Ethics and informed consent

This multicentric study was conducted at the Nanal Clinic and Life Veda Treatment and Research Centre, Mumbai, India. The trial was registered on 31/07/2017 with Clinical Trial Registry, India (CTRI/2017/07/009170; http://ctri.nic.in) and approved by Intersystem Biomedica Ethics Committee (ISBEC/NR-06/KM-VM/2016; IHS/UBL/01/16; 29/03/2016) and executed as per the guidelines of Declaration of Helsinki and ICMR, India. Patient signed informed consent forms were obtained by giving detailed oral and written information including risk and benefits associated with the current trial. The trial details can be accessed athttp://ctri.nic.in/Clinicaltrials/showallp.php?mid1 = 15233&EncHid = &userName = CTRI/2017/07/009170.

Study design and selection of patients

This was a randomized, double blind, placebo controlled trial design with 2 week single blind or open-label screening/run-in period. The randomization was done in 1:1 ratio and generated by statistical analysis system (SAS) version 9.4. It consisted of four phases: screening, baseline visit (week 0 ± 5 days), visit 1 (week 4 ± 5 day), visit 2 (week 8 ± 5 days) and visit 3 (week 10 ± 5 days, telephonic follow-up) (Table S8).

A total of 153 patients (male/female, 18–60 years) with IBS, fulfilling Rome III criteria2 i.e. abdominal discomfort/pain associated with two or more of the following at least 25% of the time: improvement with defecation, onset associated with change in frequency of stool/and or in the form (appearance) of stool were supplemented with placebo during a 2 week screening period. Moreover, physical examination, vital signs (pulse rate, respiratory rate, blood pressure, and temperature), complete medical history and medications were assessed.

After establishing the eligibility on screening (80% compliance to the presence and persistence of trial entry criterion), patients were called for baseline visit (randomisation/day 0). The randomization was done on the basis of qualification to inclusion and exclusion criteria. The investigation product (B. coagulans Unique IS2, 2 billion CFU/capsule) or placebo (identical in size and appearance to the probiotic capsule but contained only excipient, maltodextrin) was administered to qualified patients for up to 8 weeks, followed by observation and telephonic follow-up (Table S8). The complete blood count, serum creatinine, serum glutamate pyruvate transaminase (SGPT), and the levels of serum cytokines (pro-inflammatory IL-6, 12, TNF-α, INF-γ and anti-inflammatory IL-10) were measured. Physical and vital examinations were recorded. Diaries were provided to patients for the assessment of pain logs for complete spontaneous bowel movement (CSBM), severity of symptoms (abdominal pain/discomfort, bloating/distension, urgency, incomplete evacuation, straining, passage of gas, bowel habit satisfaction, overall assessment of IBS symptoms, and change in severity of symptoms) and stool consistency. Adverse events, if any were documented and usage of any rescue or concomitant medication was reviewed and documented.

Inclusion criteria

(a) patients of either sex in the age group of 18–60 years, (b) fulfilling Rome III criteria of IBS, (c) no evidence of inflammatory, anatomic and metabolic or neoplastic process, (d) weekly, average worst abdominal pain score of ≥3.0 on 11 point scale, (e) average of less than 3 CSBMs per week (not due to the laxatives), and (f) able to provide informed consent.

Exclusion criteria

(a) patients with Bristol stool scale score of 7 or 6 for >25% of their bowel movements during the 12 weeks before screening or, during the run-in period (except laxative induce effect). (b) disease that may affect bowel motility other than IBS, (c) presence of rectal bleeding, recent weight loss (>5 kg in the past month) or iron deficiency anemia, (d) history of lactose intolerance and other malabsorption syndromes (e.g. fructose), (e) previous abdominal surgery and severe systemic diseases, (f) use of probiotic within 3 months of screening visit, (g) pregnant or breast-feeding or planning on becoming pregnant/women of child-bearing potential not using effective contraception, (h) use of any antibiotics (e.g. neomycin, rifaximin) within 1 month of screening, (i) daily use of laxative within one month of screening/current usage, or usage from the past 3 months, of narcotics or other medications for IBS management (e.g. alosetron, tegaserod and lubiprostone).

Discontinuation criteria

(a) patients were free to drop out from the study at any time without stating any reason (b) investigator could withdraw the patients from the study at any time due to adverse event or laboratory abnormality, non-compliance to visit requirements per protocol (±5 days), <80% drug compliance, intake of any prohibited medications, pregnancy, repeated and frequent non-adherence to prescribed dosing regimen (window period of 7 days), and worsening of condition or disorder.

Sample size determination

Sample size was calculated using SAS software. The following assumption was made to detect presence of proportion difference. A minimum of 134 patients were required to be screened and 98 patients required evaluating the primary endpoint, which will provide 80% power to reject the null hypothesis (H0 = Test (%) – Placebo (%) = 0 verses Ha = Test (%) – Placebo (%) ≠ 0) when the true overall response is minimum 30% at a significant level of 0.05.

Randomization, treatment allocation and procedures

Patients were randomized into two treatment arms (B. coagulans Unique IS2 and placebo) according to standard operating procedures. The study medications (B. coagulans Unique IS2 and matching placebo) and randomization code were kept blinded. The patients were instructed to consume one capsule post meal (as per the randomization schedule) per day for 8 weeks. During the trial, patients were prohibited to take medications/therapies (Table S9). Rescue medications that could be taken in case a need arose (mild infections or allergies) were listed (Table S10). The blinded information (in sealed envelopes) was supplied to each sites in case of emergency. The compliance during the run-in phase was maintained to 80%.

Efficacy and safety measurement criteria

The primary efficacy outcomes were measured by assessing, (a) pain intensity on 11-point numerical rating scale (NRS)44 and (b) frequency of CSBM/SBM. The secondary efficacy outcomes were measured by (a) severity of symptoms on 6-point Likert scale45, (b) stool consistency on Bristol stool scale46, (c) patient and physician global assessment47,48, and (d) serum biomarker (TNF α, γ, and IL 6, 10, 12) levels (pictograms/ml) using ELISA (Diaclone Research, France). The safety was assessed by adverse event reporting, physical examination, monitoring of vital signs (heart rate, respiratory rate, blood pressure and temperature) and laboratory investigations (complete blood count).

Statistical analysis

The SAS software (SAS®, Version 9.4, USA) was used for statistical evaluations. Chi-square/two sample t-test was performed to calculate statistical significance for pain intensity, CSBM, serum cytokine profile and severity of symptoms. Physician and patient global assessment was evaluated as frequency distribution and significance was assessed with chi-square test. A p value < 0.05 was considered as statistically significant.