Helicobacter pylori and enteric parasites co-infection among diarrheic and non-diarrheic Egyptian children: seasonality, estimated risks, and predictive factors

Abstract

Helicobacter pylori (H. pylori) and intestinal parasites are known for their high prevalence in children. Both of them infect the gastrointestinal tract with overlapping clinical pictures. This study was conducted to determine H. pylori prevalence and its association with intestinal parasites in children, moreover to estimate risk and predictive factors for their detection in stool samples. Single fecal samples were collected from 226 Egyptian pediatric patients (125 diarrheic and 101 non-diarrheic) attending gastroenterology outpatients’ clinics, from February 2016 to June 2017. All stool specimens were microscopically examined to search for ova and parasites. Copro-DNAs detection of H. pylori and Cryptosporidium were performed using nested-PCR assays. H. pylori was detected molecularly in 36.8% of the total study population, with a higher prevalence in diarrheic than in non-diarrheic children. Intestinal parasites were detected in 27.4% of the total study populations, of these, 43.9% had co-existence with H. pylori colonized patients and was significantly associated with Cryptosporidium spp. and G. intestinalis. Estimated risk of the presence of H. pylori was in January. Our data provide a better understanding of the epidemiology of H. pylori infection when associated with intestinal parasites. H. pylori co-existence with G. intestinals and Cryptosporidium may suggest the association of H. pylori infection with markers of fecal exposure. Whether H. pylori provides favorable conditions for intestinal parasitosis or vice versa, still further investigations are needed with an emphasis upon determining correlation with gut microbiomes.

Full text

ORIGINAL ARTICLE
Helicobacter pylori and enteric parasites co-infection
among diarrheic and non-diarrheic Egyptian children:
seasonality, estimated risks, and predictive factors
Asmaa Ibrahim
1,2
•Yasser B. M. Ali
2
•Amal Abdel-Aziz
2
•Ayman A. El-Badry
1,3
Received: 14 November 2018 / Accepted: 15 December 2018 / Published online: 1 January 2019
ÓIndian Society for Parasitology 2019
Abstract Helicobacter pylori (H. pylori) and intestinal
parasites are known for their high prevalence in children.
Both of them infect the gastrointestinal tract with over-
lapping clinical pictures. This study was conducted to
determine H. pylori prevalence and its association with
intestinal parasites in children, moreover to estimate risk
and predictive factors for their detection in stool samples.
Single fecal samples were collected from 226 Egyptian
pediatric patients (125 diarrheic and 101 non-diarrheic)
attending gastroenterology outpatients’ clinics, from
February 2016 to June 2017. All stool specimens were
microscopically examined to search for ova and parasites.
Copro-DNAs detection of H. pylori and Cryptosporidium
were performed using nested-PCR assays. H. pylori was
detected molecularly in 36.8% of the total study popula-
tion, with a higher prevalence in diarrheic than in non-
diarrheic children. Intestinal parasites were detected in
27.4% of the total study populations, of these, 43.9% had
co-existence with H. pylori colonized patients and was
significantly associated with Cryptosporidium spp. and G.
intestinalis. Estimated risk of the presence of H. pylori was
in January. Our data provide a better understanding of the
epidemiology of H. pylori infection when associated with
intestinal parasites. H. pylori co-existence with G. intesti-
nals and Cryptosporidium may suggest the association of
H. pylori infection with markers of fecal exposure. Whe-
ther H. pylori provides favorable conditions for intestinal
parasitosis or vice versa, still further investigations are
needed with an emphasis upon determining correlation
with gut microbiomes.
Keywords Helicobacter pylori Intestinal parasites 
Risk factors Diarrhea Children Egypt
Introduction
Helicobacter pylori (H. pylori) is a ubiquitous, helical
shaped, motile, gram-negative bacillus bacterium, which
colonizes the gastric mucosa (Rafeey et al. 2007). Colo-
nization is generally acquired during the first 5 years of
childhood (Rajindrajith et al. 2009). H. pylori prevalence in
children ranges from 30 to 80%, with a predominance in
developing countries and its prevalence differs from one
region to the other in the same country (Suerbaum and
Michetti 2002; Salih 2009). The mode of transmission of
H. pylori is still unclear. Proposed H. pylori transmission
modes include direct contact (fecal–oral increased among
immunocompromised children and children suffering from
diarrhea, vomiting, fever, and dehydration. H. pylori sea-
sonality in our cohort of children showed a circannual
pattern with peaking in winter, drinking contaminated
water and ingestion of contaminated food (Frenck and
&Asmaa Ibrahim
chemistasmaain@gmail.com
Yasser B. M. Ali
yassermb@yahoo.com
Amal Abdel-Aziz
amalmo15@yahoo.com
Ayman A. El-Badry
aaelbadry@iau.edu.sa
1
Diagnostic and Research Unit of Parasitic Diseases (DRUP),
Department of Medical Parasitology, Kasr Al-Ainy Faculty
of Medicine, Cairo University, Cairo, Egypt
2
Genetic Engineering and Biotechnology Research Institute,
University of Sadat City, Sadat City, Egypt
3
Department of Microbiology, Faculty of Medicine, Imam
Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
123
J Parasit Dis (Apr-June 2019) 43(2):198–208
https://doi.org/10.1007/s12639-018-1075-y

Clemens 2003). H. pylori infection diagnosis is generally
divided into invasive and non-invasive approaches. A
combination of at least two tests is commonly used as a
gold standard (Sethi et al. 2013). Parasitic infections,
including intestinal parasites, are distributed worldwide
and are endemic in tropical and subtropical countries.
Globally about 3.5 billion individuals are infected with
intestinal parasites, the majority of them being children.
Diarrhea is the most commonly presented gastro- intestinal
symptom and is mainly caused by intestinal parasites,
bacterial pathogens, and viruses. Diarrheal diseases are
globally estimated to be 1.7 billion annual cases (Brooker
et al. 2009; Bhutta et al. 2013; WHO 2017). Giardia
intestinalis (G. intestinalis), Cryptosporidium spp., and
Entamoeba histolytica (E. histolytica) complex are the
most common intestinal protozoan parasites which cause
acute diarrheal diseases in children (Thompson and Ash
2016;WHO2017).
PCR is considered a reliable test; it is performed rapidly
and is cost-effective. Also, it can identify different types/
strains of bacteria and protozoa for pathogenic and epi-
demiologic studies as well as for detection of antibiotic
resistance (Mehmood et al. 2010). Both H. pylori and
intestinal parasites share a common mode of transmission
and may share the same risk and predictive factors, where
one of them supports the colonization of the other. In
addition, protozoa may transmit pathogenic bacteria and
viruses (Yakoob et al. 2005).
There are few studies, which investigated co-infection
between H. pylori and certain protozoa (G. intestinalis,E.
histolytica, and Blastocystis spp.) (Torres et al. 2003;
Moreira et al. 2005; Marini et al. 2007; Zeyrek et al. 2008;
Escobar-Pardo et al. 2011; Sabah et al. 2015). The primary
objective of the present study was to evaluate H. pylori
prevalence and its co-existence with intestinal parasites
among diarrheic and non-diarrheic Egyptian children.
Additionally, we estimated risk and predictive factors,
which are thought to influence the prevalence of this co-
infection.
Subjects and methods
Study design and individuals
This cross-sectional study was carried on 226 Egyptian
children (125 diarrheic which include both immunocom-
petent and immunocompromised and 101 non-diarrheic)
attending gastrointestinal outpatients’ clinics, Kasr Al-
Ainy Pediatric hospitals, Cairo University, ranging from 0
to 16 years, from February 2016 to June 2017.
Stool specimen processing
Fresh single stool specimens were collected from each
individual. The related socioeconomic, demographic, envi-
ronmental and clinical data were collected with each sample.
Each sample was examined microscopically and using PCR
for detection of H. pylori and Cryptosporidium spp.
Copro-parasitological examination
All collected fecal samples were microscopically examined
for detection of intestinal parasite and associated elements
like pus, rbcs sand Charcot–Leyden crystals by direct wet
mount before and after formal ether concentration tech-
nique (Chesbrough 2006). Fecal smears were stained by
Kinyoun modified acid-fast stain for coccidian protozoa
detection (Garcia et al. 1983).
Copro-PCR assay
Genomic DNA extraction
Thermal shocking was done for each fecal specimen to
disrupt the oocyst wall, then genomic copro-DNA extrac-
tion from each sample was done with the Favor Stool DNA
Spin Columns Isolation Kit (cat. no. FAST1; Favorgen
Biotech Corporation, Taiwan) following the manufac-
turer’s instructions.
Helicobacter pylori nested polymerase chain reaction
(nPCR) assay
Helicobacter pylori extracted DNA amplification was
performed by nPCR targeting the H. pylori UreA gene with
two sequential PCR reactions. The first reaction amplified
the 293 bp fragment by using the 81external primers set;
2F2 50-ATATTATGGAAGAAGCGAGAGC-30and 2R2
50-ATGGAAGTGTGAGCCGATTTG-30. The second
reaction amplified the 200 bp fragment by internal primers
set; 2F3 50-CATGAAGTGGGTATTGAAGC-30and 2R3
50-AAGTGTTGAGCCGATTTGAACCG-30. Amplifica-
tion in each reaction was done following directions of
Sasaki et al. (1999). The amplified nPCR products were
stained with ethidium bromide and electrophoresed on
agarose gel (1.5%) in TAE buffer and were visualized
under a UV transilluminator.
Cryptosporidium spp nPCR assay
Cryptosporidium extracted DNA amplification was per-
formed by nPCR that targeted the COWP gene, which
included two sequential PCR reactions. The primary
reaction amplified the 769- bp fragment by using
J Parasit Dis (Apr-June 2019) 43(2):198–208 199
123

BCOWPF: 50-ACCGCTTCTCAACAACCATCTTGTCC
TC-30; and BCOWPR: 50-CGCACCTGTTCCCACTCA
ATGTAAACCC-30. The secondary reaction amplified the
553-bp fragment by internal sets -Cry-15: 50-GTAGAT
AATGGAAGAGATTGTG-30and Cry-9: 50-GGACT-
GAAATACAGGCATTATCTTG-30. Amplification in each
reaction was done according to steps carried out by Spano
et al. (1997) and, Pedraza-Dı
´az et al. (2001). The amplified
nPCR products were stained with ethidium bromide and
electrophoresed on agarose gel (1.5%) in TAE buffer and
were visualized under a UV transilluminator.
Analysis of Restriction-fragment length polymorphism
(RFLP) was conducted following the manufacturer’s
instructions using RsaI to fragment Cryptosporidium PCR
products for genotyping (product no. ER1121; Thermo
Scientific). Fragmented PCR products were elec-
trophoresed in Metaphor agarose gel (3%) after staining
with ethidium bromide, and gels were visualized using UV
transillumination.
Statistical analysis
The statistical package SPSS 17 (Chicago, IL, USA) was
used to statistically analyze the data with Fisher’s exact test
and multiple logistic regressions. Study variables, where
associated with statistical significance with the prevalence
of the bacterium H. pylori in the univariate analysis, were
subjected to multivariate logistic regression. The H. pylori
seasonality was performed by analysis of the number of
positive cases of H. pylori per number of presenting
patients per month, for duplicated months the mean was
calculated.
Results
Helicobacter pylori DNA was detected in 36.8% (82/226)
of total study population using PCR targeting H. pylori
UreA gene (Fig. 1), with a higher occurrence in diarrheic
(68.3% [56/82]) than in non-diarrheic patients (31.7% [26/
82]) (Table 1).
Intestinal parasites were detected in 27.4% (62/226) of
the study groups with Cryptosporidium being the pre-
dominant parasite (8.8%), followed by G. intestinalis
(8.4%), Blastocyst spp. (4.4%) and E. histolytica complex
(3.5%) (Table 4). Both Cryptosporidium genotypes, the
anthroponotic Cryptosporidium hominis (C. hominis) and
the zoonotic Cryptosporidium parvum (Fig. 2), were
detected with a predominance of C. hominis genotype
(80%) (Table 1; Fig. 3).
Intestinal parasites co-existed in 43.9% (36/82) of the H.
pylori colonized patients, with a statistically significant
association. H. pylori colonized half of the stool samples
that were collected from diarrheic children (28/56)
(Table 4). Polyparasitism (concurrent infection with mul-
tiple intestinal parasites species) occurred in six diarrheic
cases (Table 5). They were significantly associated with
the presence of H. pylori in the stool (P\0.05).
Fig. 1 Showing agarose gel electrophoresis for the products of the
nPCR targeting UreA gene of H. pylori at 200 bp. Lane 1: 100 bp
DNA molecular weight marker ‘‘ladder’’. Lanes 2–4, 6, 7, 9 and 11:
Positive samples. Lanes 5, 8 and 10: Negative samples. Lane 11:
Negative control. Lane 12: Positive control
Table 1 Results of molecular detection of H. pylori and Cryp-
tosporidium spp and genotypes among study population
H. pylori result using PCR
Positive Negative Total
Non-diarrheic
Cryptosporidium
Positive (Genotype)
C. hominis 00 0
C. parvum 00 0
Total 0 0 0
Negative 26 (25.7%) 75 (74.3%) 101 (100%)
Total 26 (25.7%) 75 (74.3%) 101 (100%)
Diarrheic
Cryptosporidium
Positive (genotype)
C. hominis 10 (8%) 6 (4.8%) 16 (12.8%)
C. parvum 2 (1.6%) 2 (1.6%) 4 (3.2%)
Total 12 (9.6%) 8 (6.4%) 20 (16%)
Negative 44 (35.2%) 61 (48.8) 105
Total 56 (44.8%) 69 (56.2%) 125 (100%)
Total
Cryptosporidium
Positive (genotype)
C. hominis 10 (4.4%) 6 (2.6) 16 (7%)
C. parvum 2 (0.9) 2 (0.9%) 4 (1.8%)
Total 12 (5.3%) 8 (3.5%) 20 (8.8%)
Negative 70 (31%) 136 (60.2%) 206 (91.2%)
Total 82 (36.3%) 144 (63.7%) 226 (100%)
200 J Parasit Dis (Apr-June 2019) 43(2):198–208
123

Helicobacter pylori was detected throughout the year, in
both study groups, peaking in December only for non-di-
arrheic children (Fig. 4) with statistical significance
(P\0.05).
In an effort to identify prospective shared risk factors that
could elucidate the positive association between H. pylori and
certain intestinal protozoan parasites, a number of the studied
variables such as consumed milk, immune status (immuno-
competent/immunocompromised) (Table 2), gastrointestinal
symptoms (diarrhea, vomiting, fever, and dehydration)
(Table 3), co- existence of G. intestinalis and Cryptosporidium
parasites (Table 4) and polyparasitism were significantly
associated (P\0.05) with detection of H. pylori in the stool
(Table 5). These study variables were subjected to multivariate
analysis by logistic regression and revealed an estimated
increase in the risk of H. pylori within immunocompromised
children, children presenting diarrhea, vomiting, fever, dehy-
dration, and children who had G. intestinalis,Cryptosporidium
spp. or multiple parasites in their stool (Table 6).
Discussion
Helicobacter pylori is the most prevalent human bacteria;
its infection is a serious worldwide health problem, espe-
cially in developing countries. The infection is mainly
acquired in early childhood, which can lead to gastritis in
children and adults and may cause peptic ulcer (Whitney
et al. 2000; Gallo et al. 2003; Mansour-Ghanaei et al.
2010). In our study, the overall H. pylori infection preva-
lence was 36.3%, rendering it the most prevalent pathogen
detected in the stool of our study population. This finding is
confirmed by a previous study from Egypt (33%) (Frenck
et al. 2006) as well as the reported global average preva-
lence in children (32.6%) (Zamani et al. 2018).
There was a circannual seasonal variation of H. pylori
for both diarrheic and non-diarrheic children, with peaking
in mid-winter in non-diarrheic children. Though we
reported seasonality of H. pylori in Egyptian children for
the first time, this seasonal pattern with an increase in the
rate of transmission in winter than in summer has been
previously reported (Savarino et al. 1992; Raschka et al.
1999). Although we did not include peptic ulcer in our
study variables, the seasonal variation in H. pylori was
found to be parallel to peptic ulcer periodicity (Savarino
et al. 1992; Raschka et al. 1999). Co-infections between H.
pylori and protozoa namely, G. intestinalis,E. histolytica,
and Blastocystis spp. have rarely been studied. The few
Fig. 2 Showing agarose gel electrophoresis for the products of the
nPCR targeting COWP gene of Cryptosporidium spp. at 553 bp. Lane
1: 50 bp DNA molecular weight marker ‘‘ladder’’. Lane 2: positive
control. Lanes 3–10: positive samples
Fig. 3 Showing agarose gel electrophoresis for the products of the
nPCR targeting COWP gene of Cryptosporidium spp. after digestion
by RsaI. Lane 1: 100 bp DNA molecular weight marker ‘‘ladder’’,
lanes 2–6: Positive C. hominis samples (285, 125, 106 and 34 bp).
Lanes 7–11: positive C. parvum samples (410, 106 and 34 bp)
Fig. 4 Seasonal distribution of cases of H. pylori (%) among
diarrheic and non-diarrheic children positive by PCR
J Parasit Dis (Apr-June 2019) 43(2):198–208 201
123

Table 2 Distribution of studied variables among study population in relation to diarrhea and H. pylori colonization
Non-Diarrhoeic Diarrhoeic All study individuals
H. pylori negative H. pylori positive Total P. value H. pylori negative H. pylori positive Total P. value H. pylori negative H. pylori positive Total P. value
Age group
0–1 years 0 0 0 15 (12) 7 (5.6) 22 (17.6) 15 (6.6) 7 (3.1) 22 (9.7)
[2–5 years 46 (45.5) 16 (15.8) 62 (61.4) 0.72 33 (26.4) 23 (18.4) 56 (44.8) 0.24 79 (35) 39 (17.2) 118 (52.2) 0.34
[5–12 years 28 (27.7) 9 (8.9) 37 (36.6) 18 (14.4) 24 (19.2) 42 (33.6) 46 (20.4) 33 (14.6) 79 (35)
[12–16 years 1 (0.9) 1 (0.9) 2 (1.8) 3 (2.4) 2 (1.6) 5 (4) 4 (1.7) 3 (1.3) 7 (3)
Gender
Female 41 (40.6) 14 (13.8) 55 (54.4) 31 (24.8) 23 (18.4) 54 (43.2) 72 (31.85) 37 (16.3) 109 (48.2)
Male 34(33.6) 12 (11.9) 46 (45.5) 1.00 38(30.4) 33(26.4) 71 (56.8) 0.77 72(31.85) 45 (20) 117 (51.8) 0.49
Residence
Urban 27(26.7) 12 (11.9) 39 (38.6) 0.36 35 (28) 22 (17.6) 57 (45.6) 0.21 62 (27.3) 34 (15) 96 (42.4) 0.89
Rural 48 (47.5) 14 (13.9) 62 (61.4) 34 (27.2) 34 (27.2) 68 (54.4) 82 (36.3) 48 (21.3) 130 (57.6)
Water source
No 71 (70.3) 25 (24.8) 96 (95) 1.0 67 (53.6) 52 (41.6) 119 (95.2) 0.41 138 (61.1) 77 (34.1) 215 (95.1) 0.53
Yes 4 (4%) 1 (1%) 5 (5) 2 (1.6) 4 (3.2) 6 (4.8) 6 (2.7) 5 (2.2) 11 (4.9)
Animal at the house
No 72 (71.3) 24 (23.8) 96 (95.1) 68 (54.4) 54 (43.2) 122 (97.6) 140 (62) 78 (34.6) 218 (96.5)
Yes 3 (3%) 2 (2%) 5 (5%) 0.6 1 (0.8) 2 (1.6) 3 (2.4) 0.59 4 (1.7) 4 (1.7) 8 (3.5) 0.47
Feeding
Milk
Fresh 68 (67.3) 20 (19.8) 88 (87.1) 19 (15.2) 18 (14.4) 37 (29.6) 87 (38.5) 38 (16.8) 125 (55.3)
Canned 3 (3%) 2 (2%) 5 (5%0 12 (9.6) 20 (16) 32 (25.6) 15 (6.6) 22(9.7) 37(16.3) 0.0001*
Breast 3 (3%) 1 (0.9) 4 (3.9) 0.110 26 (20.8) 6 (4.8) 32 (25.6) 0.002* 29 (12.8) 7 (3.1) 36 (15.9)
Pasteurized 0 0 0 5 (4) 1 (0.8) 6 (4.8) 5 (2.2) 1(0.4) 6 (2.6)
Not 1 (0.9) 3 (3%) 4 (3.9) 7 (5.6) 11 (8.8) 18 (14.4) 8 (3.5) 14 (6.2) 22 (9.7)
202 J Parasit Dis (Apr-June 2019) 43(2):198–208
123

Table 3 Associated clinical symptoms and immunity status among study population
Non-diarrhoeic Diarrhoeic All study individuals
H. pylori negative H. pylori positive Total P. value H. pylori negative H. pylori positive Total P. value H. pylori negative H. pylori positive Total P. value
Diarrhea
Yes 0 0 0 No
a
69 (55.2) 56 (44.8) 125 (100) No
a
69 (55.2) 56 (44.8) 125 (100) 0.003*
No 75(74.3) 26(25.7) 101(100) 0 0 0 75 (74.3) 26 (25.7) 101 (100)
Vomiting
Yes 4 (3.96) 5 (4.95) 9 (8.9) 0.047* 17 (13.6) 29 (23.2) 46 (36.8) 0.003* 21 (9.3) 34 (15) 55 (24.3) 0.0001*
No 71 (70.3) 21 (20.8) 92(91.1) 52(41.6) 27(21.6) 79 (63.2) 123 (54.4) 48 (21.2) 171 (65.6)
Fever
Yes 3 (3) 8 (7.9) (10.9) 15 (12) 10 (8) 25 (20) 18 (8) 18 (8) 36 (15.9)
No 72 (71.3) 18 (17.8) 90 (89.1) 0.001* 54 (43.2) 46 (36.8) 100 (69) 0.657 126 (55.8) 64 (28.3) 190 (84.1) 0.088
Abdominal pain
Yes 64 (63.4) 23 (22.8) 87 (86.1) 1.000 60 (48) 54 (43.2) 114 (91.2) 124 (54.9) 77 (34.1) 201 (88.9) 0.081
No 11 (10.9) 3 (3%) 14 (13.9) 9 (7.2) 2 (1.6) 11 (8.8) 0.109 20 (8.8) 5 (2.2) 25 (11.1)
Dehydration
Yes 5 (5%) 8 (7.9) 13 (12.9) 10 (8) 1 (0.8) 11 (8.8) 15 (6.6) 9 (4) 24 (10.6) 1.000
No 70 (69.3) 18 (17.8) 88 (87.1) 0.004* 59 (47.2) 55 (44) 114 (91.2) 0.022 129 (57.1) 73 (32.3) 202 (89.4)
Alternating constipation
Yes 3 (3) 2 (2%) 5 (5%) 3 (2.4) 3 (2.4) 6 (4.8) 6 (2.7) 5 (2.2) 11 (4.9) 0.533
No 72 (71.3) 24 (23.8) 96 (95) 0.601 66 (52.8) 53 (42.4) 119 (95.2) 1.000 138 (61.1) 77 (34.1) 215 (95.1)
StatusImmuno
Immuno-competent 75 (74.3) 26 (25.7) 101 (100) No
a
46 (36.8) 31 (24.8) 77 (61.6) 0.20 121 (53.6) 57 (25.2) 178 (78.8) 0.02*
Immuno-
compromised
0 0 0 23 (18.4) 25 (20) 48 (38.4) 23 (10.2) 25 (11) 48 (21.2)
Total 75 (74.3) 26 (25.7) 101 (100) 69 (55.2) 56 (44.8) 125 (100) 144 (63.7) 82 (36.3) 226 (100)
J Parasit Dis (Apr-June 2019) 43(2):198–208 203
123

existent studies had different objectives and non-conclu-
sive outcomes.
Both H. pylori and intestinal parasites colonize the
human gastrointestinal tract and are the most common
childhood infections (Torres et al. 2003; Moreira et al.
2005; Marini et al. 2007; Zeyrek et al. 2008; Escobar-Pardo
et al. 2011; Sabah et al. 2015). There was a 28.6%
prevalence of intestinal parasitic infections in our study
populations, predominantly anthroponotic Cryptosporid-
ium and G. intestinalis, of which 43.9% co-existed with H.
pylori with statistical significance (pvalue, 0.02 and 0.05,
respectively).
Our study revealed that more than half of cryptosporid-
iosis (60%) and/or giardiasis (58%) cases coexisted and
showed a duplicated risk for H. pylori (O.R 2.9 and 2.6,
respectively) with statistical significance. Escobar-Pardo
et al. (2011) and Moreira et al. (2005)reportedanassocia-
tion between detection of G. intestinalis microscopically and
H. pylori with two different method Elisa to determine anti-
H. pylori IgG antibodies and using the
13
C urea breath test
among children. To our knowledge, the present study is the
first study to include Cryptosporidium protozoa in associa-
tion with H. pylori using molecular assays.
Co-existence of H. pylori and intestinal parasites mostly
occur in low income developing countries and may be
linked mechanically or pathologically. H. pylori shares the
associated gastrointestinal symptoms of intestinal parasites
and shares the same mode of transmission. This may sug-
gest the association of H. pylori infection with markers of
fecal exposure.
This hypothesis may be supported by our findings of a sta-
tistically significant association between presence of H. pylori
and polyparasitism of intestinal parasites in diarrheic children.
Polyparasitism may increase human susceptibility to H. pylori
and other intestinal microbial infections. Both H. pylori and
gastrointestinal parasites share thesameestimatedriskfactors,
including poor sanitation and hygiene, low socioeconomic
conditions and overcrowded populations (Cheng et al. 2009).
These factors affect the dynamics of pathogen transmission and
are the main drivers of the seasonal distribution of infectious
enteric diseases (Lal et al. 2012).
In addition, H. pylori may support Cryptosporidium spp.
and G. intestinalis colonization in human gastrointestinal
tract by producing urease enzyme to overcome gastric
acidity (Suerbaum and Michetti 2002; David and William
2006; Rodriguez et al. 2011). On the other hand, gas-
trointestinal parasitic infection may affect inflammatory
response to H. pylori (Whary et al. 2011). This significant
co-existence may suggest that H. pylori could be a risk
factor for intestinal parasitic infection or vice versa, which
still needs further investigations. Co-existence of H. pylori
and intestinal parasites might interact synergistically
leading to serious health consequences which could be
Table 4 Associated parasites with H. pylori colonization among study individuals
Non-Diarrhoeic Diarrhoeic All study individuals
H. pylori
negative
H. pylori
positive
Total P. value H. pylori
negative
H. pylori
positive
Total P. value H. pylori
negative
H. pylori
positive
Total P. value
Microscopy
G. intestinalis 5 (5.0) 5 (5.0) 10 (9.9) 0.12 3 (2.4) 6 (4.8) 9 (7.2) 0.297 8 (3.5) 11 (4.9) 19 (8.4) 0.05*
Hymenolepis nana 000No
a
0 3 (2.4) 3 (2.4) 0.252 0 3 (1.3) 3 (1.3) 0.56
Entrobius vermicularis 000No
a
0 2 (1.6) 2 (1.6) 0.119 0 2 (0.9) 2 (0.9) 0.13
E. histolytica complex 1 (1.0) 1 (1.0) 2 (2.0) 0.45 3 (2.4) 3 (2.4) 6 (4.8) 1.000 4 (1.8) 4 (1.8) 8 (3.5) 0.46
Blastocystis spp. 4 (4.0) 2 (2.0) 6 (6.0) 0.65 2 (1.6) 2 (1.6) 4 (3.2) 1.000 6 (2.7) 4 (1.8) 10 (4.4) 1.00
Cryptosporidium spp.
by PCR
000No
a
8 (6.4) 12 (9.6) 20 (16) 0.107 8 (3.5) 12 (5.3) 20 (8.8) 0.02*
Total 10 (9.9) 8 (7.9) 18 (17.8) – 16 (12.8) 28 (22.8) 44 (35.2) – 26 (11.5) 36 (15.9) 62 (27.4) –
No parasite 65 (64.4) 18 (17.8) 83 (82.2) 0.07 53 (42.4) 28 (22.8) 81 (64.8) – 118 (52.2) 46 (20.4) 164 (72.6) –
Total 75 (74.3)c 26 (25.7) 101 (100) – 69 (55.2) 56 (44.8) 125 (100) – 144 (63.7) 82 (36.3) 226 (100) –
204 J Parasit Dis (Apr-June 2019) 43(2):198–208
123

influenced by hosts and environmental factors (Torres et al.
2003; Marini et al. 2007).
Intestinal parasites and H. pylori colonized more than
half of the stool samples collected from diarrheic children
with statistical significance. Though many pathogens such
as bacteria, viruses, and intestinal parasites can cause
diarrhea, a large proportion of cases is caused by parasitic
protozoan (Kotloff et al. 2013). Diarrhea is currently con-
sidered the second cause of death in children during the
first 5 years of life; rotavirus being the most deadly
infectious agent, followed by Cryptosporidium (Striepen
2013; Vos et al. 2016). We classified our study population
into diarrheic and non-diarrheic groups of children. Diar-
rhea represented 55.3% of the total study population. G.
intestinalis,Cryptosporidium spp and E. histolytica are
known to be the most prevalent protozoan parasites that
cause acute diarrhoeal disease in children (WGO 2012),
they were also the most prevailing parasites in our study
populations (Table 4).
Although a previous study reported that infection with
H. pylori had a protective role in reducing frequency of
diarrhoeal illness in children (Chen et al. 2003), in our
study, there was a higher H. pylori prevalence in diarrheic
children (44.8%) than non-diarrheic children (25.7%). This
may be due to co-infection with intestinal protozoa (Bhan
et al. 2000).
Cryptosporidium spp. was one of the top diarrhea
associated pathogens in children (Kotloff et al. 2013).
Cryptosporidium is the second most common organism
causing diarrhea and death in children, with a higher death
rate in immunocompromised than immunocompetent
patients (Sow et al. 2016). In our study, Cryptosporidium
was the most prevailing parasite with a predominance of C.
hominis species, which agrees with the result of other
studies in Egypt (Abd El Kader et al. 2012; Helmy et al.
2013; El-Badry et al. 2015).
Similarly, G. intestinalis is a common protozoan para-
site causing diarrhea worldwide (Einarsson et al. 2016).
Based upon the microscopic examination, G. intestinalis
was the second most common parasite in the present study;
however, if the molecular method had been used, it might
have revealed a higher prevalence.
Helicobacter pylori was associated with vomiting with
statistical significance in both diarrheic and non-diarrheic
children. Fever and dehydration were statistically signifi-
cant symptoms in non- diarrheic children and could be
Table 5 Cases showed polyparasitism
Case 1 Case 2 Case 3 Case 4 Case 5 Case 6
H. pylori ?????-
Cryptosporidium spp. ?????-
G. intestinalis ??----
E. histolytica complex ----??
Blastocystis spp. ----??
Entrobius vermicularis --??--
Table 6 Multivariate analysis for nPCR H. pylori positive cases
OR 95% CI Pvalue*
Immunity
Immunocompetent/immunocompromised
All study group 2.3 (1.2–4.4) 0.017*
Diarrhoea
Yes/no
All study group 2.3 (1.3–4.1) 0.003*
Non-diarrhoeic group 4.2 (1.0–17.2) 0.047*
Associated symptoms
Vomiting
Yes/no
Diarrhoeic group 3.3 (1.5–7.0) 0.003*
All study group 4.1 (2.2–7.9) 0.0001*
Fever
Yes/no
Non-diarrhoeic group 10.7 (2.6–44.3) 0.001*
Dehydration
Yes/no
Non-diarrhoeic group 6.2 (1.8–21.3) 0.004*
Associated parasitic infection
G. intestinalis
Yes/no
All study group 2.6 (1.0–6.8) 0.048*
Cryptosporidium spp
Yes/no
All study group 2.9 (1.1–7.5) 0.02*
Polyparasitism
Yes/no
Diarrhoeic group 2.1 (1.3–4.2) 0.01*
Data presented as n, with (*) Pvalue for OR \0.05 is significant
J Parasit Dis (Apr-June 2019) 43(2):198–208 205
123

predictors for suspecting H. pylori in these patients. This
finding agrees with Jacoby and Porter (1999)and Shahinian
et al. (2000).
Many socio-behavioral, demographic and environmental
variables in association with H. pylori were previously
studied with controversial results (Moayyedi et al. 2002;
Rodrigues et al. 2004; Tanih and Ndip 2013). Our study
results showed no significant association between age,
gender, residency, and source of drinking water, however
consumption of raw animal (cow, goat, and sheep) milk
was linked as one of the major sources of H. pylori
infection (Vale and Vitor 2010). Drinking milk in our study
was significantly associated with the presence of H. pylori
in the stool; however, after being subjected to multivariate
analysis by the logistic regression test, consuming milk was
not estimated for the presence of H. pylori in children’s
stool.
Conclusion
Our results documented significant association of H. pylori
with G. intestinals and Cryptosporidium species. This co-
existence may suggest the association of H. pylori infection
with markers of fecal exposure. Furthermore, our study
documented the circannual pattern of H. pylori seasonality
in Egyptian children. Our findings would indicate that in
addition to searching for H. pylori in gastrointestinal
symptomatic children, screening for cryptosporidiosis and
giardiasis in diarrheic children is recommended.
Helicobacter pylori may support the colonization by
intestinal parasites or vice versa. The interaction between
H. pylori and intestinal parasites may have serious health
consequences. This point needs further investigations with
an emphasis upon determining correlation with gut
microbiomes. The findings of the present study provide a
better understanding of the epidemiology and the estimated
risks of H. pylori infection when associated with intestinal
parasites. Further research is needed to provide better
insight into their co-infection and ensure future improve-
ments in clinical practice, testing, and development of
therapies to these pathogens.
Authors contribution AI: corresponding author, participate in all
stages from study design to manuscript writing and revision, YBMA:
participate in study design and manuscript revision); AA-A provide
technical help; AAE-B: participated in Study design, supervised the
lab work, analysis and interpretation of data and involved in drafting
the manuscript.
Funding This research was self-funded and did not receive any
grants from any funding agency.
Compliance with ethical standards
Conflict of interest The authors have declared that no competing
interest exists.
Ethical approval Ethical board of University of Sadat City, Genetic
Engineering, and Biotechnology Research Institute, Egypt approved
the study. Parents of all the children included in the study were
verbally informed about the study’s aims, and collection of the
specimens was done after their consent was obtained.
References
Abd El Kader NM, Blanco MA, Ali-Tammam M, El Ghaffar Abd,
Ael R, Osman A, El Sheikh N, Rubio JM, de Fuentes I (2012)
Detection of Cryptosporidium parvum and Cryptosporidium
hominis in human patients in Cairo, Egypt. Parasitol Res
110:161–166
Bhan MK, Bahl R, Sazawal S, Sinha A, Kumar R, Mahalanabis D,
Clemens JD (2000) Association between Helicobacter pylori
infection and increased risk of typhoid fever. J Infect Dis
186(12):1857–1860
Bhutta ZA, Das JK, Walker N, Rizvi A, Campbell H, Rudan I, Black
RE (2013) Interventions to address deaths from childhood
pneumonia and diarrhoea equitably: what works and at what
cost? Lancet 381:1417–1429
Brooker S, Kabatereine NB, Smith JL, Mupfasoni D, Mwanje MT,
Ndayishimiye O, Lwambo NJ, Mbotha D, Karanja P, Mwan-
dawiro C, Muchiri E, Clements AC, Bundy DA, Snow RW
(2009) An updated atlas of human helminth infections: the
example of East Africa. Int J Health Geogr 8:42
Chen TL, Chan CC, Chen HP, Fung CP, Lin CP, Chan WL, Liu CY
(2003) Clinical characteristics and endoscopic findings associ-
ated with Blastocystis hominis in healthy adults. Am J Trop Med
Hyg 69:213–216
Cheng H, Hu F, Zhang L, Yang G, Ma J, Hu J, Wang W, Gao W,
Dong X (2009) Prevalence of Helicobacter pylori infection and
identification of risk factors in rural and urban Beijing, China.
Helicobacter 14:128–133
Cheesbrough M (2006) District laboratory practices in tropical
countries. Part London Cambridge University Press, Cambridge,
pp 184–215
David TJ, William AP (2006) Markell and Voge’s medical parasitol-
ogy. 9th edn. Saunders Elsevier, New York
Einarsson E, Ma’ayeh S, Svard SG (2016) An up-date on giardia and
giardiasis. Curr Opin Microbiol 34:47–52.
https://doi.org/10.1016/j.mib.2016.07.019 PMID:27501461
El-Badry AA, Al-Antably AS, Hassan MA, Hanafy NA, Abu-Sarea
EY (2015) Molecular seasonal, age and gender distributions of
Cryptosporidium in diarrhoeic Egyptians: distinct endemicity.
Eur J Clin Microbiol Infect Dis 34:2447–2453
Escobar-Pardo ML, de Godoy AP, Machado RS, Rodrigues D,
Fagundes Neto U, Kawakami E (2011) Prevalence of Heli-
cobacter pylori infection and intestinal parasitosis in children of
the Xingu Indian Reservation. J Pediatria 11:87–105
Frenck RW Jr, Clemens J (2003) Helicobacter in the developing
world. Microbes Infect 5:705–713
Frenck RW Jr, Fathy HM, Sherif M, Mohran Z, El Mohammedy H,
Francis W, Rockabrand D, Mounir BI, Rozmajzl P, Frierson HF
(2006) Sensitivity and specificity of various tests for the
diagnosis of Helicobacter pylori in Egyptian children. Pediatrics
118:1195–1202
Gallo N, Zambon CF, Navaglia F, Basso D, Guariso G, Grazia Piva
M, Greco E, Mazza S, Fogar P, Rugge M, Di Mario F, Plebani M
(2003) Helicobacter pylori infection in children and adults: a
206 J Parasit Dis (Apr-June 2019) 43(2):198–208
123

single pathogen but a different pathology. Helicobacter
8(1):21–28
Garcia LS, Bruckner DA, Brewer TC, Shimizu RY (1983) Techniques
for the recovery and identification of Cryptosporidium oocysts
from stool specimens. J Clin Microbiol 18:185–190
Helmy YA, Krucken J, Nockler K, von Samson-Himmelstjerna G,
Zessin KH (2013) Molecular epidemiology of Cryptosporidium
in livestock animals and humans in the Ismailia province of
Egypt. Vet Parasitol 193(15–24):2
Jacoby EB, Porter KB (1999) Helicobacter pylori infection and
persistent hyperemesis gravidarum. Am J Perinatol 16:85–88
Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, Farag TH,
Panchalingam S, Wu Y, Sow SO, Sur D, Breiman RF, Faruque
AS, Zaidi AK, Saha D, Alonso PL, Tamboura B, Sanogo D,
Onwuchekwa U, Manna B, Ramamurthy T, Kanungo S,
Ochieng JB, Omore R, Oundo JO, Hossain A, Das SK, Ahmed
S, Qureshi S, Quadri F, Adegbola RA, Antonio M, Hossain MJ,
Akinsola A, Mandomando I, Nhampossa T, Aca
´cio S, Biswas
K, O’Reilly CE, Mintz ED, Berkeley LY, Muhsen K,
Sommerfelt H, Robins-Browne RM, Levine MM (2013)
Burden and aetiology of diarrhoeal disease in infants and
young children in developing countries (the Global Enteric
Multicenter Study, GEMS): a prospective, case–control study.
Lancet 382(9888):209–222.
https://doi.org/10.1016/S0140-6736(13)60844-2
Lal A, Hales S, French N, Baker MG (2012) Seasonality in human
zoo-notic enteric diseases: a systematic review. PLoS ONE
7(4):e31883
Mansour-Ghanaei F, Taefeh N, Joukar F, Besharati S, Naghipour M,
Nassiri R (2010) Recurrence of Helicobacter pylori infection
1 year after successful eradication: a prospective study in
Northern Iran. Med Sci Monitor 16(3):CR144–CR148
Marini E, Maldonado-Contreras AL, Cabras S, Hidalgo G, Buffa R,
Marin A, Floris G, Racugno W, Pericchi LR, Castellanos ME,
Gro
¨schl M, Blaser MJ, Domı
´nguez-Bello MG (2007) Helicobac-
ter pylori and intestinal parasites are not detrimental to the
nutritional status of Amerindians. Am J Trop Med Hyg
76:534–540
Mehmood A, Akram M, Shahab-uddin AA, Usmanghani K, Hannan
A, Mohiuddin E, Asif M (2010) Helicobacter pylori: an
introduction. Int J Appl Biol Pharm Technol 1(3):1337–1351
Moayyedi P, Anthony TR, Feltbower R, Duffett S, Crocombe W,
Braunholtz D, Richards ID, Dowell AC, Forman D, Leeds HELP
Study Group (2002) Relation of adult lifestyle and socioeco-
nomic factors to the prevalence of Helicobacter pylori infection.
Int J Epidemiol 31:624–631
Moreira ED Jr, Nassri VB, Santos RS, Matos JF, de Carvalho WA,
Silvani CS, Santana e Sant’ana C (2005) Association of
Helicobacter pylori infection and giardiasis: results from a study
of surrogate markers for fecal exposure among children. World J
Gastroenterol 11:2759–2763
Pedraza-Dı
´az S, Amar C, Nichols GL, McLauchlin J (2001) Nested
polymerase chain reaction for amplification of the Cryptosporid-
iumoocyst wall protein gene. Emerg Infect Dis 7:49–56
Rafeey M, Ghotaslou R, Nikvash S, Hafez AA (2007) Primary
resistance in Helicobacter pylori isolated in children from Iran.
J Infect Chemother 13:291–295 PMID: 17982716
Rajindrajith S, Devanarayana NM, de Silva HJ (2009) Helicobacter
pylori infection in children. Saudi J Gastroenterol 15(2):86–94
Raschka C, Schorr W, Koch HJ (1999) Is there seasonal periodicity in
the prevalence of Helicobacter pylori? Chronobiol Int
16(6):811–819
Rodrigues MN, Queiroz DM, Bezerra Filho JG, Pontes LK, Rodrigues
RT, Braga LL (2004) Prevalence of Helicobacter pylori
infection in children from an urban community in north-east
Brazil and risk factors for infection. Eur J Gastroenterol Hepatol
16(2):201–205
Rodriguez OL, Ortiz-Princz D, Cavazza ME, Lo
´pez E, Hagel I (2011)
Evaluation of the possible association between the presence of
intestinal parasites and Helicobacter pylori in children of ethnic
Warao Venezuela. Bol Mal Salud Amb 11:41–50
Sabah AA, Gneidy MR, Saleh NM (2015) Prevalence of Helicobacter
pylori infection among adult patients with different gastrointestinal
parasites in Tanta City district. J Egypt Soc Parasitol 45:101–106
Salih BA (2009) Helicobacter pylori infection in developing coun-
tries: the burden for how long? Saudi J Gastroenterol
15(3):201–207
Sasaki K, Tajiri Y, Sata M, Fujii Y, Matsubara F, Zhao M, Shimizu S,
Toyonaga A, Tanikawa K (1999) Helicobacter pylori in the
natural environment. Scand J Infect Dis 31:275–279
Savarino V, Mela GS, Zentilin P, Lapertosa G, Cutela P, Mele MR,
Mansi C, Dallorto E, Vassallo A, Celle G (1992) Are duodenal
ulcer seasonal fluctuations paralleled by seasonal changes in
24-hour gastric acidity and Helicobacter pylori infection? J Clin
Gastroenterol 22(3):178–181
Sethi A, Chaudhuri M, Kelly L, Hopman W (2013) Prevalence of
Helicobacter pylori in a first nations population in northwestern
Ontario. Can Fam Physician 59(4):e182–187
Shahinian ML, Passaro DJ, Swerdlow DL, Mintz ED, Rodriguez M,
Parsonnet J (2000) Helicobacter pylori and epidemic Vibrio
cholerae O1 infection in Peru. Lancet 355:377–378
Sow SO, Muhsen K, Nasrin D, Blackwelder WC, Wu Y, Farag TH,
Panchalingam S, Sur D, Zaidi AK, Faruque AS, Saha D,
Adegbola R, Alonso PL, Breiman RF, Bassat Q, Tamboura B,
Sanogo D, Onwuchekwa U, Manna B, Ramamurthy T, Kanungo
S, Ahmed S, Qureshi S, Quadri F, Hossain A, Das SK, Antonio
M, Hossain MJ, Mandomando I, Nhampossa T, Aca
´cio S, Omore
R, Oundo JO, Ochieng JB, Mintz ED, O’Reilly CE, Berkeley
LY, Livio S, Tennant SM, Sommerfelt H, Nataro JP, Ziv-Baran
T, Robins-Browne RM, Mishcherkin V, Zhang J, Liu J, Houpt
ER, Kotloff KL, Levine MM (2016) The burden of cryp-
tosporidium diarrhoeal disease among children \24 months of
age in moderate/high mortality regions of Sub-Saharan Africa
and South Asia, utilizing data from the global enteric multicenter
study (GEMS). PLoS Negl Trop Dis 10(5):e0004729
Spano F, Putignani L, McLauchlin J, Casemore DP, Crisanti A (1997)
PCR-RFLP analysis of the Cryptosporidium oocysts wall protein
(COWP) gene discriminates between C. wrairi and C. parvum,
and between C. parvum isolates of human and animal origin.
FEMS Microbiol Lett 150:209–217
Striepen B (2013) Parasitic infections: time to tackle cryptosporid-
iosis. Nature 503:189–191
Suerbaum S, Michetti P (2002) Helicobacter pylori infection. N Engl
J Med 347(15):1175–1186
Tanih NF, Ndip RN (2013) A South African perspective on
Helicobacter pylori: prevalence, epidemiology and antimicrobial
chemotherapy. Review article. Afr J Microbiol Res
7(21):2430–2437
Thompson RCA, Ash A (2016) Molecular epidemiology of Giardia
and Cryptosporidium infections. Infect Genet Evol 40:315–323
Torres J, Perez GP, Ximenez C, Mun
˜oz L, Camorlinga-Ponce M,
Ramos F, Gomez A, Mun
˜oz O (2003) The association of
intestinal parasitosis and Helicobacter pylori infection in chil-
dren and adults from a Mexican community with high preva-
lence of parasitosis. Helicobacter 8:179–185
Vale FF, Vitor JM (2010) Transmission pathway of Helicobacter
pylori: does food play a role in rural and urban areas? Int J Food
Microbiol 138:1–12
Vos T, Allen C, Arora M, Barber RM, Bhutta ZA, Brown A, Carter A,
Casey DC, Charlson FJ, Chen AZ, Coggeshall M, Cornaby L
(2016) Global, regional, and national incidence, prevalence, and
J Parasit Dis (Apr-June 2019) 43(2):198–208 207
123

years lived with disability for 310 diseases and injuries,
1990–2015: a systematic analysis for the Global Burden of
Disease Study 2015. Lancet 388(10053):1545–1602
Whary MT, Taylor NS, Feng Y, Ge Z, Muthupalani S, Versalovic J,
Fox JG (2011) Lactobacillus reuteri promotes Helicobacte
hepaticus-associated typhlocolitis in gnotobiotic B6.129P2-
IL10(tm1Cgn) (IL-10(-/-)) mice. Immunology 133:165–178
Whitney AE, Guarner J, Hutwagner L, Gold BD (2000) Helicobacter
Pylori gastritis in children and adults: comparative histopatho-
logic study. Ann Diagn Pathol 4(5):279–285
World Gastroenterology Organisation (WGO) (2012) World gas-
troenterology organisation global guidelines: acute diarrhoea in
adults and children: a global perspective. WGO, Milwaukee
World Health Organization (WHO) (2017) Diarrhoeal diseases: WHO
fact sheet on diarrhoeal diseases. http://www.who.int/
news-room/fact-sheets/detail/diarrhoeal-disease. Accessed 15
Sept 2018
Yakoob J, Jafari W, Abid S, Tafari N, Hamid S, Shah HA, Rizvi L,
Islam M, Shaikh H (2005) Giardiasis in patient with dyspeptic
symptoms. World J Gastroenterol 11(42):6667–66705
Zamani M, Ebrahimtabar F, Miller WH, Alizadeh-Navaei R, Shokri-
Shirvani J, Derakhshan MH (2018) Review with meta-analysis:
the worldwide prevalence of Helicobacter pylori infection.
Aliment Pharmacol Ther 47:868–876
Zeyrek D, Zeyrek F, Cakmak A, Cekin A (2008) Association of
Helicobacter pylori and giardiasis in children with recurrent
abdominal pain. Turkiye Parazitol Derg 32:4–7
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
208 J Parasit Dis (Apr-June 2019) 43(2):198–208
123