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Organization Regional Center for Food and Nutrition), and WHAT THIS STUDY ADDS: Lactobacillus reuteri DSM17938 may
cFaculty of Medicine, Division of Pediatric Gastroenterology,
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ARTICLE
TABLE 1 Composition of LC and RC Milk and Probiotic Straws have ended on the last day of diarrhea
Composition LC RC Casei Reuteri followed by 2 diarrhea-free days.27
UHT milk (per 100 mL) Duration of diarrhea was dened as
Energy, kcal 93.8 98.0 98.0 98.0 number of days from rst until last
Fat, ga 3.5 3.9 3.9 3.9
excretion of the loose or liquid stool
Protein, ga 3.9 3.8 3.8 3.8
Total carbohydrate, ga 11.7 12.0 12.0 12.0 that was not followed by another ab-
Lactose, g 0.07 0.09 0.09 0.09 normal stool in each episode.26,28
Vitamin A, g 32 30 30 30
Calcium, mga 15 129 129 129 The presence of an ARTI was dened as
Phosphor, mg 32 77 77 77 when a child had $1 respiratory tract
Magnesium, mg 6 6 6 6 symptom(s) (runny nose, cough, or
Iron, mg 0.30 0.30 0.30 0.30
Zinc, mg 0.14 0.14 0.14 0.14
sore throat) and/or $1 additional re-
Straw probiotic, CFU/day spiratory tract symptom(s) or 1 con-
L casei CRL431 5 3 108 stitutional symptom (fever, headache,
L reuteri DSM17938 5 3 108
restless, aphony, shortness of breath,
UHT, ultra-high temperature.
a Based on chemical analyses. acute ear pain, or discharge).29,30 These
symptoms were conrmed with a
physicians diagnosis of acute-upper
Mothers were instructed to provide the nonooding), age (,57 and $57 (rhinitis, pharyngitis, sinusitis, otitis,
children with 180 mL of milk twice daily months), and gender. A randomization and common cold) and lower (pneu-
(not with a meal) by using the straws table with treatment codes and a block monia, bronchitis, and bronchiolitis)
provided. Mothers were requested to size of 8 was generated by using SAS respiratory tract infection.31 ARTI du-
maintain the childs habitual diet but to version 9.1 (SAS Institute, Inc, Cary, NC) ration was the number of consecutive
exclude probiotic, prebiotic, or high- by an independent individual at Wage- days with $2 dened signs and
calcium foods/drinks other than the ningen University. Twin siblings of sub- symptoms, with a 7-day symptom-free
supplied ones. The amount of milk jects (n = 3) were allocated to the same interval before a new episode could
consumed was measured by using a treatment group. Researchers, moth- occur.32
calibrated stick put into the tetra paks ers, children, and laboratory personnel
to score the remaining volume by using were unaware of the treatment until all
a pretested 5-point scale. The eld biochemical and data analyses were Data Collection
workers observed the children drink- nished and until after the blind review Field workers collected fecal samples
ing milk at least once a week, and meeting. The data safety monitoring before and at the end of the inter-
empty packages had to be shown dur- board (DSMB) and an independent vention, as well as during diarrheal
ing visits. During diarrheal episodes, person at SEAMEO RECFON kept 3 sets episodes. Diarrheal samples were col-
children continued or restarted drink- of sealed envelopes allowing deblinding lected from onset of diarrhea until
ing milk as soon as possible but after per subject without disclosing other maximally 3 days later. Stools contam-
being rehydrated with oral rehydration childrens treatments. inated with urine or that had fallen into
solution according to World Health Or- the toilet or the childs underwear were
ganization (WHO) guidelines.23 We fol- Outcomes discarded. Collected stools were kept
lowed the local standard for outpatient The primary outcomes were the num- cool (20C) at the eld workers house
and hospital care for diarrhea and ARTI, ber and duration of diarrheal episodes. until storage in a freezer (70C) at the
which were per WHO guidelines.2325 The main secondary outcomes were the laboratory. Stools were freeze-dried
Liability insurance was provided for number and duration of ARTI episodes. and analyzed for calcium8 and rotavi-
the children during the study. Activities Diarrhea was identied according to rus (diarrheal samples).33
with creative and educational contents the WHO denition ($3 loose/liquid Before and at intervention end, non-
were implemented to maintain com- stools in 24 hours).23 In addition, all fasting venous blood was drawn in the
pliance of both mothers and children. reported diarrhea (broader denition: morning by trained phlebotomists. A
$2 loose/liquid stools in 24 hours) was study physician examined the health
Randomization and Blinding evaluated. Stool frequency was coun- status of the children, and eld workers
Eligible children were admitted to the ted when there was at least a 1-hour performed anthropometric measure-
study on enrollment basis and stratied interval since the previous defeca- ments. Lightly clothed children were
according to area of living (ooding and tion.26 An episode was considered to weighed without shoes by using an
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ARTICLE
DISCUSSION
Neither calcium nor L casei CRL431
affected any of the diarrheal outcomes.
In contrast, L reuteri DSM17938 sup-
plementation signicantly reduced the
incidence of all reported diarrhea
(32% in $2 loose/liquid stools in 24
hours) and nonsignicantly reduced
the incidence of WHO-dened diarrhea
(24% in $3 loose/liquid stools in 24
hours). Notably, for both diarrhea out-
FIGURE 1 comes, the protective effect of L reuteri
Flow diagram of study subjects. ITT, intention-to-treat; TB, tuberculosis.
DSM17938 was signicant in children
with lower nutritional status (below-
children with below-median weight-for- The percentages of diarrheal samples median height-and-weight-for-age z
age z score (RR for WHO-dened di- positive for rotavirus according to study score). None of the interventions af-
arrhea compared with RC group: 0.44 group were as follows: LC, 28%; RC, 25%; fected incidence or duration of ARTIs.
[95% CI: 0.210.92]; RR for all reported casei, 28%; and reuteri, 19%. Differ- No serious adverse events related to
diarrhea: 0.54 [95% CI: 0.310.94]) and ences were not signicant. the interventions were reported.
in children with below-median height- The incidence, number of episodes, and We applied the WHO denition of di-
for-age z score (RR for WHO-dened duration of ARTIs were not signicantly arrhea to collect data on the primary
diarrhea: 0.44 [95% CI: 0.210.90]; RR different among treatments (Fig 4, Table 3). outcome. Because the WHO considers
for all reported diarrhea: 0.53 [95% CI: Reported adverse events (International fecal consistency more important than
0.300.92]). In children above the me- Classication of Diseases, 10th Revi- the number of stools23,43 and their
dian z scores, the results for the reu- sion codes) were comparable among denition leaves room for registration
teri group were not signicantly groups, except for change in bowel of any increase in normal stool fre-
different from the RC group. The prev- habits (less regular defecation) and quency, we also evaluated the outcome
alence of underweight and stunting asthma. Nine children in the reuteri of all reported diarrhea (a broader
was not signicantly changed by the group experienced a change in bowel denition of diarrhea). Although the
interventions (data not shown). habits, compared with 2 in the RC group. WHO denition is the best validated, it
may not be generalizable to different Previous evidence on the preventive ef- The rationale for using calcium in chil-
settings such as our intervention, fect of probiotics on diarrhea and ARTIs dren is based on a proof-of-principle
which included children of older age has been limited to small studies, mainly study with adults orally challenged
and in an urban community setting.44 hospital or day care center based, with with live but attenuated enterotoxi-
Moreover, mothers in the study area a short follow-up period and performed genic Escherichia coli. Dietary calcium
usually reported diarrhea when their in developed countries.1113,40,48 There- strongly reduced infection-induced di-
child defecated $2 loose/liquid stools, fore, our study in a low socioeconomic arrhea in that study.8 Animal studies show
and broader diarrhea denitions were community of a developing country, with protective effects against Salmonella as
applied by other clinical trials.4547 a much higher number of subjects and well,49,50 but human verication for that
We did not only rely on mothers per- longer follow-up, provides critical nding is still lacking. Rotavirus is re-
ception but implemented an active data to help establish the relevance sponsible for 60% of hospitalized and 41%
surveillance program to verify mothers of these interventions for the pre- of outpatient clinic diarrheal cases in In-
daily records with twice-a-week visits of vention of diarrhea in developing donesian children.51 Important bacterial
trained eld workers and twice-a-month countries. To our knowledge, our pathogens among children in developing
visits of eld supervisors. The physician study is the rst large randomized countries are E coli (10%20%), Salmo-
and monitoring expert accompanied controlled trial, focusing on the effect nella (,5%), Shigella (5%10%), Cam-
the eld workers on several of their of calcium with or without 1 of 2 spe- pylobacter, and Vibrio cholerae (exact %s
home visits. All end points were as- cic probiotics to reduce diarrhea and unknown).52 The absence of a benecial
sessed by using structured and pre- respiratory tract infections in these effect of calcium in our trial may indicate
tested forms as applied by others.26,29,37, settings. Our results indicate that the a difference in efcacy between children
38 The forms were adapted to the local effect of a probiotic, such as L reuteri and adults and/or that protective effects
situation and were used by eld work- DSM17938, on diarrhea is modied by are pathogen dependent.
ers who were rigorously trained and nutritional status and is conned to The application of probiotics to prevent
supervised on their application. children with lower nutritional status. or treat acute diarrhea is based on the
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ARTICLE
TABLE 3 Effects of Probiotics and Calcium on Incidence of Diarrhea and ARTIs Among Indonesian Children
Outcome Measures LC RC Casei Reuteri
(n = 124) (n = 126) (n = 120) (n = 124)
WHO-dened diarrhea episodes ($3 loose/liquid stools in 24 h)
Mean incidence/child per year 0.91 0.86 1.05 0.67
No. of episodes, mean 6 SD 0.40 6 0.81 0.38 6 0.78 0.47 6 0.87 0.30 6 0.56
Adjusted RR (95% CI)a 1.00 (ref) 0.99 (0.621.58)
1.00 (ref) 1.21 (0.761.92) 0.76 (0.461.25)
Duration of episodes, mean 6 SD, d 3.06 6 4.43 2.94 6 3.25 2.37 6 2.68 2.68 6 3.05
All diarrhea episodes (2 and $3 loose/liquid stools in 24 h)
Mean incidence/child/y 1.73 1.86 2.04 1.28
Number of episodes, mean 6 SD 0.73 6 1.14 0.77 6 1.38 0.87 6 1.32 0.56 6 0.77
Adjusted RR (95% CI)a 1.00 (ref) 1.10 (0.771.59)
1.00 (ref) 1.06 (0.741.53) 0.68 (0.460.99)
Duration of episodes, mean 6 SD, d 2.57 6 4.09 2.03 6 2.84 2.08 6 2.40 1.91 6 2.52
ARTIs episodes
Mean incidence/child/y 7.22 7.52 7.07 7.45
No. of episodes, mean 6 SD 2.41 6 1.59 2.43 6 1.61 2.36 6 1.62 2.48 6 1.56
Adjusted RR (95% CI)b 1.00 (ref) 1.00 (0.861.18)
1.00 (ref) 0.97 (0.821.14) 0.99 (0.841.16)
Duration of episodes, mean 6 SD, d 4.87 6 4.05 4.90 6 3.70 4.96 6 3.71 4.58 6 3.43
ref, reference group of comparison.
a Negative binomial model, adjusted for area of living, gender, age, diarrhea and ARTI 2 weeks before the study, household expenditure, and weight-for-height z score.
b Poisson model, adjusted for area of living, gender, age, diarrhea and ARTI 2 weeks before the study, household expenditure, and weight-for-height z score.
CONCLUSIONS
Supplementation of L reuteri, at least on
a diet including regular calcium milk, is 1
of the potential interventions to reduce
the burden of acute infectious diarrhea in
children. These results need to be con-
rmed by at least 1 other independent
study in a comparable community.
ACKNOWLEDGMENTS
We thank Dr Christien van Beusekom, Mr
Peter Spiekstra, Mr Jan van der Leij, and
Ms Vicky Valentina (FrieslandCampina
Research) for their contribution to
FIGURE 4 study milk production, and Martin Jkel,
Adjusted Cox survival curve of ARTI episodes. Adjusted for area of living, gender, age, diarrhea and ARTIs 2
weeks before the study, household expenditure, and weight-for-height z score. Probability of survival MD (Unilever Research and Develop-
without acute respiratory infections in relation to duration of episodes (days) for 4 groups. No sig- ment) for his advice on adverse-event
nicant differences between interventions were observed. analysis. We thank the highly dedicated
and motivated children, parents, physi-
cians, and research team members, es-
the incidence of diarrhea in children,60 CRL431 (without other strains) was pecially Ms Ratna Wulanti, Ms Imas
eliminated diarrhea due to post- without effect. The dosage of our pro- Maliha, Ms Siti Mulyani, Santi Sinarwati,
gastroenteritis syndrome of mal- biotic strains (5 3 108 CFU/day) is within MD, and Ms Devy Davelyna. We acknowl-
nourished hospitalized children,61 and the effective dosage recommended by edge the support of the head, elders,
signicantly reduced the number of daily the Food and Agriculture Organization of women leaders, and volunteers in
stools, diarrheal duration, and vomiting the United Nations/WHO.62 Kampung Melayu and Rawabunga.
of children with persistent diarrhea.22 The major strength of this study was its We also thank Prof Purwantyastuti,
Our results underline that probiotic ef- focus on prevention in contrast to most Prof Arini Setiati, Sri Lestari, MD, Dr
fects are strain specic, as we found previous studies, which aimed at Moesijanti Soekarti, Ms Yulianti Wibowo,
protective effects of L reuteri DSM17938 treatment of institutionalized children. Mr Jeroen Sterken, Iwan Setiawan, MD,
against acute diarrhea in children, Additional strengths were its double- Dr Miren Iturriza-Gmara, and Mr Ahmad
whereas supplementation of L casei blind design, the strict adherence to Sadariskar.
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ARTICLE
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Randomized Trial of Probiotics and Calcium on Diarrhea and Respiratory Tract
Infections in Indonesian Children
Rina Agustina, Frans J. Kok, Ondine van de Rest, Umi Fahmida, Agus Firmansyah,
Widjaja Lukito, Edith J. M. Feskens, Ellen G. H. M. van den Heuvel, Ruud Albers
and Ingeborg M. J. Bovee-Oudenhoven
Pediatrics 2012;129;e1155; originally published online April 9, 2012;
DOI: 10.1542/peds.2011-1379
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