Gut microbiota profiles of treatment-naïve adult acute myeloid leukemia patients with neutropenic fever during intensive chemotherapy

Thanawat Rattanathammethee; Pimchanok Tuitemwong; Parameth Thiennimitr; Phinitphong Sarichai; Sarisa Na Pombejra; Pokpong Piriyakhuntorn; Sasinee Hantrakool; Chatree Chai-Adisaksopha; Ekarat Rattarittamrong; Adisak Tantiworawit; Lalita Norasetthada

doi:10.1371/journal.pone.0236460

Peer Review History

Original SubmissionJuly 7, 2020
20 Aug 2020 Decision Letter - Christopher Staley, Editor PONE-D-20-20999 Gut microbiota profiles of treatment-naïve adult acute myeloid leukemia patients with neutropenic fever during intensive chemotherapy PLOS ONE Dear Dr. Rattanathammethee, 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. The reviewers and I feel that the manuscript, as currently presented, suffers from a lack of clinical connection with the microbiome data. Further, revision of statistical approaches and presentation of the data are suggested. Due to the small sample size and primarily descriptive findings, revision to a shorter format is also encouraged. Please submit your revised manuscript by Oct 04 2020 11:59PM. 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For a full list of recommended repositories, see http://journals.plos.org/plosone/s/data-availability#loc-omics or http://journals.plos.org/plosone/s/data-availability#loc-sequencing. [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 Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: No ****** 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 ****** 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: No Reviewer #2: No ****** 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: The authors present a report in which they follow the microbiological flora ny NGS sequencing of bacterial DNA/rRNA in the guts of patients with AML . They compare finds pretreatment , the time of neutropenic fever (F/N) as well as upon recovery. They make some observations concerning the changes noted; in particular, they seem to show that bacterial diversity declines between pre-treatment and F/N Comments 1. The abstract and the entire paper suffer from severe problems with grammar and syntax; these need to be addressed, most likely by a native English speaker. 2. Abstract. This needs to be sufficiently re-worked to give a better idea to the reader of the quantitative findings. Further no mention is made of antibiotic usage, particularly prophylaxis, which could obviously affect the results. 3. The Introduction should state what advantage the sequencing technology has over culturing methods to determine gut bacterial diversity. 4. Is the fact that all patients had invasive pulmonary aspergillosis unusual ( or so that is stated in the text, but not related in the table)? 5. What does 3 patients … microbiologically defined mean? 6. The pretreatment neutrophil count seemed remarkably homogeneous for a group of patients with AML. The AML characteristic should be provided. 7. Table 2 what do the numbers in the leftmost column represent? 8. In the discussion section the authors really don't give a good explanation of why the flora of the gut changed despite the lack of use of antibacterial prophylaxis (and before any specific therapy for fever neutropenia was given) Reviewer #2: The authors present a study examining the bacterial composition of stool samples collected from patients undergoing initial induction chemotherapy treatment for AML. Three time points were analyzed from 10 patients, with 100% and 90% of samples successfully analyzed at baseline and at onset of febrile neutropenia, and 50% of samples analyzed at bone marrow recovery. The methodologies reported are appropriate and the manuscript is overall well-written. I have the following comments: - The study overall is fairly limited. The sample size is only 10 patients, with significant drop-off of samples collected at the 3rd time point. Such a small sample size doesn't allow for exploration of microbiome associations with different clinical outcomes. Larger retrospective cohorts have already been published, and it is a but unclear what this current study adds to the field. - Comparisons of microbiome parameters (abundances of taxa or diversity) should be adjusted for multiple comparisons, using for example the Benjamini-Hochberg method. They currently don't appear to be adjusted. - Conveying bacterial abundances using tables is probably not as effective as graphically. Consider preparing stacked bar graphs for individual samples. - Data underlying the findings described are not fully available in the manuscript, supplementary materials, or in an online repository. - Consider asking a native English speaker to edit the manuscript. ****** 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 [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.0236460.r001
Revision 1
5 Sep 2020 Author Response Response to reviewers ========= Reviewer #1: The authors present a report in which they follow the microbiological flora ny NGS sequencing of bacterial DNA/rRNA in the guts of patients with AML . They compare finds pretreatment , the time of neutropenic fever (F/N) as well as upon recovery. They make some observations concerning the changes noted; in particular, they seem to show that bacterial diversity declines between pre-treatment and F/N Comments 1. The abstract and the entire paper suffer from severe problems with grammar and syntax; these need to be addressed, most likely by a native English speaker. Response: The manuscript has already been revised by English language team. 2. Abstract. This needs to be sufficiently re-worked to give a better idea to the reader of the quantitative findings. Further no mention is made of antibiotic usage, particularly prophylaxis, which could obviously affect the results. Response: Addition of quantitative findings and usage of antibiotics have been addressed. 3. The Introduction should state what advantage the sequencing technology has over culturing methods to determine gut bacterial diversity. Response: The advantage of sequencing technology over traditional culturing methods has been addressed. 4. Is the fact that all patients had invasive pulmonary aspergillosis unusual ( or so that is stated in the text, but not related in the table)? Response: Only 3 patients had invasive pulmonary aspergillosis. I have rewritten this statement into " Three patients (33.3%) were defined as microbiologically significant with invasive pulmonary aspergillosis confirmed by typical radiologic finding and serum galactomannan (indicated as patient's code: P4, P6 and P10)". 5. What does 3 patients … microbiologically defined mean? Response: I have rewritten this statement into "Three patients (33.3%) were defined as microbiologically significant with invasive pulmonary aspergillosis confirmed by typical radiologic finding and serum galactomannan (indicated as patient's code: P4, P6 and P10)". 6. The pretreatment neutrophil count seemed remarkably homogeneous for a group of patients with AML. The AML characteristic should be provided. Response: I have provided the AML patients' characteristic in Table 1. The median of pretreatment absolute neutrophil count was 2.85 x 109 /L (1.42-7.67) with the range of 1.42 x 109 /L (minimum) to 7.67 x 109 /L (maximum). 7. Table 2 what do the numbers in the leftmost column represent? Response: The leftmost column in table 2 represent the name of each bacterial phyla and next column is the percentage of microbial relative abundance among five most abundant stool bacterial phyla. 8. In the discussion section the authors really don't give a good explanation of why the flora of the gut changed despite the lack of use of antibacterial prophylaxis (and before any specific therapy for fever neutropenia was given) Response: I have explained more detailed on the changes of gut microbiota in the discussion section. (We hypothesized that even the first cycle of intensive chemotherapy, which would contribute to the NF, and without antibiotic prophylaxis might reveal evidence of changes in intestinal bacteria flora as all of febrile neutropenia patients require empirical treatment with broad spectrum antibiotics. Gut barrier is a vulnerable site of injury after receiving highly intensive chemotherapy and resulting microbiota pattern disruption. The antibiotic therapy may eradicate particular taxa which affect host immunity and the chemotherapy can extensively damage the gut barrier, the neutropenia itself also potentially affecting the intestinal bacteria of the patients.) ========= Reviewer #2: The authors present a study examining the bacterial composition of stool samples collected from patients undergoing initial induction chemotherapy treatment for AML. Three time points were analyzed from 10 patients, with 100% and 90% of samples successfully analyzed at baseline and at onset of febrile neutropenia, and 50% of samples analyzed at bone marrow recovery. The methodologies reported are appropriate and the manuscript is overall well-written. I have the following comments: - The study overall is fairly limited. The sample size is only 10 patients, with significant drop-off of samples collected at the 3rd time point. Such a small sample size doesn't allow for exploration of microbiome associations with different clinical outcomes. Larger retrospective cohorts have already been published, and it is a but unclear what this current study adds to the field. Response: This study conducted solely treatment-naïve AML patients who homogenously experienced the first episode of neutropenic fever and currently not received antimicrobial prophylaxis. Unfortunately, the limitation of clinical correlation cannot be eliminated but to point out the microbial composition changes even in the first dose of AML treatment which microbiota diversity remained constant despite recovery of bone marrow. Moreover, the study also expand the dataset of gut microbiota profiles in adult Asian ethnicity with AML. - Comparisons of microbiome parameters (abundances of taxa or diversity) should be adjusted for multiple comparisons, using for example the Benjamini-Hochberg method. They currently don't appear to be adjusted. Response: In terms of correction of multiple comparisons, Benjamini-Hochberg method has been introduced to the manuscript. - Conveying bacterial abundances using tables is probably not as effective as graphically. Consider preparing stacked bar graphs for individual samples. Response: I have converted table 3 to the stacked bar graph for individual sample and this table has been moved to supplementary data (Table S1). - Data underlying the findings described are not fully available in the manuscript, supplementary materials, or in an online repository. Response: All of the sequencing results had been provided as an online accession of Sequence Read Archrive (SRA), as BioProject number PRJNA661595 (https://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA661595), and supplementary material has been added. - Consider asking a native English speaker to edit the manuscript. Response: The manuscript has already been revised by English language team. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0236460.r002
14 Oct 2020 Decision Letter - Christopher Staley, Editor Gut microbiota profiles of treatment-naïve adult acute myeloid leukemia patients with neutropenic fever during intensive chemotherapy PONE-D-20-20999R1 Dear Dr. Rattanathammethee, 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, Christopher Staley, Ph.D. 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 #2: 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 #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: 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 #2: 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 #2: 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 #2: The authors have responded to the comments and the manuscript is improved. I have no further concerns. ****** 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 #2: No https://doi.org/10.1371/journal.pone.0236460.r003
Formally Accepted
19 Oct 2020 Acceptance Letter - Christopher Staley, Editor PONE-D-20-20999R1 Gut microbiota profiles of treatment-naïve adult acute myeloid leukemia patientswith neutropenic fever during intensive chemotherapy Dear Dr. Rattanathammethee: 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. Christopher Staley Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0236460.r004

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