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Lack of Efficacy of Lactobacillus reuteri DSM 17938 for the Treatment of Acute Gastroenteritis

A Randomized Controlled Trial

Szymański, Henryk MD*; Szajewska, Hania MD

The Pediatric Infectious Disease Journal: October 2019 - Volume 38 - Issue 10 - p e237–e242
doi: 10.1097/INF.0000000000002355
Original Studies
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Objective: To assess the efficacy of Lactobacillus reuteri DSM 17938 (L. reuteri) for the treatment of acute gastroenteritis in children.

Study design: Children younger than 5 years with acute diarrhea, defined as a change in stool consistency to a loose or liquid form and/or an increase in the frequency of evacuations (≥3 in 24 hours), lasting for no longer than 5 days, were eligible for inclusion. Participants (n = 100) were recruited from the pediatrics department of a Polish hospital and randomly assigned to receive L. reuteri in a dose 2 × 108 colony-forming units or placebo, for 5 days, in addition to standard rehydration therapy. The primary outcome measure was duration of diarrhea.

Results: Ninety-one of the 100 children randomized were included in the intention-to-treat analysis (L. reuteri n = 44; placebo n = 47). The duration of diarrhea after randomization in both groups was similar (P = 0.6). The groups were also similar with respect to all secondary outcome measures, with one exception. Compared with the placebo group, patients in the L. reuteri group had a shorter duration of hospitalization (P = 0.048). Adverse events were similar in both groups.

Conclusions: Among children with acute gastroenteritis who were younger than 5 years of age, L. reuteri compared with placebo, as an adjunct to rehydration therapy, did not reduce the duration of diarrhea; however, it reduced the duration of hospitalization.

From the *Department of Paediatrics, St. Hedwig of Silesia Hospital, Trzebnica

Department of Paediatrics, The Medical University of Warsaw, Warsaw, Poland.

Accepted for publication March 28, 2019.

BioGaia provided the study products; the manufacturer had no role in the conception, design, or conduct of the study, or in the analysis or interpretation of the data. There was no external funding. H. Szajewska has served as a speaker for BioGaia, the manufacturer of Lactobacillus reuteri DSM 17938. The other author has no conflicts of interest to disclose.

H. Szajewska initially conceptualized this study. Both authors contributed to the study protocol. H. Szymański was responsible for data collection, data analysis, data interpretation and preparation of the report, and assumed the main responsibility for the writing of the first draft of this manuscript. Both authors contributed to (and agreed upon) the final version. No honorarium or other form of payment was given to anyone to produce the manuscript.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (www.pidj.com).

Address for correspondence: Hania Szajewska, MD, Department of Paediatrics, The Medical University of Warsaw, Żwirki i Wigury 63A, 02-091 Warsaw, Poland, E-mail: hania@ipgate.pl.

A number of scientific societies have provided recommendations on the use of probiotics for the treatment of acute gastroenteritis (AGE). Most, albeit not all, have agreed that use of probiotics with documented efficacy may be considered in the management of children with AGE in addition to oral rehydration therapy.1–5 Probiotics such as Lactobacillus rhamnosus GG (LGG) and Saccharomyces boulardii are among those with the best-documented efficacy, although the effectiveness of LGG has been recently questioned.6 Less compelling evidence is available for L. reuteri DSM 17938 (hereafter, L. reuteri).3,7 A 2015 meta-analysis of 3 randomized controlled trials (RCTs; n = 256) found that compared with placebo or no treatment, L. reuteri DSM 17938 administration significantly reduced the duration of diarrhea and increased the chance of cure on day 1 and day 2.8 However, heterogeneity and wide confidence intervals called for caution in interpreting these results and indicated that further research is needed. One of the proposed mechanisms of action of L. reuteri is its antimicrobial activity. L. reuteri strains produce reuterin, a broad-spectrum antibacterial substance,9,10 which is capable of inhibiting the growth of a wide spectrum of microorganisms such as Gram-positive or negative bacteria, yeast, fungi, or parasites.11L. reuteri strains may also regulate immune responses.12–14 Our aim was to assess the efficacy of L. reuteri for the management of AGE in children.

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METHODS

We conducted a randomized, double-blind, placebo-controlled trial. Enrollment occurred from January 2017 to November 2018 at the Pediatric Department of St. Hedwig of Silesia Hospital, Trzebnica, Poland. The Ethics Committee of the Lower Silesia Medical Chamber approved the study. Written informed consent was obtained from parents or legal guardians before enrollment. The trial was registered at ClinicalTrials.gov (NCT02989350), and the protocol was published in a peer-reviewed journal (both before enrollment of the first patient).15

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Inclusion and Exclusion Criteria

Eligible participants were children younger than 5 years with AGE, defined as a change in stool consistency to a loose or liquid form [according to the Bristol Stool Form (BSF) scale, or, in the case of infants, the Amsterdam Stool Form (ASF) scale] and/or an increase in the frequency of evacuations (typically ≥3 in 24 hours), lasting for no longer that 5 days. Exclusion criteria included the use of antibiotics, gelatin tannate, diosmectite, probiotics, racecadotril or zinc [including zinc-containing oral rehydration solution (ORS)] within a week before enrollment as well as exclusive breast-feeding; chronic diarrheal gastrointestinal disease; immunodeficiency disease and malnutrition (weight/height/length under third percentile, according to World Health Organization Child Growth Standards).16

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Intervention

Children were randomly assigned to receive either L. reuteri in a daily dose of 2 × 108 colony-forming units (CFU) or placebo, for 5 days, in addition to standard rehydration therapy. The study products (L. reuteri and placebo) were manufactured and supplied by BioGaia (Lund, Sweden) as bottles with drops, free of charge. The manufacturer did not have any role in the conception, protocol development, design, or conduct of the study, or in the analysis or interpretation of the data.

The intervention was started immediately after recruitment of the participants into the study. Caregivers were instructed to administer the study products at the same time each day. All study participants were followed up for the duration of the intervention (5 days), and then for an additional 72 hours. The caregivers were asked to bring the remaining study product and diary to the study site at the end of the intervention period. Compliance was checked by measuring the volumes left of the unused study products. Participants receiving <75% of the recommended doses were treated as being noncompliant.

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Outcome Measures

The primary outcome measure was the duration of diarrhea; this outcome was defined as the time until the normalization of stool consistency according to the BSF or ASF scale (for BSF scale, numbers 1, 2, 3, 4 and 5; for ASF scale, letters B or C), or the time until the normalization of the number of stools (compared with the period before the onset of diarrhea), and the presence of normal stools for 48 hours. The secondary outcome measures included the need for and duration of intravenous rehydration, the duration of hospitalization, the need for hospitalization of outpatients, the numbers of watery stools, vomiting, recurrence of diarrhea (48 hours after the intervention), severity of diarrhea according to the modified Vesikari Scale, use of concomitant medications and adverse events (whether or not they were considered to be related to the study products).

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Allocation Concealment and Blinding

The computer-generated randomization scheme used random permuted schemes of 6 children to ensure the comparable assignment of eligible patients to L. reuteri or placebo during the study. The randomization lists were stratified according to rotavirus vaccination status [nonvaccinated and vaccinated (defined as having received at least one dose of rotavirus vaccine)]. The allocation sequence was secured until enrollment and all decisions regarding data analysis had been finalized. The study products (active and placebo) were packaged in identical bottles and had the same look and taste. Researchers, caregivers, outcome assessors and a person responsible for the statistical analysis were blinded to the intervention until the completion of the study and analysis of the data.

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Sample Size Calculation

The primary outcome of the study was the duration of diarrhea. We assumed that a clinically significant difference in the effectiveness of L. reuteri versus placebo would shorten the duration of symptoms by 24 (±12 hours). To detect such a difference in the duration of diarrhea between the study groups with a power of 90% and α = 0.01, a sample of 60 children was needed. Assuming approximately 20% loss to follow-up, we aimed to recruit a total of 72 children for this study. A similar sample size for rotavirus-vaccinated and -nonvaccinated children was planned.

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Statistical Analysis

All analyses were conducted on data from the intention-to-treat population, including all patients in the groups to which they were randomized and for whom at least primary outcomes were available (including dropouts and withdrawals). Descriptive statistics were used to summarize baseline characteristics. Data were tested for normality using the Shapiro–Wilk test. Nonnormally distributed data were log-transformed or square-root-transformed and retested; nonparametric analysis was applied if data still did not conform to a normal distribution after transformation (8 transformation methods were tested). Descriptive statistics included number (% of total) for nominal data and the mean (SD) or median (interquartile range) for continuous data. To detect significant differences between groups, group comparisons were conducted with the use of χ2 test or Fisher exact test for nominal data and an independent-samples t test or nonparametric Mann-Whitney U test, as appropriate, for continuous data. For continuous outcomes, differences in the mean or median (depending on the distribution of data) with the 95% confidence interval (CI) were calculated; for nominal outcomes, the relative risk with the 95% CI was calculated.

Significance tests of baseline differences were not reported in line with the CONSORT.17 However, we noted differences between groups in the duration of diarrhea before randomization. analysis of covariance adjusted for baseline variables that were significantly different between groups was not conducted because of numerous violations of normality assumptions among primary and secondary outcomes, not solved by data transformation, as well as based on existing recommendations.18 All tests were two-tailed, and differences were considered significant at the level of P <0.05. Two independent reports (rotavirus-vaccinated and nonvaccinated children) were planned. Because of the small number of children vaccinated against rotavirus enrolled in the study, the planned sample size for that population was not achieved. Here, we report summary results for both groups. Subgroup analyses based on the vaccination status are also reported. Statistical analysis was carried out with the use of R-package v.3.4.4 (http://cran.r-project.org).

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RESULTS

During the enrollment period, a total of 289 children with AGE were potentially eligible, 100 of whom met all inclusion criteria; 50 of these children were randomly assigned to receive L. reuteri, and 50, to receive placebo. Included children were predominantly hospitalized (96 of 100). For a flow diagram, see Figure 1. Eight children were lost to follow-up; one ineligible patient who was mistakenly randomized into a trial was excluded after randomization. Ninety-one children (91%) completed the intervention and were included in the analysis. All participants were compliant (ie, received >75% of the recommended doses). Baseline demographic and clinical characteristics are shown in Table 1. The 2 groups were comparable at study entry, except for the duration of diarrhea before enrollment in the study, which was shorter in the placebo group compared with the L. reuteri group.

TABLE 1

TABLE 1

FIGURE 1

FIGURE 1

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Primary and Secondary Outcomes

The primary and secondary outcome measures for the overall study population are presented in Table 2. The duration of diarrhea after randomization was similar in both groups (mean difference: 8.3 hours; 95% CI: −17.8–22 hours; P = 0.6). There were also no significant differences between the groups with regard to any of the secondary outcomes, with one exception. Compared with the placebo group, the duration of hospitalization was shorter (mean difference: 6.1 hours; 95% CI: 0.1–17.7; P = 0.048) in the L. reuteri group. Adverse events were similar in both groups.

TABLE 2

TABLE 2

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Rotavirus Nonvaccinated and Vaccinated Populations

The primary and secondary outcome measures for rotavirus nonvaccinated (n = 67) and rotavirus-vaccinated (n = 24) populations are presented in the Supplemental Digital Content 1 and 2, http://links.lww.com/INF/D480 and http://links.lww.com/INF/D481, respectively. For nonvaccinated children, the duration of diarrhea after randomization was similar in both groups (P = 0.6). Compared with the placebo group, the duration of hospitalization was shorter (P = 0.025) in the L. reuteri group. For vaccinated children, the groups were similar with respect to the primary and all secondary outcome measures.

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DISCUSSION

Principal Findings

Our randomized, double-blind, placebo-controlled study showed that among children with AGE younger than 5 years of age, L. reuteri compared with placebo as an adjunct to rehydration therapy did not reduce the duration of diarrhea; however, it reduced the duration of hospitalization.

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Strengths and Limitations

This study was an RCT. The protocol was published in a peer-reviewed journal.15 Adequate methods for the generation of the allocation sequence and allocation concealment were used. Blinding was maintained throughout all phases of the study. For assessment of the consistency of stools, we used the validated BSF scale or the ASF scale, depending on the age of the participants. The sample size was predefined. These features minimize the risk of bias. The trial, however, has some limitations. No measures were taken to formally confirm the identity of the organism or the number of CFU in the study product. However, in another study,19 both study products were blindly tested using genetic methods, and it was confirmed that the active product contained L. reuteri DSM 17938. One can also hypothesize that the lack of an effect of L. reuteri may be due to the insufficient colonization rate of L. reuteri, thus an inability to exert its probiotic effects. However, colonization is not needed for probiotics to exert beneficial effects, and other mechanisms, such as the production of active compounds, may play a role.9,10 We did not assess stool volume as the primary outcome measure, which is a clinically meaningful but technically difficult endpoint. While our follow-up rate was excellent, we recruited only approximately 35% of potentially eligible patients. One of the main reasons for nonparticipation was one of our exclusion criteria, specifically the use of probiotics within a week before enrollment. Considering that probiotics are widely used in our setting, the lack of an effect also may be due to the unreported use of probiotics (or other antidiarrheal drugs). Moreover, our trial does not allow one to draw firm conclusions on the efficacy of L. reuteri in relation to specific enteropathogens. Finally, the recruitment of rotavirus-vaccinated children was inadequate due to low rotavirus vaccination coverage (likely due to the relatively high vaccine cost to be covered by caregivers). Thus, the findings with regard to the rotavirus-vaccinated population remain inconclusive. The lack of an effect of L. reuteri in our rotavirus-vaccinated subgroup does not suggest differences in the efficacy of L. reuteri in nonvaccinated and vaccinated populations.

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Comparison With Other Studies

Our findings contrast with results of 3 previously published, positive trials on L. reuteri for the treatment of AGE in children.20–22 However, there are possible reasons for the lack of an effect in our trial. Compared with our study, in a 2012 study by Francavilla et al,20 the total daily dose of L. reuteri was higher (2 × 108 CFU vs. 4 × 108 CFU, respectively) and the intervention was longer (5 vs. 7 days, respectively). On the other hand, the 2 other studies21,22 with positive findings used a dose that was even lower than that used in our study (1 × 108 CFU). However, both studies, even if randomized, were single-blinded studies, which increase the risk of performance and outcome assessment biases. Last but not least, in all of these studies, compared with our study, the end of diarrhea was defined differently. More recently, in line with our findings, a randomized, double-blind, placebo-controlled trial (n = 51) found similar effectiveness of ORS supplemented with L. reuteri DSM 17938 and zinc and unsupplemented ORS for the management of acute diarrhea in well-nourished, nonhospitalized children.23

The effectiveness of L. reuteri has also been evaluated in children with other types of diarrheal diseases. In preventive trials carried out in hospitalized children, based on the findings from 2 RCTs (n = 290), there was no significant reduction in the risk of nosocomial diarrhea, rotavirus diarrhea or diarrhea of any origin with L. reuteri administration.8 One recent, double-blind randomized trial involving 250 children showed no effect of L. reuteri on the risk of antibiotic-associated diarrhea.19 In preventive studies carried out in apparently healthy children, L. reuteri reduced diarrheal outcomes in one RCT;24 the evidence from another trial was less convincing.25 Taken together, the role of L. reuteri for treating or preventing diarrheal diseases remains unclear.

Other probiotics often used for treating AGE include LGG and S. boulardii. Although one recent trial negated the efficacy of LGG,6 a recent systematic review with meta-analysis showed that, overall, LGG reduced both the durations of diarrhea (with a higher impact in European countries) and hospitalization in inpatients.26 These findings should be viewed in the context of the high heterogeneity and methodologic limitations of the included trials. Other strains or combinations of strains have been tested, but evidence of their efficacy is weak or preliminary.3

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CONCLUSIONS

This randomized, double-blind, placebo-controlled trial showed that in children with AGE younger than 5 years of age, L. reuteri compared with placebo as an adjunct to rehydration therapy did not reduce the duration of diarrhea. L. reuteri reduced the duration of hospitalization; however, it had no effect on other diarrhea-related outcomes. Our findings, together with those of other recently published null studies on use of probiotics in this population,6,27 are important for the updating of recommendations on the use of probiotics for the management of AGE in children.

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ACKNOWLEDGMENTS

We thank Mrs Urszula Maciejewska for her help with the statistical analysis. We also thank physicians working at the Department of Paediatrics, St. Hedwig of Silesia Hospital for their help in the recruitment of children into the study.

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REFERENCES

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23. Maragkoudaki M, Chouliaras G, Moutafi A, et al. Efficacy of an oral rehydration solution enriched with Lactobacillus reuteri DSM 17938 and zinc in the management of acute diarrhoea in infants: arandomized, double-blind, placebo-controlled trial. Nutrients. 2018;10: E1189.
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Keywords:

probiotics; diarrhea; infants; randomized controlled trial

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