Dear Dr. Schuelke,

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Mohammed Misbah Ul Haq, Pharm-D

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: No

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

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Reviewer #1: The study hypothesized that “the use of carbogen (5 % CO2 plus 95 % oxygen) in the home environment would be an effective and safe treatment for recurrent febrile seizures”. It used RCT design to test the abovementioned hypothesis.

Here are my comments on the manuscript

1) The primary outcome measure (Seizure-cessation efficacy endpoint) was measured within three minutes of medication application, but the authors did not justify choosing a three-minute window. Why three minutes and not say two or any time greater than 3 minutes? This explanation must be provided in the manuscript. What would have been the implication if the outcome was measured at different time windows??

2) Please include the measurement scales of the secondary outcome measures (binary, count, multinomial, etc.).

3) The section titled statistical analysis is a bit confusing. The content is more of a power analysis (sample size estimation). There should be a section titled Power Analysis to capture the details of sample size estimation, and another section titled Statistical Analysis to highlight the statistical methods used in analysing the data and generating the results in the manuscript.

4) It also does not explicitly state the statistical method(s) that was/were used in quantifying or assessing the relation between the primary outcome measure (Seizure cessation) and the secondary endpoint and the use of carbogen compared to control-100% oxygen. These must be clearly stated

5) In Table 1, the authors indicated, “Age at febrile seizure, median [months]” but rather reported the mean and standard deviation instead of the median and interquartile range. Once again, the t-test does not test the median difference. In addition, normality of the outcome measure required to test mean of a distribution was not assessed and reported

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Reviewer #1: No

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Dear Dr. Schuelke,

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 Sep 27 2025 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.

• 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 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,

Dr. Mohammed Misbah Ul Haq, Pharm-D

Academic Editor

PLOS ONE

Journal Requirements:

1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 

2. 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.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: The authors have addressed all my previous comments and the content of the revised manuscript has improved significantly

Reviewer #2: The use of carbogen for the interruption of febrile seizures - the randomized controlled CARDIF trial

Summary

The authors did a clinical trial based on animal studies. They targeted one of the most common pediatric diseases, which has significant long-term and short-term complications. The study also aligns with other promising studies being conducted to assess the effectiveness of carbogen in treating absence seizures and status epilepticus. They clearly outlined the rationale for doing the study.

Questions

1. What proportion of the patients in your study had complex febrile seizures, and what percentage had simple febrile seizures?

2. Using parent-reported seizure duration, benzodiazepine use, and any serious adverse events is understandable, but wouldn't it be better to also add home video recording since at times patients can have a seizure attack but the parents may not notice it if they are not around, or if they are sleeping.

3. You need to add to the manuscript why the 5% carbondioxide and 95% oxygen are used. What were your reasons to avoid 7%/93% or other proportions?

4. When the low-pressure can was used, what precautions were taken to avoid rebreathing? The reason that I asked this is that even if the carbogen composition is 5% CO2 and 95% O2, the expiratory air has 4% CO2 and 16% O2 composition, which can affect the results of your study.

5. What the caregivers were advised, they might forget, so giving written advice will be beneficial. Was written instruction given for the caregivers?

6. How can we reliably say that the abnormal body movement the child has at home is a febrile seizure? What if the child is having shivering, and the parents think it is a seizure? What if the child is not having a fever at the time of the seizure that occurred at home? What if the child is having a central nervous system infection?

7. What if the child has seizures and the family didn't report? How do you make sure that the families report and apply the carbogen or 100% oxygen whenever their child has a febrile seizure?

8. How is the safety profile of the equipment? Is there a possibility that the equipment is malfunctioning and stopping air flow, which can potentially lead to rebreathing or suffocation?

9. In line 106, you stated that the study was done between 2012 and 2015. What was the reason for not submitting the result and conclusion earlier?

10. What was the reason for making the face mask loose-fitting?

Comments

The conclusion is not well suited for the results and the discussion of the study. A better conclusion would be: The study didn't show any superior benefit of carbogen over pure oxygen in ceasing febrile seizures within 3 minutes. There are possible different reasons that might mask the benefit of carbogen, such as the alkalosis reversal was not confirmed with blood gas analysis, diagnosing febrile seizure can be difficult for parents, and knowing when the seizure has stopped is difficult for the parents.

Reviewer #3: Review to paper “The use of carbogen for interruption of febrile seizures - the randomized controlled CARDIF trial”

As a general comment, I want to say that this work meets all the criteria I was asked to evaluate. It is original, unpublished, and carried out with a high level of technical rigor, with the methods, analyses, and conclusions clearly described and supported by the data. The paper is well written, ethically sound, and follows the expected reporting standards. More importantly, I found it both relevant and informative, and I truly appreciate how creative and thorough the team has been in tackling the complex logistics, preparation, and follow-up for such a challenging study. Even as an interim analysis, the findings have real potential to move the field forward. However, I do have some minor and a few major comments that I believe could be of value to the authors.

Minor comments

1. In the abstract, on page 3, lines 43–44, it is not clear whether you are referring to the number of children or the number of febrile seizures: “The febrile seizure was terminated in 5/15 children on carbogen and in 8/11 children on oxygen.” I understood that the study medication was used in n = 30 febrile seizures (15 episodes in children within the verum arm, 11 episodes in children within the placebo arm, and 4 episodes in children within the verum open arm), in a total of 20 children. Check whether this could be clarified.

2. On page 6, line 113, the sentence could be improved because it contains two actions (“have been published” and “are added”) that are not connected by a linking word such as “and” or “which.”

3. In Table 1, page 9, line 233, the number of patients in the verum arm indicates that 32 [69.6%] have one FS in history and 15 [32.6%] have more than one FS in history, which does not add up to 46. Verify if this is correct, since cases per gender indeed add up to 46.

4. It would be stronger and clearer if, in the Methods section corresponding to “Randomisation and allocation,” the final number of patients randomised and followed, as well as those with recurrent febrile seizures, were stated. Additionally, it would be beneficial to refer to the CONSORT flowchart provided.

5. The section on power analysis, in my opinion, should not be part of the statistical analysis since it is mostly related to sample size determination, either as part of the explanation on how a desired sample size was decided or to contextualize the limitations faced during the interim phase of the clinical trial. It is not an actual analysis of the data obtained. An easy solution would be to put it as an individual subsection within the Methods.

6. What is the reason for carrying out a conventional chi-squared test instead of a Fisher’s exact test? You have a small sample size; usually, Fisher’s exact test works better under such conditions (small sample size or when any expected cell count is <5). However, looking at your results, it would not make much of a difference. However, since you actually followed that kind of reasoning when you applied the Mann–Whitney U test, you should consider this.

7. When comparing the patients in the crossover part of the study (6 patients in each group), the reasoning behind using a statistical test (regardless of whether it is a chi-squared test or a Mann–Whitney U test) is even more confusing. In my opinion, it is not worth relying on a formal null-hypothesis test with n = 6 vs. 6. You can run one, but the result will be practically uninterpretable and may mislead readers. It would be better to treat the data as descriptive/exploratory and focus on effect sizes, exact confidence intervals, and raw data. Formal testing is not useful here since the expected power is extremely low for any but enormous effects, so a nonsignificant p-value mostly reflects small n, not “no effect.” You already followed this logic in Table 2.

8. You include in your paper the CONSORT flowchart for participation; however, in no other place in the paper do you mention adherence to the CONSORT guidelines. If you adhered to any reporting guidelines, and also to guidelines for designing the trial (or good practice guidelines), you should mention it.

Major comments

1. In relation to how the hypothesis is declared

I have some issues with the way the hypothesis is stated (page 6, lines 117–119), especially from a reader’s standpoint before reading the rest of the Methods section.

You stated: “…Six liters of carbogen gas administered over a period of 3 minutes through a low-pressure can with an attached loose-fitting face mask is safe and more effective than placebo (100% oxygen) in interrupting recurrences of febrile seizures…”

I assumed, based on what you explained throughout the paper, that the intention is to communicate that you want to assess whether the intervention stops the seizure during the 3-minute window while receiving the intervention.

However, in my opinion, readers could understand that the intention is to communicate that the intervention’s goal is to prevent another seizure episode after receiving the intervention (3 minutes’ exposure to carbogen). The wording “interrupting recurrence” may imply this. If you say that the outcome of interest is “interrupting recurrences of febrile seizures” to a clinician, it would usually be understood as preventing another seizure from happening after the episode under treatment.

For the reader, this could mean both stopping the ongoing seizure and giving a treatment that reduces the likelihood of subsequent seizures. I am sure your intention is to communicate as the outcome “stopping of an ongoing seizure” rather than preventing a new one; in that case, you are measuring termination.

I think readers would benefit from a clearer statement of the hypothesis, similar to how the aim is stated: to determine whether 6 liters of carbogen gas (5% carbon dioxide + 95% oxygen) delivered from a low-pressure can would suppress acute febrile seizures (page X, lines 102–104).

2. In relation to the power analysis

If, in the most conservative scenario, you expected a power of 86% for a difference of 50% (75% vs. 25%), then when, in the preliminary phase, you obtained 15 episodes in one arm and 11 in the other, that would indeed impact power. This should be addressed either in the power analysis section (my recommendation) or somewhere in the Results (as a limitation within the interim results).

3. In relation to reason for not observing the expected effect

In the Discussion, you are right to discuss the reasons why you did not observe a similar effect as previously reported by small hospital-based studies or animal studies, and I agree with you that difficulties in the administration of carbogen are a major determinant of not achieving proper exposure under such a critical situation. However, you should briefly discuss the possibility that carbogen (with 5% CO₂) is not actually effective in children. Is there any reason you may think this could be possible?

Steven N. Cuadra

Steven.cuadra@gmail.com

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes:  Steven Napoleón Cuadra

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The use of carbogen for interruption of febrile seizures - the randomized controlled CARDIF trial

PONE-D-25-19281R2

Dear Dr. Schuelke,

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Kind regards,

Dr. Mohammed Misbah Ul Haq, Pharm-D

Academic Editor

PLOS ONE

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