PONE-D-20-36680

Use of hyperbaric oxygen therapy for preventing delayed neurological sequelae in patients with carbon monoxide poisoning: a multicenter, prospective, observational study in Japan

PLOS ONE

Dear Dr. Fujita,

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.

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Tai-Heng Chen, M.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?

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: Yes

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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

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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 study by Dr. Fujita and his team is a case series that included 255 patients with CO poisoning of a total 311 initially registered. In the group of 255, 171 received hyperbaric oxygen (HBO2) 1-3 times in the first 24 hours; the remaining patients (84) received normobaric oxygen (NBO2). Delayed neurological sequelae (DNS) occurred in 7.6% of the HBO2 group and 3.6% of the NBO2 group. The authors conclude that HBO2 therapy has no advantage over NBO2 therapy for the prevention of DNS or improving prolonged impaired consciousness. Interestingly, a factor associated with the incidence of DNS in this study is the number of HBO2 sessions in the first 24 h, suggesting that more HBO2 may worsen outcome. The finding of this retrospective analysis is surprising given the opposite conclusion of several randomized, blinded studies with HBO2 administered at what are believed to be therapeutic doses (2.4-2.8 ATA). If correct, the current paper could be an important finding that may affect current practice.

This is a moderately large case series, of the size needed to address this important question, and the paper is easy to read. There are a few minor points that need to be addressed. In addition, the data do not adequately support the authors’ conclusions.

The authors have made an effort to elaborate the patients’ initial conditions. However there are important differences in the two groups that may have affected outcomes. For example, there were differences in the percentages of patients who lost consciousness (42.3% vs. 48.0%, NBO2 vs. HBO2), SpCO at the scene (26.4% vs. 30.1%). These differences were not statistically significant but that does not exclude a possible effect on outcome. In this case the authors used multivariable logistic regression to sort that our, however they need to state how they chose the variables that were in their regression model. It would be helpful to have a table that includes both univariable and multivariable risk factors for outcome.

Another important factor that suggests selection bias is that only 44% of patients in the NBO2 group were transferred to hospitals where HBO2 was available. It is therefore plausible that initial evaluation at those hospitals triaged to NBO2 those patients who were a priori expected to do well without HBO2, thus enhancing the outcome of the NBO2 group. It is also conceivably there may have been a selection bias on the part of the EMS to send less severely affected patients to NBO2 hospitals. Please could the authors comment. Among those patients who were referred to a hospital with hyperbaric facilities, it would be useful for information on the indications for treatment in that subgroup.

The fact that SpCO wat the scene was 26.4% (NBO2) and 30.1% (HBO2), yet despite long times between exposure and hospital arrival (average of around 4 half times if the patients were breathing oxygen), COHb was decreased only to 19.3% and 18.7% in the two groups. Please could the authors comment.

In addition for the potential for selection bias, it is also possible that the relatively low numbers of adverse outcomes indicates that the study is underpowered to detect a difference. DNS was assessed either during a hospital visit or by phone. The relatively low rate of sequelae in this study compared with previous reports (e.g. Gorman et al, Anaesth Intensive Care. 1992;20:311-6, refs. 1 and 4) may have been because phone assessment is less sensitive than formal neuropsychological testing).

There were differences between groups regarding CT findings, which should be described, along with information on abnormal MRI findings. Previous studies have observed imaging abnormalities as risk factors for DNS (e.g. Jeon, et al. JAMA Neurol. 2018;75:436-443). It would be helpful to include an analysis of the subset of patients with CT or MRI abnormalities should be included.

In any retrospective analysis it is impossible to account for therapeutic decisions made based upon real-time assessment of patients, for example the observation that a greater number of HBO2 sessions in the first 24 hours was associated with a greater incidence of DNS. From this, the authors conclude: “Therefore, multiple rounds of HBO2 therapy should be administered with caution, because it is possible that symptoms will worsen.” Of the two factors here (number of treatments and clinical outcome), it is important to consider which one is cause and which one is effect. Contrary to the authors’ conclusion, it is more plausible that additional HBO2 was administered to patients who were not responding well.

The following is an overstatement: “Moreover, Scheinkestel et al. [6] reported harmful effects of HBO2 therapy in patients suffering acute CO poisoning.” It is also noteworthy that while this study reported fewer sequelae with normobaric vs. hyperbaric oxygen, if their protocol is to be the basis for treating CO poisoning, then 3-6 days of inpatient treatment are required.

The following statement, “Experimental data have shown that HBO2 induces oxidative stress in healthy rat brains, measured as the lipid peroxidation products in brain cortex homogenates [24–26]” should be amended to include the observation that in CO poisoning HBO2 actually reduces oxidative stress (Thom SR. Toxicol Appl Pharmacol 1993;123:248-56). It is also of note that other animal studies of CO poisoning have revealed other beneficial effects of HBO2, including inhibition of leukocyte beta-2 integrins (reference 28), reversal of CO-cytochrome c oxidase binding (Adv Exp Med Biol 1989;248:747-54) and recovery of energy metabolism (J Clin Invest 1992;89: 666-72).

The review of the literature in Conclusions suggests that all studies of HBO2 are equivalent, which they are not. Raphael’s and Annane’s studies were performed at 2 ATA, which as the authors point out is considered by many experts to be inadequate. In Raphael’s study the only comparison between NBO2 and HBO2 was in mild cases. Scheinkestel’s study utilized 3-6 days of therapy, which is usually considered impractical. On the other hand, studies at 2.5-2.8 ATA by Thom, Mathieu and Weaver have shown better outcome for HBO2. These details need to be included explicitly in the Discussion. The following statement pertains to a meta-analysis, “A recent meta-analysis of the therapeutic effects of different numbers of HBO2 sessions found that HBO2 therapy was associated with a lower risk of memory impairment than NBO2 therapy, but that two sessions of HBO2 therapy were associated with a higher risk of memory impairment than one session [30].” It is noteworthy that this meta-analysis also assumed equivalence of the various doses.

Reviewer #2: Thank you for allowing me to review this study by Motoki Fujita et al. I previously reviewed this manuscript for another journal. I feel like the manuscript is much improved from the prior version I reviewed.

The question of hyperbaric oxygen therapy efficacy to prevent delayed neurological sequelae in CO poisoning is very important. There has been much controversy over the topic. As the manuscript refers to, probably the best designed study - reported in Weaver et al NEJM 2002 - showed quite a positive benefit of using HBO2 in CO poisoning patients. When looking at the most up-to-date meta-analysis, however, combined, 6 studies show no benefit. The most recent one - performed in 2011 - actually showed some trend towards harm.

Regardless of the stance on HBO2 therapy effectiveness, even presuming it is effective, time delays to therapy (>5 hours in the Weaver study) could certainly limit the benefit of the therapy. Many centers do not offer emergent HBO2. Further studies in this situation are critical.

The authors present a prospective, multi-center clinical study on the use of HBO2 in acutely CO poisoned patients. This is valuable and a fairly large size.

There are some major limitations to the study in its design that could limit further applicability – this was not a randomized trial, there was not set HBO2 protocol (and many patients received non standard therapy previously tested it appears), and the definition of neurocognitive deficits was a bit murky. That said, this was an interesting study that is important to publish.

I am going to reveal my prior critiques and comment on responsiveness to those. I have some additional comments based on the new manuscript.

Prior review:

Major Issues:

1) How did providers determine who was to receive HBO2 therapy? This is not specified. I think if it was up to the provider, more analysis of the decisions/trends of the patients need to be identified. Did they use a general criteria for the trial? Did they go with a professional organization recommendation? The American College of Emergency Physicians does not provide specific recommendations, of note.

-> There was quite a bit of discussion around how many patients received what therapy. It was quite non standard but it was sufficiently reported here. Some were very strange (15 HBO2 sessions over a week), so not sure how to interpret these.

2) Mechanically ventilated patients were not included in the analysis. This is a major limitation. By my calculation, only 4.6% of the HBO2 patients required mechanical ventilation, whereas, 19.5% of the NBO2 group required mechanical ventilation. These patients were then not analyzed. This is a major limitation as these would be the most severely ill patients. Ideally, there is some sensitivity analysis that would include these patients. This may be why the delayed neurological sequelae are so low in the NBO2 group - the sickest were eliminated (19.5%). I do agree to not include patients that had an arrest upon arrival for this study.

-> The authors included mechanical ventilation patients in the analysis. This was a great improvement in the study and makes it much more applicable.

3) The definition of DNS is very vague. Page 7 refers to patients were "checked by a physician if any doubt". What does this mean? This needs to be listed very specifically how DNS was determined as it is a primary outcome of the study. If some of the list or discussion of the DNS determination algorithm needs to be in supplemental materials, that is fine. The exact method needs to be reported.

-> Clearer algorithm certainly. Still some limits. Is the screening questions asked ever been standardized or validated previously? With the 4x mental status exams, did the patient need to have an abnormality on any one of these or multiple tests? If it is only one deficit on a single test this could be a biased definition. The opposite is true as well – if very strict on defining (for instance, patients had to have all 4 with a deficit, then could be biased the other way (not reporting the DNS enough). I would clarify. This is much improved already though.

4) No reports on mortality in this group. I think it would be helpful. The size of the study and exclusion of cardiac arrest upon arrival patients does not warrant this being an outcome measure; however, the numbers should be reported.

-> better commentary on mortality overall

5) Exposure distribution was unique - the most common causes of CO poisoning are generally motor vehicle exhaust and fires. Here, >50% of patients were exposed to burning charcoal. Is this the most common form of poisoning in Japan? This should be discussed more. Related, a lot more of the NBO2 group was exposed to fire, generally felt as the most severe form of exposure. This would provide that the effect seen is even more surprising.

-> much clearer. I would use term “intentional” not “suicide”. For the statistical comparison, I would do statistics for each exposure type (fire, exhaust, coal) instead of exposure in general.

6) Why were the DNS events so low. Most reported prevalence is between 20-40% long term. Why was there only one DNS event in the NBO2 group? Is this because mechanical ventilated patients are excluded from the analysis? Again, I think this data needs to be reported at least in a sensitivity analysis if available. Else, should be listed as a major limitation.

-> Now addressed mechanical ventilation issue. Still quite low though. Any reason why you suspect? Perhaps points above about definition of DNS would be helpful to learn.

7) Why was the PaO2 higher in the HBO2 group? When was this lab taken? Was it prior to HBO2 initiation?

-> This was discussed a bit. Not sure why it is higher but not sure clinical meaningfulness.

8) The issue of heterogeneity of therapy needs to be addressed as a major limitation - no standard intervention, multiple different pressures/number of hyperbaric treatments for the patients.

-> this was well discussed.

Applicable Minor issues:

4) 35% of facilities did not have access to HBO2 - this is actually a high percentage of HBO2 capable facilities. The US only has 250-350 nationwide. Is there a lot more hyperbaric centers in Japan or is it more that centers with HBO2 were more likely to enroll in this study? Is there data for how many centers in Japan?

-> related, I would include how many patients required intrafacility transfer for HBO2.

6) Page 10, what does "period of oxygen administration" mean? I am confused by this term. Does that mean time before presentation to hospital on oxygen?

-> I am still not sure about this term. It is clearer how it is used now, but I think general length of stay in hospital is more meaningful. Could probably drop it. We can presume most of the patients received oxygen I think.

7) When discussing controversy around HBO2 being effective or not, I would include the discussion of some of the large retrospective studies that suggest mortality or ADL benefit to HBO2:

a. Rose JJ, Nouraie M, Gauthier MC, et al. Clinical Outcomes and Mortality Impact of Hyperbaric Oxygen Therapy in Patients With Carbon Monoxide Poisoning. Crit Care Med. 2018;46(7):e649-e655. doi:10.1097/CCM.0000000000003135

b. Nakajima M, Aso S, Matsui H, Fushimi K, Yasunaga H. Hyperbaric oxygen therapy and mortality from carbon monoxide poisoning: A nationwide observational study. Am J Emerg Med. 2020;38(2):225-230. doi:10.1016/j.ajem.2019.02.009

-> I would still include this

Additional comments:

1) CK-MB is used for cardiac involvement. What was your cut off? How many had “High” values? Do you have troponin data available?

2) What were the ECG abnormalities seen? 20-22% incidence is quite high.

3) What were the CT abnormalities seen? Head CT or Chest CT?

4) What were MRI abnormalities seen, quite a few in the HBO2 group.

5) Not sure you need a paragraph describing vital signs, we can read table

6) Why was there bed rest prescribed and what is the meaning of it? I did not think we prescribe this in clinical care anymore very much.

7) Give % mortality of overall cohort (included and excluded) when you report the number I think.

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Reviewer #1: Yes: Richard Moon

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.]

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PONE-D-20-36680R1

Use of hyperbaric oxygen therapy for preventing delayed neurological sequelae in patients with carbon monoxide poisoning: a multicenter, prospective, observational study in Japan

PLOS ONE

Dear Dr. Fujita,

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 Jun 23 2021 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: http://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,

Tai-Heng Chen, M.D.

Academic Editor

PLOS ONE

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)

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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

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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: Dr. Fujita and his team have responded appropriately to many of the reviewers’ comments, but several concerns remain.

Lines 325-338: the authors are attempting to make the point that HBO2-induced oxidative stress could the cause of worse outcome in the HBO2 group. In favor of this hypothesis they cite evidence that HBO2 induces oxidative stress. Of course it does. However, so does carbon monoxide exposure, and published evidence indicates that HBO2 reduces it (reference 26). The conclusion in lines 335-338 therefore needs to be amended.

Lines 350-354: “A recent meta-analysis of the therapeutic effects of different numbers of HBO2 sessions, including a different range of therapeutic pressures from 2.0 to 2.8 ATA, found that HBO2 therapy was associated with a lower risk of memory impairment than NBO2 therapy, but that two HBO2 sessions was associated with a higher risk of memory impairment than one session [33].” This particular conclusion of Wang’s meta-analysis is based upon two studies (Raphael and Annane) in which sub-therapeutic HBO2 doses were used, and both mild and moderate sequelae (including memory impairment) were defined only by a patient questionnaire (no formal testing of memory); only 4 patients out of 170 had severe sequelae defined as objective abnormalities. All studies using HBO2 at 2.0 ATA have shown no benefit, while all studies using HBO2 at 2.8-3.0 ATA have shown benefit. Please add a caveat to this discussion.

There were 171 patients in the HBO2 group, however the number who received a first HBO2 treatment (Table 2) totaled only 165. Please explain.

From the previous review: It would be helpful to include an analysis of the subset of patients with CT or MRI abnormalities. What were the outcomes for those individuals with brain abnormalities on either CT or MRI?

The following points need to be addressed in a ‘Shortcomings’ section after the Discussion.

(1). This is not a randomized study, therefore, as pointed out in the previous review, the possibility of selection bias is large, both at the EMS level and within hospitals where HBO2 was available. Indeed, there is evidence that those patients who were treated with hyperbaric oxygen (HBO2) were more severely affected than those who were not. In addition to differences pointed out in the previous review (percentages of patients who lost consciousness, SpCO at the scene), the percentage of patients with abnormal CT, basal ganglia lesions) and MRI (basal ganglia lesions) was higher in the HBO2 group. The lack of statistical significance does not exclude a clinical relationship, particularly since fewer than 60% of patients who received HBO2 in this series were treated at a pressure considered to be therapeutic (≥2.4 ATA).

(2). There is no evidence in the manuscript that the assessors were blinded as to the treatment group (NBO2 vs. HBO2).

(3). An apparent dose-response between number of HBO2 treatments and incidence of delayed neurological sequelae (DNS) is incorrectly interpreted as evidence of possible harm due to HBO2. There is no statistical way to control retrospectively for a clinical observation related to improvement/lack of improvement during therapy. For example, a study of seriously ill infected patients might reveal a relationship between duration of antibiotic therapy and mortality. However on this basis it would be incorrect to conclude that antibiotic therapy is harmful; rather, longer antibiotic therapy was most likely prescribed because of lower clinical response to therapy in more severe infections.

(4). The relatively low statistical power to exclude an effect given the low rate of DNS and the fact that that many patients were not formally tested (the numbers are not stated) and 16% were lost to follow-up.

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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: Richard Moon

[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.

Use of hyperbaric oxygen therapy for preventing delayed neurological sequelae in patients with carbon monoxide poisoning: a multicenter, prospective, observational study in Japan

PONE-D-20-36680R2

Dear Dr. Fujita,

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.

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Kind regards,

Tai-Heng Chen, M.D.

Academic Editor

PLOS ONE

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: No

**********

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: Dr. Fujita and colleagues have done a tremendous amount of work in this study, however modifications are still needed to communicate the differences between this retrospective study and a randomized, blinded prospective study.

The following paragraph has been edited by the authors, but in a way to focus on possible adverse effects of oxidative stress due to administration of hyperbaric oxygen for CO poisoning (lines 334-346):

“Oxidative stress is a key mechanism in DNS [20–25]…Although HBO2 therapy has beneficial effects, it should be considered that there are concerns about adverse effects of HBO2 therapy such as HBO2-induced oxidative stress.”

However, the biochemical data from animals and humans overwhelmingly point to a reduction in oxidative stress in CO poisoning after hyperbaric oxygen. A more appropriate wording of that paragraph would be:

“Oxidative stress is a key mechanism in DNS [20–25]. However, there have been reports that HBO2 therapy itself induces oxidative stress [29–32]. Experimental data have shown that HBO2 induces oxidative stress in healthy rat brains, measured as the lipid peroxidation products in brain cortex homogenates [29–31]. This HBO2-induced oxidative stress is related to the HBO2 pressure [29] or the exposure time [30]. It has also been reported that a 342 single session of HBO2 (2.4 kPa, 131 min) reduced plasma vitamin C and increased plasma lipid peroxides and urinary 8-oxo-deoxyguanosine excretion in healthy volunteers [32]. Although there are concerns about adverse effects of HBO2 therapy such as HBO2-induced oxidative stress, measurement of oxidative stress in animal models of CO poisoning has demonstrated reduced lipid peroxidation [26], inhibition of leukocyte beta-2 integrins [18], reversal of CO-cytochrome c oxidase binding [27] and recovery of energy metabolism [28]. It should also be noted that markers of oxidative stress in humans poisoned by carbon monoxide are reduced after treatment (https://pubmed.ncbi.nlm.nih.gov/21424975/).”

Lines 364-368 have been edited to address the issue of selection bias: “Therefore, multiple HBO2 sessions with insufficient therapeutic pressure should be administered cautiously because of the possibility of worsening symptoms. However, the present data could not rule out the possibility that more severely affected patients had received more HBO2 sessions because the HBO2 therapy protocols were not consistent and depended on each institutions’ policies [8].” The manuscript should also note that a randomized blinded study of three hyperbaric oxygen treatments vs. one revealed no evidence of increased adverse outcomes (page 155 of Weaver LK. Carbon monoxide poisoning. Undersea Hyperb Med 2020;47(1):151-69).

Lines 439-441: “There were several issues with assessing DNS, including non-blinded evaluators, 13.8% of loss of follow-up, and the possibility of oversight of patients with mild symptoms.” In the manuscript it is stated that 41 patients were lost to follow-up (lines 406, 409). The total number of patients is 255 (Table 1), thus percentage lost is not 13.8% but 16.1%.

The shortcomings section should be formatted per the prior review. Although some of the shortcomings were stated earlier in the manuscript, the reader expects a complete list in the Shortcomings paragraph. It is also not clear what is meant by, “the possibility of oversight of patients with mild symptoms”.

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Reviewer #1: Yes: Richard Moon