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

Mortimer Gierthmuehlen; Petra Christine Gierthmuehlen

doi:10.1371/journal.pone.0315606

. 2025 May 9;20(5):e0315606. doi: 10.1371/journal.pone.0315606

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

Mortimer Gierthmuehlen ^1,^*, Petra Christine Gierthmuehlen ²

Editor: Usman Ghafoor³

PMCID: PMC12063903 PMID: 40343901

Abstract

Background: Up to 80% of patients who develop coronavirus disease-2019 (Covid-19) infection subsequently experience long covid/post-covid syndrome. The World Health Organization (WHO) has estimated that >770 million patients have been infected with Covid-19 globally.https://data.who.int/dashboards/covid19/cases Even if only 10% of these patients develop long covid, > 75 million patients will suffer for a long period. Among the various symptoms of post-covid syndrome, fatigue is common, affecting up to 60% of the patients. As observed in other viral infections, elevated levels of inflammatory cytokines may play a role. Transcutaneous auricular vagal nerve stimulation (taVNS) is a noninvasive method that modulates the immune system via the central nervous system and has shown promising effects in autoimmune diseases and improving fatigue. In this pilot study, we investigated the feasibility of daily taVNS in patients with long covid-related fatigue. Additionally, the effects of taVNS on fatigue and quality of life will be analyzed. Methods: A total of 45 adult patients with long covid associated fatigue syndrome will be enrolled in this study, and will be randomized to the above-threshold-stimulation, below-threshold-stimulation, or sham-stimulation arms, after being informed that they will feel the stimulation. The above-threshold-group will receive a 4-week-long left-sided cymba conchae taVNS with 25 Hz, 250 µs pulse width 28s/32s on/off paradigm for 4 h throughout the day. The below-threshold group will receive stimulation below the sensational threshold, whereas the sham group will receive no stimulation following application of a non-functional electrode. The daily stimulation protocol will be recorded either manually or using the provided app. Three well-established questionnaires, the Multidimensional-Fatigue-Inventory-20, Short-Form-36, and Beck-Depression-Inventory, and the newly established Post-Covid-Syndrome-Score will be completed both before and after 4 weeks of stimulation. Discussion: The primary endpoint has been set as the patients’ average daily stimulation time after 4 weeks, while secondary endpoints include the effects of taVNS on fatigue and Quality of Live (QoL). As a non-invasive treatment option, taVNS may be a notable alternative for patients with post-covid related fatigue. Trial registration: This study was approved by the local ethics committee (23/7798) and registered (DRKS00031974) (see supporting information files). Ethics & Dissemination: The ethical justifiability of this study was supported by prior research demonstrating the safety of taVNS. Patients will be recruited by general practitioners, and written informed consent will be obtained. All data will be pseudonymized for collection and storage. The study results will be published in peer-reviewed journals with the aim of providing evidence of the potential of taVNS in long covid management. The study will be conducted in accordance with the principles of the Declaration of Helsinki.

Introduction

Up to 80% of patients suffer from long-term consequences after recovering from a SARS-CoV2 infection, that hinder their reintegration into daily work and family life [1,2]. These limitations have prompted the recent publication of the German “Standing Guideline Commission of the Association of Scientific Medical Societies in Germany”(“Arbeitsgemeinschaft der Wissenschaftlichen Medizinischen Fachgesellschaften” - AWMF) guidelines for the rehabilitation of patients with coronavirus disease-2019 (Covid-19) [3]. In one study, fatigue was observed in 53% of 143 patients [4]. Fatigue is a well-known symptom of viral infections [5] which can significantly impact patients, particularly in patients who have had a Covid-19 infection [6]. This symptom can continue to affect patients for months after recovery from the infection [2,7]. The fatigue severity scale is a frequently used screening tool for fatigue [8], commonly used to diagnose chronic fatigue.

Invasive vagus nerve stimulation (iVNS) has been approved for the treatment of epilepsy and depression since the early the 1990s and 2000s [9]. Vagal activation through vagal nerve stimulation (VNS) leads to immunomodulation, which seems to have a positive effect on autoimmune diseases such as chronic inflammatory bowel disease [10], rheumatoid arthritis [11], and pancreatitis [12] in experimental settings.

Based on the anatomical observation that a specific part of the ear, the cymba conchae, is solely innervated by the vagus nerve [13–15], electrical stimulation of this region has been investigated as a noninvasive alternative to iVNS. Studies have shown that this transcutaneous auricular VNS (taVNS) leads to comparable brain activation as iVNS [16,17]. Furthermore, taVNS has been shown to positively influence epilepsy [18], depression [19], and cytokines in rheumatoid arthritis [20]. Further large-scale studies and compliance with the defined standards [21] are required to further investigate the effectiveness of taVNS under these conditions.

Owing to the pandemic, further research on the causes of fatigue has not yet been conducted, while the underlying causes of fatigue remain inconclusive. Although conflicting results have been published [22,23], existing research indicates that the development of 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 [24]. Studies have further shown that Covid-19 infection leads to a massive increase in cytokines, known as the cytokine storm [25,26], resulting in elevated levels of interleukin (IL) 2, IL-4, and tumor necrosis factor-alpha [27]. Consequently, one Cochrane review demonstrated that IL-6 blocking medication could have a positive impact on the course of acute Covid-19 infection [28]. Overactivation of mast cells also seems to play a possible role in the development of long-term Covid [29], while D-dimer and C-reactive protein levels also appear to correlate with the occurrence of fatigue during the long-term Covid phase [30].

TaVNS has been shown to reduce inflammatory cytokines [31–33], modulate cardiac vagal tone [34], modulate arousal through central pathways [35] and positively influence depressive disorders [36,37], which may play a role in the development of long-covid associated fatigue. This modality has already been applied in pilot studies of patients with acute Covid-19 infection, where it has been shown to help lower cytokine levels [38], thus emphasizing the urgent need for further studies on non-invasive VNS in Covid-19 [39]. The effectiveness of taVNS on fatigue syndromes in systemic lupus erythematosus [40] and Sjögren’s syndrome [35,41] was also investigated in a recent clinical trial FatiVa [42]. However, whether taVNS reduces fatigue by reducing inflammatory cytokine levels or through a direct central mechanism remains unclear [43]. However, several studies have discussed the potential use of noninvasive vagal nerve stimulation in the treatment of long-covid, showing promising results [44–47]. The influence of VNS on olfactory function is currently being investigated; however, further research is warranted [48,49].

In summary, vagus nerve stimulation appears to have the potential to reduce the occurrence of fatigue and depression in long covid syndrome by modulating the immunosystem and influencing the central arousal pathways. As reintegration into daily life and work is of paramount importance in patients’ quality of life, and the healthcare or retirement system, we aimed to evaluate whether taVNS, which has few side effects, exerts a positive influence on the rehabilitation capacity and acceptance of treatment among long covid patients with fatigue syndrome.

Materials and methods

Inclusion criteria:

History of prior Covid infection within the last 36 months
Fatigue persisting for ≥ 3 months, with a score ≥4 on the FSS (Fatigue Severity Scale)
Age > 18 years
No or stable treatment for depression for at least 4 weeks

Exclusion criteria:

Severe psychiatric illness, such as schizophrenia
Acute Covid infection within the last 14 days
>8 points on the « Nurses’ Global Assessment of Suicide Risk » questionnaire
Implanted vagus nerve stimulator or history of vagotomy
Significant heart disease: bradycardia (e.g., sick sinus syndrome), heart failure, or a history of myocardial infarction
Active implants include pacemakers, defibrillators, neurostimulators, cochlear implants, and drug delivery devices
Inability to understand the study plan
Progressive neurological disease (e.g., Parkinson’s, Multiple Sclerosis, epilepsy)
Pregnancy
Other acute illnesses or conditions associated with fatigue (e.g., cancer, chemotherapy treatment, and autoimmune diseases)
Polyneuropathy
Presence of skin conditions, such as infection, psoriasis, or eczema in the treatment area
Presence of any anatomical anomaly that prevents successful placement of the ear electrode
Presence of any serious illness that prevents successful participation in the study.

Questionnaires

Three well-established questionnaires will be used: the ShortForm36 (SF36), the Beck Depression Inventory (BDI), and the Multidimensional Fatigue Inventory (MFI20). In addition, the newly established Post-Covid-Syndrome-Score (PCS Score) of the “National Pandemic Cohort Network” Consortium [50] will be included.

Abortion criteria

Patients will discontinue participation if they present with any of the exclusion criteria.

Endpoints

The present study aims to investigate whether taVNS can influence fatigue and the associated quality of life in patients with long covid. To achieve this, the following endpoints will be evaluated:

The primary endpoint is acceptance of taVNS in patients with a long stimulation duration, measured by the average daily stimulation duration.
Secondary endpoints include the effects of taVNS on the MFI20, BDI, quality of life (SF36), and PCS-Score.

Selection of patients and data collection

A total of 45 patients will be recruited and divided into three groups. Patients will be informed about the study through flyers and by their general practitioners, and will be provided with access to a websitehttps://redcap.kk-service.de/surveys/?s=CHMDH8LYY94LLDJY https://t1p.de/sa518. Additionally, local support groups will be contacted and informed. On the website, questions regarding the inclusion and exclusion criteria will be asked anonymously, without data storage. At the end of the questionnaire, patients will be informed of whether they qualified for the study. In the case of study qualifications, patients will be provided with an email address where they can sign up to participate. Patients will be informed about the study aims and procedures by an investigator or certified medical professional. After obtaining written informed consent, data collection will be pseudonymized in the RedCap electronic case report form (eCRF) system of Ruhr-University-Bochum and will be General Data Protection Regulation (GDPR)-compliant. The RedCap system automatically randomizes patients into Verum-1, Verum-2, or Sham groups (n = 15 each). The patients will be instructed on how to use the simulator for 4 h per day, with at least 1 h of consecutive stimulation in the left ear. Patients will be asked to log all stimulation times manually in a diary. Because patients in the sham group will also receive the device’s user manual, which states that they should feel a tingling sensation in the ear, they will be instructed to use the highest stimulation intensity that is not perceptible to them. After adjusting the stimulation to this level, sham patients will receive a non-functional electrode that does not perform any stimulation. This approach is justified as the principle “more is better” does not apply to vagus nerve stimulation, and stimulation below the perception threshold could still, theoretically, have an effect.

Patient and public involvement statement

Local support groups, regional long-term outpatient clinics, and general practitioners all participated in the study design. They were informed that the study would take place, and encouraged to disseminate this information among their patients and study flyers.

Sample size calculation

The group size was based on the aforementioned studies on taVNS in other forms of fatigue [31,35,41], our aforementioned FatiVa study [42], and an ongoing similar study in the USA which enrolled 40 patients (NCT05630040). Recommendations for group size made in previous pilot studies were also considered [51,52]. Based on the expectation of the superiority of the two target groups (low- and high-threshold stimulation) of at least 5 and 10 MFI units, respectively, compared to the placebo (shift), and a type I error of alpha = 0.05, a total of 3 × 12 cases = 36 cases were deemed to be required to achieve a study power of 80% for the most important secondary endpoint, the MFI20 questionnaire. The mentioned numbers were secured with a safety margin of approximately 10%–20% (depending on the dropout rate), resulting in a total of 3 × 15 cases = 45 cases, if necessary. The calculation will be performed using the R Package “MultNonParam” [53].

Data analysis

The two-sided p-value of Fisher’s exact test will be used to evaluate the difference between the three groups (primary outcome is an incidence rate with patients who exceed a usage-time-threshold). Additionally, the two treatment groups will be descriptively compared with the placebo group, and between themselves. The rationale to use Fisher’s exact test is to compare binary data between groups (in total three two-group comparisons). The rationale for the Kruskal-Wallis rank analysis of variance is to avoid the potential influence of outliers and to handle unknown data distributions (normally distributed data are not expected).

Since three tests (each group compared with each other) are performed and the threshold for the p-value was set at an overall (studywise) alpha of 0.05, then three tests result in an Bonferroni-adjusted (comparisonwise) threshold of p/ 3 = 0.017. p-values below this threshold denote a statistical significant difference.

Secondary outcome measures included the MFI20, BDI, SF-36, and PCS-score, which were modified similarly to the primary outcome measures. BDI, SF36 and PCSS are other secondary measures which will be analyzed descriptively. Since those parameters are continous rank-analses of variance (Kruskal wallis tests) will be used. Descriptive statistics, such as means, medians, quantiles, and respective case numbers of the available data, will be reported. The between-group contrast will be reported as the mean difference with 95% confidence intervals. Additionally, a nonparametric estimation of the group effect (shift) using the Hodges-Lehmann estimator shift will be provided.

Successful acceptance and compliance will be defined as achieved when the acceptance criteria (average taVNS for 4 h/day) are met by at least 80% of patients. A total of 45 patients will be enrolled, with 15 assigned to each of the three groups: “sham”, “Verum-1”, and “Verum-2.” The decision to have 15 patients per group was based on feasibility, as well as from influence by studies that examined noninvasive VNS for sleep deprivation, lupus, and Sjoegren’s syndrome. Furthermore, the selected sample size was aligned with the recommended sample sizes for feasibility studies [51,52].

Study plan (Fig 1)

Verum stimulation.

This study included two verum-stimulation groups. In both groups, taVNS will be administered according to the device approval at the cymba conchae of the left ear. Because establishing a placebo group in stimulation studies is generally difficult, two Verum groups were planned: one will receive stimulation above the sensory threshold, and one below. If subthreshold stimulation also leads to a positive effect, future studies may omit the suprathreshold stimulation group.

Stimulation will be conducted using the commonly chosen parameters in the literature, which have been successfully applied over a period of 20 weeks [54]. These parameters include a frequency of 25 Hz and a pulse width of 250 µs. These stimulation parameters will be preset in the commercially available, CE (Conformité Européenne, European equivalent to FDA) tVNS-L stimulator and cannot be changed. Stimulation will be applied throughout the day for 4 h using a 28s on/ 32s on/off schedule. Patients will be asked to download available Android and iOS apps, which will be paired with the stimulator via Bluetooth to document the daily stimulation time. Based on these records, patients will be able track when the recommended 4 h daily stimulation target day is reached. Although the stimulator will be turned off after 4 h of continuous stimulation, there is no automatic blocking of the stimulator after 4 h of cumulative stimulation, as only patients who understood the study protocol will participate, in accordance with the exclusion criteria. Additional negative effects of a stimulation lasting more than 4 hours have not been reported in literature – however, the likelihood of known side effects such as headache, ear pain, skin irritation or nausea [55] increases if stimulation lasts longer than 60min [56].

Verum-1 stimulation.

The intensity will initially be set to produce a mild tingling sensation in the ear that should not be painful. Subsequently, the intensity will be reduced to a level at which the patient no longer feels the stimulation. The patient will then be asked to repeat this procedure every time stimulation is initiated to ensure that the stimulation is always subthreshold.

Verum-2 stimulation.

The intensity will be adjusted according to the instructions provided to produce a mild, non-painful tingling sensation in the ear. The patients will be able modify the stimulation intensity during the study to ensure that it remains above this threshold.

Sham stimulation.

The intensity will initially be set to produce a mild tingling sensation in the ear that should not be painful. Subsequently, the intensity will be reduced to a level at which the patient no longer feels any stimulation. The stimulator will be maintained at this level throughout the study. However, participants in the sham group will receive an ear electrode provided by the manufacturer, which is based on a regular electrode but does not establish an electrical connection between the stimulator and ear contact, ensuring no actual stimulation occurs.

Study procedure

The study commenced on 1^st September 2023. As there is no central point of contact for patients with long covid, all participants were/will be informed about the study by their general practitioners. Contact will be established between general practitioner networks and local support groups. General practitioners can identify suitable patients based on the inclusion and exclusion criteria, and referred them to the study investigator. The two visits (“Visit 0” and “Visit 1”) and the telephone interview will subsequently be scheduled following consultation with the patient.

During the study period, participants will use the “tVNS Patient” app developed by tVNS Technologies GmbH (available for free in the respective app stores) to automatically document the frequency and duration of stimulations. Compliance documentation will be provided, as recommended in the 2020 consensus paper on noninvasive VNS [21]. Only the patient can access these data, which will be finally reviewed by a physician at the end of the stimulation period. All data will be stored exclusively on participants’ smartphones. The questionnaires will be entered and analyzed as electronic case report forms (eCRFs) in the RedCap system of the Department of Medical Informatics.

Visit 0.

During the initial visit, patients will receive informational documents, sign informed consent forms, and complete the questionnaires. Personal data such as age, sex, height, weight, therapy time, duration, and type will also be documented. The patients will receive the stimulator and be instructed regarding its use by the investigator, according to the guidelines of the German Medical Devices Act (MPG/MPDG). Additionally, group assignment (SHAM, Verum 1, or Verum 2) will be performed. Subsequently, the 4-week stimulation phase will be initiated.

Telephone interview after 7 days.

After 7 days, patients will be contacted by phone or email (if preferred) to inquire about their use of the stimulator, any side effects, or any potential questions they may have.

Visit 1

After 4 weeks, the patients will complete the questionnaires again. Personal data, including weight, will be collected, while data regarding the duration of stimulation will be retrieved from a smartphone. Subsequently, the stimulators will be returned. Patients who cannot attend visit 1 because of their health conditions will receive online access to the questionnaire section of the eCRF to complete it from home.

Optional Visit 2 (optional cross-over-design).

Patients enrolled in “Sham” or “Verum 1” will be offered to receive “Verum 2” stimulation for 4 weeks at the end of the study.

The study will be terminated here.

Discussion

Safety

Several studies have shown that healthy patients undergo acute taVNS to investigate scientific questions in behavioral research [57–59]. Although mild side effects may occur, none of the existing studies on this topic have reported any severe side effects. TaVNS was also investigated in a medical doctoral thesis involving healthy patients without relevant side effects [60]. In total, the cited studies included >1,200 healthy patients who underwent taVNS without any difficulty. Although the occurrence of side effects correlates with the duration of stimulation [61], even during chronic taVNS, which lasts several weeks, only a few adverse effects and no severe events have been observed [13,56,62].

A recent review of transcutaneous VNS (including both cervical and auricular stimulations) reported skin redness (16.7%), headache (3.3%), nasopharyngitis (1.6%), and nausea/dizziness (1.1%) as the most common side effects among 1,322 patients. Only 2.6% of all patients had to discontinue the study because of side effects, while facial palsy occurred only with cervical vagus nerve stimulation [55]. Long covid and its associated fatigue syndrome pose a challenging burden for both patients and healthcare systems. Investigating a noninvasive method to improve this devastating condition may be extremely beneficial, and this goal could justify these known side effects. The results of this pilot study will be analyzed with respect to subsequently conducting a full multicenter interventional study, and will provide information regarding future inclusion and exclusion criteria, recruitment rate, sample size, expected adherence, and dropout rates among patients with long covid-associated fatigue.

Ethics and regulatory aspects

This study was approved by the local ethics committee of Ruhr-University-Bochum under registration number 23/7798 on 05/12/23, with amendment acceptance on 06/14/23. It was further registered in the DRKS-System under the number DRKS00031974 (https://drks.de/search/de/trial/DRKS00031974) on 05/25/23. The accepted version was version 3.0, from 06/14/23, and the sponsor was the University Medical Center Knappschaftskrankenhaus Bochum. The study will be performed under the §47(3) of the new MPDG law defining clinical studies involving medical devices. As the tVNS-L-System, owing to the fixed set of stimulation parameters, possesses a CE mark for vagal nerve stimulation, and this study involves no invasive procedures, ethical approval was relatively simple and rapid to obtain. Thus, if the results indicate feasibility, a larger study could be quickly initiated with a CE-certified system, in contrast to a system that does not have a CE mark.

Sham intervention

Defining the sham group in a stimulation study is challenging. Several different approaches have been discussed in the literature, including stimulation of the ear lobe instead of the cymba conchae, or stimulation of other parts of the body [63–66]. However, an informed patient may realize that such stimulation is different from normal taVNS, and could thus identify themselves as being in the sham group. To address this, patients will alternately provide with information that they will either feel or do not feel the stimulation. Once the tingling sensation has been demonstrated to the patient, the intensity will be reduced until the patient does not feel any stimulus. The sham group will then be provided with a non-functional electrode, while the verum-1-group will maintain their functional electrode throughout the study phase. If the analysis shows that both the verum-1 and verum-2 interventions have a significant effect on fatigue, future studies will need only one subthreshold group and one sham group, leaving the patients blinded to which group they have been included in.

Within this feasibility trial, no invasive procedures, such as blood sampling, has been planned, in order to maintain the regulatory requirements as low as possible. The stimulation system tVNS-L® is manufactured by tVNS GmbH in Erlangen, Germany. This system allows modification of the stimulation intensity but not of the pulse width or frequency. As such, it is a CE-mark for transcutaneous auricular vagal nerve stimulation. The variant tVNS-R® (R for research) allows modification of all parameters; consequently, it does not have a CE-mark, making it extremely difficult to obtain insurance and ethical approval for a scientific study.

The results of this study will be published in scientific, probably open-access, journals with a scope for public health. If taVNS, as a very safe procedure, can improve fatigue and the challenging sequelae of long covid, it would significantly improve the quality of life of such patients. This potential improvement in the quality of life justifies the extremely low risk of taVNS therapy in this study.

Data protection, management, and monitoring

Only pseudonymized data will be stored on the RedCap®-System in our hospital, while a table containing patient records will be stored on a different drive within the hospital network. Only the investigator and study nurses will have access to the database, and the rules of the GDPR will be applied. Data monitoring will be performed by the Department of Medical Informatics, Biometry, and Epidemiology at Ruhr-University Bochum, which runs and maintains the RedCap system. The results of the study will be made available in peer reviewed journals.

Supporting information

S1. SPIRIT-Checklist.

(XLSX)

pone.0315606.s001.xlsx^{(14.8KB, xlsx)}

Acknowledgments

Not applicable.

Data Availability

No datasets were generated or analysed during the current study. All relevant data from this study will be made available upon study completion.

Funding Statement

This study was financed by the Department of Neurosurgery at the University Medical Center Knappschaftskrankenhaus Bochum.

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PLoS One. 2025 May 9;20(5):e0315606. doi: 10.1371/journal.pone.0315606.r001

Author response to Decision Letter 0

12 Dec 2023

PLoS One. doi: 10.1371/journal.pone.0315606.r002

Decision Letter 0

Mohammadreza Pourahmadi

10 Jul 2024

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

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Reviewer #1: Yes

Reviewer #2: Partly

**********

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Reviewer #1: Partly

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #2: No

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Reviewer #2: Yes

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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.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: Yes: Gansheng Tan

**********

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PLoS One. 2025 May 9;20(5):e0315606. doi: 10.1371/journal.pone.0315606.r003

Author response to Decision Letter 0

31 Aug 2024

We thank the reviewers and the editor for the interesting and thoughtful comments and especially the mentioned internet resource OSF.

Editor:

When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process.

This is only a study protocol, so there is no additional data which could be published at this point. Once the study is completed and analyzed, the results will be made available within the publication of the results.

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.

Statistically, the difference between the two groups is checked in continuous data (main target parameters and ranked judgment scales) using exact rank variance analysis according to Kruskal and Wallis (R package “coin”) to produce a 2-sided p-value. The threshold for the p-value is an alpha of 0.05, i.e. if the test value falls below the threshold, the null hypothesis can be rejected and a significant general difference between the three groups (placebo, low-threshold, higher-threshold group) can be assumed. In addition, the two treatment groups are each compared descriptively with placebo (and with each other). Secondary target parameters are the BDI, the SF-36 and acceptance; these are upgraded in analogy to the main target parameter. Since exactly one main target parameter (MFI20 change) is evaluated for confirmatory purposes, no adjustment is necessary Type 1 risk is necessary. Based on the expectation of superiority of the two target groups (low and higher threshold stimulation) of at least 5 or 10 MFI units compared to placebo (shift) and a type 1 error of alpha = 0.05, a total of 3 x 12 cases = 36 cases in total needed to achieve a study power of 80%. The numbers mentioned are minimum numbers and should be secured by a safety surcharge (depending on the drop-out rate) of around 10% - 20%, so that (if the surcharge is necessary) this results in a total of 3 x 15 cases = 45 cases in total. The calculation was carried out using the R package “MultNonParam” (Kolassa & Seifu, 2013). The remaining parameters (BDI, SF-36, acceptance judgment) are evaluated purely descriptively at the threshold of p = 0.05 (same statistical test as the main target parameter ). The calculated p-values are always two-tailed. Means, medians, quantiles and the respective sample numbers of the available data are given descriptively. For the between-group contrast, the mean difference and its 95% confidence intervals are given as the effect measure. In addition, a nonparametric estimate of the group effect (shift) using the Hodges-Lehman estimator shift (Lehmann, 1998) is given

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

Since there is a single primary target parameter the confirmatory analysis will not be adjusted for multiple testing. All other parameters will be tested descriptively only (generating hypotheses only).

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

We had the manuscript proofread.

Reviewer #2: Summary

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.

We corrected this accordingly and added respective notes and explanations.

We corrected this accordingly.

The reason to investigate feasibility is to find out whether patients with severe fatigue are even able to use taVNS for 4 hours per day. Our experience with the first patients is that having something to do for 4 hours can be a burden for these patients.

We corrected this.

We added the scale and the respective explanatations as suggested. Our ethics committee required us to define the acceptance rate as the primary outcome and the questionnaires as the secondary outcome.

The patients will be instructed, we added a respective paragraph. MFI20 is a secondary outcome – the primary outcome is acceptance.

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?

Please refer to the yellow section above, there specifics and details of the sample size estimation.

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?

Yes, we corrected that.

We corrected that – the patients will repeat the procedure to actively downregulate the intensity below threshold.

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?

We have not yet performed any analysis as the data is still “sealed” in our Recap-System. It will be analyzed once the study is completed.

This statedment was added.

The only side effect of taVNS close to fatigue is dizziness, which means vertigo in this context. Fatigue is no known side effect of taVNS.

We corrected this, it was our fault – our system stimulates the cymba conchae which is innervated by the auricular branch of the vagal nerve. We added the respective literature.

Minor comments

We added a respective link to the WHO, stating that 775 mio. Patients suffered from Covid-19. If only 10% of them get long covid, it is more than 77mio patients.

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.

We corrected this an just referred to one study with 143 patients.

We deleted them.

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

We rephrased the sentence.

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

There are other transcutaneous systems stimulating the vagle nerve directly through the skin. We change “transcutaneous” to “non-invasive” VNS to make this more clear.

6. What does Verum stand for?

“Verum” is the latin word for “true”, which means in this context “true stimulation group”.

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

Yes, sorry, we corrected that.

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

The patients receive the official manual of the stimulator and a verbal instruction of the system. The stimulator is very easy to use so the patients don’t really need any written instruction.

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

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

We rephrased this sentence.

11. What is CE in “CE-certi

Attachment

Submitted filename: Comments to the Reviewer.docx

pone.0315606.s004.docx^{(27.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0315606.r004

Decision Letter 1

Mohammadreza Pourahmadi

5 Nov 2024

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,

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

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:

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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'.

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.

[Note: HTML markup is below. Please do not edit.]

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

**********

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

Reviewer #2: Partly

Reviewer #3: Yes

**********

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

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

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?

**********

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

**********

Attachment

Submitted filename: PONE-D-23-38498 Review.docx

pone.0315606.s003.docx^{(12.7KB, docx)}

PLoS One. 2025 May 9;20(5):e0315606. doi: 10.1371/journal.pone.0315606.r005

Author response to Decision Letter 1

28 Nov 2024

We thank the reviewers and the editor for their helpful comments

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

Done.

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

We corrected that.

The test statistic will evaluate the difference between the three groups in terms of binary data (primary outcome is an incidence rate with patients who exceed a usage-time-threshold) using the Fisher-test (exact test), to obtain a two-sided p-value. Additionally, the two treatment groups will be descriptively compared with the placebo group, and between themselves.

Since three tests (each group compared with each other) are performed and the threshold for the p-value was set at an overall (studywise) alpha of 0.05, then three tests result in an Bonferroni-adjusted (comparisonwise) threshold of p / 3 = 0.017. p-values below this threshold denote a statistical significant difference.

There are three groups in total, two treatment groups and a placebo group. 5. P219, I assume the calculation has been performed in R and will not change in the future.

That is correct.

BDI, SF36 and PCSS are secondary measures which will be analyzed descriptively. Since those parameters are continous rank-analses of variance (Kruskal wallis tests) will be used.

You are right – this sentence was modified in the wrong way, I apologize. I wanted to say that in literature there is no evidence for negative effects in case the stimulation is performed for more than 4 hours.

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

Done

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

That is correct. We changed it to “application”.

We modified the sentence that the development of fatigue is linked to cytokines, while its severity seems to correlate with the initial cytokine levels.

Indeed. We corrected the sentence and added the literature.

Line 306: type “able”

We corrected this.

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

Done

We corrected that paragraph.

Since other studies also do not impose such requirements and we are aiming for maximum patient compliance, we did not specify a fixed schedule for when the stimulation should take place.

PLoS One. doi: 10.1371/journal.pone.0315606.r006

Decision Letter 2

Usman Ghafoor

26 Feb 2025

<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,

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'.

We look forward to receiving your revised manuscript.

Kind regards,

Usman Ghafoor

Academic Editor

PLOS ONE

Journal Requirements:

[Note: HTML markup is below. Please do not edit.]

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

**********

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

Reviewer #2: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

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

Reviewer #2: Yes

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?

Reviewer #2: No

Reviewer #4: Yes

Reviewer #5: Yes

**********

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

Reviewer #2: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

6. Review Comments to the Author

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.

**********

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

**********

PLoS One. 2025 May 9;20(5):e0315606. doi: 10.1371/journal.pone.0315606.r007

Author response to Decision Letter 2

11 Mar 2025

Comments to the reviewers

We thank the reviewers for their valuable comments.

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.

We changed the second “Study plan” to “Study procedure”. We also added a statement that the results will be published in peer reviewed journals.

Reviewer #4:

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

We corrected this accordingly.

This is correct, we changed that.

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

We added the respective information.

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.

Corrected.

We corrected that, too.

Reviewer #5:

This was added.

The power analysis is now in line 208. The drop-out-rate was estimated with the numbers of Redgrave et. Al. 2018 in mind, stating that throughout all studies in the meta-analysis around 3% of all participants drop out. In patients with long-covid we estimated this number a bit higher.

We have three groups: Sham, Verum-1 and Verum-2. Verum-2 is standard stimulation. Verum-1 is stimulation below the threshold, but still a stimulation. Sham is no stimulation at all, guaranteed by a non-functional plastic-electrode with no stimulation capability. This is mentioned in line 298.

We added a respective sentence. However, the stimulator we use has a CE-certificate as a medical product in Germany and is certified for recommended use of 4h/day. Both software and integrated timer are calibrated to this recommendation.

Thank you, this is an interesting point, indeed. To our knowledge, no study has so far addressed this aspect, also the manual of the stimulator does not recommend any specific time. However, the time of stimulation is logged both by the app and the patient’s stimulation diary and will consider analyzing this aspect.

Attachment

Submitted filename: Comments to the reviewers.docx

pone.0315606.s006.docx^{(21.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0315606.r008

Decision Letter 3

Usman Ghafoor

2 Apr 2025

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.

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PLoS One. doi: 10.1371/journal.pone.0315606.r009

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pone.0315606.s004.docx^{(27.5KB, docx)}

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Data Availability Statement

No datasets were generated or analysed during the current study. All relevant data from this study will be made available upon study completion.

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

Mortimer Gierthmuehlen

Petra Christine Gierthmuehlen

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Abstract

Introduction

Materials and methods

Inclusion criteria:

Exclusion criteria:

Questionnaires

Abortion criteria

Endpoints

Selection of patients and data collection

Patient and public involvement statement

Sample size calculation

Data analysis

Study plan (Fig 1)

Fig 1. SPIRIT schedule of enrollment of the CoViva Study.

Verum stimulation.

Verum-1 stimulation.

Verum-2 stimulation.

Sham stimulation.

Study procedure

Visit 0.

Telephone interview after 7 days.

Visit 1

Optional Visit 2 (optional cross-over-design).

Discussion

Safety

Ethics and regulatory aspects

Sham intervention

Data protection, management, and monitoring

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Author response to Decision Letter 0

Decision Letter 0

Mohammadreza Pourahmadi

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Author response to Decision Letter 0

Decision Letter 1

Mohammadreza Pourahmadi

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Author response to Decision Letter 1

Decision Letter 2

Usman Ghafoor

Roles

Author response to Decision Letter 2

Decision Letter 3

Usman Ghafoor

Roles

Acceptance letter

Usman Ghafoor

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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RESOURCES

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