Multispecies Probiotics For The Prevention of Antibiotic - Associated Diarrhoea in Children

Research
JAMA Pediatrics | Original Investigation
Multispecies Probiotic for the Prevention

of Antibiotic-Associated Diarrhea in Children
A Randomized Clinical Trial
Jan Łukasik, MD; Thomas Dierikx, MD; Isolde Besseling-van der Vaart, MSc; Tim de Meij, MD, PhD;
Hania Szajewska, MD, PhD; for the Multispecies Probiotic in AAD Study Group
Supplemental content
IMPORTANCE The efficacy of multispecies probiotic formulations in the prevention of
antibiotic-associated diarrhea (AAD) remains unclear.
OBJECTIVE To assess the effect of a multispecies probiotic on the risk of AAD in children.
DESIGN, SETTING, AND PARTICIPANTS This randomized, quadruple-blind, placebo-controlled

trial was conducted from February 2018 to May 2021 in a multicenter, mixed setting
(inpatients and outpatients). Patients were followed up throughout the intervention period.
Eligibility criteria included age 3 months to 18 years, recruitment within 24 hours following
initiation of broad-spectrum systemic antibiotics, and signed informed consent. In total,
646 eligible patients were approached and 350 patients took part in the trial.
INTERVENTIONS A multispecies probiotic consisting of Bifidobacterium bifidum W23,

Bifidobacterium lactis W51, Lactobacillus acidophilus W37, L acidophilus W55,
Lacticaseibacillus paracasei W20, Lactiplantibacillus plantarum W62, Lacticaseibacillus
rhamnosus W71, and Ligilactobacillus salivarius W24, for a total dose of 10 billion
colony-forming units daily, for the duration of antibiotic treatment and for 7 days after.
MAIN OUTCOMES AND MEASURES The primary outcome was AAD, defined as 3 or more
loose or watery stools per day in a 24-hour period, caused either by Clostridioides difficile
or of otherwise unexplained etiology, after testing for common diarrheal pathogens. The
secondary outcomes included diarrhea regardless of the etiology, diarrhea duration, and
predefined diarrhea complications.
RESULTS A total of 350 children (192 boys and 158 girls; mean [range] age, 50 [3-212] months)
were randomized and 313 were included in the intention-to-treat analysis. Compared with
placebo (n = 155), the probiotic (n = 158) had no effect on risk of AAD (relative risk [RR], 0.81;
95% CI, 0.49-1.33). However, children in the probiotic group had a lower risk of diarrhea
regardless of the etiology (RR, 0.65; 95% CI, 0.44-0.94). No differences were observed
between the groups for most of the secondary outcomes, including adverse events.
CONCLUSIONS AND RELEVANCE A multispecies probiotic did not reduce the risk of AAD in
children when analyzed according to the most stringent definition. However, it reduced the
Author Affiliations: Department of
overall risk of diarrhea during and for 7 days after antibiotic treatment. Our study also shows Pediatrics, The Medical University
that the AAD definition has a significant effect on clinical trial results and their interpretation. of Warsaw, Warsaw, Poland
(Łukasik, Szajewska); Department of
TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT03334604 Pediatric Gastroenterology,
Emma Children’s Hospital,
Amsterdam UMC,
Academic Medical Centre,
Amsterdam, the Netherlands
(Dierikx, de Meij);
Winclove Probiotics B.V.,
Amsterdam, the Netherlands
(Besseling-van der Vaart).
Group Information: A complete list
of the members for the Multispecies
Probiotic in AAD Study Group
appears in Supplement 3.
Corresponding Author: Jan Łukasik,
MD, Department of Pediatrics,
The Medical University of Warsaw,
JAMA Pediatr. 2022;176(9):860-866. doi:10.1001/jamapediatrics.2022.1973 Żwirki i Wigury 63A, Warsaw, Poland
Published online June 21, 2022. Corrected on July 5, 2022. 02-091 (jan.lukasik@wum.edu.pl).
860 (Reprinted) jamapediatrics.com
© 2022 American Medical Association. All rights reserved.

Multispecies Probiotic for the Prevention of Antibiotic-Associated Diarrhea in Children Original Investigation Research
A
ntibiotic-associated diarrhea (AAD) is a common com-
plication of antibiotic treatment.1,2 Several different Key Points
definitions of AAD have been proposed, including
Question What is the efficacy of a multispecies probiotic in
“diarrhea that occurs in relation to antibiotic treatment with the prevention of antibiotic-associated diarrhea in children?
the exclusion of other etiologies.”3,4 In clinical practice and in
Findings In this placebo-controlled randomized clinical trial
most clinical trials, microbiological tests are not routinely per-
of 350 children, a multispecies probiotic had no significant
formed to exclude an infectious origin of AAD, confirming
effect on the risk of antibiotic-associated diarrhea caused by
its etiology.5 AAD is considered to result from gut dysbiosis by Clostridioides difficile or of unknown etiology, but it reduced
antibiotics, which may provoke overgrowth of specific patho- the overall risk of diarrhea regardless of the etiology from
gens, most prominently Clostridioides difficile, and lead to 32% to 20%, a statistically significant difference.
altered function of the microbiota.6,7
Meaning The use of the studied probiotic formulation may be
The most thoroughly studied preventive intervention for considered for diarrhea prevention during antibiotic treatment
AAD is the administration of probiotics, defined as “live mi- in children.
croorganisms, that when administered in adequate amounts,
confer a health benefit on the host.”8 According to a 2019 Coch-
rane review,2 probiotics as a group have a moderate protec- spectrum oral or intravenous antibiotic therapy, and signed in-
tive effect on the prevention of pediatric AAD. The results of formed consent. The exclusion criteria were as follows: use of
individual studies in this review varied depending on the dose antibiotics within the previous 4 weeks; use of probiotics, pro-
of probiotic, with higher doses of 5 billion colony-forming units ton pump inhibitors, laxatives, or antidiarrheal drugs within
(CFU) or more per day demonstrating a better effect. Among the previous 2 weeks; severe infection or life-threatening ill-
the 33 included studies, only 6 randomized clinical trials (RCTs) ness at recruitment (ie, indicated or probable admission to an
of limited size investigated combinations of more than 3 pro- intensive care unit); preexisting diarrhea within the previous
biotic strains, with varied results.9-14 Thus, the effect of mul- 4 weeks based on patient’s or caregiver’s report; severe chronic
tispecies probiotic supplementation on AAD incidence in disease (eg, cancer, inflammatory bowel disease, short-
children remains in question. In adult patients, one of the pre- bowel syndrome); diagnosed primary or secondary immune
viously studied multispecies probiotics consisted of 9 bacte- deficiency; required tube-feeding; exclusive breastfeeding; and
rial species.15,16 In the current study, we aimed to assess the known allergy or hypersensitivity to any component of the
efficacy of a comparable multispecies probiotic mixture in study product.
the prevention of AAD in a pediatric population.
Randomization and Masking
A block randomization in blocks of 4 was performed centrally
in a 1:1 ratio by Winclove Probiotics B.V. with use of a com-
Methods puter random-sequence generator, by a person not other-
Study Design wise involved in the study. The randomization lists were stored
A parallel-group, randomized, quadruple-blind placebo- in sealed, opaque envelopes at the study centers. The partici-
controlled RCT (trial protocol can be found in Supplement 1) pants, caregivers, and all investigators, including data collec-
was conducted in pediatric clinical and outpatient wards of tors and outcomes assessors, were blinded until the primary
3 Dutch and 2 Polish hospitals (eTable 1 in Supplement 2). The data analysis was performed. Probiotic and placebo were
study was prospectively registered in ClinicalTrials.gov packed identically and had the same appearance, taste, and
database (NCT03334604), and the protocol was published in smell.
a peer-reviewed journal.17 Consolidated Standards of Reporting
Trials (CONSORT) guidelines were followed for reporting Procedures and Interventions
trial results.18 The parents were instructed to administer 2 sachets of the study
product daily to their children for the duration of antibiotic
Ethics treatment and for 7 days after, up to a maximum of 17 days,
The study was approved by the Bioethics Committees of the starting within 24 hours of the first antibiotic dose. The mul-
Medical University of Warsaw (KB/198/2017) and Amsterdam tispecies probiotic (Ecologic AAD 612; Winclove Probiotics
UMC (2019.227). Written informed consent was obtained by B.V.) contained 8 bacterial strains: Bifidobacterium bifidum
the parents or the legal guardians of all participants. During W23, Bifidobacterium lactis W51, Lactobacillus acidophilus
the study, 2 changes in the study protocol were introduced W37, L acidophilus W55, Lacticaseibacillus paracasei
in response to an unsatisfactory inclusion rate. First, recruit- W20, Lactiplantibacillus plantarum W62, Lacticaseibacillus
ment in additional centers was started, as planned in the study rhamnosus W71, and Ligilactobacillus salivarius W24 (for-
protocol. Second, the lower age limit of the participants was merly known as Lactobacillus salivarius W24), for a total dose
adjusted from 6 months to 3 months. of 5 billion CFU per sachet (10 billion CFU daily).
The data on outcomes were collected using study diaries
Participants during antibiotic treatment and for 7 additional days. The con-
Eligibility criteria included age from 3 months to 18 years, re- sistency was reported according to the Amsterdam Infant Stool
cruitment within 24 hours following initiation of broad- Scale (AISS)19 or Bristol Stool Form Scale (BSFS),20 depending
jamapediatrics.com (Reprinted) JAMA Pediatrics September 2022 Volume 176, Number 9 861

Research Original Investigation Multispecies Probiotic for the Prevention of Antibiotic-Associated Diarrhea in Children
on participant’s age. In case of diarrhea occurrence, the par- (ITT) analysis was performed on the available participants. Ow-
ticipants’ caregivers were requested to provide stool samples ing to the completeness of our baseline data, no imputation
for testing for rotavirus, adenovirus, and norovirus by immu- methods were used in ITT analysis.23 Sensitivity analyses
noassay; Campylobacter species, Salmonella species, Shigella with plausible assumptions regarding patients lost to fol-
species, and Yersinia species by isolation from stool cultures; low-up as described by Akl et al24 were performed. Addition-
and C difficile in children older than 1 year by detection of ally, per-protocol analysis was performed on the participants
glutamate dehydrogenase in conjunction with toxins A and B who ingested at least 75% of the study formula based on care-
with immunoassay. Additionally, stool samples for micro- givers’ reports and the counting of unused sachets. For the all
biota and metabolomics analysis were collected from a of the calculations, StatsDirect, version 3.3.5 (StatsDirect Ltd)
subset of patients at 4 timepoints: at baseline, on the day of was used.
antibiotic discontinuation, at the end of the intervention
period, and 1 month after the intervention period. The results
of microbiota and metabolomics analysis will be reported in
a separate publication.
Results
Between February 2018 and May 2021, 350 participants (192
Outcome Measures boys and 158 girls; median age: 28 months; mean [range] age,
The primary outcome measure was AAD, defined as 3 or more 50 [3-212] months) were consecutively enrolled. Among
loose or watery stools (a score of A on the AISS or 5-7 on the them, 202 participants were included in Poland and 148 in
BSFS) per day in a 24-hour period, caused either by C difficile the Netherlands. Available case analysis was carried out in
or of otherwise unexplained etiology, after testing for com- 313 participants and per-protocol analysis in 229 compliant par-
mon, predefined diarrheal pathogens. Secondary outcomes in- ticipants (Figure). Participants’ characteristics were compa-
cluded diarrhea, defined as 3 or more loose or watery stools rable between the 2 groups (Table 1). Patients from the Neth-
per day in a 24-hour period regardless of the etiology, mild AAD, erlands differed from the Polish patients mainly in terms of
defined as 2 or more loose or watery stools per day for a mini- class of used antibiotics, antibiotic administration route, and
mum of a 24-hour period caused by C difficile or of otherwise setting. Also, loss to follow-up frequency in Poland was al-
unexplained etiology, severe AAD defined as 3 or more loose most 4 times higher than in the Netherlands (15.1% vs 4.1%,
or watery stools per day for a minimum of a 48-hour period respectively) (eTable 2 in Supplement 2). The characteristics
caused by C difficile or of otherwise unexplained etiology, of the patients lost to follow-up were similar in the placebo
diarrhea duration, defined as the interval until normalization and probiotic groups (eTable 3 in Supplement 2) and similar
of stool consistency according to the BSFS (1, 2, 3, or 4) or AISS to characteristics of the remaining study participants (Table 1).
(B, C, or D) and the presence of normal stools for 48 hours, di- Among 83 patients who developed diarrhea, stools from
arrhea caused by C difficile, discontinuation of the antibiotic 10 children tested positive for rotavirus, 3 for norovirus, 1 for
treatment owing to diarrhea, hospitalization caused by diar- adenovirus, and 1 for Salmonella enterica; 6 patients in the
rhea, need for intravenous rehydration owing to diarrhea, and probiotic group and 11 patients in the placebo group did not
adverse events. provide a stool sample for the etiology testing. The reasons for
the stool sampling failures were difficulties in communicat-
Sample Size Calculation ing with patients after discharge from the hospital. All of these
Based on the pooled risks of AAD determined from the previ- patients were not qualified as AAD cases for the primary out-
ous studies conducted at the Medical University of Warsaw,21,22 come measure. In the ITT analysis (Table 2), AAD incidence
as well as those reported in a Cochrane review,2 we expected was comparable between the probiotic and placebo groups
that the incidence of AAD would be 16% among children re- (23 of 158 [14.6%] vs 28 of 155 [18.1%,] respectively; RR, 0.81;
ceiving placebo. To detect a difference of 11% between the arms 95% CI, 0.49-1.33). The frequency of AAD according to the al-
at a 5% significance level and with 80% power, we deter- ternative definitions (mild, severe) was also similar between
mined that 350 participants (175 in each arm) were needed both study groups. The patients in the probiotic group had a
assuming potential loss to follow-up of 20%. significantly lower risk of developing diarrhea than those in
the placebo group when analyzed regardless of its etiology (33
Statistical Analysis of 158 [20.9%] vs 50 of 155 [32.3%], respectively; RR, 0.65; 95%
Descriptive statistics were used to present the participants’ char- CI, 0.44-0.94; NNTB = 9; 95% CI, 5-64; P = .02); they were also
acteristics. For the dichotomous outcomes, relative risk (RR) less likely to require intravenous rehydration owing to diar-
was calculated with 95% CIs, along with number needed to ben- rhea (0 of 158 [0%] vs 5 of 155 [3.2%], respectively; NNTB = 32;
efit (NNTB), if appropriate. Presented P values were derived 95% CI, 14-125; P = .03). We found no significant difference be-
from χ2 test or Fisher exact test where appropriate. For the con- tween the groups in the other outcomes. Effect sizes in the per-
tinuous outcome, Man Whitney U test was performed. All of protocol analysis were similar to the ones observed in the ITT
the statistical tests were 2-tailed and performed with a 5% level analysis; however, because of a smaller sample size, they were
of significance. The primary outcome was also analyzed by lo- not statistically significant (eTable 4 in Supplement 2).
gistic regression, controlling for 5 prespecified potential risk fac- To investigate whether the country-related differences
tors for AAD (age, sex, antibiotic type, duration of antibiotic might have had an effect on the results, we performed a sub-
treatment, and duration of hospital stay). Intention-to-treat group analysis. The effect sizes for AAD, diarrhea, and diar-
862 JAMA Pediatrics September 2022 Volume 176, Number 9 (Reprinted) jamapediatrics.com

Figure. CONSORT 2010 Flow Diagram
777 Individuals assessed for eligibility
427 Excluded
296 Declined to participate
131 Did not meet inclusion criteria
350 Randomized
176 Randomized to probiotic 174 Allocated to placebo

174 Received intervention 0 Did not receive allocated
as randomized intervention
2 Did not receive intervention
as randomized because consent
was withdrawn on day 2;
children did not receive any
doses of study product
18 Lost to follow-up (including 19 Lost to follow-up

2 participants who did not receive 10 Lost or did not fill diary
intervention) 9 Lost contact with the participant
13 Lost contact with the participant
11 Discontinued intervention
5 Lost or did not fill diary
4 Difficulties with administering the
12 Discontinued intervention study product owing to the child’s
5 Difficulties with administering the illness
study product owing to child’s 4 Intervention discontinued by parents
illness because of loose stools in child
4 Vomiting or gag reflex immediately 2 Parents not willing to continue
after ingestion of the study product intervention with no specific reason
2 Intervention discontinued by 1 Vomiting or gag reflex immediately
parents because of loose stools after ingestion of the study product
in child
Other noncompliant participants
1 Parents not willing to continue
18 Negligence or unintentional omission
intervention with no specific reason
of >25% of the study product doses
Other noncompliant participants 16 Child’s refusal to ingest >25% of study
14 Child refused to ingest >25% of the product doses
study product
13 Negligence or unintentional omission
of >25% of the study product
158 Intention-to-treat 155 Intention-to-treat analysis

119 Per-protocol analysis 110 Per-protocol analysis
rhea duration were similar in Poland and in the Netherlands, associated with younger age and diarrhea was associated with
and only small differences were observed in the effect sizes allocation to the placebo group, younger age, and use of amoxi-
for mild AAD and severe AAD outcomes between the coun- cillin with clavulanic acid (eTable 7 in Supplement 2).
tries. None of these differences between groups were statis-
tically significant (eTable 5 in Supplement 2).
To examine which subgroup(s) of patients contributed to
the difference between the effect sizes for AAD and diarrhea
Discussion
outcomes, we performed sensitivity analyses with modified In this RCT, a multispecies probiotic did not significantly re-
outcomes: (1) patients with AAD combined with the patients duce the risk of AAD when analyzed according to the most strin-
with diarrhea who did not provide a stool sample, (2) infec- gent definition. However, the participants in the probiotic group
tious diarrhea with the exclusion of C difficile diarrhea, and had a significantly lower overall risk of diarrhea during the an-
(3) infectious diarrhea caused by specific pathogens (eTable 6 tibiotic treatment and 7 days after when the groups were ana-
in Supplement 2). For all of these outcomes, the effect size was lyzed regardless of diarrhea etiology. The studied probiotic did
larger than that for the AAD outcome, especially for rotaviral not demonstrate a beneficial effect on most other secondary
diarrhea (RR, 0.11; 95% CI, 0.02-0.65; NNTB = 19; 95% CI, outcomes, with the exception of the need for intravenous re-
10-63; P = .01). In the sensitivity analysis with plausible as- hydration due to diarrhea, which was less common in the pro-
sumptions about missing data, the effect size for the diarrhea biotic group. In the per-protocol analysis, the results were simi-
outcome was either no longer significant, of borderline sig- lar to those in the ITT analysis. Our results did not change after
nificance, or statistically significant depending on the as- an adjustment for potential AAD risk factors.
sumed risk of diarrhea among patients lost to follow-up It remains unclear why the studied probiotic had no signifi-
(eTable 6 in Supplement 2). In the logistic regression, AAD was cant effect on the AAD outcome, despite its beneficial effect in

Table 1. Characteristics of Participants
No. (%)
Placebo Probiotic Total
Characteristic (n = 174) (n = 176) (N = 350)
Age, median (range), mo 27 (3-204) 32 (3-212) 28 (3-212)
Sex
Female 76 (43.7) 82 (46.6) 158 (45.1)
Male 98 (56.3) 94 (53.4) 192 (54.9)
Setting
Inpatient 135 (77.6) 136 (77.3) 271 (77.4)
Outpatient 39 (22.4) 40 (22.7) 79 (22.6)
Reason for antibiotic treatment
Lower respiratory tract infection 54 (31) 56 (31.8) 110 (31.4)
Upper respiratory tract infection 52 (29.9) 49 (27.8) 101 (28.9)
Urinary tract infection 35 (20.1) 24 (13.6) 59 (16.9)
Skin infection 8 (4.6) 16 (9.1) 24 (6.9)
Lymphadenitis 6 (3.4) 7 (4) 13 (3.7)
Nervous system infection 3 (1.7) 4 (2.3) 7 (2.0)
Gastrointestinal infection 5 (2.9) 5 (2.8) 10 (2.9)
Joint infection 3 (1.7) 2 (1.1) 5 (1.4)
Other 8 (4.6) 13 (7.4) 21 (6.0)
Antibiotic administration route
Only oral 71 (40.8) 73 (41.5) 144 (41.1)
Only intravenous 25 (14.4) 28 (15.9) 53 (15.1)
Intravenous followed by oral 78 (44.8) 75 (42.6) 153 (43.7)
Antibiotic type
Cephalosporin
Second generation 25 (14.4) 26 (14.8) 51 (14.6)
Third generation 33 (19) 36 (20.5) 69 (19.7)
Aminopenicillin 69 (39.7) 71 (40.3) 140 (40)
Amoxicillin with clavulanic acid 67 (38.5) 55 (31.3) 122 (34.9)
Clindamycin 14 (8) 17 (9.7) 31 (8.9)
Cloxacillin/flucloxacillin 0 6 (3.4) 6 (1.7)
Gentamicin 1 (0.6) 3 (1.7) 4 (1.1)
Other 6 (3.4) 6 (3.4) 12 (3.4)
Two concomitant antibiotics 15 (8.6) 24 (13.6) 39 (11.1)
Change of antibiotic class 26 (14.9) 20 (11.4) 46 (13.1)
Treatment duration, median (range), d 10 (2-21) 10 (1-36) 10 (1-36)
Hospital stay duration, median (range), d 5 (1-35) 5 (1-45) 5 (1-45)
Table 2. Main Results of the Available Case Analysis
Events, No. (%)

Absolute risk
Outcome Probiotic group Placebo group Relative risk (95% CI) reduction, % NNTB (95% CI)
AAD 23 (14.6) 28 (18.1) 0.81 (0.49-1.33) 3.5 NA
Severe AAD 18 (11.4) 19 (12.3) 0.93 (0.51-1.69) 0.9 NA
Mild AAD 40 (25.3) 38 (24.5) 1.03 (0.7-1.52) −0.8 NA
Diarrheab 33 (20.9) 50 (32.3) 0.65 (0.44-0.94)a 11.4 9 (5-64)a
Clostridioides difficile diarrhea 1 (0.6) 3 (1.9) 0.33 (0.05-2.26) 1.3 NA
Hospitalization owing to diarrhea 1 (0.6) 2 (1.3) 0.49 (0.06-3.71) 0.7 NA
Antibiotic cessation owing to diarrhea 0 0 NA 0 NA
Intravenous rehydration owing 0 5 (3.2) NA 3.2 32 (14-125)a
to diarrhea
Adverse eventsc 16 (10.1) 10 (6.5) 1.57 (0.75-3.3) −3.6 NA
c
Abbreviations: AAD, antibiotic-associated diarrhea; NA, not applicable; Including readmission to hospital owing to reasons other than diarrhea (5 in
NNTB, number needed to benefit. probiotic group; 4 in placebo group), rash (2 in probiotic group; 3 in placebo
a
P < .05. group), vomiting (3 in probiotic group; 1 in placebo group), gag reflex (2 in
b probiotic group), abdominal pain (3 in probiotic group; 2 in placebo group),
Diarrhea duration in days, median (IQR) for probiotic group (5 [3-7]) and
trace of blood in the stool (1 in probiotic group).
placebo group (4 [3-7]).

the prevention of diarrhea when analyzed regardless of the eti- Strengths and Limitations
ology. One could speculate that a trial involving a larger group Our study had a number of strengths. To our knowledge, this is
might have shown significant results for the primary outcome. the largest trial investigating the effect of a probiotic contain-
Nevertheless, considering the satisfactory incidence of AAD in ing more than 3 species of microorganisms on the incidence of
the placebo group, our study was adequately powered to detect AAD in children. The number of participants is almost 3 times
a clinically significant difference in this outcome and even more higher than that in the second largest study of which we are
than adequately powered for assessing the diarrhea outcome. In aware.11 It was designed with an intent to answer an unambigu-
the sensitivity analyses, we investigated which subgroup(s) of ous research question with a choice of clearly predefined out-
patients contributed to this difference in outcome effect sizes to comes. The study was conducted in settings of international co-
the highest extent. We found that the effect was highest for vi- operation, which enabled verification of the collected data by
ral gastroenteritis, especially caused by rotavirus. Another sig- comparison between the different populations and recruitment
nificant result, ie, the number of children requiring intravenous centers. However, there are also some limitations. Loss to follow-
rehydration due to diarrhea, was also related to this finding, as up was relatively high, which is reflected by the range of uncer-
all of these patients received intravenous fluids owing to rota- tainty demonstrated in analyses with plausible assumptions
virus infection. There is evidence supporting a role of the micro- about missing data. To search for indications of imbalances be-
biota in rotavirus infection,25,26 as well as for a preventive effect tween the trial arms owing to selective missing data,31 we inves-
of certain probiotics.27 One could speculate that our study de- tigated the number and characteristics of participants lost to
tected a similar effect of the studied probiotic on diarrhea caused follow-up in both arms. We found them to be comparable with
by rotavirus. However, caution is needed when interpreting this each other, as well as with the rest of the study participants. We
finding, as this trial was not designed to answer this specific re- also compared the outcome data between the Polish and the
search question. Moreover, since the participants were not tested Dutch participants, who differed greatly in terms of loss to follow-
for the presence of diarrheal pathogens at baseline, some of them up, and we found mostly similar effect sizes. We assume that the
might have already been within the incubation period of infec- missing data were unlikely to have introduced a significant bias
tious diarrhea on hospital admission. to our study; nevertheless, no method of testing can rule out
In our study, we used a rather stringent definition of AAD, such a possibility completely.32 As mentioned, there was a puz-
which allowed us to differentiate between clinically relevant con- zling difference between loss to follow-up in Poland and in the
ditions and clinically unimportant changes in the consistency Netherlands. All but 4 of the participants were recruited and
of stools. It also considered the most common etiology of diar- followed-up by 3 researchers (J.Ł., T.D., and T.d.M.) who were
rhea related to antibiotic administration and assumed that in a regular contact with each other to standardize the study con-
common nosocomial infections, such as norovirus or rotavirus duct. Therefore, this difference may be explained by country-
gastroenteritis,28,29 are not directly associated with antibiotic specific attitudes of patients and overlooked differences in the
treatment. However, the definitions of AAD in published stud- researchers’ practice. Another study limitation is a potential mis-
ies vary, and in many studies it was similar to the definition of di- classification between the AAD and diarrhea outcomes, owing
arrhea, as applied in current study. To illustrate, a 2020 review to the limited diagnostic accuracy of immunoassay tests,33 the
found that microbiological tests were not performed to identify limited number of diarrheal pathogens tested, and the number
AAD outcomes in 28 of 33 previous studies on probiotic supple- of patients who failed to provide stool samples. Additionally, the
mentation during antibiotic treatment in children.5 While this limited study follow-up duration might have led to an omission
approach may pose a question as to whether the researchers really of some diarrhea cases occurring later than a week after antibi-
measured AAD or rather diarrhea during antibiotic treatment re- otic cessation.7
gardless of the etiology, it also represents a much more pragmatic
point of view. Etiology testing is not routinely recommended for
cases of acute diarrhea in children,30 and for both the patient and
the physician, what caused the diarrhea may not be relevant as
Conclusions
long as the preventive intervention is effective. The multispecies probiotic used in this trial did not reduce the
Why the effect sizes in the ITT analysis were similar to risk of AAD when analyzed according to the most stringent defi-
those observed in the per-protocol analysis is unclear. This find- nition. However, we found a beneficial effect of the formula-
ing may reflect misclassification of compliance data, as it was tion on the overall risk of diarrhea during and 7 days after an-
collected only by indirect methods, ie, study diaries and count- tibiotic therapy (NNTB = 9). The latter outcome corresponds well
ing of unused sachets. Another possible explanation is that the with the standard approach to AAD in clinical practice. There-
studied probiotic is effective even if not taken regularly. Ad- fore, the use of the studied probiotic may be considered for di-
ditionally, participants deemed as overall noncompliant might arrhea prevention during antibiotic treatment in children. Our
have been compliant during a specific time period crucial for study also shows that the AAD outcome definition has a sig-
diarrhea, eg, during the first days of antibiotic therapy. nificant effect on clinical trial results and their interpretation.
ARTICLE INFORMATION Published Online: June 21, 2022. Author Contributions: Dr Łukasik had full access
Accepted for Publication: March 20, 2022. doi:10.1001/jamapediatrics.2022.1973 to all of the data in the study and takes responsibility
Correction: This article was corrected on July 5, for the integrity of the data and the accuracy of the
2022, to fix 3 probiotics that were misspelled. data analysis.

Concept and design: Łukasik, 7. McFarland LV. Antibiotic-associated diarrhea: 21. Ruszczyński M, Radzikowski A, Szajewska H.
Besseling-van der Vaart, Szajewska. epidemiology, trends and treatment. Future Microbiol. Clinical trial: effectiveness of Lactobacillus
Acquisition, analysis, or interpretation of data: 2008;3(5):563-578. doi:10.2217/17460913.3.5.563 rhamnosus (strains E/N, Oxy and Pen) in the
All authors. 8. Hill C, Guarner F, Reid G, et al. Expert consensus prevention of antibiotic-associated diarrhoea
Drafting of the manuscript: Łukasik. document. The International Scientific Association in children. Aliment Pharmacol Ther. 2008;28(1):
Critical revision of the manuscript for for probiotics and prebiotics consensus statement 154-161. doi:10.1111/j.1365-2036.2008.03714.x
important intellectual content: Dierikx, on the scope and appropriate use of the term 22. Kotowska M, Albrecht P, Szajewska H.
Besseling-van der Vaart, de Meij, Szajewska. probiotic. Nat Rev Gastroenterol Hepatol. 2014;11 Saccharomyces boulardii in the prevention of
Statistical analysis: Łukasik, Szajewska. (8):506-514. doi:10.1038/nrgastro.2014.66 antibiotic-associated diarrhoea in children:
Obtained funding: de Meij, Szajewska. 9. Ahmad K, Fatemeh F, Mehri N, Maryam S. a randomized double-blind placebo-controlled trial.
Administrative, technical, or material support: Probiotics for the treatment of pediatric Aliment Pharmacol Ther. 2005;21(5):583-590.
Dierikx, Besseling-van der Vaart, de Meij. helicobacter pylori infection: a randomized double doi:10.1111/j.1365-2036.2005.02356.x
Supervision: de Meij, Szajewska. blind clinical trial. Iran J Pediatr. 2013;23(1):79-84. 23. Jakobsen JC, Gluud C, Wetterslev J, Winkel P.
Conflict of Interest Disclosures: Dr Łukasik 10. Saneeyan H, Layegh S, Rahimi H. Effectiveness When and how should multiple imputation be used
of probiotic on treatment of Helicobacter pylori for handling missing data in randomised clinical
reported nonfinancial support from Winclove
infection in children. Majallah-i Danishkadah-i trials—a practical guide with flowcharts. BMC Med
Probiotics B.V. Dr Dierikx reported grants and
Pizishki-i Isfahan. 2011;146(29):882-889. Res Methodol. 2017;17(1):162. doi:10.1186/s12874-
nonfinancial support from Winclove Probiotics B.V.
017-0442-1
Dr de Meij reported grants and nonfinancial 11. Merenstein DJ, Foster J, D’Amico F.
24. Akl EA, Briel M, You JJ, et al. Potential impact
support from Winclove B.V. Dr Szajewska reported A randomized clinical trial measuring the influence
on estimated treatment effects of information lost
nonfinancial support from Winclove B.V. of kefir on antibiotic-associated diarrhea: the
to follow-up in randomised controlled trials
Measuring the Influence of Kefir (MILK) study.
Funding/Support: Both the placebo and the (LOST-IT): systematic review. BMJ. 2012;344:e2809.
Arch Pediatr Adolesc Med. 2009;163(8):750-754.
probiotic preparation were manufactured and doi:10.1136/bmj.e2809
doi:10.1001/archpediatrics.2009.119
provided for study purposes by Winclove Probiotics 25. Gozalbo-Rovira R, Rubio-Del-Campo A,
B.V. (Amsterdam, the Netherlands), free of charge. 12. Conway S, Hart A, Clark A, Harvey I. Does eating
Santiso-Bellón C, et al. Interaction of intestinal
The Medical University of Warsaw and Amsterdam yogurt prevent antibiotic-associated diarrhoea?
bacteria with human rotavirus during infection
UMC received a donation for the statutory a placebo-controlled randomised controlled trial
in children. Int J Mol Sci. 2021;22(3):1010.
in general practice. Br J Gen Pract. 2007;57(545):
purposes. doi:10.3390/ijms22031010
953-959. doi:10.3399/096016407782604811
Role of the Funder/Sponsor: The funders had no 26. Uchiyama R, Chassaing B, Zhang B, Gewirtz AT.
13. Dharnai S, Nirmala P, Ramanathan R, Vanitha S.
role in the design and conduct of the study; Antibiotic treatment suppresses rotavirus infection
Comparative study of efficacy and safety of
collection, management, analysis, and and enhances specific humoral immunity. J Infect Dis.
azithromycin alone and in combination with
interpretation of the data; preparation, review, 2014;210(2):171-182. doi:10.1093/infdis/jiu037
probiotic in the treatment of impetigo in children.
or approval of the manuscript; and decision to Int J Curr Pharm Res. 2017;9(6):52-55. 27. Hojsak I, Szajewska H, Canani RB, et al;
submit the manuscript for publication. doi:10.22159/ijcpr.2017v9i6.23429 ESPGHAN Working Group for Probiotics/Prebiotics.
Group Information: A complete list of the Probiotics for the prevention of nosocomial
14. Zakordonets L, Tolstanova G, Yankovskiy D,
members of the Multispecies Probiotic in AAD diarrhea in children. J Pediatr Gastroenterol Nutr.
Dyment H, Kramarev S. Different regimes of
2018;66(1):3-9. doi:10.1097/MPG.
Study Group appears in Supplement 3. multiprobiotic for prevention of immediate
0000000000001637
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In children. Res J Pharm Biol Chem Sci. 2016;7(3): 28. Załęski A, Banasiuk M, Karpierz K, Kuchar E,
2194-2201. Podsiadły E. The clinical course of gastroenteritis
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risk factors of oral antibiotic-associated diarrhea and immunocompetent children—single center
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in an outpatient pediatric population. J Pediatr experience. Przegl Epidemiol. 2020;74(1):23-31.
microbiota and bowel movements in healthy
Gastroenterol Nutr. 2003;37(1):22-26. doi:10.1097/ 29. Ogilvie I, Khoury H, Goetghebeur MM,
volunteers taking the antibiotic amoxycillin. Am J
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Johnston BC. Probiotics for the prevention of gastroenteritis in the pediatric population of
16. Koning CJ, Jonkers D, Smidt H, et al. The effect
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the faecal microbiota and bowel habits in chronic
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obstructive pulmonary disease patients treated
3. Szajewska H, Canani RB, Guarino A, et al; with antibiotics. Br J Nutr. 2010;103(10):1452-1460. Lo Vecchio A, Shamir R, Szajewska H; European
ESPGHAN Working Group for ProbioticsPrebiotics. doi:10.1017/S0007114509993497 Society for Pediatric Gastroenterology, Hepatology,
Probiotics for the prevention of antibiotic- and Nutrition; European Society for Pediatric
17. Łukasik J, Szajewska H. Effect of a multispecies
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42.3.1203-1206.2004 (4):423-433. doi:10.1586/eri.12.21

NL69225.029.19 IPAD study
Research protocol
The Influence of Probiotics on Pediatric

Antibiotic-associated Diarrhoea
IPAD study
Version 3
04-06-2019
The Influence of Probiotics on Pediatric Antibiotic-associated Diarrhoea –

IPAD study
Protocol ID NL69225.029.19
Short title IPAD study
EudraCT number Not applicable
Version 3.0
Date 04-06-2019
Project leaders Dhr. dr. T.G.J. de Meij
Amsterdam UMC, location VUmc, Amsterdam
Department of Pediatric Gastroenterology
Room: ZH 9D 38
Postbus 7057, 1007 MB Amsterdam, The Netherlands
t.demeij@vumc.nl
Dhr. prof. Dr. M.A. Benninga

m.a.benninga@amc.uva.nl
PhD student Dhr. T.H. Dierikx
Room: ZH 9D 36
t.dierikx@vumc.nl
Principal investigator Dhr. dr. T.G.J. de Meij
Room: ZH 9D 38
t.demeij@vumc.nl
Participating centres: Amsterdam UMC, location VUmc
De Boelelaan 1117, 1081 HV, Amsterdam
Amsterdam UMC, location AMC

Meibergdreef 9, 1105 AZ, Amsterdam
Onze Lieve Vrouwe Gasthuis, locatie Oost

Oosterpark 9, 1091 AC, Amsterdam
Onze Lieve Vrouwe Gasthuis, location West

Jan Tooropstraat 164, 1061 AE, Amsterdam
Sponsor (in Dutch: Amsterdam UMC, location VUmc,

verrichter/opdrachtgever) De Boelelaan 1117, 1081 HV, Amsterdam
Subsidising party Winclove Probiotics B.V.
Hulstweg 11, 1032 LB, Amsterdam
0031 (020) – 435 02 35
service@winbiotic.nl
Independent expert (s) Dr. Chantal J.M. Broers
Department of Pediatrics
CJM.Broers@vumc.nl
Laboratory sites Laboratory of Amsterdam UMC, location VUmc
Pharmacy Pharmacy of Amsterdam UMC, location VUmc
TABLE OF CONTENTS
1. LIST OF ABBREVIATIONS AND RELEVANT DEFENITIONS .............................................. 7

2. SUMMARY ....................................................................................................................... 9
3. ABSTRACT .....................................................................................................................11
4. INTRODUCTION AND RATIONALE .................................................................................12
5. OBJECTIVES ..................................................................................................................14
6. STUDY DESIGN..............................................................................................................14
7. STUDY POPULATION .....................................................................................................14
7.1 Population (base) ...................................................................................................14
7.2 Inclusion criteria .....................................................................................................14
7.3 Exclusion criteria.....................................................................................................15
7.4 Sample size calculation ...........................................................................................15
8 TREATMENT OF SUBJECTS ............................................................................................15
8.1 Investigational product ...........................................................................................16
8.2 Use of co-intervention (if applicable) ......................................................................16
8.3 Escape medication (if applicable) ...........................................................................16
8.4 Intolerance to investigational product .....................................................................17
9 INVESTIGATIONAL PRODUCT........................................................................................17
9.1 Name and description of investigational product(s) ...............................................17
9.2 Summary of findings from non-clinical studies ........................................................18
9.3 Summary of findings from clinical studies ...............................................................18
9.4 Summary of known and potential risks and benefits ...............................................20
9.5 Description and justification of route of administration and dosage .........................21
9.6 Dosages, dosage modifications and method of administration ...............................23
9.7 Preparation and labelling of Investigational Product ...............................................23
9.8 Drug accountability .................................................................................................23
10 NON-INVESTIGATIONAL PRODUCT............................................................................24
11 METHODS ..................................................................................................................24
11.1 Study parameters/endpoints ...................................................................................24
11.1.1 Main study parameter/endpoint .......................................................................24
11.1.2 Secondary study parameters/endpoints ...........................................................24
11.1.3 Other study parameters ...................................................................................25
11.2 Randomisation, blinding and treatment allocation ..................................................25
11.3 Study procedures....................................................................................................26
11.4 Withdrawal of individual subjects ............................................................................28
11.5 Specific criteria for withdrawal ................................................................................30
11.6 Replacement of individual subjects after withdrawal ...............................................30
11.7 Follow-up of subjects withdrawn from treatment .....................................................30
11.8 Premature termination of the study.........................................................................30
12 SAFETY REPORTING ..................................................................................................31
12.1 Temporary halt for reasons of subject safety ..........................................................32
12.2 AEs, SAEs and SUSARs ...........................................................................................32
Version number: 3. Studyprotocol for IPAD study

Date 04-06 2019 5 of 56
12.2.1 Adverse events (AEs).......................................................................................32

12.2.2 Serious adverse events (SAEs) ........................................................................32
12.2.3 Suspected unexpected serious adverse reactions (SUSARs) ............................33
12.3 Annual safety report ...............................................................................................33
12.4 Follow-up of adverse events...................................................................................33
12.5 Data Safety Monitoring Board (DSMB) ...................................................................33
13 STATISTICAL ANALYSIS .............................................................................................34
13.1 Primary study parameter(s) ....................................................................................35
13.2 Secondary study parameter(s) ...............................................................................35
13.3 Other study parameters ..........................................................................................36
13.4 Interim analysis ......................................................................................................36
14. ETHICAL CONSIDERATIONS ...........................................................................................36
14.1 Regulation statement..............................................................................................36
14.2 Recruitment and consent ........................................................................................37
14.3 Objection by minors or incapacitated subjects ........................................................37
14.4 Benefits and risks assessment, group relatedness .................................................37
14.5 Compensation for injury .........................................................................................38
14.6 Incentives ...............................................................................................................39
15 ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION ................................39
15.1 Handling and storage of data and documents ........................................................39
15.2 Monitoring and Quality Assurance ..........................................................................40
15.3 Amendments ..........................................................................................................40
15.4 Annual progress report ...........................................................................................40
15.5 Temporary halt and (prematurely) end of study report ............................................40
15.6 Public disclosure and publication policy..................................................................41
16 STRUCTURED RISK ANALYSIS ...................................................................................41
16.1 Potential issues of concern.....................................................................................41
16.2 Synthesis ................................................................................................................45
17 REFERENCES..............................................................................................................49
18 APPENDIX A: CONTACT DATA PARTICIPATING CENTRES .........................................52
19 APPENDIX B: WINCLOVE CERTIFICATE FOR SAFETY STATEMENT ............................54
20 APPENDIX C: SPECIFICATION OF INVESTIGATIONAL PRODUCT (Winclove 612) ......55

Date 04-06 2019 6 of 56
1. LIST OF ABBREVIATIONS AND RELEVANT DEFENITIONS
AAD Antibiotic Associated Diarrhoea

ABR General Assessment and Registration form (ABR form), the
application form that is required for submission to the
accredited Ethics Committee; in Dutch: Algemeen
Beoordelings- en Registratieformulier (ABR-formulier)
AE Adverse Event
AISS Amsterdam Infant Stool Scale
AR Adverse Reaction
BSF Bristol Stool Form
CA Competent Authority
CCMO Central Committee on Research Involving Human Subjects; in
Dutch: Centrale Commissie Mensgebonden Onderzoek
CDAD Clostridium difficile-associated diarrhoea
CDI Clostridium difficile infection
CFU Colony Forming Unit
CONSORT Consolidated Standards of Reporting Trials
CRB Clinical Research Bureau
CRF Case record form
CV Curriculum Vitae
DSMB Data Safety Monitoring Board
ESPGHAN European Society of Paediatric Gastroenterology, Hepatology
and Nutrition
EU European Union
EudraCT European drug regulatory affairs Clinical Trials
FAO Food and Agriculture Organisation
FOS Fructo-oligosaccharides
GCP Good Clinical Practice
GDPR General Data Protection Regulation; in Dutch: Algemene
Verordening Gegevensbescherming (AVG)
IB Investigator’s Brochure
IC Informed Consent
IMP Investigational Medicinal Product
IMPD Investigational Medicinal Product Dossier
METC Medical research ethics committee (MREC); in Dutch: medisch-
ethische toetsingscommissie (METC)
MSP Multispecies probiotic preparation
QoE Quality of Evidence
RCT Randomised Controlled Trial
Date 04-06 2019 7 of 56
RR Relative Risk
(S)AE (Serious) Adverse Event
SPC Summary of Product Characteristics; in Dutch: officiële
productinformatie IB1-tekst
SPIRIT Standard Protocol Items: Recommendations for Interventional
Trials
Sponsor The sponsor is the party that commissions the organisation or
performance of the research, for example a pharmaceutical
company, academic hospital, scientific organisation or
investigator. A party that provides funding for a study but does
not commission it is not regarded as the sponsor, but referred
to as a subsidising party.
SUSAR Suspected Unexpected Serious Adverse Reaction
UAVG Dutch Act on Implementation of the General Data Protection
Regulation; in Dutch: Uitvoeringswet AVG
WHO World Health Organisation
WMO Medical Research Involving Human Subjects Act; in Dutch: Wet
Medisch-wetenschappelijk Onderzoek met Mensen

Date 04-06 2019 8 of 56
2. SUMMARY
Rationale: Antibiotic Associated-Diarrhoea (AAD) is a common complication of
antibiotic treatment in children. Certain probiotic strains are proven to be effective in
reducing the risk on AAD, however, there is not enough evidence to recommend use
of any of the multispecies probiotic preparations (MSP). Winclove 612 (Winclove
Probiotics B.V., the Netherlands), the MSP used in this trial, is a preparation consisting
of 8 probiotic strains. In one placebo-controlled randomized clinical trial with a
comparable preparation in a group of 41 healthy adults receiving antibiotics, the group
receiving the probiotic had significantly reduced rate of diarrhoea-like bowel
movements. To date, no randomized controlled trials have been performed in larger
groups or in children to assess effectiveness of the preparation in the prevention of
AAD.
Objective: The aim of the study is to assess the efficacy and safety of a multispecies-
strain probiotic in the prevention of AAD in children.
Study design: The study is an international, multicentre, randomized, double-blind,
placebo-controlled, parallel group trial with an allocation ratio of 1:1, performed in
Poland and the Netherlands.
Study population: In total 350 children receiving broad-spectrum antibiotics who
are otherwise healthy, will be included. Of them, the aim is to include half of the
population (180 children) in the Netherlands, more specifically in AUMC and OLVG.
Intervention: The participants will be randomly assigned to one of two subgroups
in which they will receive either the MSP at a dose of 1010 CFU per day in two doses
or a placebo product, starting at the same day as antibiotics are started, and
continuing use for one additional week after cessation of antibiotic use (up to
maximum 17 days). The MSP used in this study is called Winclove 612 (Winclove
Probiotics B.V., the Netherlands).
Main study parameters/endpoints: The primary outcome measure will be the
incidence of AAD. AAD will be defined as three or more loose or watery stool (a score
of A on the AISS or 5-7 on the BSF scale) per day in a 24-hour period, either caused
by C. difficile infection or of otherwise unexplained aetiology (after testing for common
diarrhoeal pathogens), occurring during the intervention period.
Nature and extent of the burden and risks associated with participation,
benefit and group relatedness: Participants will use the investigational product
for a maximum of seventeen days. The use of the investigational product is
considered to be safe in children. There is a chance on side effects, however side
effect are mild gastro-intestinal symptoms such as bloating and flatulence and will
disappear in a couple days.
Besides using the investigational product, all participants need to collect stool
samples at 6 times during. In case of diarrhoea during the intervention period or the
following seven days, we ask for one more stool samples. Furthermore, a diary has

Date 04-06 2019 9 of 56
to be kept where participants log the consistency of their stool during the
intervention period and the seven following days.
No extra (invasive) physical examinations or other tests are needed, nor are
participants required extra site visits to the study site.
Participation in this trial could potentially benefit the patients in the probiotics-group.
We hypothesize that probiotics can restore dysbiosis caused by antibiotics and
consequently prevent AAD. Patients randomly assigned to the placebo-group will
receive standard of care and will not benefit from participating in this study. The
knowledge obtained about the effects of probiotics could potentially benefit future
patients receiving antibiotics.

Date 04-06 2019 10 of 56
3. ABSTRACT
Background
Antibiotic Associated-Diarrhoea (AAD) is a common complication of antibiotic
treatment in children. Certain probiotic strains are proven to be effective in reducing
risk of AAD, however, there is not enough evidence to recommend use of any of the
multispecies probiotic preparations. Winclove 612 is a preparation consisting of 8
probiotic species. Two studies in patients receiving antibiotics demonstrated a positive
effect of probiotics on the incidence of AAD. A placebo-controlled randomized clinical
trial with a comparable preparation in a group of 41 healthy adults receiving antibiotics
confirmed these findings; the group receiving probiotics had a significantly reduced
rate of diarrhoea-like bowel movements. To date, no randomized controlled trials have
been performed in larger groups or in children to assess effectiveness of the
preparation in the prevention of AAD.
Aim
The aim of the study is to assess the efficacy and safety of this multispecies probiotic
in the prevention of AAD in children.
Method:
350 participants will be randomly assigned to one of two subgroups in which they will
receive either a multispecies probiotic at a dose of 1010 CFU per day in two doses or
a placebo product, starting at the same day as antibiotics are started, and continuing
use for one additional week after cessation of antibiotic use (up to maximum 17
days). Primary outcome will be the incidence of AAD.
Discussion
Since no trials have assessed the effectiveness of this specific formulation in the
prevention of AAD in children, results of this trial may contribute to the development
of future guidelines. Several issues, like uncertain AAD incidence or reluctance of
caregivers to consent for the study, may negatively influence it’s course. however,
certain adjustments were made to overcome or prevent these problems.

Date 04-06 2019 11 of 56
4. INTRODUCTION AND RATIONALE

Antibiotics are well known to cause disturbances in the composition of the intestinal
microbiota, leading to the development of gastrointestinal (GI) symptoms (1).
Antibiotic-associated diarrhoea (AAD), defined as diarrhoea that occurs in relation to
antibiotic treatment with the exclusion of other causes, is a common complication of
antibiotic use in children (2). In a Cochrane meta-analysis, published in 2015, the
pooled risk of AAD in children was 19% (3). However, this risk varied greatly between
individual studies, ranging from 2.1% (4) to 80% (5), depending on factors such as
the adopted definition of diarrhoea, the study population and the type of antibiotic
treatment (6). According to the World Health Organisation (WHO), diarrhoea is defined
as passage of loose or liquid stools usually at least three times per day, or a more
frequent passage than is normal for the individual (7). In small children, especially in
infants, a change in stool consistency is more important in the definition of diarrhoea
rather than the number of stool movements (8), hence the applied diagnostic criteria
for diarrhoea differ from each other in paediatric population studies. The pathogenesis
of AAD is not fully understood. It may be caused by a specific enteric pathogen (e.g.,
Clostridium difficile, Clostridium perfringens, Staphylococcus aureus, Candida
albicans), metabolic consequences of altered intestinal microbiota or a direct effect of
antibiotics on the mucosa (9). AAD may vary both in severity (from uncomplicated
diarrhoea to pseudomembranous colitis) and in incubation time (from the first day of
antibiotic treatment to 8 weeks after discontinuation) (10). The impact of antibiotics
on the abundance of commensal micro-organisms in the gut underlines the hypothesis
that administration of probiotics could reduce the incidence of AAD.
Probiotics are ‘live micro-organisms that, when administered in adequate amounts,

confer a health benefit on the host’ (11). Data from earlier studies suggest that doses
of >5×109 colony forming units (CFU) of probiotic micro-organisms are more effective
than doses <5×109 CFU in preventing AAD (12). According to the European Society of
Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) and FAO/WHO,
probiotic properties are species-specific and strain-specific, so each strain or their
combinations should be examined separately (2, 13). There is presumptive evidence
for the effect of probiotics in prohibiting AAD. However, probiotic preparations
consisting of more than one strain, multispecies probiotic preparations (MSP), are not
yet routinely recommended to reduce the incidence of AAD in children, because
sufficient evidence is still lacking (2). Nonetheless, there are certain studies focusing
on other strains and combinations of strains, suggesting evidence of their effectiveness
(3, 14). Saccharomyces boulardii and Lactobacillus rhamnosus GG are single probiotic
strains proven to be effective in the prevention of AAD (15, 16). In the international
ESPGHAN protocol these probiotics are considered safe for use in otherwise healthy
populations and can be considered using for preventing AAD in children. Results from
these studies have resulted in the ESPGHAN recommending L. rhamnosus GG
Date 04-06 2019 12 of 56
(moderate quality of evidence (QoE), strong recommendation) and S. boulardii

(moderate QoE, strong recommendation) for preventing AAD in children if the use of
probiotics is considered. In case of Clostridium difficile-associated diarrhoea (CDAD),
it is recommended to use S. boulardii (low QoE, conditional recommendation) (2).
The investigational product used in this study is a MSP called Winclove 612. There are
no studies yet investigating the effectiveness of this specific MSP. However, some
studies have been performed with Ecologic AAD, a very resembling MSP. Ecologic AAD
is a preparation consisting of nine different probiotic strains, including two strains of
Bififobacterium, six strains of Lactobacillus and Enterococcus faecium W54. Winclove
612 has a similar composition to Ecologic AAD. However, in contrast to Ecologic AAD,
Winclove 612 does not contain Enterococcus faecium W54. The species E. faecium is
not recommended for use in children by the European Society for Paediatric
Gastroenterology Hepatology and Nutrition (ESPGHAN) due to unclear safety issues
(17) and, therefore is excluded from the current used formulation.
In one RCT conducted in 41 healthy adult volunteers receiving amoxicillin with either
Ecologic AAD or placebo, subjects in the experimental group had a significantly lower
rate of diarrhoea-like bowel movements compared with those in the placebo group
(48% vs 79%, respectively, relative risk (RR)=0.61, p<0.05) (18). Another RCT
conducted in 45 adult patients with a chronic obstructive pulmonary disease
exacerbation who were treated with antibiotics did not reveal a difference in the rate
of diarrhoea-like bowel movements between the Ecologic AAD and placebo groups
(77% vs 70%, respectively, RR=1.1, p>0.05)(19). However, this study was carried out
in a very specific group of patients, consisting of subjects with a history of frequent
and prolonged antibiotic use. In another cohort study with 199 patients receiving
probiotics next to their antibiotic treatment the incidence of AAD was 0,5%. This is
much lower than the expected incidence of 5-39% in that study population suggesting
a beneficial role of Ecologic AAD (20). In a retrospective case report series of 10
Clostridium difficile infection (CDI) patients, 5 of whom experienced recurrent CDI, all
patients received, besides antibiotics before the treatment of CDI, two times daily
Ecologic AAD, which resulted in complete recovery (21).
So far, there have been no RCTs using Winclove 612 or Ecologic AAD carried out in
larger groups of participants or in children.
This will be the first randomized trial evaluating the efficacy of a probiotic preparation
with the above-mentioned composition in the prevention of AAD in children. AAD is a
frequent complication of treatment that extends the stay of patients in the hospital,
requires medical checks and increases the costs incurred by healthcare centres and
patients. Since no multispecies probiotic is currently recommended for the prevention
of AAD, this study has a chance to influence the shape of official guidelines. The study
will be conducted in accordance with the applicable clinical trial rules (registration of
the protocol in the clinicaltrials.gov registry, publication of a report prepared in
Date 04-06 2019 13 of 56
accordance with the SPIRIT statement, reporting in accordance with the CONSORT
statement), which further increases the chance of including its results in systematic
reviews and induce alterations of current guidelines.
5. OBJECTIVES
Study aim
The primary study objective of the study is to assess the effectiveness of a specific
probiotic preparation (Winclove 612 (Winclove Probiotics, Amsterdam, the
Netherlands)) in reducing the incidence of AAD in children (see chapter 11.1.1).
Secondary outcomes will include AAD based on other definitions, duration of diarrhoea,
hospitalization and adverse events and the influence on intestinal microbiota
composition (for a more detailed description of secondary outcomes, see chapter
11.1.2).
6. STUDY DESIGN
Trial design
The study is an international, multicentre, randomized, double-blind, placebo-
controlled, parallel group trial with an allocation ratio of 1:1, performed in Poland and
the Netherlands. The study protocol has already been approved by the bio-ethical
commission of the Medical University of Warsaw. This study protocol is available online
(22) and is registered at clinicaltrials.gov (NCT03334604).
7. STUDY POPULATION
7.1 Population (base)
Hundred seventy-five (175) participants in this study will be recruited among both the
inpatient and outpatient clinics from the department of Paediatrics at the Amsterdam
UMC, locations AMC and VUmc (Amsterdam) and the Onze Lieve Vouwe Gasthuis,
location Oost and West (Amsterdam). Other hospitals and medical care centres would
also be plausible sources of participants, providing the presence of adequately trained
personnel and depending on number of inclusions, this will be evaluated after 6
months.
The recruited participants will all be children with an age between 3 months and 18
years old. The included children have to be receiving antibiotics, but should otherwise
be healthy.
Hundred seventy-five (175) other participants necessary for this study will be included
from the ongoing trial at the Medical University of Warsaw.
7.2 Inclusion criteria

In order to be eligible to participate in this study, a subject must meet all of the
following criteria:

Date 04-06 2019 14 of 56
‚ Age between 3 months and 18 years old

‚ Therapy with oral or intravenous antibiotics for common infections
‚ Ability to start the probiotic intervention within 24 hours after the start of
antibiotic intake
‚ Use of broad-spectrum antibiotics
‚ Signed informed consent
7.3 Exclusion criteria

A potential subject who meets any of the following criteria will be excluded from
participation in this study:
‚ Use of antibiotics or probiotics in the last 4 weeks
‚ Presence of a severe or generalised infection
‚ History of pre-existing diarrhoea within the previous 4 weeks
‚ Exclusive breastfeeding
‚ Tube feeding
‚ Use of proton-pump inhibitors, laxatives or antidiarrheal drugs during the last 2
weeks or during the intervention period
‚ History of severe chronic disease (e.g.; cancer, inflammatory bowel disease,
tuberculosis)
‚ Critical or life-threatening illness, including systemic symptoms of sepsis,
pancreatitis, multi-organ failure or admission to intensive care unit
‚ Immunodeficiency
‚ Allergy or hypersensitivity to (ingredients) of the intervention product
7.4 Sample size calculation

The pooled risk of AAD as a consequence of using broad-spectrum antibiotics,
determined from previous studies conducted at the Medical University of Warsaw (23-
25) is 13.5%. This is less than the incidence of 19% from a Cochrane meta-analysis
on the protective effect of probiotics on AAD (3). There are no well documented data
on the incidence of AAD in the Netherlands. We have chosen to perform a sample size
calculation based on an expected AAD risk of 16%. To show a difference of 11% in
the treatment effect in the study groups with =0.05% and 80% power, and assuming
a 20% withdrawal rate, a total of 350 participants will be needed. Sample size
calculations were performed with StatsDirect (V.3.1.4, StatsDirect statistical software;
StatsDirect, Chesire, UK) (22).
8 TREATMENT OF SUBJECTS
Interventions
The participants will be randomly assigned to one of the two subgroups in which they
will receive either the MSP at a dose of 1010 CFU per day in two doses or a placebo

Date 04-06 2019 15 of 56
product. The MSP used in this study (Winclove 612) contains the following eight
strains:
‚ Bifidobacterium bifidum W23
‚ Bifidobacterium lactis W51
‚ Lactobacillus acidophilus W37
‚ Lactobacillus acidophilus W55
‚ Lactobacillus paracasei W20
‚ Lactobacillus plantarum W62
‚ Lactobacillus rhamnosus W71
‚ Lactobacillus salivarius W24
Those strains are based on a carrier material consisting of maize starch, maltodextrins,
fructo-oligosaccharides (FOS) P6, maize dextrin P9, mineral mix (potassium chloride,
magnesium sulphate, manganese sulphate), hydrolysed rice protein and enzymes
(amylase). The control group will receive a placebo product (Winclove Probiotics,
Amsterdam, the Netherlands) that is indistinguishable in colour, smell and taste from
the used MSP. Both the MSP and placebo are a powder, which have to be dissolved in
water or milk before use. The interval between antibiotic intake and probiotic
consumption has to be at least two hours. Both study products (MSP and placebo) will
be manufactured and supplied free of charge by Winclove Probiotics B.V. (Amsterdam,
the Netherlands).
8.1 Investigational product

The intervention used in this study will be a food supplement; the MSP, Winclove 612
(for more detailed information about the MSP, see section 9). The control group will
receive a placebo.
The role of the producer of the investigational product in the course of the
study
The producer of the product had the opportunity to comment on the first draft of the
protocol, but any final decisions regarding its shape were made by the research team.
The producer will have no influence on the course of the study as well as the analysis
and interpretation of the data. The results of the study will be published, regardless of
whether they are positive or negative.
8.2 Use of co-intervention (if applicable)

Not applicable.
8.3 Escape medication (if applicable)

Not applicable.

Date 04-06 2019 16 of 56
8.4 Intolerance to investigational product

Intolerance to the investigational product is unlikely. Participants with a known
allergy or hypersensitivity to (ingredients) of the study product will be excluded.
However if patients are intolerant, they will be treated according to physicians
discretion. Study follow-up will continue according to the intention-to-treat principle.
9 INVESTIGATIONAL PRODUCT
9.1 Name and description of investigational product(s)
The investigational product used in this study will be a MSP called Winclove 612. It is
a multispecies probiotic formulation, developed to prevent and restore antibiotic-
induced disturbances of the microbiota and subsequently the risk of antibiotic
associated side effects. Together with the Maastricht University Medical Centre
(MUMC+) in the Netherlands, Winclove Probiotics has selected the strains from the
commercially available probiotic strain collection of Winclove Probiotics based on
their capacity to restore the intestinal microbiota. More specifically, they were
selected on their capacity to inhibit Clostridium difficile and its toxins, being one of
the most important factors in development of AAD/CDAD, their inhibition of Candida
spp. and their in vitro survival of the GI-tract to reach the gut in proper state.
The formulation consists of 8 specifically selected probiotic strains: Bifidobacterium

bifidum W23, Bifidobacterium lactis W51, Lactobacillus acidophilus W37, Lactobacillus
acidophilus W55, Lactobacillus paracasei W20, Lactobacillus plantarum W62,
Lactobacillus rhamnosus W71 and Lactobacillus salivarius W57 and is based on a
carrier material consisting of maize starch, maltodextrins, amylases, fructo-
oligosaccharides (FOS) P6, maize dextrin P9, mineral mix (potassium chloride,
magnesium sulphate and manganese sulphate), vanilla flavor and vegetable protein.
The mixture is 2 years stable at room temperature, no refrigeration needed.
There are no studies yet investigating the effectiveness of this specific MSP. However,
some studies have been performed with Ecologic AAD, a very comparable product.
Ecologic AAD is a preparation consisting of nine different probiotic strains. It is
available in many countries, and on the Dutch market under the brand name Winbiotic
Pro.AD. Ecologic AAD consists of the exactly the same trains as Winclove 612.
However, in contrast to Winclove 612 it does contain Enterococcus faecium W54 too.
The species E. faecium is not recommended for use in children by the European Society
for Paediatric Gastroenterology Hepatology and Nutrition (ESPGHAN) due to unclear
safety issues (17) and, therefore is excluded from our current used formulation. It is
very plausible to presume that effects found for Ecologic AAD will apply to Winclove
612 too. The specification of the investigational product can be found in Appendix C.

Date 04-06 2019 17 of 56
9.2 Summary of findings from non-clinical studies

Selection criteria for development of Winclove 612 were to screen the probiotic
bacteria on their ability to inhibit the pathogens Clostridium perfringens, Enterococcus
faecalis, Escherichia coli and Bacillus subtilus. Pathogen inhibition is measured in vitro
using the well diffusion test, described by Hechard (26). Briefly, a nutritious medium
is poured into a petri-dish. In this agar medium a potentially pathogenic organism is
grown. The probiotic strain is added to the hole (8 mm diameter) in the agar. If the
probiotic strain inhibits the pathogen, a clear zone is shown around the hole. The
diameter of this clear zone is measured. The larger this zone, the better is the capacity
of the probiotic strain to inhibit the pathogenic organism. It can be concluded that
most strains in Winclove 612 are very capable to inhibit one or several of the above
mentioned pathogens.
It has been shown that Clostridium difficile is the cause of diarrhoea in 20% of AAD.
Moreover Clostridium difficile associated diarrhoea (CDAD) can lead to severe and life
threatening pseudomembranous colitis. Therefore inhibition of Clostridium difficile
growth was examined in vitro using a co-culture method. Growth of C. difficile was
compared to growth of the strain in the presence of a probiotic strain. The effect of
the probiotic bacteria on the production of the toxins A and B by C. difficile was
measured, using an ELISA assay. Results of the analyses are shown in figure 1.
Figure 1: Inhibition of growth of C. difficile over time (after 24hr and 48hr) and shows that several strains completely
inhibit the growth of C. difficile, especially after 48h of co-culture. Furthermore, it can be seen that 8 strains inhibit
the production of toxins A&B (red triangles).
9.3 Summary of findings from clinical studies

A double-blind, placebo controlled randomized controlled trial showed that Ecologic
AAD is able to significantly reduce the risk of diarrhoea-like defecation (18). The

Date 04-06 2019 18 of 56
study was executed in the Maastricht University Medical Centre (MUMC+) in the
Netherlands, where 41 healthy volunteers were given 500 mg of the antibiotic
amoxicillin twice daily for 7 days and were randomized to either 5 gram Ecologic AAD
or placebo, twice daily for 14 days.
Bowel movements with a frequency ≥3 per day for at least 2 days and/or a
consistency ≥5 for at least 2 days, were reported less frequently in the probiotic
compared to the placebo group (48% vs 79% (p<0.05)). Mean number of
enterococci increased significantly from log 4.1 at day 0 to log 5.8 (day 7) and log
6.9 (day 14) CFU/gram faeces (p<0.05) during probiotic intake. Apart from an
increase in enterococci no significant differences in microbial composition and
metabolic activity were observed in the probiotic compared with the placebo group.
However, changes over time were present in both groups, which differed significantly
between the probiotic and the placebo arm, suggesting that the amoxicillin effect
was modulated by probiotic intake.
The effect of Ecologic AAD was also investigated in COPD patients, being a target
group with a history of frequent antibiotic use (19). In this randomized placebo-
controlled double-blind study 30 COPD patients treated with antibiotics for a
respiratory tract infection received Ecologic AAD twice daily for two weeks. During
and after antibiotic treatment, DGGE-based similarity indices (SIs) were high (≥84%)
and band richness was relatively low both remaining stable over time. No difference
in SIs was observed between patients with and without diarrhea-like bowel
movements. Ecologic AAD had a modest effect on the bacterial subgroups.
Nevertheless, it did not affect the composition of the dominant faecal microbiota nor
the occurrence of diarrhea-like bowel movements. The dominant faecal microbiota
was not affected by antibiotics in this COPD population, suggesting an existing
imbalance of the microbiota, which may also have contributed to the lack of effect by
probiotic intake.
In a cohort in Austria with 199 people surgery patients were -after an antibiotic
regimen- longitudinally treated with Ecologic AAD (20). In this study, 199 patients of
the surgery ward of Landesklinikum Thermenregion Neunkirchen (Austria) received
Ecologic AAD next to antibiotic treatment, twice daily (total 1*1010 CFU/day), starting
from the first day of antibiotic treatment and until one week after cessation of
antibiotics. During the test period only 2 of the 199 patients developed AAD, which
probably had been caused by overgrowth of Clostridium difficile in one of the
patients. Based on the knowledge from literature and experiences in the
Landesklinikum the incidence of AAD ranges from 5-39% in patients using
antibiotics, in this study one might expect 10-80 patients suffering from AAD. An
observed almost complete absence of AAD incidence (0.5%) in this cohort of
Ecologic AAD treated patients strongly suggests a beneficial role of Ecologic AAD
therapy during and after antibiotic treatment.

Date 04-06 2019 19 of 56
In a retrospective case report series of 10 Clostridium difficile infection (CDI)

patients, 5 of whom experienced recurrent CDI, all patients received, besides
antibiotics before the treatment of CDI, two times daily Ecologic AAD, which resulted
in complete recovery. Moreover no adverse events were reported (21).
In their global guidelines, the World Gastroenterology Organisation (WGO) evaluates

pro- and prebiotics on their efficacy and safety. In this guideline Ecologic AAD is
evaluated as a randomized trial with dramatic effect in the prevention of antibiotic-
associated diarrhea (27). More specifically, the evidence for Winclove’s probiotic
formulation Ecologic AAD is evaluated as level 2 evidence: Randomized trial or
observational study with dramatic effect. The recommendation of Ecologic AAD as an
effective and safe probiotic for reducing antibiotic-associated diarrhea by the WGO is
very promising in implementing probiotics as a standard prescription next to
antibiotics.
It is very plausible to presume that effects found for Ecologic AAD will apply to
Winclove 612 too as the former consists of all eight strains that can be found in
Winclove 612 too. The only strain that is included in Ecologic AAD that is not present
in Winclove 612 is E. faecium since it is not recommended for use in children by the
ESPGHAN due to unclear safety issues. Consequently, we don’t expect more or other
side effects from Winclove 612 then are described for Ecologic AAD.
9.4 Summary of known and potential risks and benefits

Based on all clinical trials performed with Ecologic AAD (18-21, 28), patients can
expect that by using Winclove 612 less diarrhoea-like bowel movements will occur,
and microbiota is restored or even prevent dysbiosis.
The potential risks of participation are low, especially as probiotic intervention is
started after bacteraemia and sepsis are excluded. No serious events are expected
from administration of the test product or control product. If adverse effects occur,
they are likely to be minor gastro-intestinal symptoms, such as bloating or flatulence,
especially when one uses probiotics for the first time. These effects are expected to
disappear after a few days.
Recently the Netherlands Organization for Applied Scientific Research (in Dutch: de
Nederlandse Organisatie voor toegepast-natuurwetenschappelijk onderzoek; TNO)
published a practical guide for the use of probiotics for the prevention of antibiotic-
associated diarrhea (29). According to the Agency for Healthcare Research and
Quality (AHRQ) there is not enough information to confidently judge the safety of
probiotic-based interventions. Still, probiotic products are generally regarded as safe,
and they are used both by healthy and ill people globally. Possible safety concerns
are especially concerning for patients with a weakened or compromised immune

Date 04-06 2019 20 of 56
system. For that reason those children are being excluded from current study. The
the multi-strain formulation of Lactobacillus rhamnosus, Lactobacillus acidophilus,
Bifidobacterium lactis, which all are present in our investigational product, was found
to have a positive effect on the incidence of AAD. Furthermore, a one-star
recommendation (an effect shown in only one study, a trend supported by two or
more studies, or the presence of a strain that satisfies one of the above criteria
(showing an effect in one study or a trend in at least 2 studies) in sufficient amounts
in food supplement or dairy product with a mixed formulation) was assigned to
formulations that showed a trend supported by two or more studies, including
Bifidobacterium bifidum, Bifidobacterium lactis, Lactobacillus acidophilus,
Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus,
Lactobacillus salivarium (all strains in our product) with a minimal daily dose of 10
billion CFU. One of the multispecies probiotic products they recommend is Winbiotic
Pro-AD (containing all 8 strains of our investigational product plus Enterococcus
faecium W54) in a dose of 1x1010 CFU/day.
Even though most literature and meta-analysis (30, 31) show that probiotics are safe
and effective in preventing AAD, there are some studies questioning this. They state
that most studies on probiotics do not report adverse events or possible harms
well(32, 33). One of these trials examined the effects of multi-strain probiotics on
post-antibiotic reconstitution of the murine and human mucosal microbiome niche
(34). Compared to spontaneous post-antibiotic recovery, probiotics induced a
markedly delayed and persistently incomplete indigenous stool/mucosal microbiome
reconstitution and host transcriptome recovery toward homeostatic configuration.
This underlines the hypothesis that probiotics, if wrong species are administrated,
can be counterproductive.
There are concerns by some researchers that probiotics may cause infections,
especially in critically ill or immunocompromised patients. However, there are very
few case reports describing these infections and studies showing an absence of any
change in the prevalence of probiotic induced bacteremia (35). There is a theoretical
possibility of lateral gene transfer between probiotic organisms and other organisms
in the gut or other site, however no clinical evidence of transfer of antimicrobial
resistance has ever been seen (35).
9.5 Description and justification of route of administration and dosage

The product has a concentration of 2.5*109 CFU/gram with an advised daily dosage
of 2 grams twice daily (so 1010 CFU per day). The dosage of Winclove 612 is in
accordance with most probiotic dosages used in human studies and many
commercially available products, ranging from 109-1010 CFU/dose (K6. Probiotica
verkrijgbaar in Nederland demonstrates a list of all probiotics available in the

Date 04-06 2019 21 of 56
Netherlands). It is not possible to state a general dose for probiotics; some have
shown to be effective at lower levels, while other require substantially more. The
dosage of Winclove 612 in this study is based on prior human studies in healthy
volunteers, COPD patients and general surgery patients with the product (18-21, 28)
showing a health benefit without adverse reactions.
The concentration of probiotics needed to obtain a clinical effect is often quoted as
≥106 colony-forming units/ml (CFU) in the small bowel and ≥108 CFU/g in the colon
(36). Dose–response studies are, however, scarce, and it is not known whether the
percentage survival is stable for various ingested doses.
The survival of probiotics depends on their intrinsic resistance, on host factors and
on the vehicle in which they are ingested. The main obstacles to survival being
gastric acidity and the action of bile salts. Well-controlled, small-scale studies on
diarrhea in both adults and infants have shown that probiotics survive in sufficient
numbers to affect gut microbial metabolism (37). Survival rates have been estimated
at 20–40% for selected strains, but differ between strains (38). Approximately 1–
10% of Lactobacillus acidophilus ingested in fermented products was found to
survive until the ileum in several human studies using intestinal intubation techniques
(39). In contrast, 30% of ingested Bifidobacterium was found in the colon (40).
Other studies in healthy volunteers with different probiotic preparations showed that
the faecal concentrations of ingested Lactobacillus acidophilus, Lactobacillus
salivarius and Lactobacillus rhamnosus strains reached around 106 CFU/g. Probiotics
are usually excreted within a few days of their ingestion in faeces at the same rate as
or even more quickly than a transit marker (37).
The Cochrane review, published the 30th of April 2019, on prevention of pediatric
AAD states that studies using a dose of >5x109 (n=20) showed a significant
reduction of AAD, whereas studies using a dosage of <5x109 (n=12) did not. These
studies included children from all 3 days old up to 17 years of age. (41, 42). Despite
proven to be safe in these dosage, dose-response studies for AAD and on the
microbiome are missing. A dose of 1x1010 is generally accepted in studies with
children and this study will investigate whether this dose has the same effect on the
incidence of AAD and the microbiota of young children compared to older children.
In the supplementary file (K6. Probiotica verkrijgbaar in Nederland) all probiotics
available in The Netherlands are listed. As shown, most are recommended in a daily
dose of ≥ 10 billion (i.e. 1x1010 CFU or 10x109 CFU).
With this study we want to contribute to the knowledge on response to probiotics in
children from different ages. By performing sub analyses we want to see whether the
microbiota of young children is affected in the same amount as the microbiota of
older children.

Date 04-06 2019 22 of 56
9.6 Dosages, dosage modifications and method of administration

Each sachet contains 2 grams of probiotics with 2.5*109 CFU per gram. Participants
need to take 2 sachets per day; one in the morning and one in the evening (so 1010
CFU per day). The content of a sachet needs to be dissolved in 100mL luke-warm
water, milk or yoghurt, and not in fruit juices or hot liquids. When antibiotics are
administered orally, the investigational product needs to be taken at least two hours
prior to or two hours after the use of the antibiotics. Furthermore, the solution
containing probiotics needs to be ingested orally, preferably on an empty stomach. It
is recommended not to use disinfectant mouthwash prior to ingestion. product
9.7 Preparation and labelling of Investigational Product

Preparation and labelling will be conducted at Winclove Probiotics B.V. In the
Netherlands and European Union all probiotics are considered to be food
supplements and therefore have to be produced under HACCP regulations, the Dutch
regulation system for safety and hygiene in food and food supplements. Winclove
has been producing multispecies probiotics for over 25 years. Winclove’s ISO
22000:2005 certificate for safety statement can be found in appendix B.
Products will be transported to the experimental pharmacy of the Amsterdam UMC,
location VUmc where the products will be stored and distributed to pharmacies of the
participating centers.
9.8 Drug accountability

The boxes of probiotics will be given to subjects by the coordinating investigator
(supply for the complete project period). Boxes include probiotic sachets and
Winclove’s user instruction. The shipment, coded blank boxes and coded sachets, will
be delivered at the pharmacy of Amsterdam UMC, location VUmc and AMC and OLVG
location Oost and West by courier. The food supplements will be safely stored and
locked by key at the pharmacies and will only be accessible by the research team
and involved pharmacists. The product will be kept at room temperature. The
coordinating investigator and/or pharmacy will distribute the intervention (placebo or
probiotic mixture) to participants in person. Patients are supposed to use all sachets
provided. For compliance reasons, the patients will be asked to save all the empty
sachets, and bring everything (used and unused sachets) in return to the
investigator. After counting the empty sachets, to get numbers on compliance, all
study material can be destroyed. As this is a food supplement, there is no need for
special waste treatment.
The investigational product (Winclove 612) and the placebo product will be produced
free of charge by Winclove Probiotics B.V.

Date 04-06 2019 23 of 56
10 NON-INVESTIGATIONAL PRODUCT
The placebo is indistinguishable in color, smell and taste from the investigational
product. It contains maize starch, maltodextrin, potassium chloride, hydrolysed rice
protein, magnesium sulphate, amylase and manganese sulphate. The specification of
the products (the investigational product and placebo) can be found in Appendix C.
11 METHODS
11.1 Study parameters/endpoints
11.1.1 Main study parameter/endpoint

The primary outcome measure will be the incidence of AAD. AAD will be defined as
three or more loose or watery stools (a score of A on the AISS or 5-7 on the BSF scale)
per day in a 24-hour period, either caused by C. difficile infection or of otherwise
unexplained aetiology (after testing for common diarrhoeal pathogens), occurring
during the intervention period.
11.1.2 Secondary study parameters/endpoints

Secondary outcomes assessed during the intervention period will include AAD based
on two other definitions of diarrhoea used in previous studies:
‚ Three or more loose or watery stools per day (defined as above) for a minimum
of 48-hour period either caused by C. difficile infection or of otherwise
unexplained aetiology
‚ Two or more loose or watery stools per day (defined as above) for a minimum
of a 24-hour period either caused by C. difficile infection or of otherwise
Other secondary outcome measures will be as follows:

‚ The duration of diarrhoea (defined as the time until the normalisation of stool
consistency according to the BSF or AISS scale (on BSF scale, numbers 1, 2, 3
and 4; on AISS scale, letters B, C or D), and the presence of normal stools for
48 hours)
‚ Incidence of any diarrhoea (three or more loose or watery stools (defined as
above)) per day, for a minimum of 24 hours, regardless of its aetiology.
‚ C. difficile-associated diarrhoea (diarrhoea defined as in the primary outcome
but caused by C. difficile confirmed by the presence of toxin-producing C.
difficile in stools (positive toxin tests))
‚ Discontinuation of the antibiotic treatment due to severity of diarrhoea
‚ Hospitalisation caused by diarrhoea in outpatients
‚ Need for intravenous rehydration in any of the study groups
‚ Adverse events

Date 04-06 2019 24 of 56
‚ Intestinal microbiota composition, composition of both subgroups will be

compared
‚ Amino acid/metabolomics analysis
‚ Effects of different types of antibiotics on the incidence of AAD
‚ Effects of different types of antibiotics on the incidence of the microbiome
11.1.3 Other study parameters

Baseline characteristics and other study parameters important for this study will be
obtained at inclusion. These values and parameters will include:
- Standard demographic data
- Age and date of birth
- Gender
- Type of antibiotic used during intervention period
- If applicable: feeding type (breast feeding, formula or combination)
- Use of antibiotics in last 4 weeks
- History of diarrhoea during last 4 weeks
- Use of antibiotics, probiotics, proton pump inhibitors, laxatives,
immunosuppressive or antidiarrheal drugs during the last 4 weeks
- Use of other medication during last 4 weeks
- History of chronic disease
- History of gastro intestinal surgery and gastro-intestinal anomalies
- Inflammatory bowel disease, coeliac disease, diabetes mellitus type I/II
- (History of) critical or life-threatening illness
- Immunodeficiency
- Allergy or hypersensitivity to (ingredients of) the intervention products
- Presence of other comorbidities
11.2 Randomisation, blinding and treatment allocation

Randomization and allocation concealment
The randomisation and coding will be performed centrally by Winclove Probiotics B.V.
by a subject not involved in the study using a computerised programme. Blocked
randomisation (blocks of four) will be used to ensure a good balance of participants’
characteristics in each group. Allocation will be determined by using a computerised
random number generation process. All study products will be sequentially numbered.
Coded study products will be handed over to the researchers. When the study has
ended, participants will be divided into two blinded groups, which will be used in the
statistical analysis. After performing the analyses, code numbers will be opened by the
coordinating and principal investigator. Sealed envelopes containing the allocation of
each number will be handed to the principal investigator ensuring that if a medical
problem or emergency occurs for which the treating physician needs to know the
treatment allocation (probiotics or placebo), the code can at all times be broken.

Date 04-06 2019 25 of 56
Agreements are made with the manager of the experimental medicine pharmacy (dr.
I. Bartelink, pharmacist) and team leader of the experimental medicine pharmacy (A.
Admiraal) about the randomisation, storage and distribution of the product. Since the
pharmacy of the Amsterdam UMC location VUmc does not have enough storage
capacity to store all product and divide them to the participating centres, we decided
in joint agreement that randomisation and distribution to participating centres will be
done centrally at Winclove Probiotics B.V. as described previously. Winclove Probiotics
B.V. will manage this randomisation list and will make a sealed envelope for each
randomisation number with information for whether this randomisation number
corresponds with either probiotics or placebo. These sealed envelopes will all be put
in a bigger sealed envelope. These sealed randomisation codes will be send to and
stored in the Amsterdam UMC location VUmc, so the code can be broken 24/7 in case
of a medical emergency.
After randomisation, Winclove Probiotics B.V. will deliver the randomised and coded
study products to the (experimental) pharmacy of the Amsterdam UMC location VUmc
and pharmacies of participating centres. Products will be stored here and handed out
to participants when they are being included. A part of the products will be stored
outside of the pharmacy (on the paediatric ward or E.R.) to make sure participants can
be included when the (experimental) pharmacy is closed. The pharmacy will make sure
the products are stored in a correct way by performing audits etc. The products will
be stored in boxes containing 34 sachets of the investigational product or placebo. The
person handing out the products will be aware of the study protocol and how much
the participants need to use (2 sachets per day during the antibiotic treatment plus
the 7 following days, with a maximum of 17 days (and so a maximum of 34 sachets)).
The pharmacist or person handing out the products can give additional information on
how to use the products and can remind participants to return the empty used sachets
and the full unused sachets afterwards to the pharmacy to verify the compliance.
Blinding
The probiotic preparation and placebo will be stored in identical packages. The
contents will look, smell and taste the same. Researchers, caregivers, participants,
medical personnel and outcome assessors will all be blinded to the intervention until
the study is completed and the data analysed.
11.3 Study procedures

A total of 350 children aged 3 months - 18 years, undergoing antibiotic treatment for
common infections, will be enrolled in this double-blind, placebo controlled trial. After
obtaining written consent to participate in the study, the children will be randomized
to receive either Winclove in a dose of 1010 CFU daily (2 gram of probiotics with 2.5x109
CFU per gram twice per day), or an identical placebo. The product will be used for the

Date 04-06 2019 26 of 56
duration of antibiotic treatment and the 7 following days with a maximum of 17 days
(the intervention period). The primary outcome measures will include incidence of AAD
during intervention period and 7-day follow-up period. During this time, frequency of
bowel movements and consistency based on the Amsterdam Infant Stool Scale (AISS)
(43) for children younger than one year and Bristol Stool Form (BSF) scale (44) for
children older than one year will be recorded in a study diary, which will be handed
out to the participants after recruitment. A score of ‘A’ on the AISS or 5-7 on the BSF
scale will be considered as loose or watery stool.
Participants using the study product will be treated for their (infectious) disease
according to common guidelines from the hospital. No adjustments in initial treatment
will be made if subjects decide to participate in this study, so participating in this study
will not negatively influence the initial treatment for their (infectious) disease. Besides
the use of the study product, no further (invasive) interventions are needed. No extra
visits to the hospital are necessary for participants. The study can be performed at
home and collected samples (see next 2 paragraphs) and the diary can be picked up
by one of the investigators.
In cases of the occurrence of diarrhoea during the study period, stool samples will be
obtained and examined for presence of common diarrhoeal pathogens—rotavirus,
adenovirus, norovirus, Campylobacter spp, Salmonella spp, Shigella spp, and Yersinia
spp (—via chromatographic immunoassay for viruses or isolation from stool culture
for bacteria). Additionally, C. difficile toxins A and B will be identified in the stool
using immunoassay in cases involving children older than 1 year. Stool samples will
be collected in sterile stool containers (Stuhlgefäß 10 mL, Frickenhausen, Germany).
Additionally, stool samples will be collected at home in the freezer at -20C from each
participant at six time points: (1) at baseline preferably before the start of antibiotics,
(2) at the day of antibiotic cessation, (3) at the end of intervention and (4) 1 month,
(5) 6 months and (6) one year after the cessation of the intervention period. The
collected samples will be used to conduct microbiota analysis and metabolomics.
Besides this, demographic data, in particular participants’ weight and length will be
collected at the same time as the last stool sample and compared with data collected
at baseline in both groups since microbiota dysbiosis increases the risk on obesity
(45).
The observation period for primary and secondary outcomes will be identical to the
intervention period and the following 7 days.
All samples from participants in Dutch centres will be transported to the Paediatric
ward of the Amsterdam UMC, location VUmc and will be stored in freezers at a

Date 04-06 2019 27 of 56
temperature of -20ºC. A data and material transfer agreement is made between

providing centres (AMC and OLVG Oost and West) and receiver (VUmc).
In the event that a participant does not respond well to the antibiotics prescribed by
the physician this can roughly be because of following reasons; (1) the micro-organism
causing the infectious disease is not of bacterial origin (for example a viral infection
like an otitis) or (2) there is no response to the antibiotics because the causing bacteria
is resistant to the antibiotic prescribed or because of inhibition of effect of the
antibiotics as a result of interaction with the probiotics.
For this last reason, if a participant does not respond well to the prescribed antibiotics
and does become more ill and consults the treating physician for this reason, it will be
decided whether participation should stop. If a participant develops a severe or
generalised infection, symptoms of sepsis, or a critical or life-threatening illness (and
thus not meeting the inclusion-criteria) the intervention will be immediately stopped
and the participant will be excluded. The treating physician will inform the researcher
about the termination of this individual in the study participation. If relevant for the
regular treatment, the treating physician can ask for the code to be broken so it
becomes evident whether the patient was using probiotics or placebo.
The timeline of this study is presented in table 1 below.
Observation and adherence to protocol recommendations

Participants will be monitored during the intervention period and for the next seven
days. In cases of inpatients discharged from the hospital before the end of the
intervention as well as in outpatients, the remaining product along with the study diary
and the collected samples will be collected from the participant’s home by one of the
investigators. Compliance with the study protocol will be assessed by direct interview
with the patient and/or caregiver, by analysing information from the study diary and
by checking the number of returned non-consumed study products. Participants who
receive <75% of the recommended dose of MSP/placebo will be considered as non-
compliant and will be excluded in the further analysis.
11.4 Withdrawal of individual subjects

Subjects can leave the study at any time for any reason if they wish to do so without
any consequences, and without an obligation to give reasons for the decision. The
investigator can decide to withdraw a subject from the study for urgent medical
reasons.
In case of occurrence of serious adverse events or new circumstances affecting the
safety of the participants (e.g. difficulty in swallowing, new diagnosis of
immunodeficiency), the intervention will be discontinued.

Date 04-06 2019 28 of 56
Table 1. The timeline of the study
Intervention period
Days of antibiotic treatment Days after antibiotic treatment
n + 37 Follow up
(1 n+6
n (end of n n n n n n n
month months and
1 2 3 4 5 Etc. antibiotic + + + + + + +
after n + 1 year
treatment) 1 2 3 4 5 6 7
n+7)
Enrolment
Eligibility assessment x
Informed consent reception x
Allocation and randomisation x
Handing over study diary x
Interventions
Antibiotic treatment
Multispecies probiotic
Placebo
Data collection
Study diary
Stool tests in case of diarrhoea
Stool microbiota examination x x x x x

Reception of study diary and
unused product x

Date 04-06 2019 29 of 56
11.5 Specific criteria for withdrawal

The treating physician can deviate from study protocol for medical reasons, but study
follow-up will continue as part of the intention-to-treat analysis. Thus no specific
criteria for study withdrawal exist.
11.6 Replacement of individual subjects after withdrawal

Individual subjects will not be replaced after withdrawal. In the sample size
calculation an assumed 20% withdrawal rate was taken in account.
11.7 Follow-up of subjects withdrawn from treatment

An intention-to-treat model will be applied—data from all randomised participants will
be used in the analysis, including those with low compliance or those who drop out
or withdraw their consent. This method analyses the occurrence of outcomes in
groups to which subjects were initially assigned by randomization, regardless of
whether they ultimately underwent the planned intervention or not. This method
preserves the essence of randomization, i.e. the initial balance of known and
unknown prognostic factors between groups. Samples obtained from participants
before withdrawal will be used for the analyses too.
Per-protocol analysis will be performed as well, and it will include all participants who
finish the study according to the protocol.
11.8 Premature termination of the study

We do not expect the study to be terminate prematurely. The study can be stopped
prematurely if completing the study is no longer feasible. The steering committee,
the Data Safety Monitoring Board, will be systematically involved in every important
decision concerning conduct of the study, including discontinuation. The METC will
be informed without delay if any investigator has ethical concerns about continuation
of the trial.
Possible criteria for terminating the study prematurely:

‚ Early evidence that the investigational product is beneficial for the conditions
studied or solid statistical evidence that an the investigational product is better
than the comparator
‚ Early evidence that the investigational product is, in contrary, harmful (ADRs,
SAEs, SUSARs, etc.)
‚ If an interim statistical analysis showed that a clinical trial has no scientific
(statistical) value/power
‚ It is not feasible to reach the planned outcomes.
‚ Loss of extramural funding

Date 04-06 2019 30 of 56
‚ difficulty in completing the study as originally planned

‚ Other reasons pertaining to the efficacy, safety, or feasibility of the study
12 SAFETY REPORTING
Monitoring
The study will be carried out in accordance with the protocol, as it will be registered.
No changes in the study protocol are expected to be made after the study starts.
However, in case of any unexpected circumstances requiring alterations of the
protocol, changes will be immediately applied to the protocol registry site at
clinicaltrials.gov, and, if relevant enough, reported to the Bioethics committee. An
independent Data and Safety Monitoring Board (DSMB) will be created before the
start of the study. The DSMB will review data every half yearto assess the study
progress (including rate of recruitment, completeness of data and their appropriate
collection) and all of the SAE’s. The number of recruited patients will be monitored
and kept up to date (see section 12.5); appropriate changes (i.e., training of the
recruiting physicians, study leaflets, addition of new recruitment centres) will be
applied to the study procedure and protocol if the pace of recruitment is not high
enough to finish the study within the established time, which is 2 years.
Harms
All eight of the probiotic strains to be used in the study have the Qualified
Presumption of Safety status (46) established by the European Food Safety Authority
(EFSA). The occurrence of serious adverse events in immunocompetent populations
during oral use of probiotics is unlikely (47).
The exact same product has not been assessed in previous studies. However, several
clinical studies have been performed with a comparable product, in different
populations (healthy volunteers and chronic obstructive pulmonary disease patients)
in the Netherlands and Austria without any reported serious side effects (18-21, 28).
In addition, the preparation is commercially available in several countries (Austria,
Germany, Greece, Norway, Russia, Slovenia, Ukraine and the Netherlands) and since
the market introduction in 2007, no serious adverse effects have been reported.
In the Netherlands, probiotics are considered to be food or food supplements and,

therefore, have to be produced under Hazard Analysis and Critical Control Point
regulations, which is the Dutch regulation system for safety and hygiene in food and
food supplements. Winclove is an NSF International Certified Good Manufacturing
Practices Facility for manufacturing dietary supplements and works with the food
safety management system ISO 22000:2005.
In case of suspected serious adverse events, the project leader will immediately
notify the Ethics Committee, DSMB, all study personnel and the manufacturer of the
product about the nature of the event. The decision regarding continuation or
Date 04-06 2019 31 of 56
discontinuation of the trial will be made by the project leader in agreement with the
Ethics Committee and DSMB.
12.1 Temporary halt for reasons of subject safety

In accordance to section 10, subsection 4, of the WMO, the sponsor will suspend the
study if there is sufficient ground that continuation of the study will jeopardise
subject health or safety. The sponsor will notify the accredited METC without undue
delay of a temporary halt including the reason for such an action. The study will be
suspended pending a further positive decision by the accredited METC. The
investigator will take care that all subjects are kept informed.
12.2 AEs, SAEs and SUSARs
12.2.1 Adverse events (AEs)

Adverse events are defined as any undesirable experience occurring to a subject
during the study, in any way being considered related to the investigational product.
All adverse events reported spontaneously by the subject or observed by the
investigator or his staff will be recorded.
12.2.2 Serious adverse events (SAEs)

A serious adverse event is any untoward medical occurrence or effect that
1. results in death;
2. is life threatening (at the time of the event);
3. requires hospitalisation or prolongation of existing inpatients’
hospitalisation;
4. results in persistent or significant disability or incapacity;
5. is a congenital anomaly or birth defect; or
6. any other important medical event that did not result in any of the
outcomes listed above due to medical or surgical intervention but could
have been based upon appropriate judgement by the investigator.
An elective hospital admission will not be considered as a serious adverse
event.
The investigator will report all SAEs to the sponsor without undue delay after
obtaining knowledge of the events.
The sponsor will report the SAEs through the web portal ToetsingOnline to the
accredited METc that approved the protocol, within 7 days of first knowledge for
SAEs that result in death or are life threatening followed by a period of maximum of
8 days to complete the initial preliminary report. All other SAEs will be reported
within a period of maximum 15 days after the sponsor has first knowledge of the
serious adverse events.

Date 04-06 2019 32 of 56
12.2.3 Suspected unexpected serious adverse reactions (SUSARs)

Not applicable as we will not investigate a medicinal product. Winclove 612 is
considered to be a food supplement/product.
12.3 Annual safety report

Not applicable.
12.4 Follow-up of adverse events

All AEs will be followed until they have abated, or until a stable situation has been
reached. Depending on the event, follow up may require additional tests or medical
procedures as indicated, and/or referral to the general physician or a medical
specialist. SAEs will be reported till end of study within the Netherlands, as defined in
the protocol.
12.5 Data Safety Monitoring Board (DSMB)

An independent Data and Safety Monitoring Board (DSMB) will be created before the
start of the study. The DSMB will be established to perform ongoing safety
surveillance and to perform interim analyses on the safety data. This committee will
be an independent committee. The members have no conflict of interest with the
sponsor of the study.
The project leaders had contacted the Clinical Research Bureau (CRB) from the
Amsterdam UMC, location VUmc about the formation of the DSMB. Usually a DSMB is
only facilitated by the CRB when the study is a classified as a ‘high-risk study’. This
study is not classified as a ‘high-risk’ study. However, since it involves children it was
decided, in consultation with the CRB, that the CRB will facilitate a DSMB for this
study and make sure that interim analysis will performed appropriately according to
the protocol.
The DSMB will review data after recruitment every hald year to assess the study
progress (including rate of recruitment, completeness of data and their appropriate
collection) and all of the adverse events.
A separate Charter will describe the roles and responsibilities of the independent
DSMB, including the timing of meetings, methods of providing information to and
from the DSMB, frequency and format of meetings and relationships with other
committees.
The advice(s) of the DSMB will only be sent to the sponsor of the study. Should the
sponsor decide not to fully implement the advice of the DSMB, the sponsor will send
Date 04-06 2019 33 of 56
the advice to the reviewing METC, including a note to substantiate why (part of) the
advice of the DSMB will not be followed.
The DSMB will consist of:

1. A chair of the DSMB:
o Prof. dr. P. Huijgens
2. An independent researcher with sufficient knowledge on statistics and
epidemiology:
o Prof. dr. Boers
3. An independent doctor with sufficient clinical knowledge on the topic of this
study.
The members of the DSMB will be formed by the CRB. The CRB has confirmed that
they will provide members as described above.
No DSMB member has any conflict of interest with Winclove Probiotics B.V.
13 STATISTICAL ANALYSIS
Subject baseline and demographic data, as well as baseline disease characteristics
data will be summarized for all subjects by treatment regimen and compared
between the two groups.
For continuous variables, comparison between groups will be done using the
Student’s t-test or Mann-Whitney U test, depending on whether or not the variables
are distributed normally. The 2 test or Fisher’s exact test will be used, as
appropriate, to compare dichotomous variables. Differences between groups will be
presented for continuous outcomes as differences in means or differences in medians
(for normal or non-normal distribution, respectively) along with a 95% CI. For
dichotomous outcomes, the relative risk (RR) and number needed to treat, calculated
as the inverse of the absolute risk reduction, will be determined along with a 95%
CI. The difference between study groups will be considered significant when the p
value is <0.05.
An intention-to-treat model will be applied—data from all randomised participants will

be used in the analysis, including those with low compliance or those who drop out
or withdraw their consent. This method analyses the occurrence of outcomes in
groups to which subjects were initially assigned by randomization, regardless of
whether they ultimately underwent the planned intervention or not. This method
preserves the essence of randomization, i.e. the initial balance of known and
unknown prognostic factors between groups. Per-protocol analysis will be performed
as well, and it will include all participants who finish the study according to the
protocol.
Date 04-06 2019 34 of 56
13.1 Primary study parameter(s)

Primary outcome
The incidence of AAD, defined as three or 2 test or Fisher’s exact test with
more loose or watery stool per day in a relative risk and number needed to
24-hour period, either caused by C. treat, along with a 95% CI.
difficile infection or of otherwise
13.2 Secondary study parameter(s)

Variable distribution will be assessed by Shapiro-Wilk tests. Depending on the results
of these tests, data will be analyzed by either parametric or non-parametric statistics.
The incidence of AAD, defined as three 2 test or Fisher’s exact test with relative
or more loose or watery stools per day risk and number needed to treat, along
(defined as above) for a minimum of 48- with a 95% CI.
hour period either caused by C. difficile
infection or of otherwise unexplained
aetiology.
The incidence of AAD, defined as two or 2 test or Fisher’s exact test with relative
more loose or watery stools per day risk and number needed to treat, along
(defined as above) for a minimum of a with a 95% CI.
24-hour period either caused by C.
difficile infection or of otherwise
unexplained aetiology.
The duration of diarrhoea (in days) Student’s t-test or Mann-Whitney U test
with differences in means or differences
in medians along with a 95% CI.
Incidence of any diarrhoea, regardless of 2 test or Fisher’s exact test with relative
its aetiology risk and number needed to treat, along
with a 95% CI.
Incidence of C. difficile-associated 2 test or Fisher’s exact test with relative
diarrhoea risk and number needed to treat, along
with a 95% CI.
Discontinuation of the antibiotic 2 test or Fisher’s exact test with relative
treatment due to severity of diarrhoea risk and number needed to treat, along
with a 95% CI.

Date 04-06 2019 35 of 56
Hospitalisation caused by diarrhoea in 2 test or Fisher’s exact test with relative

outpatients risk and number needed to treat, along
with a 95% CI.
Need for intravenous rehydration 2 test or Fisher’s exact test with relative
risk and number needed to treat, along

with a 95% CI.
Adverse events 2 test or Fisher’s exact test with relative
risk and number needed to treat, along

with a 95% CI.
Intestinal microbiota composition The species diversity ( -diversity) of
faecal samples will be calculated using
Amino acid analysis Faith’s phylogenetic diversity. The
Shannon-diversity indices, absolute and
Metabolomics analysis relative abundances, unsupervised and
supervised classification methods for
diversity applying correction for the
differences in sequencing depths by
rarefaction will be used.
13.3 Other study parameters

Not applicable
13.4 Interim analysis

The DSMB will review data every half year as decribed in the additional DSMB
charter.
14. ETHICAL CONSIDERATIONS

Ethics and dissemination
The protocol of the study will be reviewed by the Ethics Committee (METc) of the
Amsterdam UMC, location VUmc. Participants (or their legal representatives) will be
fully informed about the study, and informed consent will be obtained. The full protocol
is published in a peer-reviewed journal and available online (22). The results of the
study will also be published in a peer-reviewed journal and submitted for presentation
on conferences related to the topic of the thesis. The results will be reported in
accordance with the Consolidated Standards of Reporting Trials (CONSORT).
14.1 Regulation statement

This study will be conducted according to the principles of the Declaration of Helsinki
(WMA Declaration of Helsinki – Ethical Principles for Medical Research Involving
Human Subjects Adopted by the 18th WMA General Assembly, Helsinki, Finland, June
Date 04-06 2019 36 of 56
1964 and amended by the 64th WMA General Assembly, Fortaleza, Brazil, October
2013)) and in accordance with the Medical Research Involving Human Subjects Act
(WMO).
14.2 Recruitment and consent

The supervising physician will inform researchers whether a patient is eligible to
participate or not. In the event a patient seems eligible, the treating physician will
contact one of the researchers. He or she will then further inform participants.
Participants will both orally by the researcher and in writing via a patient information
letter (PIL), be informed about the study. In the information letter participants
and/or parent(s) and/or caregiver(s) are asked for informed consent. Participants will
have 24 hours after taking the antibiotics to consider their decision in participating. It
will be clearly stated that the participant is free to withdraw from the study at any
time for any reason without prejudice to future care, and with no obligation to give
the reason for withdrawal. Participant Information Sheets will be available in Dutch.
Written Informed Consent will be confirmed by the dated signatures of the
participant and by the person who presented and obtained the informed consent.
The person obtaining consent must be suitably qualified and experienced, and be
authorized to do so by the Principal Investigator. A copy of the signed Informed
Consent will be given to the participants. The original signed form will be retained at
the study site.The patient information letter and informed consent form will be
attached as a separate document.
14.3 Objection by minors or incapacitated subjects

It is possible that a child will resist to participate. Section 4, subsection 2, of the
WMO stipulates that a minor or legally incompetent adult cannot be forced to
undergo a treatment or behave in a particular manner in the context of non-
therapeutic research against his or her will. In the patient information letter the
grounds on which a subject will be deemed to object are stated. In case of resistance
to participating from a minor, the researcher will stop the study in this particular
subject. We will adhere to The Netherlands Association for Pediatric Medicine (NVK)
‘Code of conduct relating to expressions of objection by minors participating in
medical research’ which can be found on the website of the CCMO and to the
European Medicines Agency guidelines for good clinical practice.
14.4 Benefits and risks assessment, group relatedness

Based on all clinical trials performed with comparable products, patients can expect
that by using the investigational product less diarrhoea-like bowel movements will
occur, and microbiota is restored or will even prevent dysbiosis, consequently
preventing AAD. The potential risks of participation are low, especially as probiotic
intervention is started after critical or life-threatening illness are excluded. No serious
Date 04-06 2019 37 of 56
events are expected from administration of the test product, or control product. If
adverse effects occur, they are likely to be minor gastro-intestinal symptoms, such as
bloating or flatulence, especially when one uses probiotics for the first time. These
effects are expected to disappear after a few days.
As mentioned earlier all substances of the used products are considered safe for use
in children. There have been several studies with comparable products which do not
report any serious side effects. Besides this, a quite comparable product to the
investigational product is available in many countries, and on the Dutch market since
2007 without any serious side effects being reported.
AAD is a common complication of antibiotic treatment in children. Certain probiotic
strains are proven to be effective in reducing risk of AAD, however, there is not enough
evidence to recommend use of a MSP. The benefit-risk ratio of this MSP needs to be
determined in children. With this study we want to contribute to better insight in the
effectiveness of MSP’s in the prevention of AAD this population. As response to
probiotic treatment might differ between children and adults, this study cannot be
performed in adult patients. Since this study will lead to important knowledge on
preventing AAD in children (and the risks and burden of participating are low), we
think that the risk to and burden for the subject will be in proportion to the potential
value of the research.
14.5 Compensation for injury

Insurance for the participants
Participants are insured against any potential damages incurred as a result of
participating in the study. Amsterdam UMC, location VUmc covers the WMO subject
insurance by a continuous coverage.
The investigator has a liability insurance which is in accordance with article 7 of the
WMO.
The sponsor (also) has an insurance which is in accordance with the legal
requirements in the Netherlands (Article 7 WMO). This insurance provides cover for
damage to research subjects through injury or death caused by the study.
1. € 650.000,-- (i.e. four hundred and fifty thousand Euro) for death or injury
for each subject who participates in the Research;
2. € 5.000.000,-- (i.e. three million five hundred thousand Euro) for death or
injury for all subjects who participate in the Research;
3. € 7.500.000,-- (i.e. five million Euro) for the total damage incurred by the
organisation for all damage disclosed by scientific research for the Sponsor as
‘verrichter’ in the meaning of said Act in each year of insurance coverage.

Date 04-06 2019 38 of 56
The insurance applies to the damage that becomes apparent during the study or
within 4 years after the end of the study.
14.6 Incentives
Not applicable. Participants will receive not receive any compensation for
participating. No extra visits are warranted, so there won’t be any travel or parking
costs for participants. Remaining study products and collected samples can be
transported to the study site at a regular appointment or can be picked up at home
by one of the investigators.
15 ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION

15.1 Handling and storage of data and documents
Data will be handled confidentially. The handling of patient material and data will
comply the EU General Data Protection Regulation and the Dutch Act on
Implementation of the General Data Protection Regulation. (in Dutch: Uitvoeringswet
AVG, UAVG).). All study data and human material will be handled confidentially and
coded with a unique study number to protect the subject privacy. Data will be coded
on sequence of inclusions per participating centre, e.g. AMC001, AMC002, OLVG001,
OLVG002 et cetera. A subject identification code list will be used to link the data to
the subject. Only the research team (i.e. principal investigators, research nurse,
researcher and those otherwise involved in this study) will be able to identify the
participants and will have access to the data and human material. The key to the
code will be safeguarded by the investigator. Data will be recorded on a secure
password-protected electronic case record form (eCRF) only on protected servers in
the Amsterdam UMC, location VUmc, which will be managed and checked by the
researcher. These data will be transferred to a computer system for subsequent
tabulation and statistical analysis. Data and human material will be coded and stored
during the study period. When patients and their parents/caregivers give permission,
this data will be stored for a period of 15 years. Only the principal investigator and
other investigators involved in the study will have access to the samples. These
samples will be used to answer current research questions as well as possible new
research questions that fall within the scope of this study. For future research that
falls outside the scope of this study, participants will be asked new permission to use
these samples.
Stool samples from the 175 participants in the Netherlands can be collected at home
or in the hospital when a patient is admitted to the ward. Samples will be
transported to the Amsterdam UMC, location VUmc and will be coded. A subject
identification code list will be used to link the data to the subject. Only the research
team (i.e. principal investigators, research nurse, researcher and those otherwise
involved in this study) will be able to identify the participants and will have access to
the data and human material. The key to the code will be safeguarded by the

Date 04-06 2019 39 of 56
investigator. Samples will be stored in to a special biobank of the Amsterdam UMC,

location VUmc. Samples will be stored here for a maximum of 15 years after written
informed consent is obtained to do so.
15.2 Monitoring and Quality Assurance

The DSMB will review data after recruitment from 25%, 50% and 75% of
participants to assess the study progress (including rate of recruitment,
completeness of data and their appropriate collection) and all of the adverse events.
The number of recruited patients will be monitored and kept up to date.
We refer to section 12.5 to the exact tasks of the DSMB and what will be included in
the interim analysis.
Monitoring of the coordinated investigator will be done by a GCP-certified

research nurse, PhD candidate and/or the DSMB. The monitor will provide a written
report to the coordinated investigator after each visit including a summary of the
significant findings, deviations and deficiencies, conclusions, actions taken or
recommended to secure compliance. The coordinated investigator will run
consistency checks on a monthly basis and produce queries to be resolved by the
local investigator(s). The final database will be obtained after the resolution of all
queries.
15.3 Amendments
Amendments are changes made to the research after a favourable opinion by the
accredited METC has been given. All amendments will be notified to the METC that
gave a favourable opinion.
15.4 Annual progress report

The investigator will submit a summary of the progress of the trial to the accredited
METC once a year. Information will be provided on the date of inclusion of the first
subject, numbers of subjects included and numbers of subjects that have completed
the trial, serious adverse events/serious adverse reactions, other problems, and
amendments.
15.5 Temporary halt and (prematurely) end of study report

The investigator will notify the accredited METC of the end of the study within a
period of 8 weeks. The end of the study is defined as the last patient’s last visit.
The sponsor will notify the METC immediately of a temporary halt of the study,
including the reason of such an action.

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In case the study is ended prematurely, the sponsor will notify the accredited METC
within 15 days, including the reasons for the premature termination.
Within one year after the end of the study, the investigator will submit a final study
report with the results of the study, including any publications/abstracts of the study,
to the accredited METC.
15.6 Public disclosure and publication policy

Results of this study will be presented on national and international conferences and
in national and international medical journals, within the scope of the target group,
namely (pediatric) gastroenterologists. The sponsor of this study will not have any
influence on data analysis and on publication of results. The authors have no conflict
of interest. The project will be registered at clinicaltrials.gov (NCT03334604).
The Investigators will be involved in reviewing drafts of the manuscripts, abstracts,
press releases and any other publications arising from the study. Authorship will be
determined in accordance with the ICMJE guidelines and other contributors will be
acknowledged. The trial will be registered at clinicaltrials.gov before the first patients
is recruited. Research will be conducted in accordance with the AMC-VUmc research
code.
16 STRUCTURED RISK ANALYSIS

16.1 Potential issues of concern
Based on all clinical trials performed (18-21, 28, 29), patients can expect that by using
Winclove 612 less diarrhoea-like bowel movements might occur, microbiota might be
restored or dysbiosis will even be prevented.
The potential risks of participating are expected to be low, especially as probiotic
intervention is started after bacteraemia and sepsis are excluded. No serious events
are expected from administration of the test product or control product. However, this
can never be completely be ruled out. If adverse effects occur, they are likely to related
to minor gastro-intestinal symptoms, such as bloating or flatulence, especially when
one uses probiotics for the first time. These effects are expected to disappear after a
few days. If more serious adverse events occur, or if patients become more ill than at
the start of the study the treating physician can decide that a patients need to be
excluded from further participation.
a. Level of knowledge about mechanism of action

The pathogenesis of AAD is not fully understood. It may be caused by a specific
enteric pathogen (e.g., Clostridium difficile, Clostridium perfringens, Staphylococcus
aureus, Candida albicans), metabolic consequences of altered intestinal microbiota or
a direct effect of antibiotics on the mucosa (9). The impact of antibiotics on the
abundance of commensal micro-organisms in the gut underlines the hypothesis that

Date 04-06 2019 41 of 56
administration of probiotics could reduce the incidence of AAD. Probiotics are ‘live
microorganisms that provide health benefits to the host when ingested in adequate
amounts’ (11). Probiotics are demonstrated to be used as therapeutic options for a
variety of diseases, but the mechanisms responsible for these effects have not been
fully elucidated yet. Several important mechanisms underlying the antagonistic
effects of probiotics on various microorganisms include the following: modification of
the gut microbiota, competitive adherence to the mucosa and epithelium,
strengthening of the gut epithelial barrier and modulation of the immune system to
convey an advantage to the host. Accumulating evidence demonstrates that
probiotics communicate with the host by pattern recognition receptors, such as toll-
like receptors and nucleotide-binding oligomerization domain-containing protein-like
receptors, which modulate key signaling pathways, such as nuclear factor- B and
mitogen-activated protein kinase, to enhance or suppress activation and influence
downstream pathways. This recognition is crucial for eliciting measured antimicrobial
responses with minimal inflammatory tissue damage (37, 48). It is plausible that by
using probiotics during antibiotic treatment less diarrhoea-like bowel movements will
occur, and microbiota is restored or even prevent dysbiosis, consequently preventing
AAD.
b. Previous exposure of human beings with the test product(s) and/or products with a
similar biological mechanism
Ecologic AAD has been available at drugstores and pharmacies in many countries
including the Netherlands since 2007. Since the introduction on the market, no serious
side effects have been reported.
A double-blind, placebo controlled randomized controlled trial showed that Ecologic
AAD is able to significantly reduce the risk of diarrhoea-like defectation (18). Bowel
movements and/or a loose consistency were reported less frequently. Changes over
time were present in both groups regarding microbial composition and metabolic
activity, which differed significantly between the probiotic and the placebo arm,
suggesting that the effect of antibiotics was modulated by probiotic intake.
In another RCT with 30 COPD patients the investigated MSP did not affect the
composition of the dominant faecal microbiota nor the occurrence of diarrhea-like
bowel movements. The dominant faecal microbiota was not affected by antibiotics in
this COPD population, suggesting an existing imbalance of the microbiota, which may
also have contributed to the lack of effect by probiotic intake (19).
In a study with 199 patient receiving antibiotics and a MSP, only 2 patients developed
AAD which probably had been caused by overgrowth of Clostridium difficile in one of
the patients. Based on the knowledge from literature the incidence of AAD ranges from
5-39% in the population used in that study. An observed almost complete absence of
AAD incidence (0.5%) in this cohort of Ecologic AAD treated patients strongly suggests
a beneficial role of Ecologic AAD therapy during and after antibiotic treatment (20).

Date 04-06 2019 42 of 56
In a retrospective case report series of 10 patients with Clostridium difficile infection,

5 of whom experienced recurrent CDI, all patients received, besides antibiotics Ecologic
AAD, which resulted in complete recovery. Moreover no adverse events were reported
(21).
In their global guidelines, the World Gastroenterology Organisation (WGO) evaluates
pro- and prebiotics on their efficacy and safety. In this guideline Ecologic AAD is
evaluated as a randomized trial with dramatic effect in the prevention of antibiotic-
associated diarrhea (27). More specifically, the evidence for Winclove’s probiotic
formulation Ecologic AAD is evaluated as level 2 evidence: Randomized trial or
observational study with dramatic effect. The recommendation of Ecologic AAD as an
effective and safe probiotic for reducing antibiotic-associated diarrhea by the WGO is
very promising in implementing probiotics as a standard prescription next to
antibiotics.
For a more detailed description of the studies on the efficacy and safety of we refer
to chapter 9.3.
c. Can the primary or secondary mechanism be induced in animals and/or in ex-vivo

human cell material?
The pathogenesis of AAD is not fully understood, and unfortunately, no good animal
model has been found until now. However there are some studies investigating the
effect of probiotics in vitro. It can be concluded that most strains in Winclove 612 are
very capable to inhibit one or more pathogens likely to cause AAD.
However, there are also some studies with opposite conclusions. In one study with
mice. spontaneous post-antibiotic recovery compared to probiotics, the latter induced
a markedly delayed and persistently incomplete indigenous stool/mucosal
microbiome reconstitution and host transcriptome recovery toward homeostatic
configuration, while autologous fecal microbiome transplantation induced a rapid and
near-complete recovery within days of administration. In this study, in vitro,
Lactobacillus-secreted soluble factors contributed to probiotics-induced microbiome
inhibition. These contradictory results show the need of good quality clinical studies
involving humans and are a rationale to perform the proposed study.
For a more detailed description of the studies investigating the effect of the strains in
vitro we refer to chapter 9.2.
d. Selectivity of the mechanism to target tissue in animals and/or human beings

Probiotics will be present in different concentrations at different levels of the gastro-
intestinal tract (see paragraph 16.2 f). They will not be absorbed nor target other
tissues and thus will only have an effect on tissues in the gastro-intestinal tract,
specifically in the intestines. The probiotics can also be present in a different amount
of CFU in the stool of the participants. This will be further discussed in 16.2
Synthesis.

Date 04-06 2019 43 of 56
e. Analysis of potential effect

The direct effect of probiotics is on tissues in the gastro-intestinal tract, mostly in the
gut. Here, probiotics may exert its beneficial effects by enhancing gut barrier
function, inhibiting colonization of potentially pathogenic microorganisms,
maintaining a normal intestinal milieu, synthesizing antibacterial substances and
stimulating local immunity.
Based on all clinical trials performed with Ecologic AAD as very comparable product
to the current investigational product Winclove 612 and the studies mentioned in
16.1b, patients can expect that by using Winclove 612 less diarrhoea-like bowel
movements will occur, and microbiota might restored or even prevent dysbiosis,
consequently preventing AAD.
The potential risks of participation are expected to be low, especially as probiotic
intervention is started after critically ill patients are excluded. No serious events are
expected from administration of the test product, or control product. If adverse
effects occur, they are likely to related to minor gastro-intestinal symptoms, such as
bloating or flatulence, especially when one uses probiotics for the first time. These
effects are expected to disappear after a few days (18-21, 28). So the adverse
effects are expected to be mild and the benefit of treatment seems to outweigh this
risk. As mentioned in paragraph 9.3 and 16.1b, it is thought that probiotics have a
beneficial effect on the microbiome, however there are also studies claiming
otherwise and that more data is needed to elucidate this. With this study we want to
contribute to evidence.
f. Pharmacokinetic considerations
The product has a concentration of 2.5*109 CFU/gram with an advised daily dosage
of 2 grams twice daily. The dosage of Winclove 612 is in accordance with most
probiotic dosages used in human studies and many commercially available products,
ranging from 109-1010 CFU/dose and by the recommendations of the TNO (29). It is
not possible to state a general dose for probiotics; some have shown to be effective
at lower levels, while other require substantially more. The dosage of Winclove 612 is
this study is based on mentioned studies and recommendations and on prior human
studies in healthy volunteers, COPD patients and general surgery patients with the
product (18-21, 28) showing a health benefit without adverse reactions.
Probiotic strains differ in their survival capacity at different levels of the
gastrointestinal tract (37). The concentration of probiotics needed to obtain a clinical
effect is often quoted as ≥106 colony-forming units/ml (CFU) in the small bowel and
≥108 CFU/g in the colon (36). Dose–response studies are, however, scarce, and it is
not known whether the percentage survival is stable for various ingested doses.
The survival of probiotics depends on their intrinsic resistance, on host factors and
on the vehicle in which they are ingested. The main obstacles to survival being
gastric acidity and the action of bile salts. Well-controlled, small-scale studies on
diarrhea in both adults and infants have shown that probiotics survive in sufficient
Date 04-06 2019 44 of 56
numbers to affect gut microbial metabolism (37). Survival rates have been estimated
at 20–40% for selected strains, but differ between strains (38). Approximately 1–
10% of Lactobacillus acidophilus ingested in fermented products was found to
survive until the ileum in several human studies using intestinal intubation techniques
(39). In contrast, 30% of ingested Bifidobacterium was found in the colon (40).
Other studies in healthy volunteers with different probiotic preparations showed that
the faecal concentrations of ingested Lb. acidophilus, Lactobacillus salivarius,
Lactobacillus rhamnosus strain GG reached around 106 CFU/g. Probiotics are usually
excreted within a few days of their ingestion in faeces at the same rate as or even
more quickly than a transit marker (37). Based on the best evidence, a strictly
selected combination of strains is used in this study.
g. Study population
The study population will consist of healthy children aged between 3 months and 18
years old (see inclusion and exclusion criteria). Participants will be recruited among
both the inpatient and outpatient clinics from the department of Paediatrics in the
participating centres in Amsterdam. The condition of the patients that participate in
the study is stable.
Since our study aim is to investigate the effect of probiotics on AAD in children, we will
include only children in this study. Response to probiotic treatment might differ
between children and adults, so this study cannot be performed in adult patients.
h. Interaction with other products

Participants will not be given a combination of products. There is no evidence
suggesting that oral administration of the used probiotics may alter the beneficial
effect of antibiotics or vice versa.
i. Predictability of effect
Predictive biomarkers for the effect and adverse effect of probiotics are not available.
j. Can effects be managed?

Ecologic AAD is available in drugstores in a lot of countries including The Netherlands
since 2007. Possible expected side effects from Ecologic AAD and Winclove 612 are
mild gastro-intestinal symptoms. These symptoms will disappear in a few days.
Physicians are experienced in coping with symptoms similar to the possible side
effects of probiotics.
16.2 Synthesis
In summary the overall risk of participation in this trial is thought to be low.
Probiotics are generally regarded as safe. Recent safety studies have concluded that
the consumption of probiotics is well-tolerated and generally recognized as safe

Date 04-06 2019 45 of 56
across all age groups (49, 50). However it has to be acknowledged that not all
studies on probiotics reported adverse thoroughly (32). Studies reporting side effects
and negative consequences of probiotic use showed no major safety concerns, as
none of the serious adverse events were related, or suspected to be related, to the
probiotic or synbiotic product and the study products were well tolerated. Despite
this, we took some additional safety measures mentioned later on in this synthesis to
secure participants safety.
Studies on the efficacy of probiotics on the incidence of AAD are controversial: there
are studies claiming that probiotics should be used to prevent dysbiosis and AAD,
however some studies questioning the effectivity of probiotics. One of these studies
showed that probiotics induced a markedly delayed and persistently incomplete
indigenous stool/mucosal microbiome reconstitution after antibiotic use (34). Is has to
be noted however that a probiotic strain with strains of the gena Lactococcus and
Streptococcus were used; two strains with less evidence of their efficacy compared to
Bifidobacteria and Lactobacilli strains. The hypothesis is that probiotics can be
disadvantageous if used in wrong dosage or if the wrong strains are used. Therefore,
it is even more important that good-quality studies are being performed on the impact
of probiotic strains on the microbiome.
A systematic review of relevant clinical studies for effective probiotics (the practical
guide for the use of probiotics for the prevention of AAD by TNO) mentions that studies
with Bifidobacterium bifidum, Bifidobacterium lactis, Enterococcus faecium,
Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus plantarum,
Lactobacillus rhamnosus, Lactobacillus salivarium (minimal daily dose: 1x1010 billion
CFU) show a trend to the reduction in the incidence of AAD (29). All these strains are
present in our investigational product except for Enterococcus faecium due to safety
concerns by the ESPGHAN. We are using the recommended dose by TNO. As
mentioned previously, with current knowledge, it is impossible to say what the best
dose and whether a different set of species should be used or in a different combination
of CFU per species. However, all strains were strictly selected as mentioned previously
and are being used in the recommended dose based on the best available evidence.
With this study, we want to contribute to evidence of the efficacy of these selected
probiotics on the incidence of AAD. Furthermore, the exact pathophysiology remains
unclear. Since we also perform microbiota and metabolomic analysis, we will contribute
to information on the mechanisms on how probiotics affect the microbiota. Besides
this, we will be able to tell whether the diversity is affected by the probiotic strains and
whether the microbiota of children who were given probiotics will resemble a more
healthy microbiota compared to the microbiota of the placebo group. It is possible that
the first group will have a more stable, and more healthy like microbiota. In a previous
study, the composition, stability and diversity of healthy children in our population was
investigated (51). Furthermore, they described healthy and commensal bacteria. With
this information we can investigate whether probiotics contribute to having a more
Date 04-06 2019 46 of 56
stable and diversity microbiota in children receiving antibiotics. Since dysbiosis is

associated with numerous clinical conditions, prevention or quick restoration of
dysbiosis might have long term positive consequences.
It could also be possible that the probiotic strains will be present in different
concentrations or CFU in the stool of children. Some researchers suggest that person-
, region and/or strain-specific mucosal patterns and that some people can be probiotic
resistant. However, they also acknowledge that information and studies on responders
and non-responders is scarce and controversial (52). If we do find a difference in CFU
in the stool of children treated with probiotics, we want to zoom in on these children
and compare if it correlates with other variables such as the incidence of AAD and the
overall diversity of the microbiota. This way, we want to investigate whether there are
clinical variables that can predict if a child will respond to probiotics.
All of the mentioned results could also be a rationale for future studies.
All of the 8 probiotic strains to be used in the study have the Qualified Presumption
of Safety (QPS) status established by the European Food Safety Authority (EFSA)
(53), or have an extensive safety dossier. QPS is similar in concept and purpose as
the Generally Recognized As Safe (GRAS) definition used in the USA, but modified to
take into account the different regulatory practices in Europe. Experts regard all
probiotic species with GRAS of QPS status as safe for consumption.
With probiotics, whether there is long-term replacement of indigenous microbes by

other species or inherent advantages given to the existing microbiota to repopulate is
still unclear(52, 54). With this study we want to gain more insight in the microbiota
composition, how this is affected by antibiotics and probiotics. Furthermore, we
contribute with good quality evidence on the effect of probiotics on the incidence of
AAD. By collecting and analyzing the follow up samples we hope to get more insight
in how the microbiota changes/restores after time and whether this is different
between both groups. With this knowledge, the use of probiotics in children receiving
antibiotics might be implemented in guidelines in the future.
Primary research studies and meta-analyses have suggested that probiotics decrease
the duration of antibiotic-associated diarrhea, however evidence from these studies is
of variable quality, and the mechanism underlying the clinical benefits remains
unknown (52, 54). With this blinded randomised study, we want to contribute to
good quality evidence on this topic and to get more insight whether there are
specific populations would benefit most from this therapy and whether there are
specific groups of patients that would be resistant to probiotics or where the
restoration of the dysbiosis is even delayed. In our opinion, this study will result in
valuable information an evidence whether there is a need of more studies and
whether the use of probiotics should be implemented in guidelines on not.

Date 04-06 2019 47 of 56
Even though we expect the risks of participating to be low. We took some additional
safety measurements to reduce the risks of participating. Our study population
consist of stable children (see inclusion and exclusion criteria; no critical ill children
will be included). If it becomes evident that children do not response well to the
antibiotics and become more ill, the participant will be immediately withdrawn from
the study. Besides these safety measures, we established a DSMB to perform
ongoing safety surveillance. In the Netherlands, probiotics are considered to be food
or food supplements and therefore have to be produced under Hazard Analysis and
Critical Control Point (HACCP) regulations, which is the Dutch regulation system for
safety and hygiene in food and food supplement. All components are legally admitted
as food additives or food components. Winclove works with the food safety
management system ISO22000:2005 and is certified for the development and
production of pre- and probiotics. This specific multispecies probiotic mixture was
chosen, of which multiple previous clinical studies have been performed showing no
increase in adverse events.
Possible side effects expected from the use of Winclove 612 are expected to be mild
gastro intestinal symptoms such as bloating or flatulence which are expected to
disappear after a few days. Besides this, children participating may even benefit by
participating as AAD may be prevented by using probiotics.
Since this study will lead to an important gain of knowledge on preventing AAD in
children and on the effects of antibiotics and probiotics on the microbiota and the risks
and burden of participating are expected to be low, we deem the risk to and burden
for the subject is in proportion to the potential benefit and value of the research. The
knowledge obtained about the effects of probiotics on AAD could potentially be
implemented in future guidelines and benefit future patients.

Date 04-06 2019 48 of 56
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50. Hempel S, Newberry SJ, Maher AR, Wang Z, Miles JN, Shanman R, et al.
Probiotics for the prevention and treatment of antibiotic-associated diarrhea: a systematic
review and meta-analysis. JAMA. 2012;307(18):1959-69.
51. de Meij TG, Budding AE, de Groot EF, Jansen FM, Frank Kneepkens CM,
Benninga MA, et al. Composition and stability of intestinal microbiota of healthy children
within a Dutch population. FASEB J. 2016;30(4):1512-22.
52. Zmora N, Zilberman-Schapira G, Suez J, Mor U, Dori-Bachash M,
Bashiardes S, et al. Personalized Gut Mucosal Colonization Resistance to Empiric Probiotics
Is Associated with Unique Host and Microbiome Features. Cell. 2018;174(6):1388-405 e21.
53. Ricci A, Allende A, Bolton D, Chemaly M, Davies R, Gironés R, et al.
Scientific Opinion on the update of the list of QPS-recommended biological agents
intentionally added to food or feed as notified to EFSA2017. 177 p.
54. Severyn CJ, Bhatt AS. With Probiotics, Resistance Is Not Always Futile.
Cell Host Microbe. 2018;24(3):334-6.

Date 04-06 2019 51 of 56
18 APPENDIX A: CONTACT DATA PARTICIPATING CENTRES
Amsterdam UMC, location VUmc

T.H. Dierikx
Student-researcher/PhD student, department of pediatric gastroenterology
Email: t.dierikx@vumc.nl
dr. T.G.J. de Meij

Department of pediatric gastroenterology
Email: t.demeij@vumc.nl

dr. prof. M.A. Benninga
Meibergdreef 9, 1105 AZ, Amsterdam
Email: m.a.benninga@amc.uva.nl
dr. T.G.J. de Meij

Amsterdam UMC
Email: t.demeij@amsterdamumc.nl

dr. S.R.D. van der Schoor
Department of pediatrics
Oosterpark 9, 1091 AC, Amsterdam
Email: s.r.d.vanderschoor@olvg.nl
Onze Lieve Vrouwe Gasthuis, locatie West

dr. S.R.D. van der Schoor
Department of pediatrics
Date 04-06 2019 52 of 56
Jan Tooropstraat 164, 1061 AE, Amsterdam

Email: s.r.d.vanderschoor@olvg.nl

Date 04-06 2019 53 of 56
19 APPENDIX B: WINCLOVE CERTIFICATE FOR SAFETY STATEMENT

Date 04-06 2019 54 of 56
20 APPENDIX C: SPECIFICATION OF INVESTIGATIONAL PRODUCT

(Winclove 612)
Label investigational product (probiotics)
Uitsluitend bestemd voor gebruik in wetenschappelijk studieverband.

IPAD studie, Amsterdam UMC, locatie VUmc
NL69225.029.19
Ingrediënten per sachet: maïszetmeel, maltodextrines, +/- fructo-oligosacchariden (FOS) P6, +/- dextrine P9, +/-
bacteriestammen (B. bifidum W23, B. lactis W51, L. acidophilus W37, L. acidophilus W55, L. paracasei W20, L.
plantarum W62, L. rhamnosus W71, L. salivarius W24; ≥ 2,5*10^9 kolonievormende eenheden per gram),
kaliumchloride, rijst eiwit, magnesiumsulfaat, enzymen (amylase), mangaansulfaat.
Gebruik: Neem 2x per dag de inhoud (2 gram) van 1 sachet. Los het poeder op in een glas water, borst- of
flesvoeding, en laat het 1 minuut staan. Goed doorroeren voor inname. Bij voorkeur innemen op de lege maag; 1
sachet voor het ontbijt en 1 sachet voor het slapen gaan. Indien u antibiotica gebruikt: na inname van de
antibiotica 2-3 uur wachten met gebruik van dit product.
THT: zie sachet

Op kamertemperatuur in de originele verpakking bewaren.
Geschikt voor pasgeboren kinderen.
Buiten bereik van jonge kinderen houden.
Bij vragen kunt u contact opnemen met hoofdonderzoeker: dr. T.G.J. de Meij. E-mailadres: t.demeij@vumc.nl.
Telefoonnummer: +31 (0)20-4443318
Geproduceerd door Winclove Probiotics B.V., Hulstweg 11, 1032 LB Amsterdam, +31 (0)20-4350235

Date 04-06 2019 55 of 56

Date 04-06 2019 56 of 56
Supplemental Online Content
Łukasik J, Dierikx T, Besseling-van der Vaart I, de Meij T, Szajewska H; the Multispecies Probiotic in AAD Study Group. Multispecies probiotic
for the prevention of antibiotic-associated diarrhea in children: a randomized clinical trial. JAMA Pediatr. Published online June 21, 2022.
doi:10.1001/jamapediatrics.2022.1973
eTable 1. Recruitment centres

eTable 2. Patient characteristics depending on the country of recruitment
eTable 3. Characteristics of patients lost to follow-up
eTable 4. Results of the per protocol analysis including 119 patients in probiotic group and 110 patients in placebo group
eTable 5. Available case analysis by the country of recruitment
eTable 6. Sensitivity analyses
eTable 7. Results of logistic regression analysis
This supplemental material has been provided by the authors to give readers additional information about their work.

eTable 1. Recruitment centres.
Location Amsterdam UMC, Amsterdam UMC, OLVG OLVG location University Clinical St. Jadwiga l ska
location VUmc location AMC location East West Center of the Medical Hospital
University of Warsaw,
De Boelelaan Meibergdreef 9, Oosterpark 9, Jan Tooropstraat Prusicka 53-55, 55100
1117 1105 Amsterdam, 1092 164, 1061 wirki i Wigury 63A, Trzebnica, PL
Amsterdam, NL NL Amsterdam, Amsterdam, NL 02091 Warsaw, PL
NL
Number of the 14 59 31 44 198 4
included
participants

eTable 2: Patient characteristics depending on the country of recruitment
The
Clinical values Poland Netherlands
Total 202 148
Lost to follow-up, n(%) 31 (15.1) 6 (4.1)
Compliant participants, n(%) 128 (63.4) 101 (68.2)
Median age in months (range) 27 (3-212) 32 (3-204)
Sex
Female, n(%) 100 (49.5) 58 (39.2)
Male, n(%) 102 (50.5) 90 (60.8)
Setting
Inpatient, n(%) 200 (99) 71 (48)
Outpatient, n(%) 2 (1) 77 (52)
Lower respiratory tract infection, n(%) 62 (30.7) 48 (32.4)
Upper respiratory tract infection, n(%) 83 (41.1) 18 (12.2)
Urinary tract infection, n(%) 27 (13.4) 32 (21.6)
Skin infection, n(%) 3 (1.5) 21 (14.2)
Lymphadenitis, n(%) 9 (4.5) 4 (2.7)
Nervous system infection, n(%) 2 (1) 5 (3.4)
Gastrointestinal infection, n(%) 3 (1.5) 7 (4.7)
Joint infection, n(%) 1 (0.5) 4 (2.7)
Other, n(%) 12 (5.9) 9 (6.1)
Antibiotic administration route
Only oral, n(%) 31 (15.3) 113 (76.4)
Only intravenous, n(%) 43 (21.3) 10 (6.8)
Intravenous followed by oral , n(%) 128 (63.4) 25 (16.9)
Antibiotic type
2nd generation cephalosporin, n(%) 48 (23.8) 3 (2)
3rd generation cephalosporin, n(%) 51 (25.2) 18 (12.2)
Aminopenicillin, n(%) 90 (44.6) 50 (33.8)
Amoxicillin+clavulanic acid, n(%) 36 (17.8) 86 (58.1)
Clindamycin, n(%) 29 (14.4) 2 (1.4)
Cloxacillin/flucloxacillin, n(%) 2 (1) 4 (2.7)
Gentamicin, n(%) 0 4 (2.7)
Other, n(%) 5 (2.5) 7 (4.7)
Two concomitant antibiotics, n(%) 31 (15.3) 8 (5.4)
Change of antibiotic class n(%) 28 (13.9) 18 (12.2)
Median treatment duration days (range) 10 (1-21) 7 (2-36)
Median hospital stay duration (range) 5 (2-21) 4 (1-45)

eTable 3: Characteristics of patients lost to follow-up
Clinical values Placebo Probiotic

Total 19 18
Median age in months (range) 26 (3-144) 25 (6-161)
Sex
Female, n(%) 9 (47) 9 (50)
Male, n(%) 10 (53) 9 (50)
Setting
Inpatient, n(%) 16 (84) 17 (94)
Outpatient, n(%) 3 (16) 1 (6)
Lower respiratory tract infection, n(%) 10 (53) 6 (33)
Upper respiratory tract infection, n(%) 5 (26) 7 (39)
Urinary tract infection, n(%) 1(5) 2 (11)
Nervous system infection, n(%) 1 (5) -
Lymphadenitis - 1 (6)
Other, n(%) 2 (10) 2 (11)
Antibiotic type
2nd generation cephalosporin, n(%) 3 (16) 5 (28)
3rd generation cephalosporin, n(%) 2 (11) 2 (11)
Aminopenicillin, n(%) 10 (53) 9 (50)
Amoxicillin+clavulanic acid, n(%) 4 (21) 2 (11)
Clindamycin, n(%) 4(21) 4 (22)
Two concomitant antibiotics, n(%) 4 (21) 4 (22)
Median treatment duration days (range) 10 (5-21) 10 (3-14)
Median hospital stay duration (range) 4 (3-14) 4 (2-9)

eTable 4. Results of the per protocol analysis including 119 patients in probiotic group and 110 patients in placebo group.
Probiotic group no. of events Placebo group no. of events

Outcome (%) (%) Relative Risk (95% CI)
AAD 16 (13.4) 18 (16.4) 0.82 (0.45 to 1.52)
Severe AAD 13 (10.9) 12 (10. 9) 1 (0.49 to 2.07)
Mild AAD 29 (24.4) 25 (22.7) 1.07 (0.67 to 1.71)
Diarrhea 20 (16.8) 27 (24.5) 0.68 (0.41 to 1.14)
C. difficile diarrhea 1 (0.84) 2 (1.8) 0.46 (0.06 to 3.49)
Hospitalization due to
diarrhoea 0 (0) 1 (0.9) n/a
Antibiotic cessation due
to diarrhea 0 (0) 0 (0) n/a
Intravenous rehydration
due to diarrhea 0 (0) 1 (0.9) n/a
Adverse events
Readmission to the
hospital 3 (2.5) 1 (0.9) 2.77 (0.29. 26.27)
Abdominal pain 3 (2.5) 0 (0) n/a
Vomiting 2 (1.7) 0 (0) n/a
Rash 1 (0.84) 0 (0) n/a
Trace of blood in the
stool 1 (0.84) 0 (0) n/a
Probiotic group median (IQR) Placebo group median (IQR) Median difference (95% CI)
Diarrhea duration in
days 3 (3-5.75) 4 (3-6) 1 (-1 to 2)

eTable 5. Available case analysis by the country of recruitment.
Available case analysis - Poland (probiotic n = 84, placebo n= 87)
Probiotic group no. of Placebo group no. of
Outcome events events Relative Risk (95% CI)
AAD 13 16 0.84 (0.44 to 1.62)
Severe AAD 8 7 1.18 (0.46 to 3.02)
Mild AAD 21 25 0.87 (0.53 to 1.42)
Diarrhoea 18 28 0.67 (0.4 to 1.1)
C. difficile diarrhea 1 2 0.52 (0.07 to 3.89)
Hospitalization 0 2 n/a
Antibiotic cessation 0 0 n/a
Intravenous rehydration 0 5 n/a
Adverse eventsa 10 5 2.07 (0.77 to 5.61)
Probiotic group median Placebo group median
(IQR) (IQR) Median difference (95% CI)
Diarrhea duration 3 (2 to 5,5) 4 (3 to 6) 1 (-1 to 2)
a
Including: rash (2), readmission to the hospital (2), vomiting (1) in the placebo group and vomiting (3), rash (2), readmission to the hospital (1), gag
reflex (2), trace of blood in the stool (1), abdominal pain (1) in the probiotic group.
Available case analysis - The Netherlands (probiotic n = 74, placebo n= 68)
Probiotic group no. of Placebo group no. of
Outcome events events Relative Risk (95% CI)
AAD 10 12 0.77 (0.36 to 1.63)
Severe AAD 10 12 0.77 (0.36 to 1.63)
Mild AAD 19 13 1.34 (0.73 to 2.5)
Diarrhoea 15 22 0.63 (0.36 to 1.09)
C. difficile diarrhea 0 1 n/a
Hospitalisation 1 0 n/a
Antibiotic cessation 0 0 n/a
Intravenous rehydration 0 0 n/a
a
Adverse events 6 5 1.03 (0.37 to 3.28)

Probiotic group median Placebo group median
(IQR) (IQR) Median difference (95% CI)
Diarrhea duration 5 (3-12) 6 (4-7) 0 (-2 to 3)
a
Including: readmission to the hospital (4), abdominal pain (2) in probiotic group and readmission to the hospital (2), abdominal pain (2), rash (1) in placebo group.

eTable 6. Sensitivity analyses
Probiotic group no. Of events Placebo group no. of events

Outcome (%) (%) Relative Risk (95% CI)
AAD cases + diarrhea

cases where the testing
for pathogens was not
performed 29 (18.4) 39 (25.2) 0.73 (0.48 to 1.11)
Infectious diarrhea
excluding C. difficile
diarrhoea 4 (2.5) 11 (7.1) 0.36 (0.02 to 0.65)
Rotaviral diarrhoea 1 (0.6) 9 (5.8) 0.11 (0.2 to 0.65)a
Norovirus diarrhea 3 (1.9) 0 (0) n/a
Adenovirus diarrhea 0 (0) 1 (0.6) n/a
Salmonella diarrhea 0 (0) 1 (0.6) n/a

Diarrhea: plausible
assumptionc 5:1 51 (29) 56 (32.2) 0.9 (0.66 to 1.23)
Diarrhea: plausible
assumptionc 2:1 41 (23.3) 56 (32.2) 0.72 (0.51 to 1.02)
Diarrhea: plausible
assumptionc 1,5:1 39 (22.2) 56 (32.2) 0.69 (0.48 to 0.97)b
AAD: plausible
assumptionc 5:1 36 (20.5) 31 (17.8) 1.15 (0.75 to 1.77)
AAD: plausible
assumptionc 1:1 26 (14.8) 31 (17.8) 0.83 (0.52 to 1.33)
a
p=0.01 bp=0.04
c
Explanation of plausible assumption: we performed a sensitivity analysis assuming that the incidence of events among participants lost to follow-up is equal to, or higher by a specific ratio relative to
the observed event incidence among participants followed up, For example, ‘plausible assumption 5:1’ means that we assumed the incidence of diarrhea among missing patients in the probiotic
group to be 5 times higher than that in the probiotic group patients who were followed-up, and the incidence of diarrhea among missing patients in the placebo group to be equal to the incidence of
diarrhea in the placebo group patients who were followed up.

eTable 7. Results of logistic regression analysis.
A. Logistic regression – AAD outcome
Predictor Model with covariates
Odds Ratio 95% CI p
Allocation to probiotic group 0.8 0.42 to 1.52 0.49
Age in months 0.99 0.98 to 1 0.006
Male sex 0.94 0.49 to 1.81 0.85
2nd gen. cephalosporin 0.83 0.24 to 2.91 0.78
3rd gen. cephalosporin 2.02 0.72 to 5.7 0.18
Aminopenicillin 0.76 0.24 to 2.45 0.65

Amoxicillin with clavulanic 2.07 0.68 to 6.31 0.2
acid
Clindamycin 0.61 0.17 to 2.23 0.45
Other antibiotic 0.49 0.1 to 2.57 0.4
Intravenous antibiotic 1.36 0.40 to 4.62 0.62
Oral antibiotic 0.62 0.26 to 1.49 0.29
Hospital stay duration 1.04 0.97 to 1.12 0.26
Antibiotic treatment duration 1.05 0.96 to 1.14 0.28
Model without covariates
Odds Ratio 95% CI p

B. Logistic regression – Diarrhea outcome
Model with covariates
Predictor
Odds Ratio 95% CI p

Age in months 0.99 0.98 to 0.99 <0.001
Male sex 1.05 0.60 to 1.82 0.86
2nd gen. cephalosporin 1.75 0.59 to 5.15 0.31

3rd gen. cephalosporin 2.44 0.98 to 6.05 0.05
Aminopenicillin 1.43 0.52 to 3.93 0.48
Amoxicillin with clavulanic acid 2.63 1 to 6.9 0.05
Clindamycin 0.72 0.23 to 2.24 0.57
Other antibiotic 1.65 0.45 to 6.02 0.45
Intravenous antibiotic 2.37 0.83 to 6.81 0.11
Oral antibiotic 0.78 0.38 to 1.61 0.5
Hospital stay duration in days 1.02 0.95 to 1.09 0.65
Antibiotic treatment duration in days 1 0.92 to 1.08 0.98
Model without covariates
Odds Ratio 95% CI p

Supplemental Online Content: Nonauthor Collaborators
*First name, last name, and suffix (if applicable) are required and will appear in PubMed.
*Group Name(s): the Multispecies Probiotic in AAD Study Group

*First Name and *Last Name *Suffix Academic Institution Location (city, Role or Contribution, Group (if more than 1
Middle Initial(s) (eg, Jr, III) Degrees state/province, country) eg, chair, principal Group listed in the byline)
investigator and/or Subgroup (eg,
Steering Committee)
Sophie R. D. van der Schoor MD, PhD OLVG Hospital Amsterdam, Noord- Helped with
Holland, The Netherlands recruitment of
participant in the
OLVG hospital
Malika Chegary MD, PhD OLVG Hospital Amsterdam, Noord- Helped with
Holland, The Netherlands recruitment of
participant in the
OLVG hospital
Catharina (Karen) Koning PhD Winclove Probiotics Amsterdam, Noord- contributed in design
J.M. Holland, The Netherlands and setup of the
study, as well as on co-
reading/giving input
on the final
manuscript.
v 02-22 1 of 1
Data Sharing Statement
Łukasik. Multispecies Probiotic for the Prevention of Antibiotic-Associated Diarrhea in Children.
JAMA Pediatr. Published June 21, 2022. doi:10.1001/jamapediatrics.2022.1973
Data
Data available: Yes
Data types: Deidentified participant data
How to access data: The data collected in the course of this study are available from the
corresponding author upon reasonable request.
When available: With publication
Supporting Documents
Document types: None
Additional Information
Who can access the data: The data will be available to the researchers whose proposed use
of the data has been approved
Types of analyses: The data will be available for any purpose,
Mechanisms of data availability: The data will be made available with investigator support.

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Research

JAMA Pediatrics | Original Investigation

Multispecies Probiotic for the Prevention

DESIGN, SETTING, AND PARTICIPANTS This randomized, quadruple-blind, placebo-controlled

INTERVENTIONS A multispecies probiotic consisting of Bifidobacterium bifidum W23,

860 (Reprinted) jamapediatrics.com

© 2022 American Medical Association. All rights reserved.

© 2022 American Medical Association. All rights reserved.

© 2022 American Medical Association. All rights reserved.

Figure. CONSORT 2010 Flow Diagram

777 Individuals assessed for eligibility

176 Randomized to probiotic 174 Allocated to placebo

18 Lost to follow-up (including 19 Lost to follow-up

158 Intention-to-treat 155 Intention-to-treat analysis

© 2022 American Medical Association. All rights reserved.

Table 1. Characteristics of Participants

Table 2. Main Results of the Available Case Analysis

Events, No. (%)

© 2022 American Medical Association. All rights reserved.

© 2022 American Medical Association. All rights reserved.

© 2022 American Medical Association. All rights reserved.

The Influence of Probiotics on Pediatric

The Influence of Probiotics on Pediatric Antibiotic-associated Diarrhoea –

Dhr. prof. Dr. M.A. Benninga

Amsterdam UMC, location AMC

Onze Lieve Vrouwe Gasthuis, locatie Oost

Onze Lieve Vrouwe Gasthuis, location West

Sponsor (in Dutch: Amsterdam UMC, location VUmc,

1. LIST OF ABBREVIATIONS AND RELEVANT DEFENITIONS .............................................. 7

Version number: 3. Studyprotocol for IPAD study

12.2.1 Adverse events (AEs).......................................................................................32

Version number: 3. Studyprotocol for IPAD study

1. LIST OF ABBREVIATIONS AND RELEVANT DEFENITIONS

AAD Antibiotic Associated Diarrhoea

Version number: 3. Studyprotocol for IPAD study

Version number: 3. Studyprotocol for IPAD study

Version number: 3. Studyprotocol for IPAD study

Version number: 3. Studyprotocol for IPAD study

4. INTRODUCTION AND RATIONALE

Probiotics are ‘live micro-organisms that, when administered in adequate amounts,

(moderate quality of evidence (QoE), strong recommendation) and S. boulardii

7.2 Inclusion criteria

Version number: 3. Studyprotocol for IPAD study

‚ Age between 3 months and 18 years old

7.3 Exclusion criteria

7.4 Sample size calculation

Version number: 3. Studyprotocol for IPAD study

8.1 Investigational product

8.2 Use of co-intervention (if applicable)

8.3 Escape medication (if applicable)

Version number: 3. Studyprotocol for IPAD study

8.4 Intolerance to investigational product

The formulation consists of 8 specifically selected probiotic strains: Bifidobacterium

Version number: 3. Studyprotocol for IPAD study

9.2 Summary of findings from non-clinical studies

9.3 Summary of findings from clinical studies

Version number: 3. Studyprotocol for IPAD study

Version number: 3. Studyprotocol for IPAD study