OPEN ACCESS Journal of Biological Sciences

ISSN 1727-3048
DOI: 10.3923/jbs.2019.148.154

Research Article
Identification of Serious Clinical Amebic Dysentery Cases in the
Middle Euphrates Region of Iraq
1
Hadi Fadhil Alyasari, 2Hayder O. Hashim, 2Alaa Hamady Obeid Altaei and 3Mohammed Baqur S. Al-
Shuhaib

1
College of Medicine, Babylon University, Babil 51001, Iraq
2
Department of Clinical Laboratory Sciences, College of Pharmacy, University of Babylon, Babil 51001, Iraq
3
Department of Animal Production, College of Agriculture, Al-Qasim Green University, Al-Qasim, Babil 51001, Iraq

Abstract
Background and Objective: A lot of enteric parasites are responsible for causing morbidity and mortality outcomes in the worldwide
individuals, especially in poor hygienic countries. The present study was conducted to detect amebic dysentery-causing Entamoeba
species in the middle Euphrates regions in Iraq. Materials and Methods: A total of 155 diarrhea admitted-females (aged from 10-70 years
old) who underwent parasitological examination were included in the study. After its classical confirmation, the presence of amebic
dysentery was detected by PCR. Results: It was found that the overall prevalence of amoebic species infection in the infected females
was 62% (96/155). Comparison of age groups showed that 30-39 aged females had a more susceptibility rate than other age groups since
the highest levels of amoebic infection were shown in the 30-39 aged females. Three forms of amoebic infection were observed, including
E. histolytica, E. dispar and E. coli, which, as long as other painful clinical symptoms were concentrated in rural and low educated level
of the studied areas. Conclusion: This pilot present study discovered a remarkable percentage of amoebic dysentery infection in the
females aged 30-39 years old, which may imply serious precautions for this group throughout the developing world.

Key words: Dysentery, PCR, bloody diarrhea, Entamoeba species, enteric parasites, amebic dysentery, co-infection

Received: October 19, 2018 Accepted: November 23, 2018 Published: January 15, 2019

Citation: Hadi Fadhil Alyasari, Hayder O. Hashim, Alaa Hamady Obeid Altaei and Mohammed Baqur S. Al-Shuhaib, 2019. Identification of serious clinical
amebic dysentery cases in the middle Euphrates region of Iraq. J. Biol. Sci., 19: 148-154.

Corresponding Author: Mohammed Baqur S. Al-Shuhaib, Department of animal production, College of agriculture, Al-Qasim Green University,
Al-Qasim, Babil 51001, Iraq Tel: 009647707115693

Copyright: © 2019 Hadi Fadhil Alyasari et al. This is an open access article distributed under the terms of the creative commons attribution License, which
permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

Competing Interest: The authors have declared that no competing interest exists.

Data Availability: All relevant data are within the paper and its supporting information files.
 J. Biol. Sci., 19 (2): 148-154, 2019

INTRODUCTION widely available nowadays to differentiate between extremely

related micro-organisms, such as enzyme-linked
Enteric parasites are responsible for causing intestinal immunosorbent assay (ELISA) and polymerase chain reaction
disorders, various morbidities and mortalities among a lot (PCR)12. However, several recent diagnostic methods for
number of individuals worldwide, especially in low-income E. histolytica in stool samples are costly because they need a
countries and in low hygienic levels and a large number of long time and outclass microscope experts for detection14.
infectious diseases can be transmitted by many and various However, PCR specificity is usually greater than that of other
contamination routes and eventually reach to human beings techniques, such as ELISA, which command isolation or
and their own animals to cause effective diseases1. The antigen capture procedures. The PCR tool still the method of
protozoan parasite Entamoeba histolytica is one of the main choice particularly in the low budget laboratories13,15. So that,
alimentary tract protozoa of human beings2. There area variety PCR is a highly discriminating technique between the
of species pertaining to the genus Entamoeba and all these pathogenic Entamoeba species from that of non-pathogenic
species can infect human's alimentary tract, including counterpart sinan accurate diagnosis for the causative agent
Entamoeba histolytica, Entamoeba coli, Entamoeba hartmani, of dysenteric amoebiasis. Though PCR techniques have been
Entamoeba dispar, Entamoeba gingivalis, and Entamoeba widely been used in the accurate identification of such
polecki 3. The Entamoeba histolytica caused amoebiasis, organisms, it s still limited in detecting the age of the assessed
which is a global health problem as it is responsible for more samples. On the other hand, though several reports have
than 100,000 deaths per year and it is the second leading measured several pathogenic Entamoeba species by
cause of global death due to protozoa after malaria4,5. implementing several infections-contributing factors, no
Entamoeba histolytica is an enteric protozoa parasite that lives sufficient data were available, in terms of dividing the accurate
as a colonizer in the lumen of the human intestinal tract and grouping of amoebic-infected patients6-8,16,. Therefore, the
has the ability to destroy the epithelium tissue. Most present manuscript aimed to combine the high accuracy of
frequently observed clinical symptoms of this disease are PCR detective power, with further details of the age of
dysenteric diarrhea, alternating with periods of constipation, patients, to provide an accurate time management of patients
flatulent stomach, colitis, fatigue, fever, malaise, abdominal ages regarding it as an essential contributing factor to assess
pain and weight loss6. Extra-intestinal infection of amoebiasis the susceptibility of the infected females age to develop such
is taken place by parasite when invading other organs, such as amoebic dysenteries.
lung, bone and brain to cause abscesses in these invaded
organs7. Thus, several experimental methods have been used MATERIALS AND METHODS
to obtain accurate and reliable diagnostic outcomes for
amoebiasis detection3,7-9. The diagnosis of enteric amoebiasis Study population and sampling: A cross-sectional study was
is primarily based on the detection of parasites in the smear, conducted from March 2014 to September 2016 and enrolling
but in the case of epidemiological complication by 155 amoebic dysentery-suspected females, aged from 10 to
E. histolytica that accompanied with other amoebic species or 70>years, who attended to the Al-Zahraa Hospital for
enteric parasite, the claimed infection may be fully realized Maternity and Children in Al-Musaib district/Babel city, Iraq.
only if there is an accurate method that can distinguish the The number of samples was155 of fresh stool. Out of 155
targeted species among all other closely relatedorganisms10. samples, 95 were confirmed to have amoebic dysentery on the
This notion is particularly applicable when great numbers of basis of a previous SAF-fixed sample using conventional
related organisms are encountered in the smear8,11,12. Though techniques, including the formalin-ethyl acetate
the preliminary diagnosis of this parasite relied on the concentration and iron hematoxylin staining17. All the
detection of cysts and trophozoites of E. histolytica with procedures used in the present study received prior approval
a light microscopy-based technique4, such general from the Committee on the use of Human Research Subjects
identifications are no longer reliable as it s so difficult to at University of Babylon. Oral consent was obtained from all
distinguish between two identical morphologies of two participants of the present study. All admitted infected
closely related organisms, as in the case of pathogenic females were itemized in specialized questionnaire papers.
E. histolytica and non-pathogenic E. dispar. Thus, the genetic Most clinical symptoms and signs of the study infected
option is the best applicable tool to differentiate between the females were reconfirmed by the aid of the specialist clinicians
closely related parasites that live in the same intestinal habit13. in the targeted study hospital and from all admitted hospital
Moreover, the highly specific molecular techniques have been patient s records. The samples were preserved in 5%

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potassium dichromate to avoid fungal growth and for the subjected to electrophoresis in 2% agarose gelsand visualized
preservation of protozoa cysts and oocysts. Subsequently, in a UV Transilluminator (ChemiDoc-Bio-Rad, USA).
cysts in stool samples were determined based on the
protocols4. Statistical analysis: Detection of Entamoeba species was
determined on the basis of morphological characteristics of
Extraction of Entamoeba genomic DNA: Genomic DNA was the cysts under microscopy. The data entry and analysis was
extracted from each microscopically positive faecal sample carried out using the SPSS software (Statistical Package for the
according to the manufacturers instructions (QIAGEN, Hilden, Social Sciences) program for Windows ver. 1718. Qualitative
Germany). The extracted DNA was quantified by a Nano drop data were estimated and presented as frequencies and
(BioDrop-UK), then stored at -20EC until performing PCR percentage. The prevalence and 95% confidence intervals (CIs)
amplification13. were calculated for each parasite according to Ngui et al.19.
The statistical percentages were estimated using Chi-Square
PCR: The PCR amplification was used to genetically and a p-value of 0.05 was regarded as an indicator for a
characterize E. histolytica and E. dispar according to Khairnar statistical significant differences in the analyzed samples.
9
and Parija , with several modifications. Both negative control
(DNase-free water, Sigma Cat. No. W4502), and positive RESULTS
control (Entamoeba species genomic DNA) were included in
each PCR run. The PCR was carried out in a 25 :L volume with The present study had shown a high infection ratio
the final mix containing 10×PCR buffer, 1.25 mM dNTPs, 25 caused by amoeba species to 30-39 years old (28%) and
mM MgCl2, 10 pmole of each primer, 2.5 U of Taq polymerase parallel low infection ratio (1%) at 70 years old (Table 1).
and 2.5 :L of DNA template. To detect E. histolytica (439 bp), Similarly, the amoebic infection had got the highest level
amplification was carried out using primer sets EH-1 (28%) in the age group 30-39 years old, while the low amoebic
(5 -AAGCATTGTTTCTAGATCTGAG-3 ) and EH-2 (5 - infection was at the age group of >70 years old (Table 2).
AAGAGGTCTAACCGAAATTAG-3 ). The samples were The current study has provided information about the
denatured at 96EC for 2 min, followed by 30 cycles of 92EC for clinical symptoms and signs of the patients-confirmed
1 min (denaturing), 56EC for 1 min (annealing), 72EC for 1 min dysentery of Table 3. These results indicated a high level of
30 sec (extension) and a final extension at 72EC for 7 min abdominal pain (94.7%) and a parallel low level of clinical
regarding the detection of E. histolytica (439 bp), symptoms as sporadic constipation (8.3 %). On the other hand,
amplification was carried out using the primers set ED-1 the current study had obviously revealed a prevalence of
(5 -TCT AAT TTC GAT TAG AAC TCT-3 ) and ED-2 (5 - three types of Entamoeba parasite which are E. histolytica,
TCCCTACCTATTAGACATAGC-3 ). The PCR conditions were E. dispar and E. coli. Furthermore, the presently observed
performed with the following conditions; denaturation (95EC amoebic mono-infections had a higher percentage than the
for 1 min), annealing (48EC for 1 min) and extension (72EC for amoebic co-infection (Table 4). Moreover, the present study
1 min). In both amplifications, samples were incubated in a has provided further information regarding the residence of
thermal cycler (MyCycler, Bio-Rad, Hercules, USA). The patients that infected with amoebiasis, in which patients in
generated PCR amplicons of Entamoeba species were rural areas had shown a high level of parasitic infection

Table 1: Assortment of the overall parasitic infections among study patients according to their age groups
Patients Amoeba
age group group % G. lamblia % E. coli % H. nana % A. lumbricoides % Hook worms % Co-infection %
Overall parasitic infection
10->19 18 19 2 29 2 17 4 40 3 50 2 50 5 25
20->29 23 24 3 43 3 25 2 20 2 33.3 1 25 6 30
30->39 27 28 1 14 4 33.1 2 20 1 16.7 0 0 4 20
40->49 15 16 0 0 1 8.3 1 10 0 0 1 25 4 20
50->59 8 8 1 14 1 8.3 0 0 0 0 0 0 1 5
60->69 4 4 0 0 1 8.3 1 10 0 0 0 0 0 0
70> 1 1 0 0 0 0 0 0 0 0 0 0 0 0
Sum 96 100 7 100 12 100 10 100 6 100 4 100 20 100
Co- infection refers to infection caused by amoeba group associated with other of non-amoebic parasite

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Table 2: Assortment of the overall parasite infection among study patients according to the type of parasitic infection
Type of parasitic infection (Among 155 patients)
---------------------------------------------------------------------------- -------------------------------------------------------------------------------------------------
Amoebic species Non-amoebic species Amoebic co-infection
Patients-age groups (infected number) % (infected number) % (infected number) %
10->19 18 19 13 33.3 4 20
20->29 23 24 11 28.2 5 25
30->39 27 28 8 20.5 5 25
40->49 15 16 3 8 4 20
50->59 8 8 2 5 2 10
60->69 4 4 2 5 0 0
70> 1 1 0 0 0 0
Sum 96 100 39 100 20 100

Table 3: Presentation of clinical signs and symptoms among all studied patients Table 6: Assortment of amoebiasis according to the origin of patient s residences
Symptomized number Age group of Origin of residence
------------------------------------------------------------------------ Amoebiasis Total infected ---------------------------------------------------
Clinical signs Dysentery- Dysentery- infected females number Genuine % Novel %
and symptoms suspected patients % confirmed patients % 10->19 18 8 8.3 10 10.4
Bloody diarrhea 120 77.4 86 89.5 20->29 23 10 10.4 13 13.5
Fever 125 80.6 83 86.4 30->39 27 10 10.4 17 17.7
Headache 100 64.5 81 84.3 40->49 15 8 8.3 7 7.2
Vomiting 98 63.2 70 72.9 50->59 8 3 3.1 5 5.2
Malaise 107 69 70 94.7 60->69 4 2 2 2 2
Abdominal pain 140 90.3 91 94.7 70> 1 0 0 1 1
Hepatomegaly 41 26.4 38 39.5 Sum 96 41 42.7 55 57.3
Itching 32 20.6 21 21.8
Weight loss 89 57.4 60 62.5
Table 7: Assortment of amoebiasis according to the patient s educational level
Anemia 80 51.6 71 73.9
Amoebiasis Patients educational level
Anorexia 77 49.6 50 52
(Patients age Total infected ----------------------------------------------------------
Flatulence 42 27 22 22.9
groups) number High % Middle % Low %
Dehydration 113 72.9 89 92.7
10->19 96 20 20.8 46 47.9 30 31.3
Sporadic constipation 19 12.2 8 8.3
Abdominal dilation 16 10.3 9 9.3 20->29
Sum 155 100 96 62 30->39
40->49
Table 4: Assortment the type of amebic complex-co-infection by use of PCR 50->59
technique and microscopic examination 60->69
Type of amebic complex co-infection 70>
------------------------------------------------------------------------- Sum 100
Case numbers Case numbers
solely amebic mixed amebic Table 8: Assortment of amoebiasis according to patient s social levels
Amebic species infection % co-infection % Patients social level
E. histolytica 60 62.5 20 13 Amoebiasis ------------------------------------------------------------------------
E. dispar 27 28.1 infected number High % Middle % Low %
E. coli 9 9.4 96 14 14.6 32 33.4 50 52
Sum 96 100 Sum 100

Table 5: Assortment of amoebiasis according to the patient s residence

E. histolytica parasite (57.3%), while the genuine residence was
Patients residence
------------------------------------------------------------------- only 42.7% (Table 6). The current study had observed a
Amoebiasis Urban % Rural % significant effect of the educational level can affect infection
Dysentery-confirmed 30 31.2 66 68.8 levels since it was found that people at a high, intermediate,
Infected number (96)
Sum 100 and low educational level had 20.8, 47.9 and 31.3%,
respectively (Table 7). Furthermore, the present study had
(68.8%), while patients in urban areas have shown a low level observed that the social level had affected on the level of
of infection (31.2%) (Table 5). As well, the obtained results of infection, as the parasitic infection at a high social level was
the current study had revealed that a novel residence of the lower than its level in the middle and low social levels,
population had an effect on infection percentage with respectively (Table 8).

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DISCUSSION regular intestinal parasites screening in diarrhea suspected

females with amoebic dysentery. As well, our results of higher
The present study has identified remarkable levels of levels of amoebic mono-infections were adjacent to the
amoebic infections in the females aged between 30-39 years, results of Fotedar et al.4, who found that the ratio of mono-
while other aged groups have not attained such serious levels infection was 70.8 %, while only 61.8% of the co-infection was
of infections. In agreement with our results, it was found that observed in the same study. This result may be due to
adult had a significantly higher amoebic infection rate than E. histolytica when it has been recognized as a nurse at the
children19. However, several reasons may lie behind such first, while the ability of the other accompanying species, such
sensitivity, which may be attributed to the fact that this age as E. moshkovskii 29 and E. dispar 30, to cause the disease is
group is more likely to be engaged in many regular activities unclear. On the other hand, our results of higher infection
that might increase their exposure to amoebic infection ratios in rural areas may be attributed to the type of drinking
through undergoing more interaction with many water and the use of polluted water too. Virtually, the
contaminated subjects, such as soil, water and food20,21. In population/personal low hygienic level, improper use of
contrast to several related studies that had dealt with age as latrines and contact with contaminated articles in rural areas
a suggestive factor with such infections22,23, the specific age- compared to urban areas can bring about exhibiting such
grouping that conducted by this study has provided more preceded results26.
detailed information regarding the accurate effect of age of Our observation of the presence of higher infection ratios
the patients susceptibility to amoebic infection. Such details in the novel residents may be referred to the loss of treatment
were accurately arranged, in such away all the studied time for drinking water, lack of cleaning, poor hygiene and
scales were separated from each other and each decade was education levels, contaminated animals and neglect of hand
dealt with as an individual unit. Such separation had enabled hygiene before meals at a new residence27.
us to provide confirmatory results regarding the particular
involvement of the 30-39 aged females in these infections. In CONCLUSION
addition to the age-related factors, these obtained results may
be due to the differences in the living and the geography of Amongst several observations the current study had
the position or to the socio-economic factors of the study observed, it was found that the amoebic infection is the most
populations places and regions24. prevalent in females aged between 30-39 years old. Thus, the
The causes for our obtained results of high abdominal specific age-grouping that conducted by this study has
pain and low level of clinical symptoms might due to the level provided further information regarding the accurate effect of
or nature of patient s immunity and infection period at a age on the patients susceptibility to amoebic infection.
chronic or acute stage25. The currently obtained results of the Accordingly, the current study is highly recommending this
significant effect of the level of education on the infection aged female group to take a special precaution to prevent
ratio were in accordance with the results of Hailegebriel26, who such infection as they exhibit an extra-ordinary susceptibility
found a proportional correlation between educational level to be affected with several forms of amoebic dysenteries.
and infection with intestinal parasites. This result may be
related to the association between patients knowledge and ACKNOWLEDGMENTS
their hygienic awareness, which includes the source of
drinking water, the level of environmental sanitation and the Authors like to express great appreciations and regards to
absence of cleaning or the cutting of fingernails and the all the staff members of Al-Zahraa Hospital for providing us
incorrect using of toilets, as the major factors contributing to the E. histolytica and E. dispar samples, control strains. This
high infection27. Such low educational level may be study was supported by Alfadhil Scientific training company,
considered as the best-suited environment to enable the Babil, Iraq (Grant No. 018-1).
amoebic cyst to survive for a long time5. Regarding the social
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