly classify CDI. Multiple invitro studies based on stool cultures also confirm the presence of VOCs in the stool. We want to clarify this study has not focused on classifying stool and plasma samples based on breath metabolites. On the other hand, we have studied cross-sectionally collected stool, plasma, and breath samples (all collected within a 24-hour time frame) of patients with CDI. We think the lack of signal in stool and plasma is due to using left-over samples that was not stored appropriately resulting in loss of VOCs. Reviewer #2: 1. Please provide the individual data for the patients including clinical status and values for each analyte observed in SIFT-MS. Response from authors: Thank you for your suggestion. Clinical data is provided in Table 1. This has already been deposited in a public repository at https://osf.io/5hv49/. The repository contains the analysis dataset and the analysis to reproduce the results 2. Example mass spectra for each sample type would be helpful to the reader. Response from authors: This is a very good suggestion. A sample mass spectra for each type of sample – namely breath, plasma and stool for a patient is added as a supplementary figure. 3. Representation of the data using principal component analysis, hierarchical clustering with a heat map, or some similar approach would enable the authors’ claims of separation of groups (C. diff and control) to be evaluated more effectively. At the current time, the claims for separation of the groups can’t be evaluated by the way the data are presented. Response from authors: Thank you for this suggestion. We have provided a cluster dendrogram that shows clustering of patients with CDI(in red). (Figure 3) 4. Abstract :Please add detail about why C. difficile is important and what methods are currently available for its detection to describe the need for the research presented here. Response from authors: The abstract section will be elaborated to provide more background about current status of C.difficile diagnostic strategies. Thank you for this suggestion. 5. Introduction: In the previous GC-MS work on C. difficile, which compounds could be used to differentiate infected people from controls? Are they detected with SIFT-MS? Response from authors : A recent study of stored stool samples from 53 cases and 53 controls, using thermal desorption-gas chromatography-time-of-flight gas chromatography, identified seven compounds (propan-1-ol, 3-methylbutanal, ethyl propionate, hexanoic acid, 4-methyl phenol, dodecane, and indole) indicative of CDI with a ROC >0.7. This information is provided in the introduction section. None of these molecules were part of the panel that was used in SIFT-MS. SIFT-MS is equipped to measure 22 common analytes that are present in breath in patients with disease states. 6. Table 1: Do the authors expect that the confounding variables from other conditions (e.g. cancer) will impact the differences between the groups? Data may need to be presented in the figures with color codes to indicate patients these other chronic diseases. Response from authors: Thank you for this query and suggestion ; Yes, we expect other disease states to affect VOC profile including cancer and metabolic conditions like diabetes mellitus. It is certainly possible that some of these comorbid conditions may be confounding factors. However, this was a pilot study and there were too few patients with each of these chronic conditions to evaluate if these chronic conditions were confounding factors. Future larger studies with larger sample size that is controlled for all confounding factors are needed. This point is added to the limitation section. 7. Why was the K nearest neighbors strategy selected? Please explain the rationale. Response from authors: A KNN classifier was selected because it is a simple and intuitive classification tool. This being a pilot study with a small sample size we did not evaluate and compare different classification methods . 8. Do any of the molecules detected in SIFT-MS have potential as individual biomarkers? This point is addressed in part by the comparisons and p values in the supplemental tables, but it should also be presented in the results section of the manuscript. Response from authors: Thank you for your suggestion. We have included Table 2 describing the concentration of VOCs in patients with and without CDI. None of the molecules on their own achieved statistical significance. Based on our findings, it does not appear that any of these compounds could serve as individual biomarkers that could effectively separate CDI from no CDI. but the pattern of VOC concentrations rather than individual concentrations was more meaningful. This is added to the discussion section of the manuscript. 9. Supplemental Figures and Tables need captions on the page with the data or image. 10. Supplemental Tables need headings to explain the data, which are presented as average and standard deviation but not labeled. Response from authors: Supplementary Tables 1 and 2 are labelled with headings and data explained. Thank you for this suggestion. Reviewer 3: This paper would benefit if the authors would expand the introduction and addressed concerns below. 1.Introduction: This section is very short. It almost reads like an abstract. The authors should do a bett er job introducing breath analysis, its prior use for CDI, and the various analytical techniques. The section could end on the rationale used for the selection of SIFT-MS. This section should be at least two paragraphs longer. Response from authors: Thank you for this suggestion. We have expanded the introduction section that now includes a problem statement why CDI is important, current diagnostic modalities and its limitations, VOCs in CDI and various methods that are utilized. We wanted to study usefulness of SIFT-MS as it was available in our center and no prior CDI studies have been done using this technology (mentioned in the introduction) 2.Materials and Methods: a) Why were weekend samples excluded? Please do a better job explaining the rationale. Response from authors: Research technicians worked only from Monday through Friday and hence weekend samples were not studied as it would not have been possible to have weekend samples analyzed promptly. b) Why were samples incubated before analysis? Is this common practice? Response from authors: Thanks for this question. Samples were incubated to desorb the VOC's from the surface of the bag for analysis. Yes, this is a common practice in our VOC lab. This information is added to the methods section. c) Table 1: Does not significantly contribute to the paper. I would recommend moving this to the supplemental section or shortening it for the main text. Response from authors: Thank you for this suggestion. Table 1 represents the baseline demographic data of cases and controls enrolled in this study. The authors feel that this is important to the study highlighting that these characteristics were not statistically different in 2 groups studied. But we will shorten it by removing lab features of cases and controls. Foot note is added to Table 1. 3.Supplemental Tables 1-3. Given that these numbers are at the heart of the study, I would move supplemental table 1 into the main text. For all supplemental tables, what are the units? What is pos and neg? CDI pos, CDI neg? Please label more clearly. Response from authors: Thank you for this suggestion. We have moved supplemental table to the main text as table 2. 3. Supplemental Figure 1 and 2 need figure legends. Response from authors: Thank you. We provided figure legends for supplementary figure 1 and 2 4. Results: I understand that the entire 22 VOC panel was used for the ROC curves. However, was there a subset of VOCs in the panel that was MORE predictive than the full panel for each type of sample? If so what? Response from authors: Thank you. No, we did not find a subset of VOCs that was more predictive than the full panel for each type of sample Other general comments: 5.Why was blood plasma used? Why not whole blood? The sample processing of whole blood to plasma would significantly change and or remove important VOCs. Response from authors: Thank you for this suggestion. Blood plasma was used as it was the most easily available left-over sample after daily testing. We agree that plasma extraction from the whole blood, might have affected the amount and number of VOCs and will include this as a limitation of the study. Attachments Attachment Submitted filename: Response to reviewers 2.22 Final.docx https://doi.org/10.1371/journal.pone.0256259.r002
28 Apr 2021 Decision Letter - Timothy J Garrett, Editor PONE-D-20-23689R1 Diagnosis of Clostridioides difficile infection by analysis of volatile organic compounds in breath, plasma, and stool – a cross-sectional proof-of-concept study. PLOS ONE Dear Dr. John, 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. ============================== Thank you for responding to the initial review. There was still a major concern from one reviewer regarding the VOC analysis and in particular the biological aspects of measuring VOCs across the samples types. It is critical that you carefully address this comment in you revision. ============================== Please submit your revised manuscript by Jun 12 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. 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, Timothy J Garrett, PhD Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: (No Response) Reviewer #2: (No Response) Reviewer #3: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: No Reviewer #2: Yes Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: Yes Reviewer #3: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: (No Response) Reviewer #3: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Is there a reference on how the monitored VOCs were selected and their presence are common in breath, stool and blood? This is to give justification that the same VOCs monitored in breath can be monitored in stool and blood. Physically, the classes of samples differ in composition significantly, thus it is not obvious to the reviewer how the blood and stool samples are expected to contain the same VOCs found in breath. The data presented does not support the conclusions claimed by the paper. It is egregious that the authors would attempt to monitor common breath metabolites (as stated in page 5 line 102) in stool and blood, and apply it as of same significance in presence and amount in contrast to breath samples. The previous question was not answered satisfactorily. Reviewer #2: The authors have addressed most of the criticisms from the previous review, but a few minor changes need to be made. The manuscript needs to be spellchecked to remove errors in the updated version. For Figure 2, what sample set is used for the heat map? It would be helpful to center and normalize the data per analyte, so that you use more of the color spectrum. Now, everything looks yellow except for a couple of samples at the top, which are high outliers. Note that red/green designations will not be available to the color blind; red/blue is recommended or use of another indicator (bold font or -CDI appended after the sample name for positive cases would work well. Reviewer #3: Thank you. All concerns have been addressed. If find this paper interesting and timely. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No 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.0256259.r003
Revision 2
16 Jul 2021 Author Response On behalf of the authors of the manuscript, ‘Diagnosis of Clostridioides difficile infection by analysis of volatile organic compounds in breath, plasma, and stool – a cross sectional proof-of-concept study’, we want to thank you and the reviewers for reviewing our manuscript and giving valuable suggestions. We have updated the manuscript to reflect the minor corrections suggested by the two reviewers. Below are our responses to the specific points raised by the reviewers. Comment 1: Is there a reference on how the monitored VOCs were selected and their presence are common in breath, stool and blood? This is to give justification that the same VOCs monitored in breath can be monitored in stool and blood. Physically, the classes of samples differ in composition significantly, thus it is not obvious to the reviewer how the blood and stool samples are expected to contain the same VOCs found in breath Thank you for allowing us to present the revised manuscript. Author Response: The authors want to thank the reviewer for this great suggestion. We agree that we don’t have a solid justification on why we looked for the same 22 VOCs in three biologically different samples. This made us focus on the ‘full scan data’ set that includes all molecules or product ions that comes in the range of 14-200 mass to charge ratio values that are present in the sample compared to the ‘sim scan data’ which looks at only 22 VOCs. This analysis improved the ROC curves of all the 3 clinical samples we studied. Figures are updated in the re-submission Comment 2: The manuscript needs to be spellchecked to remove errors in the updated version Author Response: Thank you for this suggestion. The manuscript has been spell checked. Comment 3: For Figure 2, what sample set is used for the heat map? It would be helpful to center and normalize the data per analyte, so that you use more of the color spectrum. Now, everything looks yellow except for a couple of samples at the top, which are high outliers. Note that red/green designations will not be available to the color blind; red/blue is recommended or use of another indicator (bold font or -CDI appended after the sample name for positive cases would work well. Author Response: Thank you for this suggestion. We used breath product ion concentration to build the heatmap. As per the suggestion from the reviewer, we changed the color spectrum to red and blue color. Thank you for allowing us to review this manuscript. Hope you find the revisions acceptable. https://doi.org/10.1371/journal.pone.0256259.r004
4 Aug 2021 Decision Letter - Timothy J Garrett, Editor Diagnosis of Clostridioides difficile infection by analysis of volatile organic compounds in breath, plasma, and stool – a cross-sectional proof-of-concept study. PONE-D-20-23689R2 Dear Dr. John, 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, Timothy J Garrett, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): I appreciate your clear and scientific valuable additions made after the second revision. The key aspect was providing clarity on VOCs in plasma, stool and breath and I think you have clearly done that. At this point, you have addressed all major concerns regarding the publication of this work. Reviewers' comments: https://doi.org/10.1371/journal.pone.0256259.r005
Formally Accepted
9 Aug 2021 Acceptance Letter - Timothy J Garrett, Editor PONE-D-20-23689R2 Diagnosis of Clostridioides difficile infection by analysis of volatile organic compounds in breath, plasma, and stool – a cross-sectional proof-of-concept study. Dear Dr. John: 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. Timothy J Garrett Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0256259.r006

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