Explanation for the choice of comparators

We choose a patient-reported outcome as primary endpoint in this study (EQ-VAS) [27, 28]. To differentiate the potential effects of Pycnogenol® from the placebo effect, this study utilizes a placebo that shares identical physical characteristics and dosing regimen with the verum. Using a placebo is ethically justified, as there is no approved treatment for PCC, and no treatment of proved effectiveness will be withheld. Additionally, participants are not required to discontinue any PCC-related treatment or medication prior to enrolment into the study, except regular intake of Pycnogenol®.

Intervention description

Table 1 provides an overview of study procedures including interventions. Following assessment of inclusion and exclusion criteria, eligible subjects are randomly allocated to the verum or placebo group.

Table 1 Overview of enrolment, assessments and outcome measures according to Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT)

Participants in the intervention group receive 200 mg Pycnogenol® per day for 12 weeks, with two capsules containing each 50 mg in the morning and two capsules in the evening. Pycnogenol® is a proprietary French maritime pine bark extract (Pinus pinaster subsp., atlantica, Pinaceae) produced by Horphag Research, Geneva, Switzerland. Pycnogenol® is standardized to contain 70 ± 5% procyanidins in compliance with the United States Pharmacopeia. Its compounds and gut metabolites are known among other actions for their antioxidant properties, anti-inflammatory activities, and stimulation of nitric oxide production in vascular endothelial cells [22].

Participants in the control group receive a placebo. The placebo capsules are manufactured in the same facility and have the same physical appearance, consistency, and weight as the verum. Like the participants in the verum group, participants in the control group take two capsules twice daily with or right after a meal.

Criteria for discontinuing or modifying allocated interventions

In general, Pycnogenol® is well tolerated [20, 21, 29]. In this study, Pycnogenol® is supplied at a dose of 200 mg per day, with two capsules containing 50 mg in the morning and two capsules in the evening taken together or right after a meal to avoid potential gastrointestinal discomfort. Gastrointestinal discomfort is the most frequently reported treatment-related side effect in previous clinical trials [29]. Further reported treatment-related adverse events are dizziness, headache, and nausea [29]. All side effects, possibly related to the study product, will be recorded as adverse events. Adjustments in study product dosage or intake are not planned.

Upon withdrawal or exclusion, early termination will be recorded in the database, and the participant will be invited for a termination visit. If the participant is not able or willing to participate in a termination visit, a phone call will be offered instead. Moreover, we will ask the participant if they are willing to complete the study specific questionnaires to keep missing data to a minimum. The participant will be asked to return leftover study products to the study center.

Strategies to improve adherence to interventions

To enhance the validity of our data and adherence to study products, we planned a short study visit six weeks and two phone calls two respectively 9 weeks after randomization (Fig. 1) to stay in close contact with study participants. During the phone calls, the importance of adhering to the study products and monitoring symptoms in the diary is discussed. Study products returned at the follow-up visits (V3, V4, Fig. 1) are counted and recorded in the database. In addition to the intention-to-treat analysis (primary analysis), we plan a per protocol analysis based on adherence to study products and investigate its potential impact on primary and secondary outcomes.

Fig. 1Relevant concomitant care permitted or prohibited during the trial

Concomitant medication including herbal medications or supplements is principally allowed. Investigators instruct participants to keep their therapy regimen unchanged during the study period. This also applies for concomitant non-pharmaceutical PCC-related therapies. Participants are instructed not to start any new therapy, new preventive measures such as COVID-19 vaccinations, or intervention during the trial that may interfere with study outcomes. Those who plan to get vaccinated against SARS-CoV-2 will get the opportunity to get vaccinated at our center prior to the baseline visit.

Provisions for post-trial care

Participants terminating the study (either regularly or prematurely) with reported ongoing serious adverse events (SAE) or any ongoing adverse events regarding laboratory values or vital signs being beyond the alert limit will be asked to return for a follow-up investigation. This visit will take place up to 30 days after termination of the treatment period.

Outcomes

Table 1 provides an overview of assessments including primary and secondary outcomes.

Primary outcome

The primary outcome is self-reported health status assessed with the EQ-VAS, also known as the “Feeling Thermometer” [27, 28]. The EQ-VAS is numbered from 0 to 100 with 0 representing the “worst imaginable health” and 100 representing the “best imaginable health.” The scale is frequently used in people with chronic respiratory conditions [30,31,32] and has a well-established minimal important difference (MID) of 5–8 units [31]. In our trial, EQ-VAS is assessed daily over seven consecutive days prior to the baseline visit (V2) and again prior to the last study visit (V4). Mean EQ-VAS scores (i.e., pre- and post-intervention) will be computed, including at least 4 days. Post-intervention, EQ-VAS scores will be compared between the intervention and placebo groups and adjusted for baseline values. In addition, self-reported health status is assessed at screening (V1) visit. These data will be used for descriptive purposes. A minimal important difference (MID) of 8 units will be used for interpretation of the magnitude and clinical relevance of between-group differences in post-intervention EQ-VAS values [31].

Secondary outcomes

All secondary outcomes are assessed at baseline (V2) and after 12 weeks (V4), except post COVID-19 related symptoms. Symptoms are assessed using online questionnaires prior to study visits (V1, V2, V4) and a symptom diary that is completed on a weekly basis between the baseline and 12-week visit.

Post COVID-19 symptoms are assessed using self-reported surveys prior to the screening visit (V1), baseline visit (V2), and end of intervention (V4) [1, 3, 11]. Participants complete a symptom diary on a weekly basis including severity grading.

Fatigue is assessed using the 13-item Functional Assessment of Chronic Illness Therapy—Fatigue Scale (FACIT-Fatigue) [33]. A cut-off score of < 34 will be used for descriptive analysis, and norm values from a German population [34] will be used for comparisons. The scale is responsive to treatment, and triangulated MIDs have been established for various patient populations [35]. In a small study (n = 32) with people attending COVID-19 rehabilitation, the MID for the FACIT-Fatigue scale has been estimated between 2.7 and 3.6 points [36]. This estimate will be used for the interpretation of between-group differences in this study.

Dyspnea is assessed using the Chronic Respiratory Questionnaire (CRQ) dyspnea domain [37,38,39]. The CRQ dyspnea domain consists of five questions rated on a 7-point scale. The instrument is valid, reliable, and responsive, with a reported MID of 0.5 units [40], which will be used to interpret between-group differences.

Cognitive function is assessed by trained personnel using the German version of the Montreal Cognitive Assessment (MoCA) test. The MoCA test is commonly used to evaluate cognitive function in people with COVID-19 and PCC [41,42,43,44], with a cut-off score < 26 for impairment [42, 45]. The standard correction for years of education will be applied.

Anxiety and depression are assessed with the Hospital Anxiety and Depression Scale (HADS) [46,47,48]. The scale includes 14 questions with a 4-point Likert-type scale. It is a valid and reliable instrument with a triangulated MID of 1.5–1.7 units change [30, 49, 50]. A cut-off value of 7 or higher will be used to report the number of participants with depression and/or anxiety [51].

Health-related quality of life is assessed with the EuroQol Questionnaire (EQ-5D-5L) questionnaire [27, 28]. The questionnaire includes five dimensions scored on five levels of severity. The Dutch value set will be used to calculate EQ-5D-5L index scores.

Functional exercise capacity is assessed with the 30-s sit-to-stand (STS) test [52,53,54]. A familiarization test is done at the screening visit (V1). Heart rate, oxygen saturation, and ratings of perceived exertion and dyspnea are recorded before and after the exercise test. A between-group difference of ≥ 2 repetitions post-intervention will be considered clinically relevant [53].

Physical activity is measured with a triaxial accelerometer (ActiGraph wGT3X-BT, Pensacola, FL, USA) worn around the hip for 8 consecutive days before baseline (V2) and study end (V4). Data will be used for the analysis if at least 4 days (including a weekend day) of measurements with a minimum of 10 h wear time per day are recorded. Physical activity will be expressed as daily step count and time spent in different intensity domains using validated cut-offs [55,56,57].

Biomarkers of endothelial health include soluble thrombomodulin, von Willebrand Factor antigen, syndecan-1, and sVCAM-1 [58,59,60,61] and are measured using commercially available enzyme-linked immunosorbent (ELISA) or Luminex (bead-based immunoassay) assays following the manufacturers instruction.

Biomarkers of inflammation include C-reactive protein (CRP), interleukin-6 (IL-6), and leukocyte levels [60, 62]. Blood levels of CRP- and IL-6, as well as leucocyte numbers as part of the hemogram, will be determined for each participant.

Biomarkers of coagulation and platelet function include soluble CD40 ligand (sCD40L) and plasma soluble P-selectin (sP-selectin) [58, 63,64,65,66] and will be measured using commercially available ELISA or Luminex assays following the manufacturers instruction. In addition, D-dimers, international normalized ratio (INR), activated partial thromboplastin time (aPTT), and platelet concentration as part of the hemogram will be assessed.

Biomarker of oxidative stress will be the total antioxidant capacity (TAC) of blood plasma using the fluorescence recovery after photobleaching (FRAP) method.

Biomarkers of kidney and liver function include serum creatinine levels to assess estimated glomerular filtration rate (eGFR), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), and gamma-glutamyltransferase (γ-GT).

Safety outcomes

At each study visit (Table 1), vital signs (heart rate, oxygen saturation, blood pressure, body temperature) are measured. Blood pressure is measured in duplicate after resting for 5 min in sitting position (legs uncrossed) with a portable blood pressure monitor (Omron M3 Comfort). The average blood pressure value will be recorded and used for the descriptive analysis. Heart rate and oxygen saturation are measured in sitting position with a portable pulse oximeter (Beurer PO 80).

At the screening visit (V1), a 12-channel resting ECG (ELI™ 280, Hechingen, Germany) is performed with the participant in supine position (after at least 5 min rest) to screen for unstable cardiac disease. Furthermore, a blood sample is drawn to screen for other underlying causes for symptoms similar as those seen in individuals with PCC. The tests include blood count, ferritin, CRP, vitamin B12 levels, and thyroid-stimulating hormone (TSH) as well as kidney function (eGFR) and liver enzyme activity (ALAT, ASAT, γ-GT). Clinically relevant abnormal diagnostic laboratory parameters will be passed on to the participants so that necessary measures can be taken such as further diagnostics or treatment delivered through the participant’s attending physician.

Other outcomes

Adherence to the study products is evaluated during the follow-up visits (V3 and V4) and the two follow-up phone calls (P1 and P2) using a standardized assessment form. During the first follow-up visit (V3), the number of capsules returned by the participant is counted and recorded in the database. New capsules are handed out to study participants. During the second follow-up visit (V4), the remaining capsules returned by the participants are counted and recorded in the database.

Evaluation of perceived study burden and study organization

Post-exertional malaise is a frequent symptom in PCC, and we are aware that the study visits may pose a burden on the participants. At each visit, starting with the baseline visit, participants are asked whether they experienced a worsening of their symptoms within 3 days after the last study visit. Upon completion of the study, participants receive an invitation to an online survey to provide feedback regarding the overall study organization (e.g., duration of visits, breaks, number of assessments). These assessments aim to gather insights from participants on how to improve the design and organization of future trials for both this vulnerable patient population and others.

Spirometry

In addition, spirometry is performed according to the standards of the American Thoracic Society and European Respiratory Society [67] using portable spirometry (PADSY Spiro, Spirosound, Medset Medizintechnik GmbH, Hamburg, Germany). Forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV1) are measured to screen for pulmonary obstruction (FEV1/FVC ratio below the lower limit of normal). Z-scores are calculated based on reference equations published by Quanjer et al. [68]. Spirometry data are used to characterize the patient cohort.

Participant timeline

A participant timeline including study visits and assessments is provided in Fig. 1. Study visits take place at the University of Zurich. In addition, we offer home visits for people who are unable to visit the study center due to their health condition. The study team considers a home visit when the distance from our institute to the patient’s home is no longer than 90 min (one way).

Sample size

Sample size calculations were based on data from a population-based prospective cohort study (Zurich SARS-CoV-2 Cohort) [3]. In this cohort (n = 1543), 208 (13.5%) study participants reported at least one symptom 6 months after the polymerase chain reaction (PCR) diagnosed SARS-CoV-2 infection [3]. The mean (SD) health status assessed with the EQ-VAS was 73.4 ± 16.5 on a 0–100 scale. Using the mean ± SD EQ-VAS data from this cohort and considering the MID of the EQ-VAS of 8 units [31] as reference, with 80% power, the estimated sample size is 136 subjects (i.e., 68 per group). This sample size allows us to detect a difference of 8 units in the EQ-VAS between the verum and placebo groups at 12 weeks. Assuming a drop-out rate of 10%, we aim to include a total of 150 subjects.

Recruitment

We are using various recruitment channels such as the ALTEA network (https://altea-network.com/en), the Long COVID Citizen Science Board [69], and the Long COVID Network Switzerland (https://long-covid-info.ch/). In addition, we share online flyers including information about the purpose of the study, the target group, and contact details of the study team with different stakeholders.