Direct assessment of possible mutations in the 23S rRNA gene encoding macrolide resistance in Chlamydia trachomatis

J. M. van Niekerk; I. H. M. van Loo; M. Lucchesi; S. A. Morré; C. J. P. A. Hoebe; N. H. T. M. Dukers-Muijrers; P. F. G. Wolffs

doi:10.1371/journal.pone.0265229

Peer Review History

Original SubmissionNovember 22, 2021
23 Dec 2021 Decision Letter - Deborah Dean, Editor PONE-D-21-37083Direct assessment of mutations in the 23S rRNA gene encoding azithromycin resistance in Chlamydia trachomatisPLOS ONE Dear Dr. van Niekerk, 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 address all concerns of reviewer 1. In addition, please address the following issues: 1) Under Clinical Evaluation in Methods, please briefly describe the methods that were used to determine CT positive clinical samples, CT quantitative load (including how IFU/mL was determined) and Ct typing, even if these methods are in reference 2, as this will facilitate a more complete understanding of the data. 2) Although Figure S1 is reproduced from reference 2, please include as a main figure with appropriate permissions. 3) Please change ‘golden’ to gold; change ‘to best of our knowledge’ to ‘to the best of our knowledge.’ Please submit your revised manuscript within one month. 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. 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Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover 4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: 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 ****** 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: Well written paper from a group with a lot of experience in the field. The investigators looked for mutations in the 23S rRNA gene associated with macrolide resistance in PCR positive specimens of C. trachomatis. These included specimens obtained and baseline and sample of clinical cases where the PCR remained positive for up to 51 days post treatment with azithromycin. No resistance associated mutations (RAMs) were discovered. Persistence of C. trachomatis DNA and RNA, but no viable organisms by culture) has been described for 3 weeks or more, which is why the CDC does not recommend retesting before 4 weeks post therapy (2021 CDC STI Treatment Guidelines ref # 6). These results are reassuring. The same RAMs have also been observed in Mycoplasma genitalium, M. pneumoniae and N. gonorrhoeae. SpeeDx has developed PCR assays to detect these mutations in M. genitalium and M. pneumoniae in addition to detecting the organisms. I would change the title to “Direct assessment of possible mutations in the 23S rRNA gene encoding macrolide resistance in Chlamydia trachomatis”, as they didn't find any mutations. These mutations also confer resistance to other macrolides including erythromycin. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes:** Margaret R. Hammerschlag, MD [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. Attachments Attachment Submitted filename: Riska macrolide R Cpn 2004.pdf https://doi.org/10.1371/journal.pone.0265229.r001
Revision 1
2 Feb 2022 Author Response January 21th, 2022 Dear Editors, We thank the editors and reviewers for their input and comments on the manuscript and we have edited the manuscript to address their concerns. In the next section, we’ll provide a point-by-point response to the comments. 1. Please address all concerns of reviewer 1. Reviewer #1: I would change the title to “Direct assessment of possible mutations in the 23S rRNA gene encoding macrolide resistance in Chlamydia trachomatis”, as they didn't find any mutations. These mutations also confer resistance to other macrolides including erythromycin. We agree with reviewer 1 and have changed the title of our manuscript, as suggested. 2. Under Clinical Evaluation in Methods, please briefly describe the methods that were used to determine CT positive clinical samples, CT quantitative load (including how IFU/mL was determined) and Ct typing, even if these methods are in reference 2, as this will facilitate a more complete understanding of the data. We have added the nucleic acid amplification test used to screen for Chlamydia trachomatis infections, we have described the CT quantification as reported by reference 2, and added the methods used to perform sequence typing. 3. Although Figure S1 is reproduced from reference 2, please include as a main figure with appropriate permissions. We have included ‘Figure S1’ as a main figure in the manuscript (Fig 2). We have emailed PLOS ONE for permission regarding reuse of this figure. In the reply we received 19.01.2022, it was stated that the content was published under an open access license (CC-BY). The original creators are credited in the Figure’s caption. 4. Please change ‘golden’ to gold; change ‘to best of our knowledge’ to ‘to the best of our knowledge’. We have edited this in the current manuscript. 5. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. We have edited the manuscript according to PLOS ONE’s style requirements, including file naming. 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. The reference list is complete and correct. One reference has been added, as described below (point 10). 7. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. We have provided all accession numbers required to reproduce Fig 2 in the supplementary file called ‘S1_File’. We also provided the accession numbers of the Chlamydia suis and Chlamydia muridarum 23S gene used to find discriminatory regions. These accession numbers have been added to ‘S2_File’. All 23S sequences generated in the current study have been submitted to GenBank. We have been provided accession numbers for these sequences (OM320821 to OM320875). Our sequences are currently being examined and processed by the GenBank annotation staff to ensure that it is free of errors or problems. 8. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. We have removed the phrase “data not shown” and added the discriminatory regions under the results section (technical evaluation). The Chlamydia suis and Chlamydia muridarum sequences used to evaluate these discriminatory regions, have been added to the supplementary ‘S2_File’. 9. While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool. All 3 main figures have been uploaded to the PACE tool, and LZW compressed. 10. Attachment ‘Riska macrolide R Cpn 2004.pdf’ Although we were uncertain about the aim of the added attachment (‘Riska macrolide R Cpn 2004.pdf’), we were reminded of this very interesting paper with indeed many similarities with our work on CT. We have added this reference in the introduction, 3rd paragraph. We look forward to hearing from you regarding our submission. We would be glad to respond to any further questions and comments that you may have. On behalf of all authors, Julius van Niekerk Attachments Attachment Submitted filename: Rebuttal.docx https://doi.org/10.1371/journal.pone.0265229.r002
28 Feb 2022 Decision Letter - Deborah Dean, Editor Direct assessment of possible mutations in the 23S rRNA gene encoding macrolide resistance in Chlamydia trachomatis PONE-D-21-37083R1 Dear Dr. van Niekerk, 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, Deborah Dean, M.D., M.P.H. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0265229.r003
Formally Accepted
2 May 2022 Acceptance Letter - Deborah Dean, Editor PONE-D-21-37083R1 Direct assessment of possible mutations in the 23S rRNA gene encoding macrolide resistance in Chlamydia trachomatis Dear Dr. van Niekerk: 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. Deborah Dean Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0265229.r004

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