ORIGINAL ARTICLE: GASTROENTEROLOGY

Effect of Probiotics on Diarrhea in Children With Severe

Acute Malnutrition: A Randomized Controlled
Study in Uganda

Benedikte Grenov, yHanifa Namusoke, yBetty Lanyero, yNicolette Nabukeera-Barungi,

Christian Ritz, Christian Mlgaard, Henrik Friis, and Kim F. Michaelsen

ABSTRACT

Objectives: The aim of the study was to assess the effect of probiotics on
diarrhea during in- and outpatient treatment of children with severe acute
malnutrition (SAM).
Methods: A randomized, double-blind, placebo-controlled study was
conducted involving 400 children admitted with SAM. Patients received 1
daily dose of a blend of Bifidobacterium animalis subsp lactis and
Lactobacillus rhamnosus (10 billion colony-forming units, 50:50) or
placebo during hospitalization followed by an 8- to 12-week outpatient
treatment period, depending on patients recovery rate. All outcomes were
reported for in- and outpatient treatment separately. The primary outcome was
number of days with diarrhea during hospitalization. Secondary outcomes
included other diarrhea outcomes, pneumonia, weight gain, and recovery.
Results: There was no difference in number of days with diarrhea between
the probiotic (n 200) and placebo (n 200) groups during inpatient
treatment (adjusted difference 0.2 days, 95% confidence interval 0.8
to 1.2, P 0.69); however, during outpatient treatment, probiotics reduced
days with diarrhea (adjusted difference 2.2 days 95% confidence interval
3.5 to 0.3, P 0.025). There were no effects of probiotics on diarrhea
incidence and severity or pneumonia, weight gain or recovery during in- or
outpatient treatment. Twenty-six patients died in the probiotic versus 20 in
the placebo group (P 0.38).
Conclusions: Bifidobacterium animalis subsp lactis and Lactobacillus
rhamnosus had no effect on diarrhea in children with SAM during
hospitalization, but reduced the number of days with diarrhea in
outpatient treatment by 26%. Probiotics may have a role in follow-up of
hospitalized children with SAM or in community-based treatment
of malnourished children, but further studies are needed to confirm this.
Key Words: diarrhea, low-income country, probiotic, severe acute
malnutrition, young children
(JPGN 2017;64: 396403)
What Is Known
 Severe acute malnutrition is responsible for 0.5 to 1
million child deaths annually and diarrhea is a com-
mon morbidity associated with mortality.
 Probiotics reduce the duration of acute diarrhea by 1
day in well-nourished or mildly malnourished chil-
dren and reduce the risk of antibiotic-associated
diarrhea.
What Is New
 The probiotics Lactobacillus rhamnosus and Bifido-
bacterium animalis subsp lactis had no effect on
diarrhea during inpatient treatment of children with
severe acute malnutrition, but reduced days with
diarrhea during outpatient treatment by 26%.
 The study results do not support using probiotics for
inpatient treatment of children with severe acute
malnutrition, but may have a role in outpatient
treatment.
susceptible and case fatality rates in many sub-Saharan hospitals are
often above 20% (3). Diarrhea is a major complication to SAM
associated with increased morbidity, longer hospitalization, and
death (4,5).
Meta-analyses have shown that probiotics reduce the
duration of acute infectious diarrhea by approximately 1 day and
reduce the risk of acute diarrhea lasting 4 days or more (6). The

S evere acute malnutrition (SAM) is a major challenge in low-

income countries and results in 0.5 to 1 million child deaths
annually (1,2). Hospitalized children with SAM are particularly
dosage and the strains used were, however, different. Investigation
of strain-specific effects have led to recommendation of 2 strains,
Lactobacillus rhamnosus (LGG) and Saccharomyces boulardii, in

Received August 29, 2016; accepted December 27, 2016.
From the Department of Nutrition, Exercise, and Sports, University of
Copenhagen, Denmark, and the yMwanamugimu Nutrition Unit, Depart-
ment of Pediatrics and Child Health, Mulago National Referral Hospital
Kampala, Uganda.
Address correspondence and reprint requests to Prof Kim F. Michaelsen, Dr
Med Sci, Department of Nutrition, Exercise, and Sports, Faculty of
Science, University of Copenhagen, Rolighedsvej 26, 1958 Frederiksberg
C, Denmark (e-mail: KFM@nexs.ku.dk).
Supplemental digital content is available for this article. Direct URL citations
appear in the printed text, and links to the digital files are provided in the
HTML text of this article on the journals Web site (www.jpgn.org).
www.isrctn.com registration number: ISRCTN16454889.
Chr. Hansen A/S, University of Copenhagen and Innovation Fund Denmark
funded the study. Chr. Hansen A/S suggested the intervention
(combination of probiotic strains and dosage) but did not have any
role in study design, data collection, data analysis, data interpretation, or
writing of the report. The corresponding author had full access to all the
data in the study and had final responsibility for the decision to submit
for publication. B.G. was employed by Chr. Hansen A/S as an industrial
PhD student with parallel enrolment at University of Copenhagen. Half
of her salary was sponsored by Innovation Fund Denmark. K.F.M., H.F.,
and C.M. received a grant from Chr. Hansen and Innovation Fund
Denmark for the current study, and K.F.M. and C.M. received a grant

396 JPGN  Volume 64, Number 3, March 2017

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JPGN  Volume 64, Number 3, March 2017
the treatment of acute gastroenteritis in children (7). Both strains
seem to reduce duration of acute diarrhea by 1 day and the risk of
having diarrhea on day 3 or 4 by approximately 50% (7). Probiotics
may also reduce the risk of antibiotic-associated diarrhea and the
duration of persistent diarrhea (810).
Pneumonia is the most frequent infectious cause of mortality
in children below 5 years and the risk of dying if severely mal-
nourished is several folds higher (11). Studies on the effects of
probiotics on pneumonia are scarce; however, a number of studies
have indicated that probiotics may reduce the risk of upper respir-
atory infections (12). Furthermore, a few probiotic studies have
shown a small improvement in growth (13).
Some of the studies mentioned above included mildly to
moderately malnourished children from low-income countries, but
most studies were conducted in well-nourished children from high-
income countries. The large ProNUT study (an intervention study
testing probiotics and prebiotics supplied in PlumpyNut for chil-
dren with SAM) investigated the effects of a mixture of pro- and
prebiotics in Malawian children with SAM (14). There was no
effect on nutritional cure, or on death, weight gain, or time to cure,
but a trend toward lower mortality (relative risk [RR] 0.65,
P 0.06) among patients receiving pro- and prebiotics in the
outpatient period was observed.
Bifidobacterium animalis subsp lactis (BB-12) and LGG are
among the most widely consumed and studied probiotic strains.
Earlier studies have indicated that these strains reduce diarrhea and
upper respiratory infections in well-nourished children (1518) and
that LGG may reduce diarrhea in mildly to moderately under-
nourished children (1921). We aimed at assessing the effect of a
combination of BB-12 and LGG on diarrhea, pneumonia, and
growth in children admitted with SAM.
METHODS
Design and Ethics
The ProbiSAM study was a randomized, double-blind, placebo-
controlled, 2-arm parallel-group study in children with SAM.
The study was conducted at Mwanamugimu Nutrition Unit
(MNU), Department of Pediatrics and Child Health, Mulago
National Referral Hospital, Kampala, Uganda. The mortality rate
at the unit is approximately 20%.
The study was performed in accordance with the principles in
the Declaration of Helsinki. Ethical approval was obtained from the
Makerere University School of Medicine Research Ethics Committee
in Uganda and a consultative approval was given by The National
Committee of Health Research Ethics in Denmark. Oral and written
information about the study was provided and written; informed
consent was obtained from all caregivers before enrolment in the
study. An independent data safety monitoring board (DSMB) was
established to monitor patient safety throughout the study.
Participants
Children of age 6 to 59 months admitted with SAM
(mid-upper-arm-circumference <11.5 cm or weight-for-height/
from Chr. Hansen for another probiotic study with Danish infants. The
remaining authors report no conflicts of interest.
Copyright # 2017 The Author(s). Published by Wolters Kluwer Health, Inc.
on behalf of the European Society for Pediatric Gastroenterology,
Hepatology, and Nutrition and the North American Society for Pediatric
Gastroenterology, Hepatology, and Nutrition. This is an open access
www.jpgn.org
Copyright ESPGHAN and NASPGHAN. All rights reserved.
Effect of Probiotics on Diarrhea in Children With SAM
weight-for-Length z score (WHZ/WLZ) <3 or bipedal pitting
edema) were eligible. Patients with shock or severe
respiratory difficulty at admission, weight below 4.0 kg, obvious
disability or significant congenital or malignant disease
and patients admitted with SAM the previous 6 months
were excluded.
Randomization, Allocation Concealment,
and Blinding
The study products were labeled with a 4-digit number. There
were 4 different 4-digit numbers, 2 for placebo and 2 for probiotics.
Only the study supply coordinator at Chr. Hansen A/S had access to
the blinding code. The randomization list was generated by a person
not involved in the study using the web site Randomization.com
(http://www.randomization.com, accessed February 6, 2014).
Randomization to probiotic or placebo treatments was performed
in a 1:1:1:1 ratio in blocks of 4 and 8 in random order. The
randomization list was kept by the head of MNU and only made
available to staff responsible for pre-packaging of study products.
These staff members were not involved in patient enrolment or
treatment. Subject allocation was performed by assigning eligible
subjects to the first available randomization number in consecutive
order. All participants, caregivers, investigators, and staff involved
in the study were blinded until the database was locked. The
appearance, taste, and smell of the products were identical, except
for the 4-digit number.
Intervention and Procedures
Patients received 1 sachet of study product daily in addition
to standard treatment of SAM. The study product was administered
from hospital admission to discharge and throughout an outpatient
treatment period of minimum 8 weeks and maximum 12 weeks,
depending on the nutritional recovery of each child. Each sachet
contained 1 g of white powder: maltodextrin with or without a
combination of the 2 probiotic strains BB-12 and LGG (dosage 10
billion colony-forming units [CFU], 50:50). Study products were
manufactured by Chr. Hansen A/S, Hrsholm, Denmark.
During hospitalization, study personnel administered, regis-
tered, and supervised consumption of study products, which were
provided together with the morning food. Standard treatment of
SAM was given in compliance with World Health Organization
(WHO) recommendations (22) and the Ugandan national protocol
(23). Standard treatment involved a stabilization phase where
children received F-75 formula (Nutriset, Malaunay, France) fol-
lowed by a rehabilitation phase with step-wise transitioning to
ready-to-use-therapeutic-food (RUTF; PlumpyNut, Nutriset) or
F-100 formula (Nutriset) if RUTF was not tolerated well. A
commercial soy-based, lactose-free infant formula (Isomil, Abbott,
Chicago, IL) was used if patients were suspected to have lactose
intolerance based on acidic stool pH and profuse diarrhea with >10
stools per day. Breast-feeding was encouraged throughout the study.
Study staff measured weight daily and assessed children daily for
article distributed under the terms of the Creative Commons Attribution-
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DOI: 10.1097/MPG.0000000000001515
397
Grenov et al
vital signs, signs of pneumonia (respiratory rate, chest in-drawing,
respiratory auscultation, oxygen saturation), degree of edema and
dehydration, and other signs of illness or adverse events. Triple
measurements of weight and length/height were performed weekly
and a thorough physical examination and anthropometry was
performed at admission and discharge. Antibiotics were adminis-
tered as part of standard treatment for minimum 5 days. Ampicillin
and gentamycin were first-line antibiotics, and second- and third-
line antibiotics included chloramphenicol, ceftriaxone, cloxacillin,
and ciprofloxacin.
Diarrhea data were collected using a stool diary in which
caregivers ticked every time their child passed stool. Each stool was
categorized as watery, abnormally loose, loose, or normal according
to a photo scale. Caregivers were thoroughly trained in how to use
the diary and nutritionists supported and evaluated caregivers
ability to score stool consistency and fill out the stool diary
correctly. Vomiting, fever, and consumption of the study product
were also recorded in the diary. Development and validation
of the stool diary is described elsewhere (24). Stool frequency
and consistency scoring had a high validity, good reliability, and
high sensitivity.
During outpatient treatment, children received RUTF at
200 kcal/kg bodyweight per day. Follow-up visits were scheduled
every second week to assess the children with respect to anthro-
pometry, medical history, physical examination, follow-up on stool
diary data, and to deliver new supplies of RUTF, study products, and
stool diaries. The study staff did not attempt to fill the diary together
with the caregiver if data were missing. Caregivers were contacted
by phone once a week to ask about the status of their child and to
remind them about study procedures and visit dates. If caregivers
missed a scheduled follow-up visit, they were again contacted by
phone or a home visit was done to assess reasons for failure to return
for a follow-up visit. Study product compliance during outpatient
care was estimated based on ticks in stool diaries, returned empty or
unused sachets, and differences between visit dates and number of
sachets supplied.
Outcomes
The primary outcome was duration of diarrhea during hos-
pitalization. Duration was defined as number of days with diar-
rhea of each patient. Diarrhea was defined as 3 loose or watery
stools per 24 hours (25) based on stool diary data. A diarrhea
episode started when the diarrhea definition was fulfilled and was
considered to have ceased when the child passed <3 loose or watery
stools per day. If diarrhea re-appeared after <48 hours, it was
considered part of the same diarrhea episode, but only days with
3 loose or watery stools were counted as diarrhea days. The
protocol was amended during the clinical study and approved by the
ethical committee. In the original protocol, the primary outcome
was phrased as duration of diarrhea episodes without any
specification of in- or outpatient treatment. We considered the total
number of days with diarrhea of each child to be clinically more
important than the duration of each episode. In addition, we decided
to split analyses in in- and outpatient treatment as the populations
and data collection were different in the 2 periods with more
critically ill patients and closer monitoring of patients during
inpatient treatment.
Secondary outcomes were, first, number of days with
diarrhea during outpatient treatment and incidence and severity
of diarrhea during inpatient and outpatient treatment. Diarrhea
incidence was defined as the proportion of children with mini-
mum 1 day of diarrhea. Severity was defined as the Vesikari score
for inpatients and a modified Vesikari score for outpatients (26).
The Vesikari score is a multidomain diarrhea episode severity
398
Copyright ESPGHAN and NASPGHAN. All rights reserved.
JPGN  Volume 64, Number 3, March 2017
score that includes grading of stool frequency, diarrhea duration,
vomit frequency and duration, temperature, dehydration, and need
of hospitalization. Dehydration or temperature during outpatient
treatment was not assessed. To be conservative, children were
considered not to be dehydrated and if the caregiver ticked
fever in the stool diary, it was considered to be the lowest
fever score on the Vesikari scale. The Vesikari scale categorizes
diarrhea episodes according to the following ranges: 7 mild, 7 to
10 moderate, and 11 severe. Second, pneumonia incidence,
duration and severity for inpatients, and pneumonia incidence
for outpatients. Pneumonia was diagnosed according to clinical
assessment by a pediatrician. Severity of pneumonia was cate-
gorized as mild-moderate or severe. Duration and severity
could not be assessed during outpatient treatment as children
were only observed at follow-up visits every second week. Third,
weight gain (g/kg bodyweight per day) for in- and outpatients,
respectively, and recovery defined as WHZ/WLZ >2 at study
termination. Fourth, other outcomes included days with fever or
vomiting during in- and outpatient treatment and duration
of hospitalization.
Safety outcomes included mortality and other adverse events.
Due to the high background morbidity and mortality in the study
population, only medical conditions that were judged by a study
pediatrician to be uncommon in this population were recorded as
other adverse events.
Statistical Analysis
Sample Size Calculation
To have 80% power at 5% significance level to detect a 0.3
SD reduction in number of days with diarrhea, 178 children were
needed per study arm. To account for loss to follow-up, 200 children
were recruited per arm. Assuming that the SD of days with diarrhea
at MNU was 3 days, it would be possible to detect a 1-day reduction
in days with diarrhea, which is similar to what is found in meta-
analyses (6).
Statistical Analyses
Data were double-entered in EpiData v.3.1 (EpiData,
Odense, Denmark) and analyzed using statistical software R version
3.1.1 (2014-07-10) (27).
The primary outcome and the secondary outcomes of diar-
rhea severity, weight gain, hospitalization, fever, and vomiting were
analyzed using linear-mixed models with subject-specific random
effects. Remaining secondary outcomes were analyzed either using
logistic regression (mixed-effects) models (incidence of diarrhea
and pneumonia, fever, recovery) or log-linear Poisson models with
adjustment for overdispersion (diarrhea days in outpatients, pneu-
monia duration, and severity inpatients). All models were adjusted
for age, sex, human immunodeficiency virus (HIV) status, baseline
edema, and WHZ/WLZ. Mortality was analyzed using a Cox-
regression adjusted for sex and age only as HIV data were missing
for a number of patients that died. In addition, analyses of inpatient
data included adjustment for duration of hospitalization and base-
line diarrhea or pneumonia. All analyses of diarrhea inpatient data
were repeated with additional adjustment for Isomil treatment and
reduction in days with diarrhea during outpatient treatment was
repeated with adjusted for duration of outpatient treatment. Effect
modification was also investigated for age, sex, HIV status, duration
of hospitalization and baseline edema, WHZ/WLZ, and diarrhea.
Model checking was based on residuals and predicted random
effects, which were evaluated visually using (cumulated) residual
plots and normal probability plots.
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JPGN  Volume 64, Number 3, March 2017
Intention-to-treat analysis was performed on all patients with
any available data related to the specific outcome, assuming that
drop-outs occurred at random. Intermittent missing values in inpa-
tient diarrhea data were imputed to obtain complete episodes, which
were needed for the primary outcome and for calculation of the
Vesikari score. Specifically, deterministic hot-deck imputation was
used: gaps in stool diaries were imputed using data from multiple
matched patients with complete data for the same days of hospital-
ization as the gap occurred and an identical diarrhea pattern before/
after the gap as the patient having the gap (28). For the primary
outcome, a per-protocol analysis was also carried out and a subgroup
analysis was performed to investigate if the subgroup of children who
were discharged and analyzed in the outpatient phase showed a
different result in the primary outcome than the total population.
At predefined, regular intervals, the DSMB monitored
safety with specific focus on mortality by evaluating unblinded
individual case fatality reports, serious adverse events, and the
reasons of patients lost to follow-up. The study was registered at
www.isrctn.com as ISRCTN16454889.
RESULTS
Of 757 children screened, 400 children were randomized to
receive probiotics (n 200) or placebo (n 200) (Fig. 1). Patients
were recruited between March 10, 2014 and July 8, 2015 and
followed until October 2015.
Baseline characteristics were comparable between the 2
study groups (Table 1). Mean age was 17.0 months, 58% were
757 screened
400 randomized
200 probiotics
37 lost to follow-up
23 deceased
13 self-discharged
1 other
187 included in
inpatient ITT-analysis
163 discharged
20 lost to follow-up
3 deceased
17 self-discharged
147 included in
outpatient ITT-analysis
143 completed
FIGURE 1. Trial profile. Dotted line: patients that were lost to follow-up were included in the intention-to-treat analysis on an available case basis.
The main reasons for patients not being included in the analyses were lack of data on HIV status or no stool diaries available (mainly during
outpatient treatment). ITT intention-to-treat.
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Effect of Probiotics on Diarrhea in Children With SAM
boys, 66% had edematous malnutrition, 14% were HIV seroposi-
tive, and 34% had an HIV-positive mother. The total loss to follow-
up, including deceased patients, was 18% (n 73/400) and 12%
(n 38/327) during in- and outpatient treatment, respectively. The
numbers were equally distributed between the 2 study groups. No
patients were discharged to outpatient treatment with edema, but
44% still had SAM with no medical complications (see Supple-
mental Digital Content 1, Table, http://links.lww.com/MPG/A882).
The mean duration of hospitalization was 181  9.2 days and most
patients (>80%) were discharged from outpatient treatment after 8
weeks. The number of patients included in intention-to-treat diar-
rhea analyses during in- and outpatient treatment was n 369
(probiotics n 187, placebo n 182) and n 289 (probiotics
n 147, placebo n 145), respectively. The main reasons for
patients not being included in the statistical models were lack of
data on HIV status or lack of stool diaries in the outpatient period.
Compliance to study product consumption was 98% for both
groups during inpatient treatment based on study staff registrations.
During the outpatient phase, compliance was estimated at 93% and
96% for probiotics and placebo, respectively, based on comparison
of the number of study products delivered and the number of days
between follow-up visits.
There was no difference in the primary outcome between the
probiotic and placebo groups with an adjusted difference of mean
number of days with diarrhea of 0.2 days (95% confidence
interval [CI] 0.8 to 1.2, P 0.69) (Table 2). During outpatient
treatment, number of days with diarrhea was lower in the probiotic
compared with the placebo group with an adjusted difference of
357 excluded
307 ineligible
17 declined
33 other
200 placebo
36 lost to follow-up
16 deceased
16 self-discharged
4 other
182 included in
inpatient ITT-analysis
164 discharged
17 lost to follow-up
4 deceased
13 self-discharged
145 included in
outpatient ITT-analysis
145 completed
399
Grenov et al JPGN  Volume 64, Number 3, March 2017

TABLE 1. Baseline characteristics subpopulation of inpatients that were included in the outpatient
analysis showed an adjusted difference of 0.1 days (95% CI 1.1
Probiotics Placebo to 1.2 days, P 0.91). There was no effect on modification of the
(n 200) (n 200) primary outcome by any of the covariates (data not shown). Days
with diarrhea during outpatient treatment resulted in similar results
Age (mo  SD) 17.5  85 16.5  84 after adjustment for duration of outpatient treatment (data not
Sex (male, %) 115 (58%) 115 (58%) shown).
Weight (kg  SD) 7.0  1.7 6.9  1.7
Height or length or height 71.1  6.6 71.0  6.1
(cm  SD) DISCUSSION
MUAC (cm  SD) 11.6  1.4 11.5  1.5 Probiotics did not reduce number of days with diarrhea
Height/length-for-age z score 3.2  1.4 3.0  1.4 during hospitalization, whereas days with diarrhea were reduced
Weigth-for-age z score 3.5  1.3 3.5  1.3 by 2.2 days corresponding to 26% of the mean number of days with
Weight-for-height/length 2.6  1.5 2.7  1.6 diarrhea in the outpatient phase. The different results may be
z score explained by more severe illness and a compromised gut barrier
Edema (n, %) 132 (66%) 132 (66%) in children during hospitalization. Children admitted to MNU were
HIV-positive children 28 (15%) 24 (13%) often ill with multiple life-threatening conditions and it may have
(serology) (n, %) affected their ability to respond to probiotic treatment. More
HIV-positive mothers (n, %) 56 (34%) 53 (34%) specifically, their gut function may have been impaired resulting
Presenting symptoms at admission n (%) in poor adhesion of the probiotics to the mucosa.
Fever 101 (50%) 110 (55%) Antibiotic use was also different during in- and outpatient
Cough 131 (66%) 131 (66%) treatment. Broad-spectrum antibiotics were administered intrave-
Diarrhea 118 (59%) 126 (63%) nously as part of standard treatment during hospitalization whereas
Vomiting 84 (42%) 87 (44%) oral antibiotics were used only when children developed respiratory
Medical treatment 182 (91%) 184 (93%) or other infections during outpatient treatment. Both oral and
Household n (%) intravenously administered antibiotics are known to cause diarrhea
Child lives with mother 160 (80%) 157 (79%) in some patients probably by disturbing the gut microbiota and
Mother education primary 94 (47%) 117 (59%) reducing the colonization resistance to pathogens. The 2 probiotic
school or lower strains were sensitive to most of the used antibiotics and the
antibiotics may therefore have influenced the effectiveness of the
HIV human immunodeficiency virus; MUAC mid-upper-arm-
circumference; SD standard deviation.
probiotics. On the contrary, probiotic studies including LGG have
been shown to reduce the risk of antibiotic-associated diarrhea after
administration of broad-spectrum antibiotics (29).
2.2 days (95% CI 3.5 to 0.3, P 0.025). Supplemental Finally, diarrhea was more severe with higher stool frequency,
Digital Content 2, Figure, http://links.lww.com/MPG/A883, shows more dehydration, fever, and vomiting during inpatient compared to
the distribution of diarrhea days in the probiotic versus the placebo outpatient treatment. Meta-analyses of probiotics in general (6) or
group during outpatient care. As seen, the proportion of patients LGG alone (30) have indicated that the effect on acute diarrhea is
with diarrhea for 20 or more days was reduced in the probiotic higher in community-based than inpatient studies. The results, how-
group. ever, vary and a study with LGG in hospitalized children with acute
The incidence of diarrhea was 89% versus 85% in the diarrhea showed effect in patients with profuse diarrhea (19).
probiotic versus placebo group during hospitalization, odds ratio The ProNUT study investigated a combination of pro- and
(OR) 1.6 (95% CI 0.8 to 3.3, P 0.17) and 70% versus 76% in the prebiotics in children with SAM and also observed differences
outpatient period, OR 0.7 (95% CI 0.4 to 1.2, P 0.17). The during in- and outpatient periods (14). They found more vomiting,
severity of diarrhea episodes measured by the Vesikari score severe diarrhea (6 stools per day) and cough, and a small,
was comparable between the study groups during in- and outpatient nonsignificant increase in mortality in inpatients, whereas they
treatment. Episodes observed during inpatient treatment were more reported a trend toward reduced mortality and fewer cases of severe
severe (592 episodes, mean score 10.0 (520)) compared with diarrhea in outpatients receiving pro- and prebiotics. The increase in
outpatient episodes (752 episodes, mean score 4.3 (313)). severe diarrhea among inpatients was suggested to be related to the
Pneumonia incidence, duration, and severity were not differ- intake of prebiotics.
ent between the study groups during inpatient treatment. The Based on our results, it is not possible to conclude if the effect
incidence during outpatient treatment was 5% (n 8) in the pro- in outpatients is depending on probiotic administration initiated
biotic group and 10% (n 16) in the placebo group, but the before discharge. However, studies on treatment or prevention of
difference was insignificant (OR 0.5, 95% CI 0.2 to 1.3, diarrhea either started probiotic treatment together with or maxi-
P 0.17). Nutritional recovery, total weight gain (g/kg bodyweight mum 2 days before exposure to the cause of diarrhea (15,16,31,32).
per day), fever, vomiting, and duration of hospitalization did not Meta-analyses of probiotic studies with LGG on treatment of
differ significantly between the groups. acute diarrhea have shown a reduction of diarrhea duration by 1 day
Forty-six patients died during the course of the study; 39 (23 (30) and a study on persistent diarrhea reduced duration by 4 days
probiotics, 16 placebo) during hospitalization and 7 (3 probiotics, 4 (9). The meta-analyses on acute diarrhea found that LGG seems to
placebo) during outpatient treatment. There was no difference have slightly higher effect in studies with a dose of 10 billion CFU/
between the probiotic and placebo groups during the entire study day compared with studies with lower doses and there was a
(hazard ratio [HR] 1.3, 95% CI 0.7 to 2.3, P 0.38). Other tendency toward lower effect in non-European countries compared
adverse events were not reported. with European countries (30). Some older studies with BB-12 found
The per-protocol analysis (probiotics n 176, placebo an effect on diarrhea (33,34), but 2 new larger studies in hospital-
n 169) resulted in an adjusted difference of the primary outcome ized and community-based children showed no effect on diarrhea
of 0.2 days (95% CI 0.8 to 1.2, P 0.68) and analysis of the (35,36).

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JPGN  Volume 64, Number 3, March 2017 Effect of Probiotics on Diarrhea in Children With SAM

TABLE 2. Results of probiotic treatment on diarrhea, pneumonia and nutritional recovery during in- and outpatient treatment

Outcome Probiotics (N 200) Placebo (N 200) Adjusted effect size (95% CI) probiotics vs placebo

Diarrhea

Duration, days
Inpatient 6.9  6.0 6.5  6.4 0.2 (0.8 to 1.2), P 0.69
Outpatient 6.0  8.2 8.5  10.9 2.2 (3.5 to 0.3), P 0.025
Incidencey n (%)
Inpatient 177 (89%) 169 (85%) OR 1.6 (0.8 to 3.3), P 0.17
Outpatient 107 (70%) 116 (76%) OR 0.7 (0.4 to 1.2), P 0.17
Severity (Vesikari score 020)z
Inpatient episodes 299 (51%) 293 (49%)
Inpatient scores 10.2  3.8 9.9  3.7 0.1 (0.6 to 0.8), P 0.74
Outpatient episodes 354 (47%) 398 (53%)
Outpatient scores 4.3  1.7 4.4  1.7 0.2 (0.5 to 0.1), P 0.16
Pneumonia
Duration, days
Inpatient 1.9  3.6 2.0  3.7 0.2 (0.7 to 0.4), P 0.51
Outpatient NA NA NA
Incidence
Inpatient 77 (39%) 80 (41%) OR 0.9 (0.6 to 1.5), P 0.80
Outpatient 8 (5%) 15 (10%) OR 0.5 (0.2 to 1.3), P 0.17
Severity (score)
Inpatient 2.6  5.5 2.7  5.3 0.3 (1.0 to 0.7), P 0.50
Outpatient NA NA NA
Nutritional recovery
Weight gain (g/kg bodyweight per day)jj
Inpatient 6.5  4.7 6.1  4.2 0.3 (0.6 to 1.3), P 0.49
Outpatient 3.0  2.1 3.2  2.3 0.1 (0.6 to 0.4), P 0.80
Total 4.1  2.2 4.2  2.2 0.02 (0.5 to 0.5), P 0.94
Recovered n (%) 133 (66%) 128 (64%) OR 1.2 (0.6 to 2.4), P 0.68
Other
Fever, days
Inpatient 7.0  5.1 6.7  4.4 0.3 (0.3 to 1.0), P 0.29
Outpatient 1.6  2.9 2.0  3.9 0.5 (1.3 to 0.2), P 0.16
Vomit, days
Inpatient 2.1  3.3 2.0  4.4 0.002 (0.7 to 0.7), P > 099
Outpatient 1.2  2.3 1.6  6.0 0.3 (1.4 to 0.7), P 0.53
Hospitalization, days
Inpatient 18.3  9.1 18.0  9.3 0.1 (1.7 to 1.9), P 0.93

Data are reported as mean  SD or n (%) unless otherwise indicated.

CI confidence interval; OR odds ratio.

Total number of days with diarrhea. Number of subjects analyzed: Inpatients: probiotics n 187, placebo n 182. Outpatients: probiotic n 147, placebo
n 145.
y
Incidence of diarrhea: Number of patients with minimum 1 day of diarrhea.
z
Vesikari score according to Lewis (23). For outpatients a modified Vesikari score was calculated, see methods.

Pneumonia was diagnosed based on clinical symptoms. Number of subjects analyzed: Inpatients: Probiotics, n 189, placebo n 183. Outpatients:
probiotics, n 148, placebo, n 149. Incidence: number of children with minimum 1 day with pneumonia. Severity: sum of daily pneumonia scores (0 no
pneumonia, 1 pneumonia, 2 severe pneumonia) during hospitalization. Pneumonia duration and severity were not possible to measure in outpatients as they
were only seen by a medical doctor every second week.
jj
Weight gain during inpatient treatment was calculated as the difference between discharge weight and the minimum weight during hospitalization and days
were total number of hospitalization days. Outpatient weight gain was calculated from discharge to week 8. Total weight gain was the difference between
minimum weight during hospitalization and week 8. Number of subjects analyzed: Inpatients probiotics n 159, placebo n 155, outpatients and total weight
gain: probiotics n 138, placebo n 142.

Recovery was defined as weight-for-length >2.

Diarrhea in the outpatient phase was usually mild according
to the Vesikari scale, but a number of patients had prolonged
periods of diarrhea, especially in the placebo group (Supplemental
Digital Content 2, Fig. http://links.lww.com/MPG/A883). This
could be associated with presence of environmental enteric dys-
function (EED), which involves increased gut permeability,
reduced absorptive capacity and inflammation (37). A reduction
in the number of children with long diarrhea periods could improve
the long-term nutritional status and reduce the risk of hospital
admissions in community-based children with SAM.
Regarding respiratory tract infections probiotics are mainly
reported to prevent upper respiratory tract infections (12). Both BB-
12andLGGhavebeenreportedtoreduceupperrespiratorytractinfection
(1517); however, sometimes with conflicting results (18,35,36).

www.jpgn.org 401

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Grenov et al
We did not find any difference in weight gain between the
probiotic and placebo groups, neither during hospitalization nor
during outpatient treatment. The overall evidence regarding effect
of probiotics on growth is scarce (38). Onubi performed a systema-
tic review of the effect of probiotics on growth in children and
evaluated 12 studies (13). Five studies from low-income countries,
including 4 studies with undernourished children, showed a positive
effect on weight gain whereas 7 studies from high-income countries
did not. Both studies on BB-12 or other strains belonging to the
same subspecies BB-12 and LGG were included in the review.
Safety of probiotics in immune compromised and critically
ill patients has been discussed mainly due to a concern about risk of
probiotic-related sepsis (39). The mortality rate was therefore
carefully followed throughout the study period by investigators
and the DSMB. In the current study, there was a small, nonsigni-
ficantly higher number of patients that died in the probiotic group
(26 patients) compared with the placebo group (20 patients)
(P 0.38). The mortality reports showed multiple severe medical
complications in most of the children, complicating assessment of
the exact cause of death. The most common causes of death,
according to the mortality reports, were respiratory failure/severe
pneumonia and shock/dehydration related to severe diarrhea. Sep-
ticemia was considered to be a direct cause of death in 4 patients in
each group and contributed to the underlying cause of death in 6
patients in each group. There were no signs of consistent differences
between the groups.
Strengths of the study include a randomized, double-blind
controlled design, use of a validated stool diary and thorough
training and monitoring of caregivers when they recorded childrens
stool pattern. Lack of data on diarrhea etiology is a limitation of the
study. In children with SAM, diarrhea can be caused by both
infectious and noninfectious agents. This includes infections with
bacteria, viruses or parasites and diarrhea due to malabsorption, for
example, secondary lactose intolerance and enteropathy. Probiotics
are likely to have different effects on these diarrhea etiologies, but
this was not assessed. The loss to follow-up may have resulted in
lower power, imprecise estimates of effects, and attrition bias. The
drop-outs were, however, equally distributed in the probiotic and
placebo groups. Finally, the results may not be generalizable to all
children with SAM as children below 6 months, children with an
admission weight below 4 kg and children in shock or with severe
respiratory distress were excluded from the study. Children with
any of these criteria belong to the most vulnerable children
with SAM.
The current results do not support using probiotics for the
treatment of hospitalized children with SAM and severe medical
complications. The reduction of days with diarrhea in the outpatient
phase, especially among children with long diarrhea duration, may,
however, be important in future community-based treatment of
children with SAM and may reduce admissions and mortality. But
further studies are needed to clarify this potential effect.
Acknowledgments: The authors thank children and caregivers
for their contribution to the study, especially for filling out stool
diaries and attending follow-up visits. The authors also thank the
study team for their persistent commitment to the study and patient
care. Funding was received from Chr. Hansen A/S, University of
Copenhagen and Innovation Fund Denmark with gratitude.
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