Clinical evaluation of a non-purified direct molecular assay for the detection of Clostridioides difficile toxin genes in stool specimens

Toshinori Hara; Hiromichi Suzuki; Tadatomo Oyanagi; Norito Koyanagi; Akihito Ushiki; Naoki Kawabata; Miki Goto; Yukio Hida; Yuji Yaguchi; Kiyoko Tamai; Shigeyuki Notake; Yosuke Kawashima; Akio Sugiyama; Keiichi Uemura; Seiya Kashiyama; Toru Nanmoku; Satoshi Suzuki; Hiroshi Yamazaki; Hideki Kimura; Hiroyuki Kunishima; Hiroki Ohge

doi:10.1371/journal.pone.0234119

Peer Review History

Original SubmissionMarch 27, 2020
6 May 2020 Decision Letter - Yung-Fu Chang, Editor PONE-D-20-08775 Clinical Evaluation of a Non-purified Direct Molecular Assay for the Detection of Clostridioides difficile Toxin Genes in Stool Specimens PLOS ONE Dear Dr. Hara, 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. Your manuscript has been reviewed by one expert in this field. A minor revision is suggested. We would appreciate receiving your revised manuscript by two weeks. When you are 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. 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PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: No ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This is a competently performed evaluation of a new molecular assay for the detection of C. difficile tcdB gene. There are some specific issues that the authors should address. 1. The use of the terms "positive and negative concordance" rather than "sensitivity and specificity" which are terms likely to be both familar and better understood by the readers seems a odd construct. I understand that when they compared the two molecular assay the "concordance" construct might seem more appropriate to the authors. However when comparing the molecular test to toxigenic culture, a widely accepted C. difficile reference methods (although one that has its own short-comings), sensitivity and specificity does seem more appropriate. 2. ln 48-49: Since GENECUBE assay was only compared to the BD MAX assay, this is an overstatement. Please modify. 3. ln 102-4: Since QUIK CHECK complete is widely used diagnostically, it would be interesting to have compared the GENECUBE performance to it in a manner similar to what was done with BD MAX with toxigenic culture used as a reference method to resolve discrepant results. 4. Ln 114-7: This seems a significant delay between sample collection and test performance. Perhaps a comment in the discussion is warranted? 5. ln 170-2: How was a "postive" test defined in the LOD portion of the study for the two molecular assays. 6. ln 262-6: One of the short-comings of NAAT assays for C. difficile is false positive when compared to toxigenic culture. These "false" positives are even more pronouced when cytotoxicity assays are used as reference methods. This point likely deserves a bit more discussion in your manuscript. 7. Ln 281: To be clear, the two assays to which you are referring are the LIAT and Xpert assays. Correct? ****** 6. 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Revision 1
18 May 2020 Author Response Dear Editor and Reviewer Thank you very much for reviewing our manuscript and offering valuable advice. We have addressed your comments with point-by-point responses, and revised the manuscript accordingly. Reviewer #1: This is a competently performed evaluation of a new molecular assay for the detection of C. difficile tcdB gene. There are some specific issues that the authors should address. 1. The use of the terms "positive and negative concordance" rather than "sensitivity and specificity" which are terms likely to be both familar and better understood by the readers seems a odd construct. I understand that when they compared the two molecular assay the "concordance" construct might seem more appropriate to the authors. However when comparing the molecular test to toxigenic culture, a widely accepted C. difficile reference methods (although one that has its own short-comings), sensitivity and specificity does seem more appropriate. Response: Thank you very much for your suggestion. We have changed the sentences as suggested. 2. ln 48-49: Since GENECUBE assay was only compared to the BD MAX assay, this is an overstatement. Please modify. Response: Thank you very much for your suggestion. We have changed the text to show it was only compared with the BD MAX assay (line 47). 3. ln 102-4: Since QUIK CHECK complete is widely used diagnostically, it would be interesting to have compared the GENECUBE performance to it in a manner similar to what was done with BD MAX with toxigenic culture used as a reference method to resolve discrepant results. Response: Thank you for this comment. C. DIFF QUIK CHEK COMPLETE was useful, especially for the analysis of discrepant results between molecular examination and toxigenic culture. 4. Ln 114-7: This seems a significant delay between sample collection and test performance. Perhaps a comment in the discussion is warranted? Response: We agree that a delay would negatively affect the test performance of the BD MAX assay. We have added this point regarding the negative effect to the limitation section of discussion (lines 296-297). 5. ln 170-2: How was a "postive" test defined in the LOD portion of the study for the two molecular assays. Response: The GENECUBE system and the BD MAX system automatically show the results of molecular analyses on a display when the assay evaluations are complete. We used the automatic analysis to determine “positive” and “negative” for the current evaluation (lines 133-135 and Table 2-c). 6. ln 262-6: One of the short-comings of NAAT assays for C. difficile is false positive when compared to toxigenic culture. These "false" positives are even more pronouced when cytotoxicity assays are used as reference methods. This point likely deserves a bit more discussion in your manuscript. Response: Thank you very much for your review and suggestions. In this study, GDH was also positive in two of the four culture-negative stool samples. We think that toxigenic culture with the alcohol shock method is a highly sensitive method for the detection of toxigenic C. difficile; however, some culture-negative stool samples can be detected by molecular examination. We have added new references and sentences to the discussion section as red text (lines 266-272). 7. Ln 281: To be clear, the two assays to which you are referring are the LIAT and Xpert assays. Correct? Response: Thank you for this question. You are correct, the two assays are the cobas Liat Cdiff assay and Xpert C. difficile assay. We revised the text from “the two assays” to the “Liat Cdiff and Xpert C. difficile assays”. To the editorial Office 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 Response: Thank you very much for this suggestion. We have modified the style requirements by referring to the above PDF. 2. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ Response: The corresponding author has obtained an ORCID iD. 3. The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Response: We added a supporting information file to show all of the individual data Attachments Attachment Submitted filename: response to reviewers.docx https://doi.org/10.1371/journal.pone.0234119.r002
20 May 2020 Decision Letter - Yung-Fu Chang, Editor Clinical Evaluation of a Non-purified Direct Molecular Assay for the Detection of Clostridioides difficile Toxin Genes in Stool Specimens PONE-D-20-08775R1 Dear Dr. Hara, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Yung-Fu Chang Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0234119.r003
Formally Accepted
26 May 2020 Acceptance Letter - Yung-Fu Chang, Editor PONE-D-20-08775R1 Clinical Evaluation of a Non-purified Direct Molecular Assay for the Detection of Clostridioides difficile Toxin Genes in Stool Specimens Dear Dr. Hara: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Yung-Fu Chang Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0234119.r004

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