Study population
A placebo-controlled, single-blind study of Pylopass™ containing L. reuteri DSM17648 versus placebo in subjects who are Helicobacter pylori carriers and show mild indigestion was conducted at the Clinical Trial Unit of University College of Cork and the Mercy University Hospital, Cork, Ireland. 115 healthy volunteers were screened for H. pylori infection using a 13C UBT test. Among them, 24 (13 female, 11 male) had positive results for H. pylori and were enrolled into the study. The sample size was calculated using the mean urea breath test values from baseline and post-test conditions as the standard deviation. The type I error rate (α) was assumed to be 0.05, and statistical power β = 0.20, it was estimated that the study required 20 subjects to achieve 80% power. To account for dropouts, an additional 20% was added to the n so that the total recruitment included 24 randomized volunteers. The subjects were between 18 and 75 years of age, with good general health or mild digestive discomfort (such as indigestion), a positive UBT (Helicobacter test δ > 1.5%) and had documented informed consent. Exclusion criteria were pregnancy/lactation, hypersensitivity to any of the components of the test product, any active gastrointestinal disorder or previous gastrointestinal surgery, significant acute or chronic condition or consumption of any medications (e.g. immunosuppressive drugs) deemed by the principle investigator to have the potential to confound the study or pose a safety risk, entry to the study, active gastrointestinal disorder or previous gastrointestinal surgery, condition or intake of any medication interfering with the objectives of the study, posing a safety risk or confounding the interpretation of the study results, intake of PPIs or gastroprotective medicines, oral intake of antibiotics in the past 3 months, prior eradication therapy with antibiotics, any major dietary changes in the past 3 months, intake of non-steroidal anti-inflammatory drugs (NSAIDs) within 2 weeks of baseline visit or for the duration of the trial, alcohol or drug abuse, participation at other clinical trials at the same time or completed not less than 60 days prior to this study and any surgical procedures, malignant disease or concomitant end-stage organ disease.
Study supplements
The active supplement consisted of 100 mg Pylopass™ (1 × 1010 spray-dried cells of Lactobacillus reuteri DSM17648) (LONZA Group Ltd., Switzerland), FOS, Sorbitol (E420i), Xylitol (E-967), Flavor, Silicium oxide (E551), Magnesium stearate (E470b) and Sucralose (E955), (Eladiet S.L., Spain) prepared as solid tablets for oral application. The daily dosage of 2 tablets corresponds to 2 × 1010 cells. The Pylopass™ and placebo tablets were identical in weight (600 mg), size color and flavor. The test products were produced in compliance with the requirements for Good Manufacturing Practices for nutrition ingredients (Food GMP) (Eladiet S.L., Spain).
Study design
The study was conducted according to the Declaration of Helsinki. All patients gave written informed consent prior to their participation in the study. The study protocol was approved by the local ethics advisory committee (Clinical Research & Ethics Committee of the Cork Teaching Hospitals, Cork, Ireland).
The study was a single-blind study. All participants began with placebo for the first 28 days, but were blinded to the product they received. During the weeks 5 through 8, all volunteers were provided with active supplement (Pylopass™), but were blinded to the product they received. An initial phone screen was performed, where subjects were asked questions regarding their age and general health. Eligible subjects were scheduled for a screening visit where demographic data, medical history and general health were recorded. Vitals, including weight, height, blood pressure and pulse were recorded, and the 13C-urea breath test (13C-UBT) was carried out. For women of childbearing age a pregnancy test was performed. After being included in the study subjects were instructed to take one tablet after breakfast and one tablet after their evening meal. The subjects completed the Gastrointestinal Symptom Rating Scale (GSRS) survey and a urease breath test was performed. A 16 ml fasting blood sample was collected to assess biochemical and haematological parameters, including lipid profile and blood glucose to test for physiological changes relevant in terms of safety. After the 28 days of supplementation with the placebo the second 13C-UBT was conducted, the GSRS was completed and the subjects were queried about any changes in their health status or medications and any adverse events were recorded. Then the subjects were provided with 28 days’ supply of Pylopass™ containing Lactobacillus reuteri DSM 17648. After 28 days’ supplementation, the subjects returned to the study site at day 56 for the last visit, where the GSRS was completed, the H. pylori load was reassessed by 13C-UBT, and the subjects were queried about any changes in their health status or medications and any adverse events were recorded (Fig. 1). Participants were instructed not to initiate any lifestyle or dietary changes throughout the duration of the study.
Outcome measures
Helicobacter assessment 13C-urea breath test (UBT)
The detection of H. pylori infection in the screening phase for confirmation of eligibility and the quantification of colonization to measure the effects of Pylopass™ supplementation was carried out by a breath test. The Urease breath test (Diabact UBT) is a rapid, non-invasive diagnostic procedure to assess the H. pylori infection status. The test is based upon the ability of H. pylori to convert urea to ammonia and carbon dioxide by urease activity. After an overnight fast, subjects swallowed urea labelled with non-radioactive carbon-13 (50 mg 13C-urea). Carbon dioxide resulting from the degradation of urea containing this isotope by H. pylori urease in the stomach is detectable by mass spectroscopy in the exhaled breath. As there is a small amount of naturally occurring 13C even in the absence of urease activity, breath samples are taken before and 10 min after the ingestion of 13C urea. The measurement considered H. pylori positive, if the difference (δ) in 13C/12C of 0-min-value and 10-min-value exceeds 1.5 ‰. If there is no difference, the test is negative, indicating no infection with H. pylori. All samples were analyzed in the Gastroenterology laboratory in the Mercy University Hospital which was accredited for the urease breath test. Three 13C urea breath tests were taken from the randomized subjects at each visit at the study site: baseline (day 1),end of placebo/start of Pylopass™ (day 28) and after completion of the Pylopass™ supplementation (day 56). The breath test analyses were represented as change in 13C-UBT (ΔδUBT) calculated as absolute differences from baseline (day 1) to end of supplementation with placebo (day 28) and after application of Pylopass™ from day 29 to day 56 (V4). The Δplacebo and ΔL. reuteri values were calculated as means ± standard deviation (day 28 - day 1 and day 56 – day 28).
Symptom assessment (GSRS)
The changes in symptoms were recorded using the Gastrointestinal Symptom Rating Scale (GSRS) at baseline (day 1), at end of placebo phase (day 28) after application of placebo and after completion of Pylopass™ supplementation at endpoint (day 56). 15 standardized questions were scored and summarized to 5 major categories: abdominal pain (Q1, 7 & 9), reflux (Q2–3), indigestion (Q4–6 & 8), diarrhea (Q11–12 &14), constipation (Q10, 13 &15).
Safety assessment (blood samples)
16 ml fasting blood samples were collected before and after the supplementation period at visit 2 (baseline), visit 3 and visit 4 to determine the levels of sodium, potassium, chloride, urea & creatinine, bilirubin (total), bilirubin (direct), alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), total protein, albumin, globulin, calcium, magnesium & phosphate, uric acid, cholesterol, triglycerides, and glucose.
Statistical analyses
All statistical analyses were carried out using SPSS Version 22 for Windows. The analyses variables were the changes in 13C-UBT values and the differences in GSRS score from baseline (day 1) to visit 3 (week 4) and from visit 3 to the end of the study (week 8).
Exploratory data analyses were conducted for the overall population and for each study period to determine statistically significant variance between the two different supplementations (placebo vs Pylopass™) for each endpoint assessed. Quantitative parameters and their changes were characterised by means and standard deviations on statistical relevance within the supplementation phases. Due to the deviation from normal distribution differences within a defined supplementation phase (e.g. pre- vs post- supplementation) were tested by the non-parametric Wilcoxon test.
Differences were considered significant when compared to a 0.05 level of significance. Cohen’s d classification was used to measure the strength of any observed difference (standardised mean difference) – i.e., the effect size of a result, where 0.2 ≤ d < 0.5 is a small effect size; 0.5 ≤ d < 0.8 is a moderate effect size; d ≥ 0.8 is a large effect size.