Evaluation of serum VIP and aCGRP during pulmonary exacerbation in cystic fibrosis: A longitudinal pilot study of patients undergoing antibiotic therapy

Maha S. Al-Keilani; Samah Awad; Hanan M. Hammouri; Tala Al Shalakhti; Basima A. Almomani; Muna M. Dahabreh; Mohammad-Jaafar Ajlony

doi:10.1371/journal.pone.0284511

Peer Review History

Original SubmissionSeptember 9, 2022
1 Nov 2022 Decision Letter - Santiago Partida-Sanchez, Editor PONE-D-22-25130Evaluation of VIP and aCGRP during pulmonary exacerbation in cystic fibrosis patients: a prospective study.PLOS ONE Dear Dr. Al-Keilani, 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. Authors should pay particular attention to the following points: Application, description and interpretation of all statistical analyses must be revised, reviewers 1 & 2 expressed major concerns on the study design, some methods, statistical data and interpretation. Please address specific changes to introduction and discussion as suggested by reviewer 2 and clarify additional inquires by reviewer 1. Please submit your revised manuscript by Dec 16 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, Santiago Partida-Sanchez 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 (1) whether consent was informed and (2) 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. If you are reporting a retrospective study of medical records or archived samples, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information. 3. 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. [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 ******** 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: 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 ****** 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 submission by Al-Keilani entitled “Evaluation of VIP and aCGRP during pulmonary exacerbation in cystic fibrosis patients: a prospective study” describes analyses serum concentrations of two potential biomarkers of inflammation from 21 people with cystic fibrosis (CF) who were treated for pulmonary exacerbation. Major comments. The authors make a reasonable argument for the potential utility of a systemic biomarker when handling pulmonary exacerbation. However, although their title suggests they have conducted a prospective study, they have not described a prospective hypothesis (that increase or decrease of a given biomarker will be associated with a given outcome) or a corresponding prospective statistical analysis plan (describing study power to detect a response given the anticipated response magnitude, the variance of a given measure, and the size of the population studied). Further, there is no description of how the investigators planned to adjust statistical tests for multiplicity. Thus, although data for this submission were collected prospectively, this is, in fact, a retrospective analysis. As such, it is problematic that statistical test results are reported/interpreted in the abstract for differences in serum biomarker concentrations a) before versus after exacerbation treatment (p>.05), b) by patient sex (p=.038), and c) as a function of Pseudomonas infection (p=.028) without acknowledging type I error inflation resulting from the simultaneous statistical testing of 36 different associations. Given the retrospective nature of these analyses, the investigators should not be reporting P values for tests, but rather providing point estimates and 95% confidence intervals and allowing readers to draw conclusions regarding associations. Given the number of point estimates generated in these analyses, the authors should consider presenting associations as forest plots, which would allow readers to study the juxtapositions confidence intervals for different tests. Minor comments The statement that “most of the time patients’ FEV1 values do not improve after antibiotic therapy (8)” which cites D.B. Sander’s 2010 epidemiologic analyses, is an incorrect extrapolation of those data. Sanders and colleagues reported the proportion of individuals with CF who had an FEV1 recorded after exacerbation treatment that was equal to or greater than their maximum FEV1 recorded in the prior year. This was not an analysis of change in FEV1 from immediately prior to immediately after treatment. With respect to the statement about FEV1 improvement with treatment, I refer the authors to the results of a large prospective study of CF exacerbation treatment [Am J Respir Crit Care Med. 2021 Dec 1;204(11):1295-1305], which show that FEV1 does increase after exacerbation treatment for most people with CF. The use of Fuchs criteria to define exacerbations for inclusion in this analysis should be clarified. As written, it would appear that any individual presenting with any of the 12 Fuchs criteria could be diagnosed with exacerbation and included in the study. In the past, Fuchs criteria have been applied as a means of defining an event as an exacerbation for the purpose of subsequent analyses after a clinician has made the decision to treat a respiratory event with antimicrobials. Further, Fuchs definitions have typically required the presence of at least 4 of 12 criteria. The authors’ suggestion in the abstract that “Future studies with larger sample size are required to investigate the clinical importance of VIP and aCGRP in cystic fibrosis patients” seems at odds with their results and conclusions. Don’t the presented analyses indicate a lack of clinical correlation for these markers, or do the authors believe that their analyses were substantially underpowered? If the latter is the case, is it appropriate to report these (negative) results? Reviewer #2: The authors explore changes in levels of VIP and ACGRP in people with CF treated for a pulmonary exacerbations. The sample size is quite small and subjects are predominantly children. Introduction - The introduction is long and contains irrelevant material to the topic of the study (e.g. refers to modifier genes), where the topic of the paper is biomarkers. The introduction should be reviewed and made more concise focusing on just background of relevance to the current study. Methods - Failure to measure FEV1 would substantially impact on the ability to assess response to treatment of pulmonary exacerbation. - C- reactive protein is a widely accepted marker of inflammation in CF – Were levels measured in this population. If so, they should be reported and comment made on whether these correlate with VIP / aCGRP - Mostly children – Mean age 8.7years - BMI probably not best measure of nutrition in children and at the least BMI z-scores should be reported - No measure of response to antibiotic therapy is reported – e.g. how many patients returned to a symptomatic baseline. Consequently, it is not possible to gauge whether the failure of VIP / aCGRP to change with treatment reflects a lack of association - No power calculation – It may be possible to determine a necessary sample size to detect a difference in these measured based on the literature in asthma exacerbations Results - Means and SEM were reported but a lot of the analysis used non-parametric measures suggesting data was not normally distributed – In which case, Median and IQR (or range) would be preferred. - Only univariate analysis has been performed and results may be subject to confounding. E.g. it is stated that Female subjects and subjects with P. aeruginosa had higher levels of aCGRP1. But, it is not known if the spread of P. aeruginosa infection was similar between male and female subjects. Multivariate analysis should be performed. - The small sample size leaves the study open toe Type I and II errors and comment should be made on this. Discussion - The discussion is confused. It is unclear whether the authors are proposing that VIP act as a modifier of CFTR function and thereby impacts on disease severity, or VIP can be used as a biomarker of inflammation in CF. As this is a study looking at its function as a biomarker of exacerbations, I would recommend that discussion is limited to it function as a biomarker. - Line 233 – “Previous reports Indicate VIP and aCGRP may be involved in the pathophysiology of CF exacerbations” – Please provide references. ****** 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.0284511.r001
Revision 1
17 Feb 2023 Author Response 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 Response: Confirmed. 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 (1) whether consent was informed and (2) 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. If you are reporting a retrospective study of medical records or archived samples, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information. Response: The following sentence was added to the Consent to Participate subsection: “All patients signed a written informed consent at the start of the study. For patients under eighteen years old, the informed consent was signed by one of their parents.” In the current study, the patients were recruited prospectively, and the data was collected from both patients and their medical records. As stated in the Ethical Approval subsection “The research was performed in accordance with the Declaration of Helsinki, and the protocol was approved by the Research and Ethics Committee at Jordan University of Science and Technology (registration number 34/111/2017, date of approval January 7, 2018), where all methods were performed in accordance with the relevant guidelines and regulations.” 3. 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. Response: caption has been added. Reviewers’ comments Reviewer #1: The submission by Al-Keilani entitled “Evaluation of VIP and aCGRP during pulmonary exacerbation in cystic fibrosis patients: a prospective study” describes analyses serum concentrations of two potential biomarkers of inflammation from 21 people with cystic fibrosis (CF) who were treated for pulmonary exacerbation. Major comments. 1. The authors make a reasonable argument for the potential utility of a systemic biomarker when handling pulmonary exacerbation. However, although their title suggests they have conducted a prospective study, they have not described a prospective hypothesis (that increase or decrease of a given biomarker will be associated with a given outcome) or a corresponding prospective statistical analysis plan (describing study power to detect a response given the anticipated response magnitude, the variance of a given measure, and the size of the population studied). Further, there is no description of how the investigators planned to adjust statistical tests for multiplicity. Thus, although data for this submission were collected prospectively, this is, in fact, a retrospective analysis. As such, it is problematic that statistical test results are reported/interpreted in the abstract for differences in serum biomarker concentrations a) before versus after exacerbation treatment (p>.05), b) by patient sex (p=.038), and c) as a function of Pseudomonas infection (p=.028) without acknowledging type I error inflation resulting from the simultaneous statistical testing of 36 different associations. Given the retrospective nature of these analyses, the investigators should not be reporting P values for tests, but rather providing point estimates and 95% confidence intervals and allowing readers to draw conclusions regarding associations. Given the number of point estimates generated in these analyses, the authors should consider presenting associations as forest plots, which would allow readers to study the juxtapositions confidence intervals for different tests. Response: The problem of retesting was solved using repeated measures multiple ANOVA (MANOVA) models. Regarding the univariate effect, we used forest plots for point estimates and 95% confidence intervals. Minor comments 2. The statement that “most of the time patients’ FEV1 values do not improve after antibiotic therapy (8)” which cites D.B. Sander’s 2010 epidemiologic analyses, is an incorrect extrapolation of those data. Sanders and colleagues reported the proportion of individuals with CF who had an FEV1 recorded after exacerbation treatment that was equal to or greater than their maximum FEV1 recorded in the prior year. This was not an analysis of change in FEV1 from immediately prior to immediately after treatment. With respect to the statement about FEV1 improvement with treatment, I refer the authors to the results of a large prospective study of CF exacerbation treatment [Am J Respir Crit Care Med. 2021 Dec 1;204(11):1295-1305], which show that FEV1 does increase after exacerbation treatment for most people with CF. Response: The reference has been replaced as recommended. 3. The use of Fuchs criteria to define exacerbations for inclusion in this analysis should be clarified. As written, it would appear that any individual presenting with any of the 12 Fuchs criteria could be diagnosed with exacerbation and included in the study. In the past, Fuchs criteria have been applied as a means of defining an event as an exacerbation for the purpose of subsequent analyses after a clinician has made the decision to treat a respiratory event with antimicrobials. Further, Fuchs definitions have typically required the presence of at least 4 of 12 criteria. Response: Diagnosis of pulmonary exacerbation was made based on the clinical decision of the pulmonologist. When recruiting patients, we made sure that every patient had at least 4 of the 12 Fuchs criteria to define exacerbation. The Methods section was adjusted for better clarification. 4. The authors’ suggestion in the abstract that “Future studies with larger sample size are required to investigate the clinical importance of VIP and aCGRP in cystic fibrosis patients” seems at odds with their results and conclusions. Don’t the presented analyses indicate a lack of clinical correlation for these markers, or do the authors believe that their analyses were substantially underpowered? If the latter is the case, is it appropriate to report these (negative) results? Response: In the current study we have limited access to patients, and we have been working on the recruitment for about a year and could only include this number due to lack of follow-up samples. Patients may admit at night, so sometimes it was not feasible to obtain the initial sample, or the patient was discharged early and so could not take the last sample. However, with the re-analysis performed. We could find statistically significant results by using the appropriate statistical test. Accordingly, the abstract has been rewritten to represent the new findings. Also we have added the term “pilot” to the title. Reviewer #2: 1. The authors explore changes in levels of VIP and aCGRP in people with CF treated for a pulmonary exacerbations. The sample size is quite small and subjects are predominantly children. Response: In the current study we have limited access to patients, and we have been working on the recruitment for about a year and could only include this number due to lack of follow-up samples. Patients may admit at night, so sometimes it was not feasible to obtain the initial sample, or the patient was discharged early and so could not take the last sample. Thus, this was a consecutive sampling method, and the study sample represents all patients with the three blood samples at the three occasions as described in the methods section, if missing one sample the patient was excluded from the study. 2. Introduction: the introduction is long and contains irrelevant material to the topic of the study (e.g. refers to modifier genes), where the topic of the paper is biomarkers. The introduction should be reviewed and made more concise focusing on just background of relevance to the current study. Response: irrelevant sentences focusing on genetics have been deleted as recommended and the introduction has been rephrased accordingly. 3. Methods A. Failure to measure FEV1 would substantially impact on the ability to assess response to treatment of pulmonary exacerbation. Response: As we mentioned in the limitations “Pulmonary function test as represented by FEV1 was only recorded for few patients so it was not included as a variable in our study and the patient response to therapy was assessed clinically by the responsible physician.” The inability to do pulmonary function test for the majority of the patients and at certain time points only was due to several factors which are described in the introduction section: “However, such testing is not feasible in young children and in patients with severe lung disease and is not accurate in mild conditions where the test is usually normal (7). Besides the fact that several months are required to notice a clinical response after initiation of anti-inflammatory therapy and most of the time patients’ FEV1 values do not improve after antibiotic therapy (8)”. B. C- reactive protein is a widely accepted marker of inflammation in CF – Were levels measured in this population. If so, they should be reported and comment made on whether these correlate with VIP / aCGRP Response: C-reactive protein levels were not measured in this study, however, investigating inflammatory markers and correlating them with VIP and aCGRP is of interest and high importance to identify the inflammatory roles of these target markers in CF. C. Mostly children – Mean age 8.7years - BMI probably not best measure of nutrition in children and at the least BMI z-scores should be reported Response: As it is mentioned in the method, BMI z-score has been calculated using CDC calculator (Chou JH, Roumiantsev S, Singh R. PediTools Electronic Growth Chart Calculators: Applications in Clinical Care, Research, and Quality Improvement. J Med Internet Res. 2020 Jan 30;22(1):e16204. doi: 10.2196/16204. PMID: 32012066; PMCID: PMC7058170.) as recommended and has been included in the analysis D. No measure of response to antibiotic therapy is reported – e.g. how many patients returned to a symptomatic baseline. Consequently, it is not possible to gauge whether the failure of VIP / aCGRP to change with treatment reflects a lack of association. Response: This was a consecutive sampling method, and the study sample represents all patients with the three blood samples at the three occasions as described in the methods section, if missing one sample the patient was excluded from the study. The patient’s response to therapy was assessed clinically by the responsible physician, and the last sample was taken after 2 weeks of therapy, which was the time when the patients returned to a symptomatic baseline. E. No power calculation – It may be possible to determine a necessary sample size to detect a difference in these measured based on the literature in asthma exacerbations. Response: It is a pilot study with limited access to patients. We have been working on the recruitment for about a year and could only include this number due to lack of follow-up samples. Patients may admit at night, so sometimes it was not feasible to obtain the initial sample, or the patient was discharged early and so could not take the last sample. The study sample represents all patients with the three blood samples at the three occasions as described in the methods section, if missing one sample the patient was excluded from the study. 4. Results A. Means and SEM were reported but a lot of the analysis used non-parametric measures suggesting data was not normally distributed – In which case, Median and IQR (or range) would be preferred. Response: The biostatistician re-analyzed the data where Q-Q plots were used to verify the normality of VIP and aCGRP variables. There was a need for log transformations to normalize VIP variables, whereas aCGRP variables were assumed to be normally distributed without requiring any transformations. Thus, we are keeping means and SEM as descriptive statistics for continuous variables. B. Only univariate analysis has been performed and results may be subject to confounding. E.g. it is stated that Female subjects and subjects with P. aeruginosa had higher levels of aCGRP1. But, it is not known if the spread of P. aeruginosa infection was similar between male and female subjects. Multivariate analysis should be performed. Response: We adjusted the analysis, and we used repeated measures multiple ANOVA (MANOVA) as described in the statistical analysis subsection. C. The small sample size leaves the study open to Type I and II errors and comment should be made on this. Response: In the current study we have limited access to patients and thus small sample size. We have been working on the recruitment for about a year and could only include this number due to lack of follow-up samples. Patients may admit at night, so sometimes it was not feasible to obtain the initial sample, or the patient was discharged early and so could not take the last sample. Thus, this was a consecutive sampling method, and the study sample represents all patients with the three blood samples at the three occasions as described in the methods section, if missing one sample the patient was excluded from the study. 5. Discussion A. The discussion is confused. It is unclear whether the authors are proposing that VIP act as a modifier of CFTR function and thereby impacts on disease severity, or VIP can be used as a biomarker of inflammation in CF. As this is a study looking at its function as a biomarker of exacerbations, I would recommend that discussion is limited to it function as a biomarker. Response: The discussion section has been rewritten according to the new results, and more focus on the role of VIP as inflammation biomarker has been further discussed. B. Line 233 – “Previous reports Indicate VIP and aCGRP may be involved in the pathophysiology of CF exacerbations” – Please provide references. Response: References have been added as requested. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0284511.r002
27 Feb 2023 Decision Letter - Santiago Partida-Sanchez, Editor PONE-D-22-25130R1Evaluation of serum VIP and aCGRP during pulmonary exacerbation in cystic fibrosis: a longitudinal pilot study of patients undergoing antibiotic therapy.PLOS ONE Dear Dr. Al-Keilani, 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 submit your revised manuscript by Apr 13 2023 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, Santiago Partida-Sanchez 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) ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know ****** 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 ****** 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 ******** 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: The authors of the PLOS ONE submission “Evaluation of serum VIP and aCGRP during pulmonary exacerbation in cystic fibrosis: a longitudinal pilot study of patients undergoing antibiotic therapy” have addressed my technical concerns regarding the use of multiple univariate comparisons. However, there remains an aspect of this submission (which I could not fully appreciate due to the previous analytical approach) that must be resolved. MAJOR COMMENTS: The authors engage in a form of circular reasoning regarding the utility of aCGRP and VIP measure as biomarkers of CF exacerbation response: First, they state that “monitoring of cystic fibrosis is difficult especially in cases of pulmonary exacerbations where patients fail to regain the baseline pulmonary function despite standard treatment” (its not clear how a treating clinician can know that an individual has failed to regain baseline without monitoring… so clearly monitoring must be possible in this instance). Then, they proceed to describe aCGRP and VIP changes during antimicrobial exacerbation treatment as a biomarker of treatment response without methodologic description of how clinical responses to treatment were assessed, recorded, or incorporated into analyses. They state that spirometry was either not available or uninformative and thus they relied on clinician evaluation… but I could not find these clinician assessments of response. How can the investigators conclude that aCGRP changes are indicative of treatment response without describing responses? MINOR COMMENTS The authors can be more disciplined in their manuscript construction. Lines 167 to 170 on page 9 of the results contain elements of Methods, Results and Discussion: “By analyzing the changes overtime of LOG VIP and aCGRP2 serum levels using repeated measures models, we investigated the factors responsible for these changes [a Method]. It was found that there were significant between-subjects effects over the LOG VIP [a Result], which indicates that the difference between the levels means which is not due to the time [a Discussion].” Further, rather than state: “Figure 3 shows the LS means 171 and p-values for these effects” after this passage, why not just append the second sentence with “(Figure 3)”? This manuscript is overly long at the expense of a clear description of observations. There is entirely too much discussion of the biology of aCGRP and VIP. A few sentences identifying and referencing key observations is more than adequate. Observations that these are interesting molecules are tangential to the matter at hand, which is how well they associate with exacerbation treatment. The figures are almost illegible. The fonts are too small, especially in Figures 3 and 4 and the point estimates for LS means in Figures 1 and 2 should have 95% CI or SE bars included. ******** 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 ******** [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.0284511.r003
Revision 2
14 Mar 2023 Author Response Reviewer #1: The authors of the PLOS ONE submission “Evaluation of serum VIP and aCGRP during pulmonary exacerbation in cystic fibrosis: a longitudinal pilot study of patients undergoing antibiotic therapy” have addressed my technical concerns regarding the use of multiple univariate comparisons. However, there remains an aspect of this submission (which I could not fully appreciate due to the previous analytical approach) that must be resolved. Response: A multivariate model with stepwise selection was used instead of the univariate model to test all variables and select those with significance without retesting or inflating alpha levels. MAJOR COMMENTS: The authors engage in a form of circular reasoning regarding the utility of aCGRP and VIP measure as biomarkers of CF exacerbation response: First, they state that “monitoring of cystic fibrosis is difficult especially in cases of pulmonary exacerbations where patients fail to regain the baseline pulmonary function despite standard treatment” (it’s not clear how a treating clinician can know that an individual has failed to regain baseline without monitoring… so clearly monitoring must be possible in this instance). Then, they proceed to describe aCGRP and VIP changes during antimicrobial exacerbation treatment as a biomarker of treatment response without methodologic description of how clinical responses to treatment were assessed, recorded, or incorporated into analyses. They state that spirometry was either not available or uninformative and thus they relied on clinician evaluation… but I could not find these clinician assessments of response. How can the investigators conclude that aCGRP changes are indicative of treatment response without describing responses? Response: Thanks for your comment. We agree that the statement “Adequate monitoring of cystic fibrosis is difficult especially in cases of pulmonary exacerbations where patients fail to regain the baseline pulmonary function despite standard treatment” was not correct and we deleted it. We replaced the statement by the following: “Objective monitoring of improvement during treatment of pulmonary exacerbation can be difficulty in children when pulmonary function testing cannot be obtained.” It was stated in the Method section lines 110-112 that the duration of treatment was 14 days, after which patients returned to baseline: “The duration of antibiotic therapy for pulmonary exacerbation was 2 weeks for all patients, which is the duration of antibiotic therapy where patients returned to a symptomatic baseline”, and then we added the phrase “and had clinically improved based on clinician’s assessment” to make it clearer that the improvement was based on clinical response. MINOR COMMENTS The authors can be more disciplined in their manuscript construction. 1. Lines 167 to 170 on page 9 of the results contain elements of Methods, Results and Discussion: “By analyzing the changes overtime of LOG VIP and aCGRP2 serum levels using repeated measures models, we investigated the factors responsible for these changes [a Method]. It was found that there were significant between-subjects effects over the LOG VIP [a Result], which indicates that the difference between the levels means which is not due to the time [a Discussion].” Response: The sentence “By analyzing the changes overtime of LOG VIP and aCGRP2 serum levels using repeated measures models, we investigated the factors responsible for these changes” has been shifted to the [methods] section, and the sentence “which indicates that the difference between the levels means which is not due to the time” has been shifted to the [discussion] section. 2. Further, rather than state: “Figure 3 shows the LS means 171 and p-values for these effects” after this passage, why not just append the second sentence with “(Figure 3)”? Response: The paragraph has been rephrased. 3. This manuscript is overly long at the expense of a clear description of observations. There is entirely too much discussion of the biology of aCGRP and VIP. A few sentences identifying and referencing key observations is more than adequate. Observations that these are interesting molecules are tangential to the matter at hand, which is how well they associate with exacerbation treatment. Response: Changes to the discussion has been performed where extra information about the molecular role of the markers has been deleted, and additional evidence of their role as potential biomarkers in CF has been discussed; lines 240-243 and lines 266-274. 4. The figures are almost illegible. The fonts are too small, especially in Figures 3 and 4 and the point estimates for LS means in Figures 1 and 2 should have 95% CI or SE bars included. Response: The figures for all factors have been updated. for overall LS means, we have included the SE in tables instead of adding them to the figures. Attachments Attachment Submitted filename: Response letter.docx https://doi.org/10.1371/journal.pone.0284511.r004
3 Apr 2023 Decision Letter - Santiago Partida-Sanchez, Editor Evaluation of serum VIP and aCGRP during pulmonary exacerbation in cystic fibrosis: a longitudinal pilot study of patients undergoing antibiotic therapy. PONE-D-22-25130R2 Dear Dr. Al-Keilani, 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, Santiago Partida-Sanchez 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 #1: 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: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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: The authors have addressed my concerns, although I remain skeptical that a systemic biomarker can supplant physical exam and communication with patients regarding treatment 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 #1: No ******** https://doi.org/10.1371/journal.pone.0284511.r005
Formally Accepted
27 Apr 2023 Acceptance Letter - Santiago Partida-Sanchez, Editor PONE-D-22-25130R2 Evaluation of serum VIP and aCGRP during pulmonary exacerbation in cystic fibrosis: a longitudinal pilot study of patients undergoing antibiotic therapy. Dear Dr. Al-Keilani: 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 Professor Santiago Partida-Sanchez Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0284511.r006

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 .