Research paper about Detection of Pathogenic Strains of Entamoeba Histolytica in Children Using ELISA Technique in Duhok

1. Feb. 2011, Volume 8, No. 2 (Serial No. 75), pp. 109-119
Journal of US-China Medical Science, ISSN 1548-6648, USA
Detection of Pathogenic Strains of Entamoeba
Histolytica in Children Using ELISA Technique in Duhok
Waleed Jameel Omar Barwari1
and Shameeran Salman Ismael2
1. Department of Microbiology, College of Medicine, Duhok University, Kurdistan Region of Iraq, Iraq
2. Department of Microbiology, College of Veternary, Duhok University, Kurdistan Region of Iraq, Iraq
Abstract: As Entamoeba histolytica and Entamoeba dispar cannot be differentiated by routine microscope. This study represents the
commercially antibody and antigen detection ELISA kits to estimate the incidence of E. Histolytica and E. Dispar in addition to routine
microscopy. The aim of this study was to detect the pathogenic strains of E. Histolytica in children presented with diarrhea. A total of
800 stool samples were collected from children of different ages of both sexes from the Hevi Pediatric Hospital from (October 2009 to
April 2010). For each stool sample, three replicates were examined microscopically for the detection of E. Histolytica cysts and
trophozoites. The result showed that the infection rate was 15% (120). Clinical features of amoebiasis showed that 65.5% presented
with vomiting, while among negative cases only 12.5% were presented with vomiting and the remaining (87.5%) were normal, 34.5 of
positive cases were with bloody diarrhea, 41.6% with mucoid diarrhea and 23.8% with mucoid and bloody diarrhea. While none of the
negative cases showed bloody diarrhea. The age group of l-5 years showed the highest rate of infection (41.6%), while children aged
11-14 years showed the lowest rate of infection (14.16%). The infection rate in males was higher (60%) than in females (40%). A total
of 92 positive stool samples were selected and examined by ELISA test for the detection of E. Histolytica/E. Dispar antigen.
Eighty-four samples were positive for E. Histolytica/E. Dispar and eight samples were negative. Blood was collected from the same
children and examined by ELISA test for the detection of IgG in the serum, only ten samples were positive for E. Histolytica and 82
were negative. The sensitivity of ELISA/IgG and ELISA/Ag test in comparison to microscopic examination was 100 and 98.75%
respectively.
Key words: Pathogenic Strains, Entamoeba histolytica, ELISA antibody, ELISA antigen.
1. Introduction
Entamoeba histolytica is a protozoan parasite that
infects humans, causing amoebiasis [1]. Three stages
of the organism are encountered: The active amoeba
(trophozoite), the intermediate pre-cyst and the
inactive cyst. The most common from of extra
intestinal infection is the amoebic liver abscess [2].
There are two species of E. histolytica
morphologically identical but genetically distinct
organisms that have been reclassified by [3] based on
cumulative biochemical, immunological and genetic
data: E. histolytica which causes invasive intestinal
amebiasis and extra-intestinal amoebiasis and E. dispar
which is considered non-pathogenic and non-invasive
Corresponding author: Waleed Jameel Omar Barwari, PhD,
lecturer, research field: parasitology. E-mail:
waleed_68@yahoo.co.uk.
[4, 5]. Two parasitic diseases are commonly
transmitted by sexual contact, which are amebiasis and
giardiasis [6]. Study done by [7], suggested that
sexually transmitted amebiasis due to E. histolytica
occurs among sexually biased males.
Members of all age groups and both genders are
infected, but there is a higher prevalence of amoebiasis
among adult men [8]. The role of breast milk ingestion
in passive and active protection of infants is very
important, some reports showed that the incidence of
intestinal and systemic E. histolytica infections
decreased in human breast milk fed infants [9].
The most accurate method in diagnosis of
amoebiasis (Both intestinal and extra-intestinal), is to
identify E. histolytica either in stool or pus from liver.
Examination of E. histolytica is performed by using the
traditional microscope [10]. Several recent diagnostic

2. Detection of Pathogenic Strains of Entamoeba Histolytica in Children Using ELISA Technique in Duhok
110
tests are now available which surpass the microscopic
detection of these parasites and facilitate a more
accurate diagnosis. These approaches include PCR and
ELISA [11].
Recently, real-time PCR has proven to be the most
sensitive test for the detection of E. histolytica in stool
compared to the sensitivities of the traditional nested
PCR and ELISA. However, real-time PCR is
cumbersome for routine diagnosis because it requires
expensive equipment and specialized personnel for
analysis of the results. For this reason, antigen and
antibody detection by ELISA is becoming the standard
method for diagnosis of E. histolytica infection [12].
The aim of the present study was to detect the
pathogenic strains of E. histolytica in children
presented with diarrhea to Hevi Pediatric Hospital
outpatient clinic (Duhok City) with diarrhea. Due to
high rate of infection among children especially in
summer season, it is necessary to differentiate between
the pathogenic strains E. histolytica and non
pathogenic E. dispar, this study included the:
(1) Determination of the infection rate of E.
histolytica in children of different age groups and both
sexes;
(2) Differentiation of the pathogenic strains of E.
histolytica from non pathogenic one by ELISA test;
(3) Measuring the specificity and sensitivity of
ELISA technique for detection of amoebic antigen in
stool samples and amoebic antibody in serum, and
compare them with the results obtained by microscopic
diagnosis.
2. Materials and Methods
2.1 Stool Samples
During this study, 800 stool samples were
investigated, which were collected from children of
both sexes and different age groups, from age less than
one year (3 month to 12 month) to 14 years living in
Duhok city and some of nearby villages, who visited
outpatient clinic (Hevi Pediatric Hospital) Most of the
samples were collected from Hevi Pediatric Hospital,
in addition; some of these samples were from children
of known families to me living in Duhok City, the
number of samples examined as were follow:
Seven hundreds and sixty five from children visited
Hevi Pediatric Hospital, 35 from children of known
families
2.2 Collection of Stool Samples
From each child approximately 10 g of fresh stool
was taken using a wooden spatula ensuring that the
sample was not contaminated with urine or water in a
clean sterile screw disposable plastic container labeled
clearly with child name, gender, age, address and date
of collection. The collected stool samples were divided
into two parts: The first part of the specimen was
preserved at -20ºC to be used later, while the second
part was processed immediately for microscopic
examination.
2.3 Blood Samples
Initially 120 blood samples were collected from
infected children; a sample of blood consisting of 2
milliliters was obtained from anticubital and/or jugular
vein by a sterile disposable syringe from each child
including the control group.
The blood sample was poured into clean test tube
without anticoagulant and left for 2-3 minutes in water
bath (37ºC), then centrifuged at 3000 rpm for 6-10
minutes. The serum was separated and transferred to
label multiple clean eppendrof tubes with child full
information then stored at -20ºC until used.
2.4 Macroscopic Examination
The stool samples were examined with the naked
eyes (physical examination) before microscopical
examination, for color, consistency and the presence of
any adult helminthes, mucus and blood.
2.5 Microscopical Examination
All stools samples were examined microscopically
by direct method for the presence of E. histolytica
trophozoite and cyst stages as follows:

3. Detection of Pathogenic Strains of Entamoeba Histolytica in Children Using ELISA Technique in Duhok 111
Direct wet film preparation was performed for each
sample by using normal saline (0.85%) for detecting of
cyst and trophozoite. A drop of normal saline placed on
the center of the microscopic slide, then a small amount
(0.2 g) of formed stool from different sites was taken
by wooden stick or a drop of diarrheic stool and mixed
with a drop of normal saline very well until it become a
homogenous mixture, then covered by a cover slip and
examined. Using low power objective lens (10 X),
followed by high objective lenses (40 X), at least three
smears were examined for each sample [13, 14].
2.6 Enzyme-Linked Immunosorbent Assay (ELISA)
2.6.1 ELISA- Direct: Antibody Coated Plate
(1) Preparation of serum
The frozen samples which were stored at -20ºC or
lower if not used immediately were used, not heat to
inactivate serum and avoid repeated freezing and
thawing of the samples.
Test samples: Made a 1:64 dilution of patients’ sera
using the dilution buffer (5 μl sera and 315 μl dilution
buffer were added).
(2) Preparation of the test
Before use, all reagents, samples were allowed to
return to room temperature before starting the test run.
Prior to commencing the assay, the distribution and
identification plan for all samples and controls was
carefully established on a form supplied with the kit.
Selecting the required number of wells and inserting
them into the holder as follows:
• 2 well A1-A2 for the negative control;
• 2 well B1-B2 for the positive control and
• 92 wells for negative and positive samples.
This means, 8 (A10-H10) wells for negative and
other 84 for positive and suspected samples which were
examined three times by direct method and confirmed
that they were all negative from any intestinal parasitic
stage.
All wells were read at 450 nm.
Positive - Absorbence reading greater than 0.4 OD
units.
Negative - Absorbence reading less than 0.4 OD
units.
A positive OD reading indicates that the patient may
be infected by E. histolytica.
A negative OD reading indicates that the patient has
no detectable level of antibodies; this may be due to the
lack of infection or poor immune response by the
patient.
2.6.2 ELISA- Indirect: Antigen Coated Plate
(1) Principle of procedure
During the first incubation, E. histolytica antigens
present in the stool supernatant are captured by
antibodies attached to the wells. The second incubation
an additional anti-E.histolytica antibody added that
“sandwiches” the antigen. The next incubation adds an
anti-second antibody conjugated to peroxidase, after
washings to remove unbound enzyme, a chromogen
was added which develops a blue color in the presence
of the enzyme complex and peroxide, the stop solution
ends the reaction and turns to yellow.
(2) Preparation of stool samples
The stool samples kept at 2-8ºC and tested within 24
hours of collection, samples that cannot be tested
within this time were frozen at -20ºC until used,
freezing does not adversely affect the test, all dilutions
of stools were made with diluted wash buffer.
(3) Preparation of test
All reagents and samples were allowed to return to
room temperature before starting the test run. Prior to
commencing the assay, the distribution and
identification plan for all samples and controls was
carefully established on a form supplied with the kit.
Selecting the required number of wells and inserting
them into the holder as follows:
• 2 well H12-E12 for the negative control;
• 2 well F12-G12 for the positive control and
• 92 wells for negative and positive samples.
This means, 8 (A10-H10) wells for negative and
other 84(A1-D12) for positive and suspected samples
(plate). Results were read visually or at 450 nm.
Washings consist of vigorously filling each well to

4. Detection of Pathogenic Strains of Entamoeba Histolytica in Children Using ELISA Technique in Duhok
112
overflowing and decanting contents three separate
times, controls must be included each time the kit is run
may be indicated for those patients that are suspected
of being positive for E. histolytica.
All wells were read at 450 nm.
Positive - Absorbence reading of 0.15 OD units and
above indicates the sample contains E. histolytica
antigen.
Negative - Absorbence reading less than 0.15 OD
units indicates the sample does not contain detectable
levels of E. histolytica antigen.
A positive OD reading indicates that the patient may
be infected by E. histolytica.
2.7 Statistical Analysis
The results of this study were statistically analyzed
by Chi- square test (X²) [15].
Validity indicators used for the detection of antibody
sera and antigen.
The validity indications used for the detection of
antibody in sera and antigens in stool samples were
sensitivity, specificity, positive predictive value (PPV),
negative predictive value (NPV), and accuracy rate.
The indicators were calculated according to following
formula [16].
3. Results
During the present study 800 stool samples were
collected during the period from beginning of October,
Table 1 Kits used in this study, company and origin.
Origin
Company
Kit
N
o.
USA
Diagnostic
automation
ELISA- Direct; antibody
coated plate
1
USA
Diagnostic
automation
ELISA- Indirect; antigen
coated plate
2
2009 to end of April, 2010 from children of both sexes
and of ages from 3 months to 14 years. On the basis of
microscopic examination, 15% (120 cases) of the
samples were positive for amoebic infection, mixed
infection was also detected.
Table 2 shows the distribution of infection among
various age groups. It was obvious, that the highest rate
of infection occurred in the age group 1-5 year which
was 35.71% from the total samples examined for this
group and 41.66% from total number of infected
samples. The rate of the infection was lower in older
ages (6-10) and (11-14) years, since it was 12 and
15.45% respectively from the total number of samples
examined for these ages, while the lowest rate of
infection was in infants less than one year (8.75%).
Table 3 shows that among the infected children,
65.5% (55) presented with vomiting. While, among
negative cases one out of 8 (12.5%) gave history of
vomiting and 7 out of 8 (87.5%) were normal, 29
(34.5%) of the infected children were with bloody
diarrhea, while none of the negative cases showed
bloody diarrhea and 35 (41.6%) of the samples contain
mucous and 20 (23.8%) were with both blood and
mucus.
Table 4 shows the distribution of amoebiasis in both
sexes. The rate of infection in boys was higher (60%),
as compared to girls in whom the rate was (40%), and
this difference was statistically significant (P < 0.05).
Table 5 shows the relationship between the age and
sex of the children enrolled in this study. The rate of
infection with E. histolytica was higher in boys than in
the girls in the age groups (less than one year), (1-5)
years, and (6-10) years, which were 4.97%, 8.37% and
2.26% respectively. On the other hand, the rate of
infection was higher in girls than in boys in the age
Table 2 Age distribution of patients presenting to the Hevi Pediatric Hospital.
Age group No. positive case Positive case Total No. exam for each age group Positive from total infected cases
Less than one years 35 8.75% 400 29.16%
1-5 years 50 35.71% 140 41.66%
6-10 years 18 12% 150 15%
11-14 years 17 15.45% 110 14.16%
Total 120 15% 800 100%

5. Detection of Pathogenic Strains of Entamoeba Histolytica in Children Using ELISA Technique in Duhok 113
Table 3 Clinical features of the studied groups.
Characteristic Positive cases Positive cases Negative cases Negative cases
Vomiting 55 65.5% 1 12.%
Without vomiting 29 34.5% 7 88%
Bloody diarrhea 29 34.5% 0 0%
Mucoid 35 41.6% 0 0%
Mucoid and bloody diarrhea 20 23.8% 0 0%
Table 4 Distribution of Entamoeba histolytica among total examined children according to the sex in Duhok city.
Sex No.of examined stool No. of positive stool
Percentage of positive
stool
Percentage of infection among total cases
Boys 442 72 16.28% 60%
Girls 358 48 13.4% 40%
Total 800 120 15% 100%
X²= 1.29 D.F= 1, at (P < 0.05)*
*Significant difference
Table 5 The percentage of E. histolytica infection in relation to sex and age groups.
Age group No. infected male infected male No. infected female infected female Total
>1 22 4.97% 13 3.6% 35
1-5 37 8.37% 13 3.6% 50
6-10 10 2.26% 8 2.2% 18
11-14 3 0.67% 14 3.9% 17
Total 72 16.2% 48 13.4% 120
X²= 6 D.F= 3, at (P < 0.05)*
*Significant difference
group 11-14 years which was 2.5%. Statistical analysis
of the results revealed the presence of significant
differences (P < 0.05) in the rate of infection according
to age and sex between boys and girls.
Table 6 shows Cysts were found in most fecal
samples (66 cases), while trophozoites were presented
in 43 samples and only 11 cases were presented
samples.
It is obvious from Table 7 that most of the infections
were with single parasites (E. histolytica) constituting
79.2% (95 cases), while double infections (E.
histolytica with G. lamblia) constitute 20.8% (25
cases).
The results of ELISA/Ag of 92 stool samples, in
which 69 were positive for E. histolytica
microscopically, 15 suspected for E. histolytica and 8
were non infected (free from any parasites). The results
of ELISA/Ag revealed that all the positive samples by
microscope were positive by ELISA test except 5
positive samples by microscope appeared negative by
ELISA. Also all suspected samples by microscope for
E. histolytica appeared positive by ELISA and only one
sample appeared to be positive by ELISA test from the
8 examined negative samples.
Antigens to E. histolytica were presented in 98.7%
of stool samples of cases infected with this parasite
(Table 8). The over-all sensitivity of ELISA was 98.7%
and specificity was 58.3%. The predictive value of a
positive ELISA was 94% and the predictive value of a
negative ELISA was 87.5%. The diagnostic accuracy
for the presence of E. histolytica Ag in stools was
93.4%. ELISA has high sensitivity, low specificity,
high positive predictive value and low negative
predictive value in comparison with microscopy.
Table 9 indicates the presence of IgG to E.
histolytica in 10 serum samples from 86 of infected
cases with this parasite. The over-all sensitivity of
ELISA was 100% and specificity 9.8%. The predictive

6. Detection of Pathogenic Strains of Entamoeba Histolytica in Children Using ELISA Technique in Duhok
114
Table 6 Stage occurred from the studied groups.
Cyst and Trophozoite
Trophozoite
Cyst
No. positive case
Age group
0
11
24
35
Less than one years
7
21
22
50
1-5 years
2
8
8
18
6-10 years
2
3
12
17
11-14 years
11
43
66
120
Total
Table 7 Distribution of single infection with E. histolytica and double infection E.histolytica with G. lamblia.
Percentage of infection
No. of positive cases
Type of infection
79.2%
95
Single infection E. histolytica
20.8%
25
Double infection
(E.histolytica + G.lamblia)
100%
120
Total
Table 8 Comparison of stool samples examined by microscopy and ELISA (Antigen).
ELISA positive ELISA negative Total No
Microscopic positive
Microscopic negative
79
1
5
7
84
8
Total 80 12 92
Table 9 Comparison of stool samples by microscope and ELISA (IgG) from serum sample.
ELISA Positive ELISA Negative Total No
Microscopic positive
Microscopic negative
10
0
74
8
84
8
Total 10 82 92
value of a positive ELISA was 10 % and the predictive
value of a negative ELISA was 100%. The diagnostic
accuracy for the presence of E. histolytica antigen in
stools was 19.5%. ELISA has high sensitivity, low
specificity, low positive predictive value and high
negative predictive value in comparison with ELISA
test.
Fig. 1 shows the comparison between general stool
examination and ELISA test for diagnosis of E.
histolytica in both stool antigen and serum (IgG) .The
same sample used in ELISA test was examined for
general stool examination. The infection rate of
ELISA/Ag test was 91.3%, while that for IgG in serum
was 10.8%.
4. Discussion
Most of the studies which identified E. histolytica
infection in Kurdistan, Iraq were performed without
distinguishing between the two separate species, E.
histolytica and E. dispar. This is possibly due to the
inability, in the past, to differentiate E. histolytica from
the morphologically similar, but non-pathogenic
species E. dispar, as E. histolytica and E. dispar cannot
be differentiated by routine microscopy. Therefore,
there is an apparent need to carry out studies which can
distinguish the two species of Entamoeba, this
investigation represents the first attempt to use
commercially antigen detection kit/Antibody kit by
using ELISA technique to estimate the proportion of
infection between E. histolytica and E. dispar in Duhok
province in addition to the use of routine microscopy.
The results of the present study indicate that the rate
of infection with pathogenic strains of E. histolytica
among children in Duhok province was 15%. This rate
was higher than the rate recorded by [17] in Duhok
province in which he recorded a rate of 10.68%. On the

7. Detection of Pathogenic Strains of Entamoeba Histolytica in Children Using ELISA Technique in Duhok 115
0%
20%
40%
60%
80%
100%
Microscopic exam. ELISA/IgG ELISA/Ag
84
10
80
8
82
12
92 92 92
No.of positive cases No.of negative cases
Fig. 1 Comparison between general stool examination and examination by ELISA test for both IgG and antigen.
other hand this rate is lower than that mentioned in
other studies performed in Kurdistan region of Iraq,
such as Ref. [18] which recorded a rate of 31.6% in
Kalar town, Sulaimani province. A higher infection
rate 49.5% was reported by Ref. [19], who reported
that 60% of cases of bloody diarrhea were associated
with the presence of E. histolytica and by Ref. [20],
who recorded a rate of 17.6% in Basra. While much
lower rates than this rate was recorded in Kurdistan
region of Iraq also by Ref. [21] in Erbil and Ref. [17] in
Duhok, which they recorded rates of 2.33% and
10.15% respectively. This rate was higher than the
rates reported worldwide by Refs. [22, 23] in Ilorin,
Kwara, who reported rates of 5.2% and 6.3%
respectively. On the other hand a higher rate (34%) was
reported by Ref. [24] in Nigeria, than the rate in this
study and he further added that the variation in the
prevalence over time may be related to size of the
population under study, the number of stool specimens
examined per patient, and the duration of the study.
The definitive diagnosis of intestinal amoebiasis
depends on the history, clinical findings and detection
of cysts or trophozoits of the pathogen in the stool
samples. In this aspect it appears that the microscopic
examination of the stool specimens is not sufficient for
differentiating between E. histolytica and E. dispar.
Microscopic examination is based on trichrome or
iron hematoxylin staining of fresh stool preparations
[25]. Therefore, differentiation of E. histolytica and E.
dispar is not possible with this method, while some
researchers suggest that microscopic examination of
the stool is sufficient to diagnose E. histolytica in the
presence of characteristic microscopic findings [26].
The higher rate of infection in children aged 1-5
years recorded in this study as compared with other
ages (6-10 years) may be due to the fact that this age
group often spends more of their leisure time out doors,
playing or foraging in garbage dumps and eating
discarded food remains on the street. Also they are
more often in contact with sand and eat
indiscriminately with unwashed hands. In contrast the
low prevalence of infection in the children of the oldest
ages (11-14) years may be due to the fact that at this
age young children become more hygiene-conscious
about their looks as compared to the lower age group
and hence they are able to avoid contact as much as
possible to what will lead to get infection.
This is close to the results recorded at by Ref. [27] in
Diwania, which demonstrated the occurrence of
amoebiasis at a rate of 66% among 1-5 years of age.
Also this agrees with the result of [28], in which he
revealed that the rate of infection was the highest
(13.8%) in the age-group 1-5 years. While the results of
this study disagree with those of Refs. [29, 30] in

8. Detection of Pathogenic Strains of Entamoeba Histolytica in Children Using ELISA Technique in Duhok
116
which they stated that the population over the age of
five years had higher rate of amoebal infection as
compared to less than that of a five-year-old
population.
The distribution of E. histolytica among sex groups
showed that more males were infected than females,
this high prevalence associated with boys may be due
to the fact that they are more often engaged in
predisposing activities such as football, barefoot and
also playing in streams or ponds.
This agree with the findings of Refs. [31, 32], which
performed epidemiological study in Benue State-
Nigeria and 33 in Sharjah United Arab Emirates, in
other hand this disagree with finding of Ref. [34], they
stated that the infection rate in women was higher than
man. While other studies indicated that both sexes have
equal rate of infection [26, 29].
Most of the cases reported in this study were infected
with cyst stages, 66 cases with cyst; this may be due to
the acute infection converted to chronic infection. The
present results were close to the studies done in Ankara,
Turkey by Refs. [35, 36] in Thailand, who found that
cysts were found in most fecal samples.
Most of the infections (79.2%) with amoebiasis were
with single parasites (E. histolytica), whereas, infection
with double parasites (E. histolytica with G. lamblia)
were few (20.8%). Same pattern of infection was
observed by other investigators, such as Refs. [37-39]
in Duhok city and done by Ref. [40] in Erbil.
In the present study the ELISA/IgG test revealed a
sensitivity of 100% and the specificity of 9.8%.The
predictive value of a positive ELISA was 11.9% and a
predictive value of a negative ELISA was 100%. The
diagnostic accuracy for the presence of E. histolytica
IgG in serum samples was 19.5%.
The test had low specificity, because the kit used was
only for detection of E. histolytica and not for other
non-pathogenic strains, or the failure of the ELISA to
detect the IgG in other samples may indicate that either
the amount of antibody is below the sensitivity of the
assay, or the infection is acquired recently in which the
antibody response has not yet appeared, since IgG
appear after 7 days or more. Moreover, possibly false
positive microscopic examination may be due to
misidentification between E. histolytica and other
nonpathogenic amoebae, according to these results the
accuracy of this test is high for differentiation between
E. histolytica and E. dispar.
Similarly, Ref. [41] found a less sensitivity of
ELISA as compared to microscopical diagnosis,
whereas, higher sensitivity was reported by Ref. [42],
which found that, Entamoeba ELISA was more
sensitive and specific than microscopy. The study also
indicates that E.histolytica-specific ELISA showed to
be a sensitive and specific method for the rapid
differentiation of the two species.
ELISA/Antigen test was based on the detection of E.
histolytica antigen in stool samples. The
ELISA/Antigen test revealed a sensitivity of 98.7% and
the specificity of 58.3%. The predictive value of a
positive ELISA was 94% and a predictive value of a
negative ELISA was 87.5%. The diagnostic accuracy
for the presence of E. histolytica /E. dispar Ag in stools
was 93.4%. ELISA has high sensitivity, low specificity,
high positive predictive value and low negative
predictive value in comparison with microscopy. But
this test has no ability to differentiate between E.
histolytica and E. dispar.
Previous studies also mentioned the same findings
[10, 43], they stated that the specificity and sensitivity
of the assay for pathogenic E. histolytica were 97% and
100%, respectively. These preliminary data offer
promise for ELISA monoclonal antibodies (MAbs) to
the galactose adhesin as a rapid and sensitive means to
detect the presence of pathogenic E. histolytica
infection in stool specimens. Ref. [44] suggested that
ELISA/Ag currently may represent the most practical
method for the identification of E. histolytica in fecal
samples.
Similarly, in Ankara, Turkey, Ref. [45] used an
enzyme-linked immunosorbent assay (ELISA) and
observed that the microscopy has low sensitivity and

9. Detection of Pathogenic Strains of Entamoeba Histolytica in Children Using ELISA Technique in Duhok 117
high specificity, low negative predictive value and high
positive predictive value in comparison with
microscopy. E. histolytica/E. dispar antigen detection
by ELISA tests are inexpensive as compared to the
specific tests, antigen detection appears to be a more
reliable and specific diagnostic method for amoebic
liver abscess [46].
E. histolytica-specific ELISA was shown to be a
sensitive and specific method for the rapid
differentiation of the two species of Entamoeba
because its results are comparable to those obtained
with the PCR [47].
5. Conclusions
From this study the following points are concluded:
The infection rate with E. histolytica was 15%
among children enrolled in this study.
Children of both sexes and different ages were
susceptible to E. histolytica infection and E. histolytica
was more prevalence in males than females, and in
young ages (1-5 years).
Giardia lablia was found to be associated with E.
histolytica infection.
ELISA test for detection of E. histolytica antigen in
stool samples was highly sensitive and specific as
compared to microscopy.
ELISA/Immuno-globulin G (IgG) test for
differentiating E. histolytica from non pathogenic
strains E. dispar was more highly sensitive than
ELISA/Antigen test.
6. Recommendations
From the previous conclusions, the following points
are recommended:
Advice the mothers to breast feed their babies, as
colostrum and human milk have lethal effect on E.
histolytica and protect the baby from infection, in
addition this method is more hygienic.
Educating parents to improve the hygiene of their
children and its role in prevention of infection.
Periodical examination of water sources is
recommended to exclude the contamination with E.
histolytica cyst and other infectious agents.
It was necessary to examine more than one
preparation of slides from each stool sample to get
more accurate diagnosis.
Examination of blood samples of infected children
after seven days from infection is more accurate for the
appearance of IgG in the serum samples.
Using PCR-based diagnostic methods to distinguish
between E. histolytica and other non pathogenic
Entamoeba species.
Epidemiological studies are needed to know the
prevalence and incidence of infection with various
pathogenic microorganisms in various parts of
Kurdistan.
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