2.1. Study design
This study is a 8-weeks randomized, double-blind clinical trial with a control group in the dialysis ward of Musabn Jafar Hospital in Quchan. Patients with ESRD undergoing hemodialysis will randomly be assigned using a permuted block randomization method to intervention and control group (30 patients in each group). In the intervention group patients receive two synthetic supplements of lactoker (500 mg) daily with lunch and dinner for 8 weeks. In the control group, patients received placebo capsules containing 500 mg of cornstarch twice a day for 8 weeks, with the same size, shape and color of synbiotic capsules.
2.2. Inclusion criteria
Participants will be eligible for this study if they meet the following criteria:
1) Having informed consent to participate in the study, 2) Age between 18, to 65 years, 3) patient with a diagnosis of chronic renal failure who undergoing hemodialysis for at least 3 months, 4) Patient with arterial fistula, permanent catheter or venous artery graft, 5) patient undergoing hemodialysis 3 times a week for 4 hours each time, 6) Patient with KT / V>1, 7) Do not suffer from hearing and visual disorders, 8) Not suffering from neurological disorders and history of hospitalization in the psychiatric ward.
2.3. Exclusion criteria
The following people will be excluded from the study:
1) Active infection during the last 30 days, 2) Pregnant or lactating mothers, 3) Taking antibiotics, prebiotics, probiotics, synbiotics, vitamin supplements or antioxidants during the last 30 days, 4) Suffering from autoimmune diseases or connective tissue disorders, 5) History of cardiovascular diseases, 6) Diabetic foot infection, 7) Gastrointestinal diseases (inflammatory bowel disease, ulcerative colitis, Crohn's disease, celiac disease, lactose intolerance or allergy, irritable bowel syndrome), 8) Liver disease (hepatitis B, hepatitis C), 9) Infection with various cancers and malignancies, 10) Smoking more than 10 cigarettes a day, 11) Participate in other drug interventions, 12) Having Hb less than 8 mg/dl, 13) PSQI(Pittsburgh Sleep Quality Index) score less than 6 (good sleep quality), 14) CFS(Chalder Fatigue Scale) score less than 18
After receiving ethics code from the committee of research and technology, the researcher referred to the "Dialysis Department of the Educational Center of Musabn-Jafar Hospital in Quchan and provided the necessary explanations about the research objectives and benefits of Synbiotics supplements, method and rights of research samples such as confidentiality of patient information, etc. written consent will be received. First, the demographic information will received. The Pittsburgh Sleep Quality and Chalder Fatigue Questionnaires complete by research samples after that they will randomly assigned by permutation block randomization method to intervention and control groups. Patients in the intervention group receive synbiotic supplement and the control group receive placebo for eight weeks. Pittsburgh Chalder Fatigue and Sleep Quality Questionnaire is completed by patients at the end of the fourth week and at the end of the eighth week after the intervention. The choice of study duration is based on the results of studies performed so that the positive results of the interventions have been proven during 6 weeks or more [9–16–18–19]. All subjects, researchers, statistical analyst and dialysis staff will be blind to the intervention.
Synbiotic and placebo capsules are packed in the same shape and size in boxes with the same appearance. The capsule will be labelled "A" and "B" by a third colleague who will not be involved in the study method. Subjects, researchers, statistical analysts, and dialysis staff will be blind to the analysis of data on box contents, allocation, and treatment.
2.5. Sample size
The sample size required for the present study was based on the data of Soleimani et al. who investigated the effect of probiotics supplementation on metabolic factors in diabetic hemodialysis patients. The sample size for each group was 26 individuals. Considering the 20% probability of falling for each group of 30 subjects was calculated (α = 0.05 and β = 20%). Equation (1).
The probable start date of sampling for this study is from the beginning of August and it is predicted that the sampling will end by the end of November 2021. A period of one month is also predicted for statistical analysis of data.
The intervention group take two synbiotic capsules daily (one after lunch and the other after dinner) and the control group take two placebo capsules (containing 500 mg of cornstarch) daily at the same time for 8 weeks. Synbiotic and placebo capsules are similar in appearance and are marked only with the label "A" or "B" on the box. Boxes are tagged by a third party not directly involved in this study. Each Lactoker Synbiotic Capsule (500 mg), produced by Bio Fermentation Company of Iran, contains high amounts of 12 beneficial and safe bacterial strains along with fructooligosacharide as a probiotic. The strains used in this product includes: (Lactobacillus rhamnosus, Lactobacillus Hlvtykvs, Lactobacillus casei, Bifidobacterium lactis, Lactobacillus acidophilus, Bifidobacterium Barwa, Lactobacillus bulgaricus, Bifidobacterium Langvm, Lactobacillus plantarum, Bifidobacterium bifidum, Lactobacillus Gsry, Streptococcus thermophilus). Each box contains 30 capsules. Participants are given four boxes for the entire 2-month intervention (two boxes at the beginning of each month). In addition, patients continue to take their daily medications throughout the study. The Ethics Committee of Sabzevar University of Medical Sciences will be responsible for following up on reports from participants regarding any possible related issues and side effects. Any possible side effects will be reported to the Medical Ethics Committee during the study. At the beginning of the study, subjects are informed consent take from the study subjects. Written and oral instructions on how to use the capsules are provided at the first visit and at subsequent visits. Patients followed for Adherence to the intervention twice a week by telephone. People are asked to take one capsule after lunch and another after dinner, which also improves their adherence to the intervention. In addition, subjects ask to record the daily intake of supplements or placebo in the delivered booklet. All subjects ask to return the capsules left in the boxes at the next visit. At the end of the 8 weeks if the number of capsules remaining for each participant is more than 10% of the total capsules prescribed, that participant will be excluded from the intervention.
Primary outcomes include evaluation of fatigue status and sleep quality in patients with ESRD undergoing hemodialysis. The feeling of reduced capacity for physical and mental work at the usual level is called fatigue . The patient's fatigue rate is assessed based on the Chalder fatigue scale. This scale has a total score between 0 and 33 and if the patient scores above 18 according to this questionnaire, it will be considered tired. The higher the score, the more severe fatigue the patient experiences . Sleep disturbances are typically known as difficulty in initiating or maintaining sleep or as excessive or uncontrollable drowsiness. Sleep disorders occur in the form of partial awakening in sleep . Sleep quality is calculated based on the Pittsburgh Sleep Quality Questionnaire. This scale has a total score between 0 and 21 and a score between 0 and 5 means good sleep quality and a higher score means poor sleep quality . Secondary outcomes include evaluation of the CRP and evaluation of patients' Hb level. All measurements will be done at the beginning, the end of the fourth week ,and at the end of the intervention.
2.8. Fatigue and sleep quality assessment
Chalder fatigue and Pittsburgh sleep quality questionnaire use to measure patients' fatigue and sleep quality score respectively.
The Chalder Fatigue Scale to assess fatigue disability was first published by Chalder et al. in 1993 and then in 2010 with further psychometric evaluation. This questionnaire contains 11 questions and two general areas for examining physical and emotional fatigue and examines fatigue during the last month. How to answer the questions is fourfold (much less than usual, less than usual, more than usual, much more than usual). The scoring system on this scale is such that each question is assigned a score between 0 and 3, which in total, the result is between 0 and 33. If the patient scores above 18 according to this questionnaire, he / she will be considered tired; the patient suffers from more fatigue with a higher score. The validity of this scale has been examined by Chalder et al. And by using the list of symptoms and clinical interview, the sensitivity was 75.5% and the specificity was 74.5%. The internal consistency coefficient was 0.85 for physical fatigue questions and 0.82 for mental questions. The reliability of this scale was calculated by Chalder et al. 0.92 .
In Iran, for the first time Nasri et al. evaluated the validity of this questionnaire, a group of patients with symptoms of fatigue were compared with a group of healthy individuals using this scale, and the results showed that this scale is valuable for measuring fatigue. Test-retest coefficient and internal consistency calculated in the study were reported to be 0.85 and 0.91, respectively, indicating the high validity and reliability of this questionnaire .
The Pittsburgh Sleep Quality Index is used to measure sleep quality, which is a validated standardized tool for assessing sleep quality. This self-monitoring questionnaire was designed in 1989 by Boris et al. And assesses sleep quality over the past month. The questionnaire includes 19 questions about 7 important components of sleep (mental quality of sleep, duration of sleep, duration of sleep, sleep efficiency, sleep disorders, use of sleeping pills, and inefficiency during the day). Each component is assigned a score between 0 and 3, so the overall PSQI score is between 0 and 21. A score between 0 and 5 means good sleep quality, while a score between 6 and 21 means poor sleep quality. The sensitivity and specificity of this questionnaire were reported by Boris et al. 89.6% and 86.9%, respectively .
Based on the study of Farhi Moghadam et al. Who first validated this questionnaire; based on cut-off point 6 for this questionnaire, sensitivity and specificity, scores of 94% and 72% were obtained, respectively .
2.9. Anthropometric assessment
Anthropometric indices will be accurately measured at the beginning. At the beginning of the study, the height of each participant standing on the wall without shoes will be measured with an accuracy of 0.1 cm with a stadiometer (seca, Hamburg, Germany). Waist and pelvis will be measured with an accuracy of 0.1 cm in the area with the smallest diameter with a non-stretching tape. Waist-to-height and waist-to-hip ratios are calculated using standard equations. The weight of the subjects in the fasting state, without shoes, and only by wearing light clothes, will be measured using a scale (seca, Hamburg, Germany) with an accuracy of 0.1 kg. Body mass index will be calculated by dividing body weight by kilograms by height per square meter.
2.10. Blood sampling, biochemical assessment
A 5 ml venous blood sample is collected from each participant at the beginning and end of the test. 3 ml of it is collected in a test tube containing clot activator and centrifuged at room temperature at 3000 rpm for 10 minutes (Eppendorf AG, Hamburg, Germany) to separate serum. Serum samples are stored at 70 ° C. Residual blood samples are collected in a tube containing ethylene diamine tetraacetic acid (EDTA) to measure Hb. To maintain patient confidentiality, all laboratory information is stored using an ID number.
2.11. Statistical analysis
Statistical analyses will be performed with SPSS software V.23.0 (SPSS Inc. Chicago, IL, USA). Normality of distribution of data will be assessed by the Shapiro-wilk test. At first, the primary information of the intervention and control groups will be compared. Continuous data will be presented as means ± standard deviation (SD), and categorical data will be expressed as numbers and percentages. The independent samples t test and the Mann-Whitney U test will be used for analysing the differences in parametric continuous and asymmetric variables between the two groups, respectively. Pearson correlation test is also used to analyse the correlation between quantitative variables. The repeated measure is used to examine the dependent variables that are measured in three turns. P values < 0.05 will be considered as statistically significant. The significance level is 95% and the confidence interval is 95%.
Because dialysis patients come for dialysis three times a week and the questionnaires are completed by the patients in the presence of the researcher's help, there will be no missing data. The results of the aforementioned analyses will be compared with each other.