Dose-Effect of Long-Snake-Like Moxibustion for Chronic Fatigue Syndrome: Study Protocol for a Randomized Controlled Trial

Chronic fatigue syndrome (CFS) is a disease with high incidence rate and great impact on life, but it lacks for specic pharmacological treatment and diagnostic indicators. In the theory of traditional Chinese medicine (TCM), fatigue is the result of Yang deciency. Long-snake-like moxibustion, as one of a special moxa therapy, has been applied in Yang deciency patients for thousands of years in China and it is used widely to relieve fatigue symptoms for its strong function of Yang warming. However, the does-effect relationship is unclear in the moxibustion research. Hence, we design this trial to assess the duration-effect of long-snake-like moxibustion through combining measurements of the subjective patient-reported scales with objective medical infrared imaging technology ─ Thermal Texture Maps (TTM).

greatly affect patients' work and life quality. Among the 40% global prevalence of CFS, most of suffers are women [2]. The medical cause for CFS is still not clear, so no speci c pharmacological treatment is recommended. Acupuncture [3], yoga [4], cognitive behavioral therapy (CBT) or graded exercise therapy (GET) [5][6][7] are promising approaches to the treatment.
In the theory of traditional Chinese Medicine (TCM), fatigue is the result of Yang de ciency, and moxibustion can produce yang-warming effect to improve the de cient yang for zang and fu. Although the mechanism of CFS treated by moxibustion is not fully explained, the moxibustion therapy is effective in relief of symptoms [8,9]. Long-snake-like moxibustion is also called as Du moxibustion. In this special moxa therapy, a large range of moxa cones or sticks over herbs or ginger are applied on the Governor Vessel from GV14 to GV2 to generate a strong warming stimulation. In our previous study [10], we improved the long-snake-like moxibustion technique through applying moxa box over ginger slices on the back from GV14 to GV2 as well as the same level of the rst lateral line of bladder meridian to increase clinical operability and enhance therapeutic effect.
A variety of factors in uence the therapeutic e cacy of moxibustion, especially moxibustion dose, which includes moxibustion duration, moxibustion frequency, moxibustion amount-size and number of moxa cone [11][12][13][14]. None studies have focused on the dose-effect of moxibustion for CFS up to now, therefore, we designed this trail to investigate the relationship of long-snake-like moxibustion duration and effect in the treatment of CFS by combining subjective scales and objective measurement for assessment.
So far, CFS trials into the e cacy and safety of therapies have employed the self-reported subjective measurements as the outcome assessments for evaluating subjective physical function, anxiety, depression, and pain improvement, such as the Chalder Fatigue Scale, Short Form 36 Health Survey Questionnaire (SF-36), Anxiety and Depression Scale and visual analogue pain rating scale [15]. In addition, biological markers and imaging techniques such as MRI are also used to aid clinical diagnoses and effect assessments [16,17]. In this trial, we try to use the Thermal Texture Maps (TTM)─a thermography technique of evaluating thermal signatures of the body through holistic interpretation of infrared images to aid CFS diagnosis and moxibustion dose-effect assessment.

Methods And Design
Design A single-center, randomized, controlled trial will be conducted at the Center of Preventive Medicine of Hospital of Chengdu University of TCM. 30 healthy women (Group C) without moxibustion therapy and 60 CFS female patients with a treatment period of 4 weeks (3 sessions per week, every other day) will be recruited. When meeting the inclusion criteria, 60 CFS patients will be randomly allocated to group of 60minute long-snake-like moxibustion (Group A), or 30-minute long-snake-like moxibustion (Group B) in a 1:1 ratio after exclusion. The study ow is depicted in Figure 1.

Randomization and allocation concealment
Eligible CFS participants will be randomly allocated to treatment in Group A or Group B. Random numbers will be generated by Statistics Analysis System (SAS), and then placed folded over in opaque envelopes. A research assistant, who is uninvolved with assessment or treatment, is responsible for keeping the original random allocation sequences, printing serial numbers on the outside of the envelopes, and sealing the envelopes. All envelopes will be put into a plastic container in numerical order. Once the patients are assessed to meet the inclusion criteria and informed consent forms, the investigators will open the envelopes in turn. Investigators who assess outcomes and analyze results are masked to treatment allocation.

Blinding
The outcome assessors and statistical analysts will be blinded to the intervention assignments throughout the trial.

Ethics and clinical registration
This trial will follow the rules of the Declaration of Helsinki and the Good Clinical Practice Guidelines, with unique registration number (ChiCTR2000041000) at http://www.chictr.org.cn. The protocol has been approved by the Ethics Committee of Hospital of Chengdu University of TCM in 2020 (NO.2020KL-046).
Written informed consent will be obtained from every patient before participation.

Participates
Healthy females aged 18 to 60 will be recruited as healthy participates. Patients will be recruited if : (1) females aged 18 to 60; (2) chronic fatigue is lasting and can't be explained by illness; (3) debilitating fatigue predates and is accompanied by at least 4 of 8 designated symptoms: post-exertional malaise lasting more than 24 hours; unrefreshing sleep; impaired short-term memory or concentration severe enough to cause substantial reduction in previous levels of occupational, educational, social, or personal activities; headaches of a new type, pattern, or severity; muscle pain; multi-joint pain without swelling or redness; sore throat; and tender cervical/axillary lymph nodes. Accompanying symptoms must have persisted or recurred during 6 or more consecutive months of illness.
Patients should also meet the inclusion criteria of the pattern of spleen and kidney Yang de ciency: (1) 3 of 5 main symptoms: being afraid of cold, limb and/or lower back coldness; fatigue; breath shortness; poor appetite; and weakness of lower back and knee. (2) 2 of 6 accompanied symptoms: cold pain of lower back; stomach and/or abdominal fullness; loose stool; frequent night urination; teeth-mark tongue; and deep and weak pulse.
Parts of participants will be recruited from the hospital. The other ways of participant recruitment are electronic posters on WeChat or posters displayed in hospitals and communities. A face-to-face interview will be held by the clinical trial coordinator to ensure all meet the inclusion criteria.

Intervention Practitioners
Certi ed acupuncturists with TCM license and more than 2-year experience in clinics will be trained to participate in this trial. The training includes the correct manipulation of long-snake-like moxibustion.
Group A: 60-minute long-snake-like moxibustion Patients in prone position with the whole back exposure will receive the ginger-indirect moxibustion on Governor Vessel and Bladder Meridian. Ginger slices for 2mm in thickness will be placed on the back from GV14 to GV2 to cover the area of the Governor Vessel and the rst lateral line of Bladder Meridian ( g 2), then ve or six three-hole Moxa boxes (the number of boxes depends on the patients' height; g 3) will be put on them. The distance between the center of nearby holes is 4.8cm to ensure the temperature of moxa sticks (pure-mugwort moxa stick; 20 cm in length and 18 mm in diameter; Hwato, Suzhou, China, PRC) cover the Governor Vessel and the rst lateral line of Bladder Meridian. During the 60-minute gingerindirect moxibustion treatment, the depth of moxa sticks will be regulated every ve minutes to maintain full combustion and obtain equal warm stimulation.
Patients will receive 12 sessions of moxibustion treatment over a period of 4 weeks (1 session per day, 3 sessions per week, every other day).

Group B: 30-minute long-snake-like moxibustion
The treatment process is as the same as in Group A, with a moxibustion duration of 30 minutes.

Outcome measures Primary
In this trial, the score improvement based on the Fatigue scale-14 (FS-14) will be used as the primary outcome measurement. As a standardized questionnaire to re ect physical fatigue and mental gure, FS-14 comprises 14 questions, each of which has two options (yes or no) with a score of 0-1 (0=no, 1=yes), with a total score ranging from 0 to 14 [18,19]. Higher scores indicate a higher level of chronic fatigue. It will be assessed at baseline and week 4 (the end of treatment).

Secondary
The secondary outcomes include the change of the Self-rating depression scale (SDS) the Self-rating anxiety scale (SAS), and the Symptoms Scale of spleen-kidney Yang de ciency.
The Symptoms Scale of spleen-kidney Yang de ciency is a four-rate scale designed to evaluate the symptoms based on the TCM syndrome differentiation (table 1). It consists of primary symptoms with a ranging score of 0-6 of cold limbs, fatigue, breath shortness, poor appetite, weakness of lower back or knees, and secondary symptoms with a ranging score of 0-3 of lower back cold pain, stomach or abdomen fullness, loose stool, frequent night urination. Higher scores indicate a higher level of spleenkidney Yang de ciency.
All secondary outcomes will be assessed at baseline and week 4 (the end of treatment).
Thermal Texture Maps (TTM) scanning process 90 eligible subjects (30 CFS female patients in Group A, 30 CFS female patients in Group B, and 30 healthy women in Group C) will receive TTM (Digital Medical Infrared Imaging System, MTI-ex pro-2013, Chongqing, China) scanning after inclusion, and only patients in Group A and B will receive the 2nd TTM scanning after 4-week treatment. To weaken the in uence of external thermo-source from moxibustion in the images, the 2nd TTM scanning will be employed 5 days after the end of treatment. On the thermal map, the temperature value or thermal signatures (irregularity, asymmetry, discontinuity) will be recorded in the typical area, meridians or acupoints, including the back, spine, chest, abdomen, four limbs, head, the governor vessel from GV14 to GV2, the conception vessel from CV22 to CV2, and GV14, CV17, CV8. Comparison between healthy subjects and CFS patients, self-comparison before and after treatment will be made.
To minimize the impact of outside temperature, all the scanning will be conducted at the health examination center of hospital of Chengdu university of TCM in the morning, with a controlled room temperature of 24℃±2℃ and humidity of 20%±10%, without obvious air ow, strong light and infrared radiation source.
The process is as below 1. Fill the personal health information form before scanning, which contains the medical history and the state of body condition in all organs; 2. Remove the ornaments, loosen the bra, and relax hair in advance. Have a rest of 30 minutes at rest room before scanning; 3. In prior to the scanning, patients will be asked to take all clothing off and expose the body in a temperature-controlled room for 10-15 minutes. Then images will be taken in front vs. back, left vs.
right to assess overall topological features of the whole body.
The notices are as below 1. Fast for at least 2 hours prior to the scanning; 2. Before the scanning, alcohol should be stopped for at least 24 hours, the use of medications as vasodilator or vasoconstrictors are stopped for at least 4 hours, vigorous exercise is forbidden for at least 4 hours, other stimulants are stopped for at least 2 hours and hand washing is stopped for at least 30 minutes; 3. Skin conditioning such as use of cosmetics and perfume should be minimized, and a dress of looser clothing is advised; 4. No scanning during menstruation.

Sample size
Based on our preliminary study [20], in which an improvement of 2.45 on the FS-14 score after 60-minutes Long-snake moxibustion therapy was detected, we expect a difference of 0.4 in the mean improvement of FS-14 score. With a desired power of 0.9 and a signi cance of 0.05, the number of required patients is 27 per group. Taking into consideration a dropout rate of 10%, we plan to include a total number of patients of 90.

Statistical methods
All of the data analysis will be based on an intention-to-treat population, replacing missing data by the last-observation-carried-forward method. Quantitative data will be shown by mean with standard deviation (SD) or median with percentile (QL-QU), while qualitative variables will be expressed by frequency and proportion. The change of outcome will be shown by mean and 95% certi cate interval (CI), by setting the absolute value at baseline as starting point. All the statistical analyses will be performed with R software version 3.6.3 (R Development Core Team, Vienna, Austrian). A two-sided test will be applied for all available data, and a P value <0.05 is considered statistically signi cant.

Data collection and quality control
The original data will be recorded in paper case report form (CRF) and then lled in a spreadsheet. An independent investigator will check the original data to ensure the consistency of CRF and spreadsheet. Only the principal investigators and clinical investigators are quali ed to review the data. Before the start of trial, all involved researchers will undergo training to guarantee the full understanding of study protocol, research process and CRF-lling. Additionally, once the eligible participants are enrolled, full communication about possible bene ts and risks will be made and comfortable treatment environment will be provided to ensure participants' compliance. For participants with poor compliance, investigators should contact them by phone to acquire the outcome data, possible reasons and encourage them to complete the study.

Discussion
Due to the non-organic pathophysiology, CFS is associated with suboptimal health status [21] and the present treatment of CFS is given based on limited or even contradictory evidence from clinical studies [22][23][24]. CFS is named fatigue syndrome in TCM theory and Yang-qi de ciency is regarded as the main reason. Yang qi is thought to be the vital motivity, so organs will become hypofunctional once Yang qi decreases. Moxa therapy has been applied in Yang de ciency patients for thousands of years in China and it is used widely to relieve fatigue symptoms [25].
The dose-effect relationship is the key issue in the moxibustion research. Key factors that determine the moxibustion dose include the treatment duration, frequency, the moxa extent and amount of moxa cones/sticks. However, the relationship between the dose of moxa stimulation and the resulting therapeutic e cacy has not yet been established, and previous studies have shown that the above dose factors affect e cacy to a different degree in different diseases. In the study of moxibustion in treating diarrhea-predominant irritable bowel syndrome, aconite cake-separated moxibustion with treatment regimen of 3 treatments/wk. and 1 cone/treatment appeared to produce better therapeutic effects compared with the regimen of 6 treatments/wk. and 2 cone/treatment [14]. Another study on suspended moxibustion for focal cerebral ischemia/reperfusion injury indicated a greater anti-apoptotic effect of 35minute moxibustion than 15-minute moxibustion [13]. When treating depression-like behavior disorder in rats, prolonged duration of moxibustion under the optimal extent could increase the level of 5-HT [12]. In the treatment of chronic neck pain with the direct moxibustion of small moxa cone, a trend of effect improvement was related to an increase of the amount of moxa cones, when the other dose factors were constant [11]. The duration of long-snake-like moxibustion is a key factor in optimizing e cacy, but the treatment duration in treating CFS varies [26,27]. In this trial, the duration-effect relationship will be studied for long-snake-like moxibustion employed from GV14 to GV2 as well as the same level of bladder meridian's rst lateral line on the back. The result will provide evidence of treatment duration choice for long snake-like moxibustion in treating CFS, and enrich the content of dose-effect research of moxibustion therapy.
As for the lack of diagnostic indicators, the diagnosis and therapeutic measurement of CFS lie on the application of patient-reported outcome scales [28,29], such as FSS ( Researches on the biomarkers of CFS have revealed that the disorder in neuro-immuno-endocrinological pattern is part of the pathophysiological mechanisms [30,31], and functional (non-structural) changes in the brain are found by structural and functional MRI [17], but all of them can't fully reveal a CFS-speci c measure. The research efforts are still made to seek biomarkers or new technology to aid aetiological understanding and treatment options for CFS.
A large body of evidence has showed that diseases or deviation from normal functioning are accompanied by temperature changes in the body, which in turn affect the temperature of the skin.
Infrared imaging (IR) was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature. As a new medical infrared imaging technology, TTM can pinpoint the abnormal heat sources inside human body by using thermography analysis through the surface temperature distribution. Since the invention of TTM, it has been applied in disease diagnosis such as oncology (breast, skin, etc.), vascular disorders (diabetes, deep venous thrombosis), and monitoring the e cacy of therapeutic drugs, etc. [32][33][34]. So, in this trail, we try to assess the dose-effect of moxibustion through combining measurements of the subjective patient-reported scales with objective TTM images.
In conclusion, this study will evaluate the effect difference of long-snake-like moxibustion with 60-minute and 30-minute duration, measured with medical infrared imaging technology─TTM and traditional scales. The result will contribute to the explore of the dose-effect relationship of moxibustion, therefor to optimize e cacy of moxibustion.

Trial status
The protocol number is ChiCTR2000041000.

Availability of data and materials
Data sharing is not applicable to this study protocol because no datasets have yet been produced. Study materials are available by contacting the corresponding author on reasonable request.
Competing interests