Probiotic containing Lactobacillus reuteri DSM 17648 as an adjunct treatment for Helicobacter pylori infection: A randomized, double‐blind, placebo‐controlled trial

Despite multiple therapy regimens, the decline in the Helicobacter pylori eradication rate poses a significant challenge to the medical community. Adding Lactobacillus reuteri probiotic as an adjunct treatment has shown some promising results. This study aims to investigate the efficacy of Lactobacillus reuteri DSM 17648 in H. pylori eradication and its effect in ameliorating gastrointestinal symptoms and adverse treatment effects.


Materials and Methods:
This randomized, double-blinded, placebo-controlled trial involved treatment-naïve H. pylori-positive patients.Ninety patients received standard triple therapy for 2 weeks before receiving either a probiotic or placebo for 4 weeks.
The posttreatment eradication rate was assessed via a 14 C urea breath test in Week 8.
The Gastrointestinal Symptom Rating Scale (GSRS) questionnaire and an interview on treatment adverse effects were conducted during this study.
Conclusions: There was a significant increase in H. pylori eradication rate and attenuation of symptoms and adverse treatment effects when L. reuteri was given as an adjunct treatment.

| INTRODUC TI ON
Helicobacter pylori is one of the most common chronic bacterial infections in humans, leading to a broad spectrum of gastrointestinal diseases, including peptic ulcer disease, gastric mucosa-associated lymphoid tissue lymphoma, and gastric adenocarcinoma. 1,2Despite the availability of global and regional guidelines on the holistic management of H. pylori infection, many countries did not meet a successful cure rate of ≥90%. 3,4Emerging antibiotics resistance, adverse antimicrobial treatment effects, high bacterial load with inoculum effect, and impaired gastric mucosal immunity challenge H. pylori management. 3,5Graham et al. recommended an evolution of the H. pylori treatment regime that adheres to the principles of antimicrobial stewardship that emphasize highly efficacious and sustainable treatment using patient-specific antimicrobial susceptibility data.[8] Traditionally, the standard triple therapy (STT) containing clarithromycin has been implemented as a first-line therapy for patients in regions with a low clarithromycin resistance rate (<15%).Alternatively, in regions with high or unknown clarithromycin resistance rates, the guideline recommends administering bismuth quadruple or nonbismuth concomitant quadruple therapies. 3The regional guidelines in East Asia have been revised due to the critical decline in the H. pylori cure rate, which is believed to be associated with the emergence of the clarithromycin-resistant H. pylori strain.Recent guidelines in East Asia recommend bismuth quadruple therapy as a first-line treatment in China but a clarithromycin-based STT with a potassium-competitive acid blocker as a proton pump inhibitor (PPI) alternative in Japan.In South Korea, there has been a transition toward individual antimicrobial susceptibility testing to tailor first-line treatment regimes. 9cally in Malaysia, there have been suboptimal H. pylori eradication rates of 71.2%-88.6%reported, where the latter resulted from a 14-day treatment with clarithromycin-based STT. 10,113][14] An expert consensus report was recently published to guide clinicians on the best clinical practice for H. pylori management relevant to the Malaysian population. 15It recommends a first-line therapy of clarithromycin-based STT for 14 days, while the alternative treatment regime includes high-dose amoxicillin-PPI dual therapy or PPI-amoxicillin-levofloxacin triple therapy.Due to limited resources and scarce accessibility, antibiotic susceptibility testing is mainly reserved for treatment failures.
Due to the alarming decline in the eradication rate, there has been an exponential growth of studies on probiotics as an alternative or adjunctive treatment for H. pylori infection.Probiotics are defined as live microorganisms that may confer a health benefit on the hosts when consumed in sufficient quantities. 16Probiotics are known to antagonize the effect of H. pylori through a direct antimicrobial effect, competitive inhibition interfering with H. pylori colonization, and improvement in the host's anti-inflammatory and barrier effects.However, only certain probiotics are helpful in H. pylori eradication, including Lactobacillus spp, Bifidobacterium spp, and Saccharomyces boulardii. 3A meta-analysis by Yu et al. on the efficacy of Lactobacillus-supplemented triple therapy showed an 8.4% improvement in eradication rate.Among all 11 randomized controlled trials, five involving Lactobacillus reuteri have shown successful results. 17Twelve clinical trials in six countries (Germany, Ireland, Russia, Romania, China, and India) investigating the efficacy and safety of L. reuteri showed improved eradication rates and reduced antibiotic side effects. 18The eradication rate ranged between 9% and 20%, with the highest rate reported by Parth et al. in India. 19 our knowledge, the efficacy of a Lactobacillus reuteri DSM 17648 probiotic as an adjunct treatment in H. pylori management has yet to be studied in Malaysian and South East Asian cohorts.In this study, we sought to determine the effect of Lactobacillus reuteri DSM 17648 on H. pylori eradication, mainly in improving the eradication rate, ameliorating gastrointestinal symptoms, and reducing adverse antibiotic effects.

| Study design
This prospective, single-center, randomized, double-blind, placebocontrolled trial involved H. pylori-positive subjects diagnosed at the Endoscopy Unit, Universiti Kebangsaan Malaysia Medical Centre.It was approved by the Research Ethics Committee of Universiti Kebangsaan Malaysia (approval number: FF-2021-004).This trial has been registered at the Clini calTr ials.gov(accession ID: NCT05399862).

| H. pylori diagnosis
H. pylori was diagnosed at baseline by either a Campylobacter-like organism (CLO) test from endoscopic gastric biopsies, 14 C urea breath test (UBT), or histopathological evidence of H. pylori in gastric biopsies.
All patients were prescribed STT as per local guidelines.The eradication therapy in this study comprised 14 days of amoxicillin (1 g), clarithromycin (500 mg), and esomeprazole (40 mg; all taken twice daily).For patients allergic to penicillin, metronidazole (400 mg) replaced amoxicillin (also taken twice daily).Then, patients were randomly assigned to receive either a probiotic or placebo using a randomization table generated using the Stat Trek random number generator software (http://www.stattrek.com).Both investigators and patients were blinded to the supplementary product, and the code was only revealed at the end of the study.STT and supplementary probiotic or placebo products were prescribed on the first day of diagnosis.

| Study supplements
The probiotic and placebo used in the study were prepared and funded by Y.S.P. Industries (M) Sdn.Bhd., Kuala Lumpur, Malaysia.
The probiotic contained a single L. reuteri DSM 17648 strain; its product specification is given in Table 1.The probiotic has been approved by the Ministry of Health Malaysia.The placebo contained corn starch with 0.12% of iron oxide yellow.Both the probiotic and placebo were prepared in indistinguishable capsule form and packed as 30 capsules in individual white boxes.The boxes had to be kept in a dry place below 30°C and away from direct sunlight.They were labeled in numerical order from 1 to 90.Patients were instructed to take one capsule (200 mg) once daily in the morning after a meal for 30 days.

| Treatment compliance and side effects
Patients' compliance was checked via capsule count during followup appointments.In addition, periodic checks were also performed via phone calls and text messages.We accepted compliance rates between 85% and 100%. 20Patients were interviewed for adverse treatment effects during the first week of starting treatment, 2 weeks after treatment, and at the end of therapy (8 weeks from baseline) via phone calls or text messages.The adverse treatment effects were defined as new symptoms or worsening of baseline symptoms after treatment initiation that resolved after treatment completion.

| Study outcomes: Primary outcome
The study's primary outcome was successful H. pylori eradication, assessed by posttreatment 14 C UBT performed in Week 8. 14 C UBT was conducted using the PYtest® Kit (Carbon-14-Urea 1.0 micro-Curie Breath Test), a rapid and noninvasive procedure for H. pylori detection.The UBT is a highly recommended procedure for H. pylori eradication posttreatment since it has high positive (>96%) and negative (>93%) predictive values. 5,21The UBT is a quick procedure involving the consumption of a 14 C-urea capsule after overnight fasting.A breath sample is collected in a special liquid-filled container, and the presence of H. pylori is assessed by the degree of urea degradation into carbon dioxide; carbon dioxide is the end product of urea degradation by the urease enzyme in H. pylori.
The UBT is considered negative with a score of <50 disintegrations per minute (DPM), positive with a score of >200 DPM, and indeterminate with a score of 50-200 DPM.The PYtest® manufacturer determined the cut-off values based on two clinical studies that compared the UBT results with histopathological examinations of gastric biopsies (the gold standard).Based on these studies, it has a sensitivity of 82%-96%, a specificity of 88%-98%, a positive predictive value of 86%-100%, and a negative predictive value of 98%-99%.The difference between both diagnostic methods was not statistically significant. 22Patients with a positive UBT were referred back to the primary treating doctor for second-line eradication therapy.Patients with an indeterminate TA B L E 1 The content of the probiotic used in this study.UBT result had a repeat UBT in 4 weeks under their respective primary team.

| Study outcomes: Secondary outcomes
The study's secondary outcomes included assessing H. pyloriassociated gastrointestinal symptoms via GSRS scores at baseline and the end of therapy and the incidence of adverse treatment effects reported by patients.The analyzed variables were the posttreatment UBT score (primary outcome), the mean pre-and post-treatment GSRS score difference, and the adverse treatment effects (secondary outcomes).

| Statistical analyses
Categorical variables were compared between groups using the chi-squared (χ 2 ) test.Pre-and post-treatment GSRS scores were compared within each group using the paired-sample t-test.Pre-to post-treatment GSRS score differences were compared between groups using the general linear model repeated measure method.
The frequencies of adverse treatment effects were compared between groups using the χ 2 test.

| Baseline characteristics
A schematic flow diagram of patients' inclusion and follow-up is presented in Figure 1.One hundred and one H. pylori-positive subjects were identified during the initial part of this study, of which 90 were recruited after assessment.All 90 subjects were randomized equally to receive intervention in the probiotic and placebo arms.
This study's final analysis was divided into intention-to-treat (ITT) analysis, which included 45 patients in each group, and per-protocol (PP) analysis, which included 44 and 45 patients in the probiotic and placebo groups, respectively (one dropout).
The subjects' baseline demographic data are summarized in

| Secondary outcome: GSRS scores
The GSRS scores for all subscales are summarized for the probiotic and placebo groups in Tables 6 and 7 and visualized in Figure 2.
A within-group analysis of pre-and post-treatment GSRS scores showed reductions in all subscales in both the probiotic and placebo groups, which were significant for all subscales in the probiotic group but only for the indigestion subscale and total GSRS score in

| Secondary outcome: Adverse treatment effects
The adverse treatment effects reported by patients during the study are listed in Table 8 and visualized in Figure 3.The adverse effects included abdominal discomfort, headache, nausea or vomiting, lethargy, diarrhea, and dysgeusia (metallic taste).Most adverse effects were experienced by patients in the placebo group, with dysgeusia and lethargy being the most reported (each reported by eight patients).The most reported adverse effect in the probiotic group was dysgeusia (three patients).The adverse effects least reported by the probiotic group were headache and abdominal discomfort (no patients).In contrast, the adverse effect least reported by the placebo group was nausea or vomiting (two patients).The frequencies of abdominal discomfort (p = 0.026) and headache (p = 0.012) symptoms differed significantly between groups.

| DISCUSS ION
This study used the probiotic Pylobalance®, which contains the patented ingredient L. reuteri DSM 17648.4][25][26] The ability of L. reuteri to coaggregate with H. pylori without interfering with the original commensal microflora helps to preserve a stable gastric microecology.This characteristic helps to reduce gastrointestinal symptoms associated with the H. pylori pathogen.The probiotic also has antimicrobial properties due to short-chain fatty acids derived from lactic acid, hydrogen peroxide, and the bactericidal substance reuterin, which directly inhibits H. pylori survival and proliferation. 26Regulating the immune response, improving the gut mucosal barrier, and producing cell-surface proteins to competitively inhibit H. pylori binding are among several other mechanisms being proposed to ameliorate the H. pylori pathogen effect in previous studies. 25,27 this study, both primary and secondary endpoints showed positive outcomes.The geographic proximity and almost similar dietary patterns of the two study regions may suggest a confined effect of this probiotic strain within a distinctive population.
Other factors that may have contributed to the much higher eradication rate in the probiotic group than the placebo group in our study were the subject's median age and medical comorbidities.The probiotic group had a slightly younger median age and fewer medical comorbidities, which could be advantageous since they presumably had a lower risk of gut dysbiosis, impaired gastric mucosa, and less exposure to aspirin and nonsteroidal anti-inflammatory drugs.In addition, the much lower eradication rate in our placebo group than in previous local studies could be explained by the marked increase in the local clarithromycin resistance rate from 6.8% to 14.8%. 12,14 contrast, the synergistic anti-inflammatory action between clarithromycin and L. reuteri may offset the effect of increasing clarithromycin resistance rate in the population. 28,29r this study's secondary endpoint, the H. pylori-associated gastrointestinal symptoms showed marked improvement in the probiotic group.A previous study by Buckley et al. found that patients that received L. reuteri as adjunct treatment had reduced GSRS scores in all subscales posttreatment, although the difference was not statistically significant. 30In our study, with a comparatively larger sample size of 89 compared to 24 subjects, we demonstrated a significant reduction in indigestion, abdominal pain, constipation, and total GSRS scores.The significant improvement in GSRS scores for indigestion and abdominal pain subscales was consistent with previous studies.Emara et al. illustrated significant improvements in upper gastrointestinal symptoms, including epigastric pain, abdominal bloatedness, increased flatus, and nausea, in H. pylori-infected patients after treatment with STT and L. reuteri.In addition, the specific L. reuteri strain successfully reduced inflammatory activity in both gastric antrum and corpus, while other strains were only efficacious toward the gastric antrum. 28,29e improvement in the GSRS constipation subscale score in the probiotic group is supported by previous studies.Murata et al.
showed a significant improvement in chronic constipation symptoms following successful H. pylori eradication.They postulated that improvements in the intestinal microflora after successful eradication helped to improve bowel frequency.However, this did not happen in patients with severe gastric atrophy. 31 constipation also reported significant results.The potential mechanism described by previous studies was the ability of L. reuteri to increase the intestinal myoelectric motility velocity and frequency. 32,33e adverse treatment effects reported by our study subjects were abdominal discomfort, headache, nausea or vomiting, lethargy, diarrhea, and dysgeusia.5][36] However, it was not significant in our study.The adverse effects were undoubtedly one of the challenging obstacles to achieving good compliance.
In our study, we frequently contacted patients, especially within the first 2 weeks of treatment, to educate them on the potential adverse effects and the importance of persevering through the treatment despite them.This continuous patient education process was key to good compliance in our study.
The probiotic used in our study contained a single strain.A systematic review by McFarland et al. concluded that in most studies, there was no significant difference between using multi-strain and single-strain probiotics in H. pylori eradication. 37They postulated that in multi-strain probiotics, the different phenotypic efficacy of each strain would exert an inter-strain competitive inhibitory effect, eventually weakening their antagonistic properties toward H. pylori.
Since there is overwhelmingly more significant data on single-strain than multi-strain probiotics in H. pylori eradication, a single-strain probiotic is preferable. 38e strengths of our study include its relatively larger sample size than previous studies, providing better statistical power.The GSRS questionnaire was an objective way of assessing patients' overall gastrointestinal symptoms due to H. pylori.Due to the clinical heterogeneity of H. pylori, all five subscales were believed to have provided a holistic and sufficient representation of various clinical symptoms.However, there were some limitations of our study.First, this was a single-center clinical trial hence the study may not be an accurate representation of the Malaysian population.Most of the patients recruited in the study were younger than 50-year-old, making it less applicable for elderly population.This study also did not objectively and/or subjectively assess some important confounding factors such as dietary patterns and social environment which are believed to have some important effect on gut microbiota.

TA B L E 7
Mean difference in GSRS score between groups.

| CON CLUS IONS
The study demonstrated that using L. reuteri DSM17648 as an adjunct treatment for H. pylori infection successfully increases the eradication rate to >90%.In addition, the probiotic improved GSRS scores mainly in indigestion, constipation, and abdominal pain subscales and reduced the incidence of adverse treatment effects.With the alarmingly high local clarithromycin resistance rate nearing the cut-off of 15%, it will be unsurprising if the incidence of persistent H.
pylori infections continues to rise.Therefore, future studies should examine the efficacy of L. reuteri as an adjunct treatment in persistent H pylori cases.

ACK N OWLED G M ENTS
We would like to thank the Y.S.P Industries (M) Sdn.Bhd. for their industrial support and the entire staff of the Endoscopy Service Center, Hospital Canselor Tuanku Muhriz, Universiti Kebangsaan Malaysia, for their help and support in conducting this research.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflicts of interest.
Inclusion criteria were subjects aged ≥18 years with confirmed H. pylori diagnosis who provided informed consent to participate in the K E Y W O R D S eradication, gastrointestinal symptom rating scale, GSRS, Helicobacter pylori, Lactobacillus reuteri, probiotics study.Exclusion criteria were any active gastrointestinal diseases such as inflammatory bowel disease, coeliac disease, or recent acute gastroenteritis within 4 weeks; previous surgical procedures affecting the gastrointestinal tract (e.g., bariatric surgery or gastrectomy); pregnancy or breastfeeding women; previous allergy to probiotics; taking a PPI, H2 antagonist, antibiotic, or traditional medication in the past 3 months; recent diagnosis of an advanced stage of malignancy (Stage III-IV); and a recent failure of first-line eradication therapy for H. pylori.The recruitment lasted for 12 months (January 2021 to January 2022).

1
Consolidated standards of reporting trials flow diagram of study participants.ITT, intention-to-treat; PP, per-protocol.A patient with a newly diagnosed Helicobacter pylori infection was assessed for eligibility (n = 101) to follow-up (n = 1) Allocated to the probiotic group (n = 45) Discontinued intervention (n = 0) Allocated to the placebo group (n =45)

solved 2
weeks after treatment completion.Of all six symptoms, only abdominal discomfort and headache differed significantly between groups.This outcome was consistent with previous studies by Poonyam et al. and Naghibzadeh et al. that demonstrated the efficacy of L. reuteri in reducing abdominal discomfort and headaches, respectively.Shavakhi et al. demonstrated the efficacy of a similar probiotic

F I G U R E 2 3
Change in mean GSRS score pre-to post-treatment for each of the five subscales (diarrhea, indigestion, constipation, abdominal pain, and reflux) and the total score.TA B L E 8Frequencies of adverse treatment effects in each group.Frequencies of adverse effects in each group.
Another study by Ojetti et al. on the effect of L. reuteri in patients with chronic functional TA B L E 3