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Clinical insight
P R O B E
• V o l . L I I I • N o . 3 • A p r – J u n 2 0 1 4
Abdel-Hafeez, et al.
Breast-feeding Protects Against Infantile Diarrhea
formula-fed infants. However,
there are little conflicting opinions
concerning breast milk and anti-
infective functions. Detailed
investigations are still limited.
IgE is considered to play a central
role in protective immunity against
parasites, not only helminths but also
some protozoa such as
G intestinalis
(synonymous with
G lamblia
and
G duodenalis
).
TNF-
a
is an important inflammatory
cytokine in immune regulation
and resistance to various microbes
including protozoa. This cytokine is
known to be of significant importance
in the pathogenesis of inflammatory
bowel diseases, and anti-TNF
agents have proven to be effective
in the treatment of chronic active
inflammatory bowel diseases and
fistulizing disease. Thus, the present
study was designed to investigate
breastfeeding in protection against
IPI by comparing breastfed with
non-breastfed infants with persistent
diarrhea.
Materials andMethods
Subjects andmethods
This study was carried out with 322
infants (146 boys and 176 girls, 2–6
months old) who presented with
persistent diarrhea (minimum of 3
loose stools/day for more than 14
days) in spite of an anti-bacterial drug,
nifuroxazide. They had been enrolled
from inpatient wards and outpatient
clinics of the Pediatric Department of
University Hospital (Minia District,
Egypt) between January 2011 and
March 2012. These infants were
divided into 2 equal groups of 161
each. The first group comprised of
161 partially breastfed infants (79
males and 82 females, mean ± SD =
4.5 ± 1.1 months) and the second an
equal number of non-breastfed infants
(some were having formula alone, and
some bovine milk, 67 males and 94
females, 4.4 ± 1.2 months old). The
sample characteristics (age, parent
socio-economic levels, and residency)
of these 2 groups were similar.
The breastfed infants had been
breastfed since birth, while non-
breastfed infants had received a
formula since 1 or 2 weeks after birth.
Formula was dissolved in tap water,
which had been boiled and cooled.
Ethical considerations
Verbal consent was obtained from
the parents of these infants. All
procedures were conducted according
to the ethical standards approved by
the Institutional Human Ethics Com
mittee, Faculty of Medicine (Minia
University, Egypt).
Stool examinations
Stool samples were collected in a clean
plastic container once per infant but
several stool samples were collected
on alternate days to detect parasites
because cysts of
G lamblia
are excreted
intermittently. Fecal specimens were
transported to the laboratory of
Parasitology Department, Faculty of
Medicine (Minia University, Egypt) to
be examined by different techniques
for parasitic infections within 1 to 3 h
after collection.
For microscopic examinations, direct
wet smear methods, namely saline wet
mount and iodine wet mount, were
applied. The technique of formol-ether
sedimentation was used to concentrate
the cysts or oocysts of the protozoa.
Same amount of stools (50 mg) from
each of all infants was mixed with
10 mL of fixation buffer (sodium-
acetate acetic acid formalin, SAF)
and incubated for 1 h for fixation
and inactivation. The suspension was
passed through 4 layers of cotton
netting and centrifuged at 2000 g
for 5 min. Two smears were dried in
air, fixed with methanol, and then
examined using 2 different stains—
acid fast stain (modified Ziehl-
Neelsen stain) for identification of
Cryptosporidium
spp and Giemsa stain
for identification of
Blastocystis
spp.
The intensity ofinfection was scored
by × 1000 magnification as 1, very
low infection (1 parasite per field); 2,
mild infection (2 parasites per field);
3, moderate infection (3 parasites per
field); 4, heavy infection (4 or more
parasites per field).
Blood sample collection
Blood samples were collected from
arm vein and sera were stored at
- 20°C until use. The serum levels
of IgE and TNF-
α
were measured
using commercially available ELISA
kits (MYM Laboratory and Medical
Supply, Inc, San Diego, CA, USA
and Anogen, Ontario, Canada,
respectively) according to the
manufacturers’ guidelines.
Statistical analysis
Data were coded and verified prior
to data entry. No data were double-
entered. As the data were distributed
as normal, results of the studies
were reported as mean ± SD. The
statistical package of SPSS version 16
for Windows was used for data entry
and analysis. Descriptive statistics
were calculated. The
Χ
2
-test was used
to compare proportions of parasite
detection between groups and of
sex difference between groups. The
Student t test was used to compare
means between groups. Regression
analysis was used for estimating the
relationships between serum levels of
IgE and TNF-
α
and the intensity of
the infection.
P
< .05 was considered
to be statistically significant.
