Study design
The study was a randomized, double-blind, placebo-controlled, equal allocation ratio, parallel-group, clinical trial performed at the BSMMU Gastroenterology department between April 2014 and August 2016. The study was approved by the ethical board of BSMMU, IRB (Institutional Review Board) BSMMU, Dhaka prior to commencement of the study; reference number BSMMU/2015/1011. All participants were informed about the objectives, methodology and purpose of the study in an easily understandable way, and those who agreed to participate were required to provide verbal and written consent prior to entry. The study was conducted in accordance with the ethical principles set out in the Declaration of Helsinki and the ICH Harmonized Tripartite Guideline on Good Clinical Practice. [Clinicaltrials.gov NCT03251625; retrospectively registered August 9, 2017].
Patients
Male and female patients aged 18 to 55 years with moderate to severe IBS-D diagnosed according to Rome III criteria [recurrent abdominal pain or discomfort (an uncomfortable sensation not described as pain) at least three days a month in the past three months, associated with two or more of the following: improvement with defecation; onset associated with a change in frequency of stool; and onset associated with a change in form (appearance) of stool. The criteria should be fulfilled for the past three months with symptom onset at least six months before diagnosis]. Patients classified as IBS-D (diarrhea predominant) had > 25% loose/wet motions (Bristol stool scale 6–7) and < 25% firm/hard motions (Bristol stool scale 1–2). The severity of IBS was determined by the IBS Severity Scoring System (IBS-SSS) as described below.
The following alarm features were required to be absent as part of screening to minimize the risk of missing important organic diseases; rectal bleeding, anemia, unexplained weight loss, nocturnal diarrhea, and a family history of organic GI diseases (e.g. colon cancer or inflammatory bowel disease). All patients agreed not to start any other drug treatment unless clinically indicated. Exclusion criteria included: treatment with probiotics within last three months; concurrent severe illness (cancer, uncontrolled diabetes mellitus, hepatic, renal or cardiac dysfunction, and hyper- or hypothyroidism); previous GI surgery; chronic organic bowel disorders (e.g. inflammatory bowel disease, tuberculosis, diverticular disease, etc); treatment with antibiotics in the two months prior to enrolment; pregnancy or lactation. To exclude other diagnoses, patients fulfilling the inclusion criteria for IBS were screened using the following tests: full blood count (FBC), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), antibody testing for coeliac disease (endomysial antibodies or tissue transglutaminase).
Procedures
Patients fulfilling the inclusion criteria in the absence of exclusion criteria or an alternative diagnosis, and who provided a written informed consent form, were included in the study and comprised the randomization group (Fig. 1). All participants were advised to maintain their usual dietary practices throughout the study.
The aim was to recruit approximately 400 IBS-D patients (see Statistical Analyses section). Randomization into two groups (equal ratios) was performed by an independent statistician using the randomizer software (www.randomizer.org). One group received the multi-strain probiotic Bio-Kult® (Probiotics International Ltd. (Protexin), Somerset, UK), two capsules twice daily (manufacturer-recommended daily dosage), while the other group received identical placebo capsules (the filler was microcrystalline cellulose in a vegetable capsule made of hydroxypropyl methylcellulose), two capsules twice daily. Bio-Kult® is a capsule formulation containing 14 different bacterial strains (2 billion CFUs per capsule) and a dosage of two capsules twice a day is equivalent to 8 billion CFUs / day. The 14 different bacterial strains in Bio-Kult® are: Bacillus subtilis PXN 21, Bifidobacterium spp. (B. bifidum PXN 23, B. breve PXN 25, B. infantis PXN 27, B. longum PXN 30), Lactobacillus spp. (L. acidophilus PXN 35, L. delbrueckii spp. Bulgaricus PXN39, L. casei PXN 37, L. plantarum PXN 47, L. rhamnosus PXN 54, L.helveticus PXN 45, L. salivarius PXN 57), Lactococcus lactis PXN 63, and Streptococcus thermophilus PXN 66]. The capsules were administered before or during a meal for a total of 16 weeks; patients were followed-up one month after this time.
An independent data monitor maintained treatment codes and allocation, which was locked until all analyses had been completed. Thus, the clinical trial was performed double-blind with all patients and clinical staff unaware of which treatment had been allocated.
Before starting treatment each individual underwent a baseline assessment during which demographic data, IBS symptoms and QoL data were recorded. During treatment patients were required to return to the clinic once a month for reassessment of IBS symptoms and QoL, and to report any adverse events (AE) that had occurred.
The IBS-Severity Scoring System (IBS-SSS) questionnaire was completed at baseline, at each monthly clinic visit and after one month’s follow-up. The IBS-SSS is a 5-item instrument used to measure severity of abdominal pain, frequency of abdominal pain (number of days with abdominal pain over the last 10 days), severity of abdominal distension, dissatisfaction with bowel habits, and interference with quality of life, each on a 100-point scale [35]. The items are summed and thus the total score can range from 0 to 500 points. IBS severity has the following defined ranges: mild 75–174, moderate 175–300, and severe > 300. The IBS-SSS was completed by the physician at each clinic visit.
The IBS-QoL questionnaire is a 34-item measure constructed specifically to assess QoL impairment due to IBS symptoms [36]. Each item is scored on a five-point scale (1 = not at all, 5 = a great deal) that represents one of eight dimensions (dysphoria, interference with activity, body image, health-related worries, food avoidance, social reactions, sexual dysfunction, and relationships). Items are scored to derive an overall total score of IBS related QoL. To facilitate score interpretation, the summed total score is transformed to a 0–100 scale ranging from zero (poor QoL) to 100 (maximum QoL). The QoL instrument was translated into Bengali and given to each patient before treatment was started; the patient completed the form each month and all data were recorded and entered onto a data sheet.
Efficacy assessments and endpoints
The aim of this study was to determine whether administration of a multi-strain probiotic was more effective than placebo at reducing GI symptoms and improving QoL in patients with moderate to severe IBS-D.
Primary endpoint
Secondary endpoints
-
The change in other GI symptom severity scores (including stool consistency, frequency, and bloating) on the IBS-SSS during treatment with a multi-strain probiotic and placebo, and compared with baseline.
-
The change in QoL parameters (using a validated IBS-QoL questionnaire) during treatment with a multi-strain probiotic and placebo, and compared with baseline.
-
To assess any AEs reported during treatment with a multi-strain probiotic and placebo.
Sample size
The sample size for this trial was determined using the formula:
$$ {\mathrm{n}}_1=2{\sigma}^2{\left({\mathrm{Z}}_{\beta }+\mathrm{Z}\alpha \right)}^2/{\left({\mu}_1\hbox{--} {\mu}_2\right)}^2 $$
Where:
$$ {\mathrm{Z}}_{\beta }=0.84\ \mathrm{a}\mathrm{t}\ 80\%\mathrm{power};\mathrm{and}\ \mathrm{Z}\alpha =1.96\ \mathrm{a}\mathrm{t}\ \mathrm{a}\ 95\%\mathrm{confidence}\ \mathrm{interval}. $$
μ1 – μ2 represents the minimum clinically important difference which was set at 30% (this is advocated as the minimal clinically significant reduction for probiotics). A standard deviation (σ) of 87.77 for IBS-SSS from a similarly designed study was reported by Sisson and colleagues and was used in the sample size calculation [29]. Based on these assumptions the sample size was calculated to be 135 per treatment group (270 in total). Allowing for dropouts and non-adherence we estimated that the sample size should be increased to a total of approximately 384 patients (192 per group). In practice the first 400 patients with IBS-D attending the BSMMU Gastroenterology department between April 2014 and August 2016 and who provided written informed consent entered the study and were randomized to treatment.
Statistical analyses
All analyses were performed in the per-protocol (PP) set, i.e. treated patients that had no major protocol violations, met the minimum protocol requirements, and who were able to be evaluated for the primary endpoint. IBS-SSS symptom scores and IBS-QoL instrument scores were expressed as means ± standard deviation (SD) and analysed using the Student’s unpaired t-test. Categorical data are presented as frequencies/percentages and were analysed using the chi-square test. The relationship between different variables was investigated using Pearson’s correlation coefficient. P values < 0.05 were considered to be statistically significant. All analyses were performed using a computer based SPSS program (version 13.0).