An Integrated Relaxation Protocol Improves Immune Biomarkers and Reduces COVID-19 Severity and Progression in Patients With COVID-19

Study procedures A randomized controlled design was used. The study was double-blinded in which both patients and assessors were blinded to study procedures. Each patient was asked to sign an informed consent before participation in this study. This study was approved by the ethics committee of our university (blinded). Patients were recruited from December 2020 to April 2021. The patients were collected by reviewing the hospital's data (blinded). Then, we called patients with COVID-19 to ask them about their welling to participate in this study. Patients who accepted to participate were interviewed through video calls using Zoom App to discuss study procedures with them. Patients who accepted to engage in this study were visited by a researcher at their home while they are in quarantine. Through the initial visit, the consent form was signed, and blood samples were taken. Any patient must have permission to perform our intervention from the physician who supervises him/her. The clinical protocol of this study was retrospectively registered at https://www.clinicaltrials.gov/ platform with this registration number NCT04998708. The study was registered retrospectively because of the vulnerability of the research team to have COVID-19 which may affect the dates and data of the trial. There were no deviations from the registered trial protocol.

The suitable sample size for this study was determined using a priori power test. The G*POWER program (ver. 3.1.9.2, Heinrich-Heine-University, Düsseldorf, Germany) was used. The criteria entered into the program included a MANOVA test using 2 groups, 2 measurements, a power level of 80%, a significance level of .05, and medium effect size (dz= .25)(Faul et al., 2007). Based on the previous assumptions, the total sample size needed for this study was 30 patients. A minimum power of 80% or more is acceptable in most studies(Kadam & Bhalerao, 2010). The flow of patients throughout the study is shown in Figure 1.

Initially, forty-five patients accepted the participation in this study. Ten patients did not meet the study inclusion criteria and additional five patients did not complete the whole study because they did not like doing exercise. Finally, thirty patients with COVID-19 completed the whole study (2-weeks intervention and 3-weeks post-intervention "follow-up"). The patients' age ranged from 25 to 45 years old. The inclusion criteria included that the patient has a recent mild or moderate COVID-19 with no or low-grade fever 99.5- 100.94 °F (37.5-38.3 °C) (Affronti et al., 2010; Zhuang et al., 2020). Mild grade COVID-19 was determined by having symptoms of acute upper respiratory tract infection (fever, cough, myalgia, runny nose, fatigue, sore throat, sneezing) or gastrointestinal symptoms (nausea, vomiting, abdominal pain, diarrhea). Moderate grade COVID 19 was determined by having mild symptoms plus pneumonia (cough, frequent fever) with no obvious hypoxemia, the presence of lesions on chest CT(Yuki et al., 2020). The exclusion criteria included that the patient is indicated for being hospitalized, has high-grade fever <100.94 °F (<38.3 °C), or other chronic diseases such as heart problems, hypertension, or diabetes. Women on contraceptives were excluded because contraceptives decrease immune function increase the risk of various autoimmune disorders(Williams, 2017).

Evaluative procedures There were three main dependent variables including blood immune markers, salivary immunoglobulin A, and Wisconsin Upper Respiratory Symptom Survey. These measurements were collected at the baseline, 2-weeks post-intervention, and 1-week as a follow-up.

A lab technician and one researcher were asked to visit the patient at home during the quarantine. The technician collected blood and saliva samples and the researcher conducted the Wisconsin Upper Respiratory Symptom Survey. They wore special protective equipment recommended by WHO(WHO, 2020b; World Health Organization (WHO), 2020). Three visits were performed including the beginning of the research procedures, 24-h post-intervention (2 weeks), and 1-week as a follow-up.