Table 3.

Trial outcome measures and instruments

	Outcome measures	Instrument
Feasibility outcomes	Patient recruitment	Patient recruitment per month patient/month recruited
	Patient screening	Patient eligibility % of patients
	Patient drop-out	Patient drop-out rate after randomisation % of patients, including patient acceptance to accomplish repetitive faecal microbiota transplantations (FMTs)
	Serious adverse events related to FMT	Hospitalisation or increase of >100 points on pain component of Irritable Bowel Syndrome Severity Scoring System (IBS-SSS) % of patients
	Stool sample collection	Patients provide all necessary stool samples % of the provided samples, morning stool samples will be collected during all study visits
Efficacy outcomes	>50% reduction of abdominal pain intensity and pain frequency compared with baseline at 12 (T3), 24 (T5) and 48 (T6) weeks after first FMT	Pain component of IBS-SSS score33 With two questions, the severity and frequency of the abdominal pain on the last 10 days are measured. The IBS-SSS is the only symptom severity scale that has been responsive to treatment effects.42 It has been recommended as a good instrument to obtain information on specific IBS-related symptoms.43
	Change in gut microbiota composition	MiSeq Illumina Sequencing Morning stool samples will be collected to profile the faecal microbiota composition by sequencing of the V4 region of the 16S ribosomal RNA gene
	Change in gut mycobiome composition	Internal transcribed spacer (ITS) sequencing Morning stool sample will be collected to profile the faecal mycobiome composition by high-throughput rDNA sequencing of fungal ITS−1 regions
	Change in gut metabolome composition	Capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS) Morning stool sample will be collected to profile the faecal metabolome composition by CE-TOF-MS
	Number of adverse events	Patient CRF
	Number of rescue medication	Patient CRF
	Total IBS-SSS score	IBS-SSS score33 The IBS-SSS is the only symptom severity scale that has been responsive to treatment effects.42 It has been recommended as a good instrument to obtain information on specific IBS-related symptoms.43
	Health-related quality of life	Irritable Bowel Syndrome—Quality of Life questionnaire44 This questionnaire is a 34-item assessment of the degree to which the IBS interferes with patient quality of life and consists of eight domains: dysphoria, interference with activities, body image, health worry, food avoidance, social reactions, sexual health and effect on relationships.44
	Generic quality of life	Medical Outcomes Study 36-item Short Form Health Survey (SF-36) The SF-36 questionnaire consists of 36 questions regarding eight dimensions of health perception: limitations in physical functioning, role limitation due to physical health problems, bodily pain, general health perception, vitality, social functioning, role limitations due to emotional limitations and mental health. A score between 0 (worst possible quality of life) and 100 (best possible quality of life) can be obtained. The reliability has been proven extensively for diverse patient groups and it is validated for the Dutch population.45 The SF-36 is described as adequate for persons 14 years of age and older.46
	Depression and anxiety	Hospital Anxiety and Depression Scale (HADS) The HADS is divided into two 7-item scales, with answers on a 4-point scale (0–3). Higher scores indicate a higher level of anxiety or depression (range 0–21). A scale score of ≥8 (cut-off score) indicates clinically significant anxiety or depression. The Dutch version of the HADS showed satisfactory validity and reliability.47
	Absence of school or work, healthcare resources and costs	Adapted version of the Dutch Health and Labor Questionnaire48 School or work absenteeism and indirect healthcare utilisation costs are measures by three items. Adolescents indicate whether they have been absent from school or work due to abdominal pain problems, and if yes, the amount of hours per week. For the indirect costs of healthcare utilisation, adolescents indicate additional costs they had due to symptoms of abdominal pain over the past 4 weeks.
	Impact of treatment	Adapted version of the Patient Satisfaction and Preference Questionnaire49 Impact of FMT treatment will be assessed using five questions, which are based on the Patient Satisfaction and Preference Questionnaire used in another RCT on FMT in patients with recurrent Clostridium Difficile infection.49The questions address thoughts on how unpleasant and how dirty participants find the idea of getting a faecal transplant.
	Adequate relief	One question: ‘’Did you have adequate relief of IBS symptoms (abdominal discomfort/pain, bowel habits, and other symptoms like nausea and bloating) over the past week?’’ (Yes/No)
	Plasma biomarkers Intestinal fatty acid-binding protein (I-FABP) Smooth muscle protein of 22 kDa (SM-22) Citrulline	Vena puncture EDTA vacuum tubes were used. All blood samples were centrifuged at 4000 revolutions/min, at 41°C for 15 min to obtain plasma. Plasma was immediately stored in aliquots at −80°C until analysis
	Safety parameters C-reactive protein Liver function Renal function	Vena puncture EDTA vacuum tubes were used. All blood samples were centrifuged at 4000 revolutions/min, at 41°C for 15 min to obtain plasma. Plasma was immediately stored in aliquots at −80°C until analysis
	Dietary intake	Dietary diary Dietary intake lists are filled out 7 days prior to each faecal sample collection.

CRF, case report form; RCT, randomised controlled trial.;