The clinical significance of inflammatory biomarkers, IL6 cytokine, and systemic immune inflammatory index in rabbit model of acute and chronic Methicillin-resistant Staphylococcus epidermidis-induced osteomyelitis

Diana-Larisa Ancuța; Arianna Barbara Lovati; Cristin Coman

doi:10.1371/journal.pone.0309145

Peer Review History

Original SubmissionJanuary 19, 2024
27 May 2024 Decision Letter - Seyed Mostafa Hosseini, Editor PONE-D-24-02543The clinical significance of inflammatory biomarkers, IL-6 cytokine, and systemic immune inflammatory index in rabbit model of acute and chronic Methicillin-resistant Staphylococcus epidermidis-induced osteomyelitisPLOS ONE Dear Dr. Larisa, 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 Jul 11 2024 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. 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Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access. 5. 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 If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references.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 If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [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 Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: 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: 1- In line 203, the selection of sampling days for hematological tests is based on what and how? 2- In line 213, What sample was used in interleukin 6 assay? 3- In line 213, What was the measurement method, type and brand of the kit used to measure interleukin 6? Reviewer #2: The study presents a good balance of scientific rigor and methodology. The use of a rabbit model of acute and chronic osteomyelitis induced by MRSE is a relevant and well-established model. The inclusion of different treatment groups, including empty titanium metal implants, peptide-enriched silk fibroin-coated implants, and vancomycin nano-functionalized peptide-enriched silk fibroin-coated implants, provides a good basis for comparing the effects of different treatments. The study measures a range of inflammatory biomarkers, including NLR, PLR, MLR, PMR, SII, and IL-6, which are relevant to the development of osteomyelitis. The analysis of these biomarkers is performed using routine blood tests, which is a practical and clinically relevant approach. However, there are some limitations to the study. The sample size is relatively small, with only 54 rabbits used in each study. This may not provide sufficient statistical power to detect significant differences between the treatment groups. Additionally, the study only measures the inflammatory biomarkers at a single time point, without following the rabbits over time to see how these biomarkers change in response to treatment. Writing The writing is generally clear and concise, with good use of headings and subheadings to organize the text. The introduction provides a good background on the clinical significance of osteomyelitis and the importance of developing new diagnostic markers. However, there are some areas where the writing could be improved. The transitions between sections could be smoother, and some sentences could be rephrased for clarity. Additionally, the text could benefit from more precise language and fewer technical jargon. Structure The structure of the article is generally good, with a clear introduction that sets out the background and aims of the study. The methods section is well-organized and provides a clear description of the experimental design and procedures. The results section is well-organized and presents the data in a clear and concise manner. However, some of the tables could be improved by adding more detail or context to help readers understand the data. The discussion section is generally well-organized and provides a good summary of the main findings. However, some of the sections could be condensed or reorganized to improve the flow of the text. Usefulness of hematological reports: The study evaluates the usefulness of hematological reports derived from neutrophils, leukocytes, monocytes, and platelet formula regarding the evolution of implant-associated osteomyelitis and treatment evaluation. The discussion section highlights the importance of these inflammatory biomarkers in diagnosing and monitoring osteomyelitis. Longitudinal analysis of inflammatory markers: The study follows the inflammatory biomarkers (NLR, MLR, PLR, PMR, SII, and IL-6) over time to understand their response to different treatments and to identify potential changes in the chronic phase of osteomyelitis. The discussion section suggests that the longitudinal analysis of these markers is crucial for understanding the evolution of osteomyelitis. Comparison of different treatments: The study compares the response of inflammatory biomarkers to different treatments, including empty titanium metal implants, peptide-enriched silk fibroin-coated implants, and vancomycin nano-functionalized peptide-enriched silk fibroin-coated implants. The discussion section highlights the importance of comparing different treatments to evaluate their effectiveness in treating osteomyelitis. Role of platelets in inflammation: The study investigates the role of platelets in inflammation and suggests that platelet augmentation is important during acute and chronic osteomyelitis. However, some limitations of the study are mentioned: 1. Lack of standardized cutoff values: The study notes that threshold values for inflammatory markers have not been reported in animal models, especially in rabbits, and that further research is needed to establish standardized cutoff values. 2. Limitations of the study design: The study mentions that the absence of a test group undergoing surgery without bacterial infection may have impacted the data obtained during the acute phase of osteomyelitis. 3. Limited investigation of CRP and ESR: The study notes that CRP and ESR were not investigated, although they could corroborate the degree of inflammation detected through the analyzed parameters. Overall, the article presents a good balance of scientific rigor and methodology, with some areas where the writing could be improved. With some revisions to address these areas, the article could be strengthened further. ****** 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: Yes: Dr SeyedMousa Motavallihaghi ******** [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: PONE-D-24-02543.docx https://doi.org/10.1371/journal.pone.0309145.r001
Revision 1
7 Jun 2024 Author Response Dear editors and reviewers, Thank you for your suggestions, comments and support in publishing our article. We have now revised the manuscript and answered exactly what you requested, as follows: REV 1 1- In line 203, the selection of sampling days for hematological tests is based on what and how? The authors thank the reviewer for the interesting question. The selection of sampling days for hematological tests was based on different stages of the induced osteomyelitis, both acute and chronic. For acute osteomyelitis, blood samples were collected on days 0 (baseline), 1, 3, 7, and 14. For chronic osteomyelitis, samples were taken on days 0, 21, 43, and 60. These time points were chosen to capture the progression and resolution of the inflammatory response over time. 2. In line 213, what sample was used in the interleukin 6 assay? The authors thank the reviewer to notice the lack of information regarding the sample and measurement method used to test IL-6. The interleukin 6 (IL-6) assay was conducted using plasma samples obtained from the blood collected in Lithium-Heparin preconditioned tubes. 3. In line 213, What was the measurement method, type and brand of the kit used to measure interleukin 6? The measurement of interleukin 6 (IL-6) was performed using an enzyme-linked immunosorbent assay (ELISA) method. The specific type and brand of the kit used for measuring IL-6 was R&D Systems™ Rabbit IL-6 DuoSet ELISA (Fisher Scientific, Germany) and the manufacturer's instructions were followed for the processing of the samples. This information and the previous one have been added to the revised version of the manuscript at lines 178-184. REV 2 The study presents a good balance of scientific rigor and methodology. The use of a rabbit model of acute and chronic osteomyelitis induced by MRSE is a relevant and well-established model. The inclusion of different treatment groups, including empty titanium metal implants, peptide-enriched silk fibroin-coated implants, and vancomycin nano-functionalized peptide-enriched silk fibroin-coated implants, provides a good basis for comparing the effects of different treatments. The study measures a range of inflammatory biomarkers, including NLR, PLR, MLR, PMR, SII, and IL-6, which are relevant to the development of osteomyelitis. The analysis of these biomarkers is performed using routine blood tests, which is a practical and clinically relevant approach. Thank you for your thorough and insightful review. We appreciate your positive feedback on the scientific rigor, methodology, and relevance of our study, as well as your constructive comments on areas for improvement. Below are our responses to your specific points: However, there are some limitations to the study. The sample size is relatively small, with only 54 rabbits used in each study. This may not provide sufficient statistical power to detect significant differences between the treatment groups. Additionally, the study only measures the inflammatory biomarkers at a single time point, without following the rabbits over time to see how these biomarkers change in response to treatment. Sample Size and Statistical Power: We acknowledge that the sample size of 54 rabbits per study is relatively small. The sample size was calculated a priori through a two-sample t-test with α error = 0.05% and 80% power, ensuring that it is statistically sufficient to detect significant differences in this context. Future studies with larger sample sizes could provide more robust data and further validate our findings. Single Time Point Measurement: We appreciate the suggestion to follow the inflammatory biomarkers over time. In our study, we did measure the inflammatory biomarkers at multiple time points (days 0, 1, 3, 7, and 14 for acute osteomyelitis and days 0, 21, 43, and 60 for chronic osteomyelitis), as detailed in the manuscript. This longitudinal approach was intended to capture the dynamic changes in biomarkers in response to treatment over time. We will ensure that this aspect is more clearly highlighted in the revised manuscript. Writing The writing is generally clear and concise, with good use of headings and subheadings to organize the text. The introduction provides a good background on the clinical significance of osteomyelitis and the importance of developing new diagnostic markers. However, there are some areas where the writing could be improved. The transitions between sections could be smoother, and some sentences could be rephrased for clarity. Additionally, the text could benefit from more precise language and fewer technical jargon. The authors revised the manuscript to improve the transitions between sections, rephrase sentences (especially in the discussion section) for clarity, and reduce technical jargon where possible. Our goal is to make the text more accessible while maintaining scientific accuracy. Structure The structure of the article is generally good, with a clear introduction that sets out the background and aims of the study. The methods section is well-organized and provides a clear description of the experimental design and procedures. The results section is well-organized and presents the data in a clear and concise manner. However, some of the tables could be improved by adding more detail or context to help readers understand the data. The discussion section is generally well-organized and provides a good summary of the main findings. However, some of the sections could be condensed or reorganized to improve the flow of the text. According to the reviewer’s suggestion, the authors reorganized the discussion to improve the flow of the text. Usefulness of hematological reports: The study evaluates the usefulness of hematological reports derived from neutrophils, leukocytes, monocytes, and platelet formula regarding the evolution of implant-associated osteomyelitis and treatment evaluation. The discussion section highlights the importance of these inflammatory biomarkers in diagnosing and monitoring osteomyelitis. Longitudinal analysis of inflammatory markers: The study follows the inflammatory biomarkers (NLR, MLR, PLR, PMR, SII, and IL-6) over time to understand their response to different treatments and to identify potential changes in the chronic phase of osteomyelitis. The discussion section suggests that the longitudinal analysis of these markers is crucial for understanding the evolution of osteomyelitis. Comparison of different treatments: The study compares the response of inflammatory biomarkers to different treatments, including empty titanium metal implants, peptide-enriched silk fibroin-coated implants, and vancomycin nano-functionalized peptide-enriched silk fibroin-coated implants. The discussion section highlights the importance of comparing different treatments to evaluate their effectiveness in treating osteomyelitis. Role of platelets in inflammation: The study investigates the role of platelets in inflammation and suggests that platelet augmentation is important during acute and chronic osteomyelitis. However, some limitations of the study are mentioned: 1. Lack of standardized cutoff values: The study notes that threshold values for inflammatory markers have not been reported in animal models, especially in rabbits, and that further research is needed to establish standardized cutoff values. 2. Limitations of the study design: The study mentions that the absence of a test group undergoing surgery without bacterial infection may have impacted the data obtained during the acute phase of osteomyelitis. 3. Limited investigation of CRP and ESR: The study notes that CRP and ESR were not investigated, although they could corroborate the degree of inflammation detected through the analyzed parameters. Overall, the article presents a good balance of scientific rigor and methodology, with some areas where the writing could be improved. With some revisions to address these areas, the article could be strengthened further. The authors updated the manuscript to reflect the points mentioned above, ensuring a clearer presentation of our longitudinal analysis of inflammatory markers, improving the clarity and flow of the text, and adding more detailed context to our discussion section. We hope that the revised version of the manuscript could satisfy the reviewer but also readers’ requirements. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0309145.r002
7 Aug 2024 Decision Letter - Seyed Mostafa Hosseini, Editor The clinical significance of inflammatory biomarkers, IL-6 cytokine, and systemic immune inflammatory index in rabbit model of acute and chronic Methicillin-resistant Staphylococcus epidermidis-induced osteomyelitis PONE-D-24-02543R1 Dear Dr. Ancuța, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. 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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 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 #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes 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 #1: (No Response) 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 #1: Yes 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 #1: (No Response) Reviewer #2: (No 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: Yes: Pezhman Karami Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0309145.r003
Formally Accepted
19 Aug 2024 Acceptance Letter - Seyed Mostafa Hosseini, Editor PONE-D-24-02543R1 PLOS ONE Dear Dr. Ancuța, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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. Seyed Mostafa Hosseini Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0309145.r004

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