Introduction 12 Saccharomyces boulardii Escherichia coli E. coli 8 8 The purpose of the present trial was to examine the efficacy and safety of an EcN suspension administered to infants and toddlers suffering from acute diarrhoea of different causes in terms of normalising the stool frequency. Materials and methods Infants and toddlers treated for acute diarrhoea in the paediatric outpatient wards of 11 centres between February and April 2005 were eligible for enrollment in this study. This was a multicentre, prospective, confirmative, randomised, double-blind, placebo-controlled, parallel group clinical trial of phase III. It was carried out in accordance with the requirements of Good Clinical Practice and the Revised Declaration of Helsinki. The study was approved by the Independent Ethics Committee (IEC) of the Federal Agency of Drugs Quality Control, Moscow, Russia, and by the IEC of the State Enterprise Centre of Immunobiological Medicines at the Ministry of Health of Ukraine. 1 1 Salmonella Campylobacter Yersinia E. coli Shigella Entamoeba histolytica Cryptosporidium parvum Table 1 Inclusion and exclusion criteria Inclusion criteria Exclusion criteria Age <4 years at the time of enrolment Dehydration (>5% loss of body weight) More than three watery or loose non-bloody stools in a 24-h period that had not persisted for more than three consecutive days Participation in another clinical trial Signed informed consent by the parents Intake of EcN within the past 3 months prior to enrolment  Intake of food supplements or drugs which contain living microorganisms or their metabolic products or components within 7 days prior to enrolment or during the trial  Other antidiarrhoeal drugs  Antibiotics  Breast-feeding, Premature birth  Severe or chronic disease of the bowel or severe concomitant diseases Fig. 1 *Final visit  The parents were asked to maintain a daily record (diary) containing information on the number of stools, stool consistency, admixtures of blood or mucus, frequency of vomiting, abdominal pain and cramps and fluid intake as well as concomitant medication and general state of health. An assessment of general health was also documented during each control visit by the investigator and parents. The randomisation schedule was generated by means of SAS, ver. 9.1 (SAS Institute, Cary, N.C.) based on seed values dependent on a random number generator. The method of randomly permuted blocks was used (block size: 4). Study medication E. coli 8 Infants <1 year 1 ml once daily Toddlers ≥1 to ≤3 years 1 ml twice daily Toddlers >3 to <4 years 1 ml three times daily The parents received a diary in which the intake of the trial medication was documented. The investigator checked the entries for completeness and plausibility. The compliance was also evaluated by comparing the amount of trial medication handed out with that returns. Outcome The primary effect criterion was the time to response. Treatment response was defined as a reduction in stool frequency to ≤ three watery or loose stools in 24 h over a period of at least 2 consecutive days. Secondary effect criteria included the response rate, stool consistency, abdominal pain and cramps, body temperature, frequency of vomiting, occurrence of adverse events and tolerance to the study medication. Statistical analysis 9 ADDPLAN Results Baseline data 2 2 Fig. 2 Diagram of participant flow  Table 2 Baseline data for the two treatment groups  n n Male gender  32 (58.2%) 32 (55.2%) Age (median) 21 months 23 months Height (median)  83 cm 83 cm Weight (median) 12.7 kg 12.6 kg BMI (median)  2 2 Mean duration of diarrhoea 1.4 days 1.6 days Mean stool frequency 5.0 per day 5.1 per day Possible causes for the current acute diarrhoea episode Previous antibiotic treatment 2 (3.6%) 4 (6.9%) Virus infections  16 (29.1%) 19 (32.8%) Bacterial infections 9 (16.4%) 4 (6.8%) Unspecified infections 25 (45.5%) 29 (50.0%) Other causes 3 (5.5%) 2 (3.4%) 2 Data analyses n Primary objective p p 3 Fig. 3 Time-to-response curves: Kaplan-Meier analysis (ITT analysis)  n n Secondary objectives p 4 Fig. 4 n n  3 Table 3 Improvement of symptoms after treatment   EcN (%) Placebo (%) Normal stool consistency 78.4 40.5 No abdominal pain 93.3 72.7 No abdominal cramps 94.4 80.8 In addition, the general state of health, as assessed by the investigator during clinical examinations or by the parents by means of the diary, of the patients in the EcN group improved more clearly than that of the patients in the placebo group (data not shown). Body temperature showed an almost identical decrease over time in both treatment groups (EcN: −0.5 ± 0.4°C; placebo: −0.4 ± 0.5°C). The number of vomiting episodes was very small at baseline in all patients and decreased to 0% in both groups. In principle, body weight and the status of dehydration did not show any changes from baseline to the end of study in either treatment group. Only one patient in the placebo group experienced mild dehydration. From baseline to study termination, pathogenic microorganisms disappeared in a similar number of patients in both treatment groups (14/27 patients in the EcN group and 12/21 patients in the placebo group). In the patients who were free from infectious agents at baseline, pathogenic microorganisms were detected at the end of study in 3/28 and 6/37 patients in the EcN and placebo group, respectively. Tolerance to treatment 4 Table 4 Tolerance to treatment in the two treatment groups   Assessed by parents Assessed by investigators   EcN Placebo EcN Placebo Very good a 4/58 (6.9) 5/55 (9.1) 4/58 (6.9) Good 44/55 (80.0) 53/58 (91.4) 50/55 (90.9) 53/58 (91.4) Poor 0/55 (0.0) 1/58 (1.7) 0/55 (0.0) 1/58 (1.7) a Discussion The aim of this multicentre, prospective, confirmative, randomised, double-blind, placebo-controlled clinical trial of phase III was to investigate the therapeutic efficacy and safety of orally administered EcN in treating acute diarrhoea in infants and toddlers. The results showed that EcN was superior to the placebo in terms of both time to response and response rate. The difference in median duration of diarrhoea – 2.3 days – was statistically significant and also clinically important. S. boulardii 4 12 14 18 19 18 4 19 12 14 7  p p p In the present trial, high initial response rates in both groups represent the spontaneous healing known for acute gastroenteritis. The superiority of the EcN treatment became increasingly noticeable from 3. The healing process was markedly faster in the EcN-treated patients than in the patients receiving placebo, a result which underlines the high efficacy of this probiotic. The relatively high number of children with unspecific diarrhoea corresponds quite well to the frequent failure to detect the responsible pathogen in routine analyses. This is the reason why the results of this study are not helpful in answering the question whether EcN is more efficient in bacterial or viral diarrhea. This question should be addressed by future studies. 3 10 11 16 competition with pathogens (for adherence to intestinal epithelium, for growth and survival in the gut) and inhibition of pathogen overgrowth; secretion of bacteriostatic/bactericidal peptides (e.g. colicins, microcins); enforcement of intestinal barrier function and reduction of microbial translocation; modulation of immune responses (local and/or systemic, e.g. stimulation of secretion of IgA by lymphocytes and defensins by enterocytes). E. coli 1 10 13 17 15 2 5 6 3 11 Conclusion In summary, EcN showed a significant superiority to placebo in the treatment of acute diarrhoea in infants and toddlers. EcN treatment also improved the general state of health and its administration was safe and well tolerated. Electronic supplementary material Below is the link to the electronic supplementary material 
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