PRESENTATION
Can Probiotics Reduce Diarrhea and Infant
Mortality in Africa?
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The Project of a Pilot Study
Mario Del Piano, MD,* Francesco Coggiola, MD,w Marco Pane, BS,z
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Angela Amoruso, PhD,z Stefania Nicola, PhD,z and Luca Mogna, PhDz
Background: Diarrhea accounts for 9% of the mortality among
children under 5 years of age worldwide, and it is significantly
associated with malnutrition. Each year, diarrhea kills around
760,000 children under 5 years of age and most of these are in sub-
Saharan Africa. In Uganda, the infant mortality rate of 58 per 1000
is unacceptably high, and the major contributors include malnu-
trition, diarrhea, pneumonia, malaria, prematurity, sepsis, and
newborn illnesses. There is an urgent need for intervention to
prevent and control diarrheal diseases.
Study Design: Our open-label, randomized controlled study has the
primary endpoint of reducing diarrhea and infectious diseases
(number of episodes/severity) and the secondary endpoint of
decreasing infant mortality. The trial is currently conducted in
Luzira, a suburb of Kampala, the capital of Uganda, and in Gulu
and Lira, in the north of Uganda. The study is projected to enroll
4000 babies (control = 2000 and treatment = 2000) who will be
followed till 1 year of life. As controls, 2000 babies of the same
community are planned to be considered. The probiotic product
selected for the trial is composed of 3 designated microorganisms,
namely Bifidobacterium breve BR03 (DSM 16604), B. breve B632
(DSM 24706), and Lactobacillus delbrueckii subsp. delbrueckii
LDD01 (DSM 22106). The concentration of the 3 bacteria is 108
viable cells/strain/daily dose (5 drops).
Perspectives: For a total sample of 4000 babies, the study has an
80% power at a 5% significance level.
Key Words: infant mortality, Uganda, probiotics, diarrhea
(J Clin Gastroenterol 2016;50:S120–S123)
D iarrhea accounts for 9% of the mortality among chil-
dren under 5 years of age worldwide, and it is sig-
nificantly associated with malnutrition.1,2 It is definitely the
second leading cause of mortality. Each year, diarrhea kills
around 760,000 children under 5 years of age and most of
these are in sub-Saharan Africa.2 Children who are mal-
nourished or have an impaired immunity as well as people
living with HIV are the most at risk of life-threatening
diarrhea.3 There is an urgent need for intervention to pre-
vent and control diarrheal diseases.
From the *Gastroenterology Department, Santa Rita Hospital—
Policlinico di Monza; wDepartment of Gynecology and Obstet-
rics, Santa Rita Hospital—Policlinico di Monza, Vercelli; and
zBiolab Research Ltd, Novara, Italy.
Sponsored by Probiotical SpA for ethical purposes.
M.P., A.A., S.N., and L.M. are employees of Biolab Research Ltd.
M.D.P. is the Chief of the Gastroenterology Department, Santa
Rita Hospital—Policlinico di Monza. F.C. is the General Manager
of Santa Rita Hospital—Policlinico di Monza.
Address correspondence to: Mario Del Piano, MD, Gastroenterology
Department, Santa Rita Hospital—Policlinico di Monza, Viale
dell’Aeronautica 14/16–13100 Vercelli, Italy
(e-mail: mariodelpiano3@gmail.com).
Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved.
DOI: 10.1097/MCG.0000000000000677
S120 | www.jcge.com J Clin Gastroenterol  Volume 50, Supp. 2, November/December 2016
Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved.
The infant mortality rate of 58 per 1000 is unaccept-
ably high in Uganda, and the major contributors include
malnutrition, diarrhea, pneumonia, malaria, prematurity,
sepsis, and newborn illnesses.4
Although there is a decline in the infant mortality,
neonatal death still accounts for 40% of the total infant
mortality.
Interventions with proven effectiveness in the pre-
vention level include water, sanitation, and hygiene inter-
ventions, breastfeeding, complementary feeding, vitamin A
and zinc supplementation, and vaccines for diarrhea
(rotavirus and cholera).5,6 However, current estimates show
that 2.5 billion people still lack access to improved sani-
tation facilities and 1 billion people lack access to improved
drinking-water sources. These statistics indicate that diar-
rhea will continue to remain a formidable but largely
neglected problem in many parts of the developing world.
Besides common treatment with medicinal products,
some natural approaches are drawing more and more
attention in the treatment of these pathologies. One of the
most investigated approach is based on beneficial bacteria,
named probiotics. The word probiotic is derived from the
Greek “probiosis” meaning “in favor of life” and generally
applies to bacteria conveying at least a positive effect on the
host.7 The probiotic concept dates back over 100 years, and
associates with the name of the Ukrainian bacteriologist and
Nobel Laureate Ilie Metchnikoff, who proposed the scientific
rationale for the beneficial effects of lactic acid bacteria.8
The principal microorganisms in use as probiotics
belong to the genera Lactobacillus and Bifidobacterium.
Lactobacilli and bifidobacteria constitute the 2 most
important probiotic groups under consideration owing to
their recognition as members of the indigenous microbiota
of humans, their history of safety, and the general body of
evidence that supports their positive roles.9
These favorable bacteria have been used for long in the
food and beverage industry for human consumption,
including confectioneries, breweries, and dairy products at
both domestic and industry levels. One other natural use of
these microbes is to provide the body with an improved
mucosal immunophysiological barrier that prevents colo-
nization and infection by virulent pathogenic bacteria.10
Evidence has shown that the administration of probiotics
early in life improves immunity and thus prevents the
frequency and the intensity of respiratory and diarrheal
diseases in children.11 Furthermore, the use of antibiotics is
associated with diarrheal diseases and pseudomembranous
colitis due to the depletion of the normal flora.12 The pre-
vention of pneumonia and diarrhea using probiotics would
reduce the mortality and morbidity attributable to these
illnesses. Studies provide testimony that they reduce food
intolerances and the incidence of necrotizing enterocolitis
 J Clin Gastroenterol  Volume 50, Supp. 2, November/December 2016
(NEC).13 A recent meta-analysis conducted in 2014 stated
that probiotics significantly reduce the incidence of NEC
and death by 30% among premature babies.14
Some randomized trials and systemic reviews have
demonstrated that probiotics significantly reduce morbidity
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and hospital stay due to acute episodes of diarrhea, thus
improving recovery.15–17 However, only a few studies have
been conducted in low-income countries.
Despite the good availability of scientific literature
concerning the acute treatment of diarrhea, there is a rel-
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ative scarcity of studies concerning the prevention of diar-
rhea in infants. A recent trial has assessed the effectiveness
of a selected strain in 3758 children ages 1 to 5 years who
were given either a probiotic drink containing Lactobacillus
casei or a nutrient drink daily for 12 weeks. They were
followed up for another 12 weeks. There were 608 indi-
viduals with diarrhea in the probiotic group and 674 indi-
viduals in the nutrient group during the study period of 24
weeks. The level of protective efficacy for the probiotic was
14% (95% confidence interval, 4-23; P < 0.01 in the
adjusted model).18
Nonetheless, despite the relatively good evidence for
the use of probiotics among preterm infants and children
with diarrhea in other countries, their use is nonexistent in a
country such as Uganda and not documented at all.
Moreover, the few studies performed had no long-term
follow-up, and the data from other countries cannot be
extrapolated to low-resource limited settings such as
Uganda.
Our study is the first with this focused approach and
aims to influence the future policy of use of probiotics in
Uganda and, more widely, in sub-Saharan Africa.
STUDY DESIGN
Our open-label, randomized controlled study has the
primary endpoint of reducing diarrhea and infectious dis-
eases (number of episodes/severity) and the secondary
endpoint of decreasing infant mortality.
Participation in this study is entirely voluntary and
there are no costs as it is fully funded by Probiotical S.p.A.
(Novara, Italy).
The trial is currently conducted in Luzira, a suburb of
Kampala, the capital of Uganda. It is located approx-
imately 12 km (7.5 miles), by road, southeast of the central
business district of Kampala. The study sites are St Francis
hospital, Nsambya, and Benedict hospital. Both centers are
faith-based private, not-for-profit organizations. St Francis
hospital, Nsambya, is a regional referral hospital and a
teaching hospital of Uganda Martyrs University, whereas
Benedict hospital is a health center IV. Both centers have
24-hour services and an outpatient department, with
maternity services inclusive. The study is also conducted in
Gulu Hospital and Lira Hospital, in the north of Uganda,
with the same method and rules.
TABLE 1. The Sample Size Needed to Detect a Statistical
Significance (5%) as a Function of the Hypothetic Reduction in
Infant Mortality (%)
Event Probability in Individuals Sample Size
Treated (%) (Each Arm)
53 33,313
55 93,644
39 2094
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Probiotics for Africa
The study population consists of all neonates at our
centers aged 6 to 21 days. The study participants are
recruited from babies brought for immunization by taking
every even number (eg, 2, 4, 6, 8). The babies are then
randomized to receive the probiotic (treatment arm) or the
placebo (control arm). A simple randomization by flipping
a coin for each selected baby is applied.
If the mother is willing to have her newborn take part
in the study, she is requested to sign an Informed Consent
Form provided in English and translated into local Swahili.
The study will enroll 4000 babies (control = 2000 and
treatment = 2000) who will be followed till 1 year of life. As
controls, 2000 babies of the same community will be con-
sidered. A placebo will not be used because people probably
cannot understand this concept.
For a total sample of 4000 babies, the study will have
an 80% power at a 5% significance level (Table 1). Table 1
illustrates that a mortality reduction from 58% to 39%
would be needed to achieve statistical significance with 2094
participants in each arm. Otherwise, a markedly higher
number of participants would be necessary if the reduction
of mortality rate is lower.
The study protocol is pending approval by the Insti-
tutional Review Board of St Raphael of St Francis Hos-
pital, Nsambya.
PROBIOTIC PRODUCT AND TREATMENT
The probiotic product selected is composed of 3 des-
ignated microorganisms, namely Bifidobacterium breve
BR03 (DSM 16604), B. breve B632 (DSM 24706), and L.
delbrueckii subsp. delbrueckii LDD01 (DSM 22106). The
concentration of the 3 bacteria is 108 viable cells/strain/
daily dose (5 drops).
The aim of this project is to support these populations
to reduce infant mortality and morbidity. At the end of this
project, we suppose to reduce both parameters of at least
5%.
Every month, a qualified health worker activates a
bottle of product and hands it to the mother.
Permission to import and to use the probiotics has
been obtained from National Drug Authority. The product
is still investigational.
The probiotic strains are able to directly inhibit dif-
ferent gram-negative bacteria, some of them isolated from
infants with gaseous colic, with particular reference to
Klebsiella pneumoniae, Enterobacter cloacae, Escherichia
coli, Salmonella enteriditis, Clostridium difficile, and Cam-
pylobacter jejuni.19–21
B. breve is a species typical of the newborn and is the
dominant species in breastfed infants.22,23 The species
promotes the intestinal colonization by bifidobacteria and
the formation of a balanced microbiota early in life.24 The
isolation of B. breve in combination with disease conditions
has never been reported in either animals or humans.
The 2 B. breve chosen for this project are derived from
a screening of over 50 bifidobacteria conducted in the
Microbiology Laboratory of the Department of Science
and Technology of Agroenvironmental DiSTA aimed to
select the organism with the best potential for an in vivo
application.
The strain B632 was isolated from the feces of new-
born infants,25 and was subject to a long series of in vitro
tests to verify its safe use. B. breve BR03 was isolated at the
University of Bologna from the feces of a healthy adult.
www.jcge.com | S121
 Del Piano et al
Both strains were not subjected to any genetic manipulation
and were characterized and typed at the level of genus,
species, and biotype. Both are deposited at International
Collections with the status of IDA (International Deposi-
tary Authority), specifically the German Deutsche Samm-
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lung von Mikroorganismen und Zellkulturen GmbH
(DSMZ).
The 2 strains B. breve B632 and B. breve BR03 dem-
onstrated the ability to colonize the human gut of healthy
children.26
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In addition, the microorganism B. breve BR03 is
characterized by a marked anti-inflammatory activity. This
was demonstrated both in vitro, by coincubation of the
strain with immune system cells isolated from the periph-
eral blood (PBMCs) of healthy individuals, and in vivo on
humans, by means of 2 intervention studies in individuals
suffering from irritable bowel syndrome. They reported a
significant reduction in discomfort and pain in different
quadrants of the abdomen as well as a normalization of the
intestinal transit time, especially in the constipation variant
of the syndrome.27–29
In contrast, the proposed activities of L. delbrueckii
include antagonism toward pathogens by competition for
binding sites and secretion of inhibitory compounds
(organic acids, hydrogen peroxide, and bacteriocins) as well
as by the stimulation of the mucosal immune system and
augmentation of the host defense against pathogenic bac-
teria and foreign antigens.30 Lactobacilli are known to
inhibit the growth of pathogenic bacteria, possibly by
producing inhibitory compounds such as organic acids,
hydrogen peroxide, and bacteriocins.31,32
FOLLOW-UP
A visit is performed at enrollment and every 3 months.
The following parameters are checked for both arms:
(1) A brief history of the baby’s well-being including
 Gastrointestinal and infectious diseases;
 Dermatologic and allergic diseases.
(2) A systemic examination: weight, height, and head
circumference. These will be plotted on a growth chart.
(3) Possible mortality.
(4) The number of admissions and visits in the outpatient
clinic.
Preterm infants in the neonatal unit are followed daily.
Preterm infants in the ward are assessed for the growth
velocity, signs of NEC, time to full feeds, length of stay in
the hospital, and discharge.
DATA MANAGEMENT AND ANALYSIS
All questionnaires are checked for completeness before
ending the interview. Case Report Forms are labeled with
the study identification number and the baby’s initials to
ensure accurate identification of study participants.
Raw data will be entered into a computer using the
EPIDATA version 3.1 (Denmark) software package by 2
independent data entrants. Data from both entities will be
merged and cleaned.
The data will be exported to STATA 11.0/IC (Stata
Inc., College Station, TX) and analyzed with the assistance
of a statistician.
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J Clin Gastroenterol  Volume 50, Supp. 2, November/December 2016
REFERENCES
1. WHO. Diarrhoea: Why Children Are Still Dying and What Can
be Done. Geneva, Switzerland: WHO; 2009.
2. Njume C, Goduka NI. Treatment of diarrhoea in rural African
communities: an overview of measures to maximise the
medicinal potentials of indigenous plants. Int J Environ Res
Public Health. 2012;9:3911–3933.
3. Hajela N, Nair GB, Ganguly NK. Are probiotics a feasible
intervention for prevention of diarrhoea in the developing
world? Gut Pathogens. 2010;2:10.
4. Uganda Bureau of Statistics (UBOS) and ICF International
Inc. Uganda Demographic and Health Survey 2011. Kampala,
Uganda; Calverton, MD: UBOS; ICF International Inc; 2012.
5. Das JK, Salam RA, Bhutta ZA. Global burden of childhood
diarrhea and interventions. Curr Opin Infect Dis. 2014;27:
451–458.
6. Ejemot-Nwadiaro RI, Ehiri JE, Arikpo D, et al. Hand washing
promotion for preventing diarrhoea. Cochrane Database Syst
Rev. 2015;9:CD004265.
7. Schrezenmeir J, de Vrese M. Probiotics, prebiotics, and
synbiotics—approaching a definition. Am J Clin Nutr. 2001;
73(suppl):361S–364S.
8. Gordon S. Elie Metchnikoff, the man and the myth. J Innate
Immun. 2016;8:223–227.
9. Pandey KR, Naik SR, Vakil BV. Probiotics, prebiotics and
synbiotics—a review. J Food Sci Technol. 2015;52:7577–7587.
10. Maldonado Galdeano C, Novotny Nuñez I, Carmuega E, et al.
Role of probiotics and functional foods in health: gut immune
stimulation by two probiotic strains and a potential probiotic
yoghurt. Endocr Metab Immune Disord Drug Targets. 2015;15:37–45.
11. Viggiano D, Ianiro G, Vanella G, et al. Gut barrier in health
and disease: focus on childhood. Eur Rev Med Pharmacol Sci.
2015;19:1077–1085.
12. Larcombe S, Hutton ML, Lyras D. Involvement of bacteria
other than Clostridium difficile in antibiotic-associated diar-
rhoea. Trends Microbiol. 2016;24:463–476.
13. Lau CS, Chamberlain RS. Probiotic administration can
prevent necrotizing enterocolitis in preterm infants: a meta-
analysis. J Pediatr Surg. 2015;50:1405–1412.
14. Yang Y, Guo Y, Kan Q, et al. A meta-analysis of probiotics
for preventing necrotizing enterocolitis in preterm neonates.
Braz J Med Biol Res. 2014;47:804–810.
15. Ahmadi E, Alizadeh-Navaei R, Rezai MS. Efficacy of
probiotic use in acute rotavirus diarrhea in children: a
systematic review and meta-analysis. Caspian J Intern Med.
2015;6:187–195.
16. Applegate JA, Fischer Walker CL, Ambikapathi R, et al.
Systematic review of probiotics for the treatment of
community-acquired acute diarrhea in children. BMC Public
Health. 2013;13(suppl 3):S16.
17. Isolauri E. Probiotics for infectious diarrhoea. Gut. 2003;52:
436–437.
18. Sur D, Manna B, Niyogi SK, et al. Role of probiotic in
preventing acute diarrhoea in children: a community-based,
randomized, double-blind placebo-controlled field trial in an
urban slum. Epidemiol Infect. 2011;139:919–926.
19. Mogna L, Del Piano M, Deidda F, et al. Assessment of the
in vitro inhibitory activity of specific probiotic bacteria against
different Escherichia coli strains. J Clin Gastroenterol.
2012;suppl:S29–S32.
20. Santini C, Baffoni L, Gaggia F, et al. Characterization of
probiotic strains: an application as feed additives in poultry
against Campylobacter jejuni. Int J Food Microbiol.
2010;141(suppl 1):S98–S108.
21. Aloisio I, Santini C, Biavati B, et al. Characterization of
Bifidobacterium spp. strains for the treatment of enteric disorders
in newborns. Appl Microbiol Biotechnol. 2012;96:1561–1576.
22. Biavati B, Mattarelli P. The family Bifidobacteriaceae In:
Falkow S, Rosenberg E, Schleifer KH, Stackebrandt E, eds. A
Handbook on the Biology of Bacteria 3rd ed. Berlin, Germany:
Springer Science+Business Media2006:322–382.
 J Clin Gastroenterol  Volume 50, Supp. 2, November/December 2016 Probiotics for Africa

23. Mikami K, Takahashi H, Kimura M, et al. Influence of
maternal bifidobacteria on the establishment of bifidobacteria
colonizing the gut in infants. Pediatr Res. 2009;65:669–674.
24. Li Y, Shimizu T, Hosaka A, et al. Effect of Bifidobacterium
breve supplementation on intestinal flora of low birth weight
infants. Pediatr Int. 2004;46:509–515.
Downloaded from http://journals.lww.com/jcge by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AW
28. Saggioro A. Probiotics in the treatment of irritable bowel
syndrome. J Clin Gastroenterol. 2004;38:S104–S106.
29. Del Piano M, Carmagnola S, Anderloni A, et al. The use of
probiotics in healthy volunteers with evacuation disorders and
hard stools: a double-blind, randomized, placebo-controlled
study. J Clin Gastroenterol. 2010;44(suppl 1):S30–S34.

25. Scardovi V, Casalicchio F, Vincenzi N. Multiple electro-
phoretic forms of transaldolase and 6-phosphogluconic dehy-
drogenase and their relationships to the taxonomy and ecology
of the bifidobacteria. Int J Syst Bacteriol. 1979;29:312–327.
26. Mogna L, Del Piano M, Mogna G. Capability of the two
nYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC4/OAVpDDa8K2+Ya6H515kE= on 10/14/2023
30. Nagafuchi S, Takahashi T, Yajima T, et al. Strain dependency
of the immunopotentiating activity of Lactobacillus delbrueckii
subsp. bulgaricus. Biosci Biotechnol Biochem. 1999;63:474–479.
31. Jacobsen CN, Rosenfeldt Nielsen V, Hayford AE, et al.
Screening of probiotic activities of forty-seven strains of

microorganisms Bifidobacterium breve B632 and Bifidobacte-
rium breve BR03 to colonize the intestinal microbiota of
children. J Clin Gastroenterol. 2014;48:S37–S39.
27. Nicola S, Mogna L, Allesina S, et al. Interaction between
probiotics and human immune cells: the prospective anti-
inflammatory activity of Bifidobacterium breve BR03. Agro-
FOOD. 2010;21:44–47.
Lactobacillus spp. by in vitro techniques and evaluation of
the colonization ability of five selected strains in humans. Appl
Environ Microbiol. 1999;65:4949–4956.
32. Loessner M, Guenther S, Steffan S, et al. A pedocin-producing
Lactobacillus plantarum in a multispecies cheese surface
microbial ripening consortium. Appl Environ Microbiol.
2003;69:1854–1857.

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