PONE-D-23-38498COVIVA: Effect of transcutaneous auricular vagal nerve stimulation on the fatigue-syndrome in patients with Long Covid – a placebo-controlled pilot study protocolPLOS ONE

Dear Dr. Gierthmuehlen,

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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Kind regards,

Mohammadreza Pourahmadi, PT, Ph.D., Postdoctoral Fellow

Academic Editor

PLOS ONE

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3. Thank you for stating the following in the Competing Interests section: "MG is founder and advisor of Neuroloop GmbH, a start-up investigating invasive vagal nerve stimulation against arterial hypertension. PG received grants from Ivoclar Vivadent, Straumann, Vita Zahnfabrik und Deutsche Forschungsgemeinschaft DFG."

We note that one or more of the authors are employed by a commercial company: Neuroloop GmbH

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

Reviewer #2: Partly

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2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Partly

Reviewer #2: Yes

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3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

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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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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

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(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Thank you for the opportunity to review this paper. This is an interesting manuscript presenting a protocol of placebo-controlled pilot study on effect of transcutaneous auricular vagal nerve stimulation on the fatigue syndrome. The primary objective of this study is to investigate the effect of taVNS on patients with long Covid fatigue syndrome.

My review mainly concerns only the statistical aspects of the study. Some questions reported below were raised and in my view, it is not acceptable in this form for the publication in this journal.

1. The sample size justification reported is incomplete in relation to primary outcame. Is not indicate the primary end-point variable on which the estimation is evaluated; the common SD estimated is not reported;

2. In the statistical analysis paragraph should be more detailed the model applied for the analysis with relative post-hoc analysis;

3. There are some error of typing. Please, verify the manuscript for English and typos.

Reviewer #2: Summary

This paper presents the COVIVA study protocol, which aims to evaluate the feasibility of transcutaneous auricular vagal nerve stimulation (taVNS) as a treatment for patients with long covid syndrome and its potential to alleviate fatigue. The description of the planned study is informative but requires additional details and justification to ensure reproducibility. For example, the statistical test employed for the sample size calculation and the theoretical effect size underlying this calculation need to be clearly outlined. The findings of this study have the potential to guide the development of taVNS as a treatment for patients with Long-COVID syndrome. Therefore, this protocol is worthy of publication, provided that the methods and materials are thoroughly revised and my major comments are adequately addressed.

Major comments

1. The theoretical foundation of developing taVNS as a treatment for covid- associated fatigue in this study is its anti-inflammatory effect. While the anti-inflammatory effect of taVNS is well-established, it remains unclear whether taVNS alleviates fatigue through the anti-inflammatory reflex. To address this, researchers must examine the relationship between fatigue and inflammatory cytokines, which is not the case in reference [15], [35] and [36]. Note that reference [10] states that elevated IL-10 and IL-6 are important at the early stage but do not affect persistent fatigue once the fatigue syndrome is established. Another study has attempted to investigate whether taVNS improves fatigue through modulating inflammatory markers but found no correlation between fatigue and proinflammatory cytokines (please take a look at their results and discussion https://www.neuromodulationjournal.org/article/S1094-7159(22)01263-6/fulltext ) They stated “it has been established that VNS including nVNS modulates EEG microstates and brain activity”, suggesting VNS improving fatigue might be a central mechanism. This is supported by a behavioral and physiological study of the effect of taVNS on fatigue (https://www.brainstimjrnl.com/article/S1935-861X(24)00060-3/fulltext ), which found that the taVNS modulates the arousal pathway and rescue fatigue at the later stage of memory tasks. This is a more direct evidence to support the use of taVNS in patients with long-covid syndrome in comparison to studies about its anti-inflammatory effects.

In the current manuscript, the link between fatigue and inflammation after COVID-19 infection and the anti-inflammation effects of taVNS is missing. I suggest that the author introduces taVNS as a potential intervention after line 84, The introduction of taVNS should include its history (current line 98 - 110) and current knowledge about its positive effects on fatigue such as the results from the aforementioned studies. The authors should then elaborate on the potential mechanism of fatigue and the mechanism of taVNS. Please verify if the inflammation persist months after the infection of COVID-19 potentially leading to fatigue because intuitively, the inflammation might disappear when the patient recovered from COVID-19. Again, based on the current literature, the effect of taVNS on neuroepinepherine is more likely to be the mechanism for its positive effect on fatigue, if there is any.

The authors state, “Due to the current pandemic, further research on the causes of fatigue has not yet been conducted, and the underlying cause of fatigue remains inconclusive.” The current flow of the introduction implies that this protocol will test whether taVNS can mitigate fatigue through the regulation of pro-inflammatory cytokines, which is not the case. Please restructure the introduction to focus more on the effects of taVNS on fatigue and its feasibility. For instance, what is the significance of establishing the feasibility of taVNS in patients with long-COVID syndrome? Does taVNS need to be administered for a specific duration to treat fatigue?

Please avoid statements such as “In summary, vagus nerve stimulation appears to have the potential to reduce the occurrence of fatigue and depression in long Covid syndrome through the reduction of cytokines, or treat these symptoms.”

2. More details about the methods need to be provided. For example, Line 130. Fatigue persisting for >= 3 months. Is it a subjective report of fatigue? Please introduce the fatigue scale in the introduction. In the exclusion criteria, how to define acute Covid infection. 14 days within the infection? What is Vagus nerve stimulation (line 139) in the exclusion criteria? Do you mean having done a surgical procedure for implanting the cervical VNS device? Please make the inclusion/exclusion criteria as objective as possible. I suggest consolidating Questionnaires: and endpoints (line 167) into one section: primary outcomes.

One of the outcomes is average stimulation duration. Is the total stimulation duration controlled? Will the patient receive instructions about the stimulation duration? Second outcome is MF120, as this is the metric that the author will use to calculate sample sizes. Please clearly state that analysis of other questionnaires is considered exploratory analysis, and the exploratory result should not be used to draw conclusion once the study is completed.

The sample size calculation needs to be more specific. To calculate the sample size, one will need to define the test that will be used, the power that needs to be reached, and the theoretical effect size. Line 202 “The group size is based on the aforementioned studies on taVNS in other forms of fatigue 202 (29,36,37), our aforementioned FatiVa study (38),” Simply using the number of patients of previous studies is not a scientific way for defining sample size because previous studies can be under-powered. “The threshold for the p-value is an alpha of 0.05,” which can be called the significance level.

Line 213 “Based on the expectation of a superiority

213 of the two target groups (low-threshold and high-threshold stimulation) of at least 5 and 10 MFI

214 units, respectively, compared to placebo (shift), and a type I error of alpha = 0.05, a total of 3

215 x 12 cases = 36 cases are required to achieve a study power of 80% in the most important

216 secondary endpoint, the MFI20 questionnaire. The” What is the statistic test that will be used? Is there any evidence for the expectation of 5-10 MFI units?

Could you explain what successful acceptance is and how it will be used? “Successful acceptance and compliance are achieved when the acceptance criteria, as defined

228 in the primary endpoint, are met by at least 80% of patients.” What is the acceptance criteria? Do you mean if more than 80% of patients use taVNS for more than 4 hours, then taVNS is feasible?

Line 264, it seems that this study includes a titration of intensity. Please state how the investigator will reduce the intensity from the initial setting, for example, with a decrement of 5 units when the patient reports feeling the stimulation. How do you ensure that the Verum -1 group uses the predefined stimulation intensity?

In study plan, the study started last year. Have any patients been recruited? Is there any preliminary data that can be used to estimate the effect size?

3. In conflict of interest, as “MG is founder and advisor of Neuroloop GmbH,” The authors need to state whether this company will manufacture products to reduce fatigue in patients with long-covid syndrome.

4. In the discussion section, fatigue is stated as one of the side effects of taVNS in a meta-analysis published in a scientific report. Please discuss whether this side effect will confound the analysis.

5. Please verify if cymba conchae, rather than tragus, is most innervated by the auricular branch of the vagus nerve. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7083568/). In reference [22], it seems that they also indicates that tragus is partly innervated by the auricular branch of the vagus nerve. The authors might need additional evidence to support stimulation of aVN through tragus, for example, tragus is easier to access.

Minor comments

1. Abstract, first sentence, I understand this data comes from study. But to provide a background for this protocol, I suggest that the authors state for example, estimated number of patients who is currently having long covid syndrome.

2. Page 3, line 70, “In addition to fatigue (58%), these patients experience headaches

(44%), anosmia, anxiety (13%), and depression (12%). Please specify the denominator used to calculate these percentages.

3. Key message, page 3, is the key message section required to be published in PLOS ONE? Key messages in the manuscript seem to be a shorter abstract. The manuscript without the key messages is complete.

4. Please rephrase sentences 81 – 83; for example, ‘fatigue can continue to affect these patients months after recovery from the infection.’

5. The use of abbreviations needs to be consistent. For example, line 115 is transcutaneous VNS taVNS?

6. What does Verum stand for?

7. Change references into English. Are the journal name and publication date in Germany?

8. Optional: provide instruction to the three groups of patients as supplementary material.

9. Lin 240, please revise “the effect between 239 suprathreshold and subthreshold

10. 240 stimulation will be investigated using two Verum groups.”

11. What is CE in “CE-certified” Line 247

12. In PLOS ONE protocol guideline, the authors would need to state how data saturation will be determined. Also, data availability statement is missing, please check PLOS ONE DATA policy.

13. I recommend registering this study on a research such as OSF.

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Reviewer #1: No

Reviewer #2: Yes:  Gansheng Tan

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PONE-D-23-38498R1COVIVA: Effect of transcutaneous auricular vagal nerve stimulation on fatigue-syndrome in patients with Long Covid – a placebo-controlled pilot study protocolPLOS ONE

Dear Dr. Gierthmuehlen,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

The manuscript has significantly improved, and I appreciate the authors' efforts. However, the statistical section still requires further revision before the article can be accepted.

==============================

Please submit your revised manuscript by Dec 20 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 . 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,

Mohammadreza Pourahmadi, PT, Ph.D., Postdoctoral Fellow

Academic Editor

PLOS ONE

Journal Requirements:

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

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #2: Yes

Reviewer #3: Yes

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2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #2: Partly

Reviewer #3: Yes

**********

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #2: Yes

Reviewer #3: Yes

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4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The manuscript, which has been improved in the current revised version, describes a study protocol investigating the feasibility and effect of applying taVNS in Long Covid patients. Most of my previous questions have been addressed. However, the section on statistical analysis, including the sample size calculation and hypothesis testing, requires further revision. Below are my detailed comments for review.

1. Line 199, Data analysis (hypothesis testing) should be separated from sample size calculation.

2. Line 211, the word ‘upgraded’ is ambiguous.

3. Line 206, I believe that general readers will understand if p-value < threshold means that the null hypothesis is rejected. If the authors want to make this statement specific, please correct ‘test value’ to ‘p-value’ and state explicitly what the null hypothesis is. Alsom, Line 203, will the author perform two tests on the primary outcome and ranked judgment scales?

4. Line 209, the treatment groups will be descriptively compared with the placebo group? This contradicts the previous statement that statistical tests will be used to compare (the primary outcome) between two groups. What are the two groups?

5. P219, I assume the calculation has been performed in R and will not change in the future.

6. Line 220, ‘analyzed descriptively using the same statistical test,’ is not clear. Does it mean the conclusion of this proposed study will not be drawn based on BDI, SF-36, and PCS-score? Are these comparisons only for generating hypotheses for future clinical trials? The authors should be cautious when making this statement as it will restrict what will be included in future publications.

7. Line 254, ‘Furthermore, no adverse effects associated with stimulation 255 durations exceeding 4 h/day will be reported.’ Do the authors only consider adverse effects occurring within the 4 hours after the stimulation?

Reviewer #3: Line numbers in track-changes revised manuscript

Line 52: What does “implantation of a non-functional electrode” refer to? Implantation implies invasive device (yours is not).

Line 106: Would consider addressing the disparity between your statement “Existing research indicates that fatigue is partly triggered by increased levels of pro-inflammatory hormones known as cytokines in the blood, while its severity seems to correlate with the levels of these cytokines in the initial phase of the disease” with the fact that your enrolled patients are not in the acute phase of the disease. This implies prevention of chronic-fatigue would be better tackled by initiating therapy early in the acute setting of the disease to take advantage of the anti-inflammatory effect of taVNS.

Line 286: Do you mean that adverse events have not been previously reported? Would consider reviewing and addressing concerns raised by a recent meta-analysis looking at the safety profile of taVNS that stated “Repeated sessions and sessions lasting 60 min or more are also shown to be more likely to lead to AEs” (PMID 36543841). In particular, this should be addressed for patients that could over deliver even longer stimulation times than described in the protocol.

Line 306: type “able”

Line 360: Would describe this an optional cross-over design

Line 370: It is a little misleading to state “Several studies have shown that healthy patients undergo acute taVNS to investigate scientific questions in behavioral research [55–57]. None of the existing studies on this topic have reported any relevant side effects.” While generally well-tolerated, there are reported adverse events for taVNS, particularly for longer stimulation durations as you mention several lines later. Your enrolled patients are also not necessarily “healthy”, as you exclude some severe physical and mental comorbidities, but patients may have other confounding diagnoses that could impact the safety profile of your study.

As previous research has indicated VNS increases arousal, and this may certainly combat symptoms of fatigue, have you considered any directions to participants for timing of the intervention. IE performed during early hours of the day rather than just before bed?

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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 #2: Yes:  Gansheng Tan

Reviewer #3: Yes:  Anna L Huguenard

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<div>PONE-D-23-38498R2COVIVA: Effect of transcutaneous auricular vagal nerve stimulation on fatigue-syndrome in patients with Long Covid – a placebo-controlled pilot study protocolPLOS ONE

Dear Dr. Gierthmuehlen,

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 12 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.

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,

Usman Ghafoor

Academic Editor

PLOS ONE

Journal Requirements:

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

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #2: Yes

Reviewer #4: Yes

Reviewer #5: Yes

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2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #2: Yes

Reviewer #4: Yes

Reviewer #5: Yes

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3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #2: Yes

Reviewer #4: Yes

Reviewer #5: Yes

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4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: No

Reviewer #4: Yes

Reviewer #5: Yes

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #4: Yes

Reviewer #5: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The revision is satisfactory, and I recommend the manuscript for publication. One of the reviewer evaluation criteria is "Have the authors described where all data underlying the findings will be made available when the study is complete?" The authors can consider adding such a statement. I noticed that there are two sections titled ‘Study Plan’ in the manuscript. These changes do not require another round of review.

Reviewer #4: This is a short trial designed to study effect of TAV nerve stimulation on fatigue-syndrome in patients with long Covid. I have some comments to improve the presentation of the protocol.

1. In line: 214: It should be PCS score. The word “score” is missing.

2. In line, 166 and 167 say that primary endpoint is measure by the average stimulation duration (which is a continuous variable). On the other hand, line 297 says that primary outcome is an incidence rate with patients who exceed a usage-time-threshold (which is a binary categorical variable). The definitions need to be consistent. See also the definition of primary end point in the discussion section of the abstract.

3. In line 218, which test statistics was used to calculate the sample size of 36 needs to be mentioned.

4. In line 223, It is better to mention that “other” secondary measures BDI, SF36 and PCSS……. This is because the first secondary measure MFI20 is already mentioned.

5. Sample size calculation section only has got the reference for sample size calculation ideas but not the exact sample size calculation for the trial. On the other hand, Data analysis section has got sample size calculation, statistical analysis as well as definition of end points. It would be better if clear definitions of primary and secondary end points are moved to the end points section. Similarly, sample size calculation and statistical analysis sections separated for clarity of presentation.

Reviewer #5: The manuscript investigates the feasibility and efficacy of transcutaneous auricular vagal nerve stimulation (taVNS) for reducing fatigue in long Covid patients. A placebo-controlled pilot study with 45 participants will compare above-threshold, below-threshold, and sham stimulation over 4 weeks. The primary endpoint is adherence to the protocol, while secondary outcomes include fatigue severity, quality of life, and depressive symptoms, assessed via questionnaires. The reviewer has the following concerns.

1. The manuscript describes the use of Fisher’s exact test and Kruskal-Wallis tests, but the rationale for selecting these statistical methods over alternatives should be explicitly justified, particularly in comparing the three groups.

2. The sample size calculation is based on prior taVNS studies, but there is no clear power analysis for the primary endpoint. Clarify whether a sensitivity analysis was conducted to account for expected dropout rates and compliance variability.

3. The sham control uses a non-functional electrode, but prior research suggests that even subthreshold stimulation may induce physiological effects. A stronger justification for this approach or a discussion on potential placebo effects and blinding validation would strengthen the study design.

4. The protocol allows up to 4 hours of daily taVNS, yet studies indicate that prolonged stimulation (>60 minutes) may lead to side effects such as headaches or nausea. The authors must comment on this.

5. Since taVNS influences arousal and autonomic function, the timing of stimulation (e.g., morning vs. evening) could impact outcomes. Clarify whether stimulation timing should be standardized or at least documented to analyze its effects on fatigue and QoL.

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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 #2: Yes:  Gansheng Tan

Reviewer #4: Yes:  Dr Shah-Jalal Sarker

Reviewer #5: No

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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.

COVIVA: Effect of transcutaneous auricular vagal nerve stimulation on fatigue-syndrome in patients with Long Covid – a placebo-controlled pilot study protocol

PONE-D-23-38498R3

Dear Dr. Gierthmuehlen,

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. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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,

Usman Ghafoor

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors have addressed the comments.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #4: Yes

Reviewer #5: Yes

**********

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #4: Yes

Reviewer #5: Yes

**********

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #4: Yes

Reviewer #5: Yes

**********

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. 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 #4: Yes

Reviewer #5: 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 #4: Yes

Reviewer #5: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #4: The authors have applied all of my comments for improvement and hence it may be accepted for publication.

Reviewer #5: The authors have addressed all my concerns comprehensively. The manuscript is now suitable for publication and I recommend acceptance without further revisions.

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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 #4: Yes:  Dr Shah-Jalal Sarker

Reviewer #5: No

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