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Comparison of microscopy, ELISA and nested PCR for
detection of Giardia isolates from Jordan
By
Yasmeen Marwan Mohammed Shaker Yasin
Supervisor
Dr. Nawal Sameeh Hasan Hijjawi
Associate professor
Co - Supervisor
Dr. Sameer Ahmad Naji Al Haj Mahmoud
Assistant professor
Submitted in Partial Fulfilment of the requirements for the
Master’s Degree of Medical Laboratory Science
Faculty of Graduate Studies at the
Hashemite University
Zarqa-Jordan
19, April, 2016
II
This thesis was successfully defended on (19/4/2016)
Examination Committee: Signature
Dr. Nawal Sameeh Hasan Hijjawi, Supervisor. -----------------
Associate professor, Parasitology.
The Hashemite University.
Dr. Sameer Naji Al Haj Mahmoud, Co-Supervisor. -----------------
Assistant professor, Molecular biology and Microbiology
The Hashemite University.
Dr Mamon Mohammad Ali Hatmal, Member -----------------
Assistant professor, Biochemistry and Molecular biology
The Hashemite University.
Dr .May Mohammad Ismael Khalili , Member ------------------
Assistant professor, Molecular Biology
The Hashemite University.
Dr . Khaled Mahmood Abdelkader Al-Qaoud ,Member ------------------
Professor, Immunology and Parasitology.
Yarmouk University.
III
Dedication
To my dear husband
For his continuous support & motivation
To my mother
To my father
For their support
To my mother and sisters in law
Relatives & friends;
Who were there for me whenever I needed them.
IV
Acknowledgment
Firstly, I’m extremely grateful to Allah the Beneficent, the Merciful.
I would like to express my deepest appreciation and intimate gratitude to my supervisor
Dr. Nawal Hijjawi, for her valuable guidance, precious advice, support, encouragement
and endurance throughout the course of this thesis. It has been a great learning experience
to work under her supervision.
Mamon Hatmal and Dr edicine,MI owe my thanks to Dr. Sameer Naji from faculty of
University for their Hashemiteat Laboratory Sciences Medicalof from Department
advices and support.
I owe my thanks also to Dr. Rami Mukbel from Faculty of Veterinary Medicine at Jordan
University of Science and Technology for providing the facilities for conduct part of my
work in their department and for his support.
My special thanks for my sister and teacher Ms Taghreed Al-Muharib , from department
of Medical Laboratory Sciences at Hashemite University, for making my work easier, for
giving hand in solving problems, and for providing a pleasant working atmosphere. Thanks
for my colleagues in Social Islamic collage and for my sisters Futoon Rawashdeh, Aya
Shorman, Doaa Abu-Rajab, Amani Istati and Alaa Ajawii for their help, support and
prays.
V
List of Abbreviations or Symbols
Number Abbreviation/
Symbol
Definition
1 G. duodenalis Giardia duodenalis
2 ELISA Enzyme Linked Immunosorbent Assay
3 PCR Polymerase Chain Reaction
4 bg beta giardin
5 PPV Positive Predictive Value
6 NPV Negative Predictive Value
8 gdh Glutamate dehydrogenase
9 tpi Triose phosphate isomerase
10 ssu-rRNA Small subunit ribonucleic acid
11 DNA Deoxyribonucleic acid
12 elf(a) Elongation factor 1alpha
13 UK United Kingdom
14 USA United States of America
15 FDA Food and drug administration
16 Taq Thermus aquaticus
17 Bst Bacillus stearothermophilus
18 IFNγ Interferon gamma
19 OR Odd Ratio
VI
List of Contents
SUBJECT Page
Committee Decision……………………………………………………...... Ii
Dedication…………………………………………………………….……. Iii
Acknowledgment………………………………………………………… .. Iv
List of Abbreviation or Symbols................................... V
List of Contents……………………..……………………….…………….. Vii
List of Tables……………………….……………………….……………… iX
List of Figures………………………..…………………….………………. X
List of Appendix............................ Xi
Abstract…………………………………………………….…………......... Xii
Chapter 1 …………………………..………………….…………………… 1
Introduction ……………………………………………………………… 1
1.1. Overview …………………………………………………………….
…………………………………………………
1
1.2. Objective ……………………………………………………………. 2
1.3. Purpose ……………………………………………………………. 2
Chapter 2………………………………………………….. 4
Literature review…………………………………. 4
2.1. History………………………………………..... 4
2.2. Giardiasis……………………………………
4
2.2. Morphology…………………….
4
2.2.1 Trophozoite . ………………………………. 5
2.2.2 Cyst:………………………………………….
6
2.2.3 Excyzoite ………………….. 6
2.4 Giardia life cycle……………………. 6
2.5: Transmission of Giardia………….. ………………………
8
VII
2.5.1 Direct transmission ……………….. 8
2.5.2 Indirect transmission ………………….. 9
2.6 Risk factors………… 9
2.7. Symptoms of giardiasis………………………………….
9
2.8 Prevalence.................................. 10
2.8.1 Prevalence in the Middle East................................. 10
2.8.2 Prevalence in Jordan......................... 11
2.9 Taxonomy..........................................
………………………………………………………
12
2.9.1 Assemblage A…………………………………………………
13
2.9.2 Assemblage B…………………………………………..
……………………………………....
13
2.9.3 Assemblage C-H..................................................... 13
2.10 Diagnosis……………………………………………………
…………………………………………………………………
15
2.10.1 Microscopic examination…………………………………………….
15
2.10.2 Immune assay ............................ 15
2.10.3 Molecular analysis............................ 16
2.10.4 Other diagnostic techniques.................... 16
2.11 Pathogenesis........................... 17
2.12 Immunity against Giardia........................ 17
2.13 Virulence.........................
18
2.14 Metabolism. ………
18
2.15: Treatment………………………………
19
Chapter 3:……………………………….. 20
Materials and Methods 20
3.1 Sample collection…….............................................................
20
3.2 Ethics statement............................................
.:…………………..
20
3.3 Microscopic examination:……………………….
20
3.4 ELISA:…………………………..
21
3.5 DNA extraction:.................................. 21
3.6 PCR……………………………………………………… 22
3.7 Statistical analysis…………………………………………………. 22
VIII
Chapter 4:……………………………………………………..
23
Results:……………………………………………………….. 23
4.1 Association between gender and giardiasis
:………………………………………………….
23
4.2 Association between age and giardiasis
:……………………………………………….
23
4.3 : Association between region and giardiasis
………………………………………………………
23
4.4: Microscopy, ELISA and bg nested PCR results…......................
25
4.5 : Comparison between ELISA sensitivity with Microscopic
Examination......... 27
4.6 : Comparison between bg nested PCR sensitivity with Microscopic
Examination
28
4.5 Comparison of ELISA and bg nested PCR results ...........................
………………………………………………………
29
Chapter 5: :…………………………………………..
……………………………………....
30
Discussion ………………………………………..
…………………………………………………………..
30
5.1 Relationship between gender and giardiasis.................
……………………………………………………
…………………………………………………………………
30
5.2 Relationship between age and giardiasis………………
31
5.3 Relationship between region and giardiasis:…………………
31
5.4 Comparison between diagnostic tests
…………………..
32
5.4.1 Comparison between ELISA and microscopy results
33
5.4.2 Comparison between bg nested PCR and microscopy results. 33
5.4.3 Comparison between ELISA and bg nested PCR results…………… 33
Chapter 6:…………………………………………
35
Conclusion ……………………………
35
Future trends:………………………………
36
References……………………………….. 37
Appendix ............................................... 47
53 الملخص بالعربية
IX
List of Tables
Number Table Caption Page
2.1 Prevalence of giardiasis in some Middle East countries. 11
2.2 Giardia species and its potential host 14
2.3 G. duodenalis assemblages and their suspected host. 14
4.1
Distribution, prevalence and number of G. duodenalis positive
samples which were diagnosed by microscopy according to
different age groups, gender and regions.
24
4.2
Result of each sample after analysed by three diagnostic tests
(Microscopic examination , ELISA, and beta giardin) . 26
4.3 Comparison between ELISA and Microscopy result. 28
4.4 Comparison between bg nested PCR and Microscopy result. 28
4.5
Comparison of microscopy and ELISA with bg nested PCR
results for their ability to diagnose G. duodenalis Numbers of
positive samples and sensitivity.
29
X
List of Figurs
Number Figure Caption Page
2.1 Life cycle of Giardia duodenalis 8
4.1
ELISA results for the 96 stool samples on A and B micro-
plates. 27
4.2 Beta giardin nested PCR result on agrose gel with band
size=511bp 27
XI
List of Appendices
Page Appendix title Appendix No
47 Questionnaire A
48 IRB approval B
49 Consent form C
51 ELISA Result-Plate A
D
52 ELISA Result-Plate B
E
XII
Abstract
Comparison of microscopy, ELISA and nested PCR for detection of Giardia
isolates from Jordan
By
Yasmeen Marwan Mohammed Shaker Yasin
Supervisor
Dr. Nawal Sameeh Hasan Hijjawi
Associate Professor
Co - Supervisor
Dr. Sameer Ahmad Naji Al Haj Mahmoud
Assistant Professor.
Background: This study aimed to investigate whether Giardia is a major public health
problem among Jordanian patients who suffer from diarrhoea and gastroenteritis.
Demographic data regarding age, sex and residency were collected in order to investigate
what are the most affected groups with giardasis regarding to these factors. Furthermore
during the present study we attempted to validate the best appropriate test (ELISA, and
beta- giardin (bg) nested PCR) based on microscopic examination which can be used for
G. duodenalis diagnosis.
Material and Methods: A total of 96 faecal samples were collected from patients from
different clinical labs in main hospitals who were confirmed initially to be infected with G.
duodenalis by microscopy. Demographic data for information regarding sex, age and
residency was obtained individually upon filling a designed questionnaire and signing a
consent form from each patient or his guardian. During the present study all confirmed
faecal specimens observed to be infected with G. duodenalis by microscopic examination
were further tested using ELISA, and bg nested PCR to investigate the best appropriate
diagnostic method to confirm the infection with this parasite through sensitivity calculation
of all tests.
Results: All the positive samples which were confirmed to be positive for G. duodenalis
by microscopic examination (96) were used throughout the present study to further
XIII
measure the infection rate of giardiasis and compare all the demographic data. The number
of G. duodenalis cases were observed to be more in males (40/64) than in females (24/64)
Younger age groups were also observed to be infected more with G. duodenalis compared
to older age groups where the highest number of giardasis samples was recorded among
children whose age ranged from 5 month to 14 years (42/64) Regarding residential areas,
the numbers of patients who came from rural areas were higher than those who came from
urban areas (51 vs 45 respectively). Upon the comparison between the more sensitive
diagnostic tests to confirm the infection with G. duodenalis depending on microscopy
result, sensitivity for the ELISA was recorded 62.5% while the sensitivity of bg nested
PCR was 66.67%. Specificity of ELISA and bg nested PCR couldn't be calculated.
Conclusion: G.duodenalis can be regarded as an important parasite which can be
frequently observed among Jordanian patients who suffer from diarrhea and gastroenteritis.
All gender and age groups are susceptible to giardiasis however; the higher positivity
percentage of giardasis appeared to be in younger age groups and more in Urban settings
than Rural. Based on microscopic examination , both ELISA and bg nested PCR show a
good sensitivity for G.duodenalis detection and can be used as an additional confirmation
tests in addition to routine microscopic examination.
Chapter 1
Introduction
1.1 Overview
The eukaryotic intestinal parasite Giardia duodenalis (syn, G. intestinalis, and G. lamblia)
was first described in 1681 by Antonie van Leeuwenhoek who observed the parasite upon
microscopic examination of his own diarrheal stool. Since that date and until the present
time several researchers have studied this interesting organism to determine its
characteristics and medical importance [1].
Giardia duodenalis is an enteric intestinal flagellated protozoan parasite which is
considered as a main cause of gastrointestinal disease known as giardiasis, which affects
humans as well as other animals including, birds, amphibians, reptiles, dogs, cats, rodents,
horses, pigs, and cattle [1, 2].
Nearly 200 million cases of giardiasis have been recorded worldwide in the past few years,
in addition to 500,000 cases which were reported to occur annually especially in children
[3, 4]. Although giardiasis represents a major public health problem in both developing and
developed countries, however it is more prevalent in developing countries especially the
poor ones such as Brazil, Thailand, Philippines, South Africa, Nepal and the Middle East
region [3]. The reason why higher prevalence of giardiasis was reported in the poor
developing countries more than developed ones might be contributed to the poor hygiene
and the low level of education in these countries [5]. In the Middle East the prevalence of
giardiasis varies among countries, for example, in Saudi Arabia the rate of infection was
low (6.5%) compared to the higher prevalence in Egypt that reached up to (34.6%) [6, 7].
Regarding Jordan, previous studies in different areas of the country revealed differences in
the prevalence of the G. duodenalis which ranged from 0.8% to 36% according to the
region and age groups [8, 9], with the highest prevalence (44.8%) observed children
between 6-9 years old [9, 10]. The clinical features of giardiasis may vary from
asymptomatic state to acute infection with mild diarrhea, and sometimes to life-threatening
cases which is characterised by chronic severe diarrhoea and malabsorption of many
elements and vitamins. In particular this devastating symptom of giardiasis usually affect
2
children and malnourished individuals [11, 12]. For example a study conducted on
Peruvian children showed that the infection with G. duodenalis in the first two years of a
child’s life affect their growth and cognition level later in their life [13]. Another study
supports the above observation where a lower IQ score was observed among children
suffering from various parasitic infections including giardiasis [14].
Based on some morphological features and host specificity, Giardia as a genus is classified
into six different species (G.agilis, G.ardeae, G muris, G.microti, G psittaci and G.
duodenalis) [15]. Giardia duodenalis is the only species which infect humans [16].
Moreover, recent genetic characterization studies revealed that G.duodenalis is divided
into eight different assemblages which are designated from A-H [15]. Assemblages A and
B are the most dominant ones and reported to be responsible for most of human infections,
as well as infecting a wide range of mammals. The other G. duodenalis assemblages (C-H)
infect various animals and appear to be host specific [17].
1.2 Objective:
Few studies were conducted in the Middle East region including Jordan in order to
investigate the influence of giardiasis on public health. Therefore, the overall objective of
this study was to investigate whether Giardia is an important prevalent parasite causing
diarrhoea and gastroenteritis among Jordanians. Therefore, clinical samples for patients
suffering from diarrhea and gastroenteritis were investigated for the presence of this
parasite where demographic data such as age and gender, region and the occurrence of
giardiasis was recorded in order to clarify the importance of this parasite and how
prevalent it might be in Jordan. Furthermore, in order to confirm that Giardia is accurately
diagnosed in the collected clinical samples, we evaluated the sensitivity of three diagnostic
tests (microscopy, ELISA and bg nested PCR) for their ability to properly diagnose
giardiasis.
1.3 Purpose of the study:
The two main goals of the present study can be summarised as follows: First: investigation
whether Giardia is a major public health problem among Jordanian patients who suffer
from diarrhoea and gastroenteritis. Also, to observe if the infection varied among various
3
age groups and different gender as well as location. This will be achieved by the statistical
analysis of the demographic data which were collected through a questionnaire which is
designed specifically for this study. The second goal was to investigate which is the best
diagnostic test (microscopy, ELISA and bg nested PCR) for its better sensitivity to
diagnose G. duodenalis in clinical specimen
Chapter 2
Literature Review
2.1. History:
Giardia was first recognised in 1681 by Anthony van Leeuwenhoek when he examined his
own diarrheal stool under a single lens light microscope and described his discovery in The
Royal Society of London Journal as" their bodies were somewhat longer than broad, and
their belly, which was flattened, provided with several feet, with which they made such a
movement through the clear medium and the globules that we might look at" [18]., but this
parasite remained not well recognised until Lambl (1859) provided a detailed
morphological description for this parasite after he observed G. intestinalis in the stool of
children with diarrhoea. However, he believed that this protozoa is not responsible for any
pathological symptoms [19]. Thereafter, this thought was dismissed when the French
scientist Alfred Giard proposed Giardia organism as a pathogenic protozoan parasite since
he recognised a clinical symptoms of gastroenteritis and diarrhea that accompanied
giardiasis in most cases. In 1888 the term lamblia intestinalis was introduced by Raphael
Anatole Émile Blanchard, and 27 years later the term Giardia lamblia was proposed for the
first time as an appreciation for both scientists: Giard and Lambl who had great
achievements in this field. Currently Giardia species that cause infection could be named
as G. lamblia, G. intestinalis or G. duodenalis interchangeably without any preference
between any of them [20].
2.2 Giardiasis:
Giardiasis is a gastrointestinal diarrheal disease which is caused by G. duodenalis parasite
and considered to be the most common intestinal enteric disease that can affect both
humans and animals, as it is considered in the top ten of all human parasitic diseases [8,
21]. Also in the last ten years, giardiasis had been added to the neglected diseases category
[21].
2.3. Morphology of Giardia;
5
Giardia is a unicellular simple eukaryotic organism but it lacks some organelles that are
generally identified in any eukaryotic cell such as mitochondria, endoplasmic reticulum,
and golgi apparatus, also it possess discriminative nuclei and microtubules in its
cytoskeleton [22]. According to some studies; the lack of mitochondria may be attributed
to the fact that G. duodenalis is one of the older types of eukaryotes, as it even possess a
double walled bound organelle mitosome, which has recently been thought to be derived
from the mitochondria [23]. Although the function of the mitosome is not fully understood
yet but several evidences prove that it has an effective role in iron sulfur assembly and in
energy production, the same function which is normally observed in the mitochondria [24].
Giardia genome usually contains 1.2 million base pair, arranged among five chromosomes
and enclosed equally in two Giardia nuclei that are normally divided during cells
replication. Giardia has a specific genetic loci such as glutamate dehydrogenase (gdh),
beta–giardin (bg), trios phosphate isomerase (tpi), small sub-unit ribosomal RNA(SSU-
rRNA) and elongation factor 1 alpha (elf-a) which is currently used for the specific
diagnosis of this parasite using different molecular based assays [22].
2.3.1 Trophozoite:
The trophozoite of Giardia has a pear shaped cell which is 12 to 15 μm long and 5 to 9 μm
in width. It is regarded as the motile, feeding, replicating stage with two rounded nuclei,
axostyle and four pairs of flagella (anterior, ventral, posterior/lateral and caudal flagella).
The two nuclei of the trophozoite usually replicate at the same time and have identical
shape, size, composition and lack nucleolus [25]. A unique structure which gives the
trophozoite its discriminative morphology is the adhesive ventricle disks (sucking discs)
which help the trophozoite to attach itself firmly to the intestinal mucosa and prevent its
disposal to the outside. Adhesive ventricle disks usually cover almost the entire ventricle
surface of the trophozoite [22]. This sucking concave disk composed of many cytoskeleton
proteins such as α-tubulin and β-tubulin along with proteins from the giardin family (α-
giardin, β-giardin, γ-giardin and δ-giardin). The Giardia trophozoite possess other
cytoplasmic organelles such as four pairs of cylindrical basal bodies which are important in
cell signalling transduction and flagella assembly [26]. Other interesting structural feature
of the trophozoite is the medium body which consists of bundles of microtubules which
6
contributes to the Giardia shape along with the adhesive disks. Other regular eukaryotes
cytostosomal structures like endoplasmic reticulum, Golgi complex, lysosomal vacuoles;
and medium bodies are absent from
Giardia [27].
2.3.2 Cyst:
The Giardia cyst ranges from 8–12 μm in length and between 7–10 μm in width with a
double walled capsule consisting of four small round nuclei gathering at one pole. The
cysts also contain a parabasal body and axoneme but lacking any motility structure which
is disassembled and stored until the parasite stage is transformed into a trophozoite stage.
The outer cyst wall composed of a network of filaments ranging from 7 to 20 nm in
diameter in addition to N-acetylgalactosamine and three different cyst wall proteins
(CWP1, CWP2 and CWP3). The robustness of the cyst wall increases its survival in the
environment for several months which in turn make Giardia spread more easily and makes
it more difficult to be destroyed by disinfectants [28]. This cyst is the infective stage which
is responsible for the transmission of giardiasis between individuals [29].
2.3.3 Excyzoite:
Excyzoite is a transitional short lived stage between trophozoite and cyst which originates
from the 4-nucleated Giardia cyst. Excyzoite differ from trophozoite by its tetraploid
nuclei and non assembled adhesive disks. Excyzoite usually undergo two cytokinesis
divisions without intervening S phases to finally producing two trophozoite from each
excyzoite. During this division, the excyzoite increases its metabolism and gene
expression, segregates its organelles, up-regulates the proteins which are associated with
its motility and assembles the adhesive disc [30].
2.4 Giardia Life cycle:
Giardia has a simple life cycle involving two stages: trophozoite and cyst.The life cycle of
Giardia begins when the cysts enter the host body with contaminated food or drink, and is
exposed to the severe internal host environmental conditions such as the presence of high
stomach acidity and the intestine trypsin which is considered as a starting point for the
7
excystation process [31]. Excystation usually takes place in the duodenum of the small
intestine where the cyst wall begins to dissolve by the action of cysteine proteases which is
secreted from the Giardia cyst itself, thus making holes into Giardia cyst wall. Also
lysosomal enzymes participate in damaging the cyst wall by destroying cyst proteins, after
that excyzoite is formed as a transitional state [20]. Eventually flagella are formed which
results in the emergence of the trophozoite stage which multiply by binary fission in order
to produce a large number of trophozoite s which colonizes the intestinal wall especially
the jejunum. Trophozoite proliferation usually takes from six to twelve hours to pass
through all the cell cycle stages (S, G1, G2, and M ) [15]. When the pH reaches 7.8,
trophozoite s start the encystation process by transforming into cysts as it cannot tolerate
the alkaline condition. Encystation process consists of two phases, the cyst wall formation
phase and the plasmalemma generation phase. The newly formed infective cysts and to a
lesser extent trophozoite s are excreted to the outside with faeces of the infected hosts and
have the ability to survive in the environment for weeks or months [16]. (See Figure 2.1
below)
8
Figure 2.1: Life cycle of Giardia duodenalis which consist of two major stages
transition of cyst to trophozoites by excystation and transition of trophozoites to cyst
through encystation. Source: (Shan Lv,et al., 2013), [23]
2.5 Transmission of Giardia:
Epidemiology of Giardia depends on the ability of Giardia cyst to be transmitted among
individuals which usually occurs by direct or indirect transmission.
2.5.1 Direct transmission:
The most common way of Giardia transmission is person to person through faecal-oral
route, especially in developing countries where there is low income and poor sanitation
which are factors that encourage dissemination of Giardia [5]. However, developed
9
countries also suffer from giardiasis which spread mostly in day care centres and
kindergartens [32]. Other route for direct transmission is through zoonosis, which is still
under investigation despite many studies reporting several cases of giardiasis due to human
animal contact, especially domestic animals that are in close contact with humans such as
dogs and cats [33].
2.5.2 Indirect transmission:
Infectious cysts are transmitted indirectly by the oral-fecal route where the disease is
established upon the ingestion of the cysts with contaminated food or water. According to
a comprehensive review of worldwide waterborne parasitic protozoan outbreaks (that
occurred between 2004 and 2010), it was found that at least 199 outbreaks of human
diarrheal diseases were due to the waterborne transmission of parasitic protozoa and that
G. duodenalis was the causative agent in 35.2% of these outbreaks [34]. The main reasons
of Giardia being a water borne organism is related to its small size and resistance to water
disinfection strategies [35].
2.6 Risk factors:
Normally risk factors are associated with increased infectivity rate of giardiasis in
developed countries which is different from those in developing countries. As indicated by
several studies which were conducted in the United Kingdom (UK) and the United States
of America (USA) the main risk factors for acquiring giardiasis include camping in rural
areas, accidentally drinking tap water during swimming, eating raw vegetables especially
lettuce [36]. On the other hand inappropriate sewage network, large family size, improper
hand washing and insufficient washing of vegetables are also considered to be the most
common risk factors in developing countries which are usually related to poor sanitation
and low health educational level in these countries [37]. But the most common risk factor
in developed countries is travelling to developing countries [36]. Animal contact is other
risk factor that is present in both developed and developing countries although more
investigations and research are required to verify this factor [37].
2.7 Symptoms of giardiasis:
01
Disease manifestation due to G. duodenalis infection might range from asymptomatic
carrier to patients who presented with mild or severe symptoms. Mild to severe symptoms
like fever, fatigue, bloating, epigastric pain, cramps, malaise, dehydration, weight loss,
bleeding, and malabsorption have been always observed in infected patients with G.
duodenalis. These symptoms usually appear within one to two weeks of the infection with
the parasite and may continue up to six weeks from the start of the infection [38].
The disease severity and symptoms varies according to the age and immunity of the
patient. Giardiasis has been reported to be worse in children under 5 years old. Many
children who suffer from giardiasis in developing countries suffer from severe
manifestations like malabsorption which is considered to be the most critical consequence
of the disease; since it can lead to deficiencies in several elements and vitamins in the body
such as zinc, iron, copper, folic acid, and vitamin B12 which might impede the growth and
development of the infected children [39].
2.8 Prevalence:
Giardia duodenalis is considered as one of the most common widespread parasite with two
million cases distributed in different continents and countries [40]. Globally, giardiasis
accounts approximately for 2-5% of the diarrheal cases in the developed countries and up
to 20-50% of the diarrheal cases in the developing countries. This difference in the
prevalence may be contributed to the poor sanitation and lower health education levels in
developing countries compared to developed ones [40, 41].
2.8.1 Prevalence in the Middle East:
Several studies reported differences in the prevalence of giardiasis in many countries in the
Middle East including Jordan. For example, in Saudi Arabia and Palestine where the
prevalence was reported to be 6.5%, and 11.6% respectively [43]. However higher
prevalence of giardiasis was reported in other Middle Eastern countries such as Egypt
,United Arab Emirates, Morocco, and Yemen where the prevalence of giardiasis for each
country was 80%, 60.4%, 30%,,73% respectively [42, 43, 48, 49, 50].
00
2.8.2 Prevalence in Jordan:
Studies in Jordan which were conducted in different areas of the country revealed
differences in the prevalence of the G. duodenalis especially between studies which were
performed in urban and rural areas. In one study which was carried out in the urban area of
Northern Jordan, 0.8% of patients suffering from diarrhea were infected with G.
duodenalis [8], while in other study, the prevalence of G.duodenalis among patients with
gastroenteritis in a rural area was higher and reached 16% [44]. Moreover, the highest
prevalence of G. duodenalis was observed in a study which was conducted on primary
school kids from different rural area where the prevalence was the highest among all
previous studies and reached up to 30% (43 cases) [9]. In a more recent study in Jordan,
which was done on the food handlers of four main hotels at the Dead Sea region for the
prevalence of parasites including G. duodenalis, it was found that around 2.44% of them
were infected with the G. duodenalis [45].
The prevalence of giardiasis in some Middle East countries including Jordan is
summarised in table 2.1 below.
Table 2.1: Prevalence of giardiasis in some Middle East countries. The highest
prevalence was observed in Egypt and UAE.
Country Prevalence References Num
Egypt 34.6% 46
Egypt 80% 47
Palestine 11.6% 48
UAE 60.4% 49
Yemen 73% 50
Morocco 30% 42
Saudi Arabia 6.5 % 43
Iran 4.7% 51
Turkey 15% 52
Jordan 0.8% 8
Jordan 16% 44
Jordan 30% 9
02
2.9 Taxonomy:
The genus Giardia belongs to kingdom Protista, phylum Sarcomastigophora, Subphylum
Trichozoa. Class Trepomonadea, Subclass Diplozoa. Order Diplomonadida and family
Giardiidae. Based on the molecular analysis, the genus Giardia is divided into six species:
G. agilis, G. ardeae, G. muris, G. microti, G.psittaci and Giardia duodenalis based on some
morphological differences which can be observed by light microscopy (mainly the medium
body structure; which appears to be fine and long in G. agilis, claw transverse in
G.duodenalis, and rounded in G. muris) and the spectrum of their infected host since each
species appeared to infect specific host [53]. The above mentioned six Giardia species
infect a wide spectrum of hosts but only G. duodenalis species has the ability to infect
humans in addition to other mammals [54]. Recently a new Giardia species: G. varani
which infect reptiles has been described, but still some scientists think that this species
should not be classified as a new Giardia species since it lacks the median bodies and has
bi-nucleated cysts [18]. G. duodenalis can be considered as a complex species, with
genetically different distinct assemblages that can't be easily discriminated based on their
morphology [55]. G. duodenalis assemblages can be assigned to eight distinct assemblages
(A to H) based on genetic analysis [16]. Assemblages A and B are the most prevalent ones,
and are reported to be responsible for most of the human infections, as well as infecting a
wide range of mammals which raises a question regarding its zoonotic potential [56]. For
example, promising studies on fish in Australia reported that many cases of giardiasis in
fish were infected with G. duodenalis assemblages A and B [56]. The other G. duodenalis
assemblages (C - H) appear to be host specific where assemblages C and D are found in
canines, E in livestock, F in cats, G in rodents and assemblages H in seals [16]. Although
there are some exceptions for this specificity such as some cases of cat giardiasis which
resulted from assemblages C, D, and E, also some human , dogs and pigs cases which
reported to be caused by assemblages F ,C, D and E [56]. Recently one promising study
identified two novel assemblages of Giardia which were discovered in Australian house
mice, however more extensive studies are required to confirm these new findings [55].
2.9.1. Assemblages A:
03
Assemblage A has been found in humans and a wide range of mammals especially livestock
animals like goat, sheep and cows in addition to a few types of wild animals [16].
According to many previous studies, assemblage A may be associated with diarrheal
symptoms more than assemblage B, which means the average shedding of cysts is higher in
hosts infected with assemblage A cases. Recently phylogenic analysis revealed that
assemblage A has three sub-assemblages AI, AII, and AIII [57]. Generally sub assemblage
AI is responsible for livestock and companion animals’ giardiasis, sub-assemblage AII is
responsible for the majority of human giardiasis while sub assemblage AIII causes wild
animal giardiasis. Recently genetic analysis identified sub-sub assemblages for assemblage
A [57, 58].
2.9.2. Assemblage B:
Like assemblages A, assemblage B has been reported to infect a board spectrum of animals
and humans but with predominance in wild animals, however assemblages B is more
prevalent than assemblages A to cause human giardiasis, as it accounts for 58% of all
cases world-wide [59]. Further studies indicated that this assemblage has four sub-
assemblages BI, BII, BIII, and BIV. Usually human giardiasis resulting from assemblage B
is not accompanied by any giardiasis symptoms such as vomiting, abdominal cramp,
nausea, and diarrhoea [42].
2.9.3 Assemblages (C-H):
These assemblages are considered to be host specific i.e. each assemblage can cause
infection to only one or two kinds of organisms, however there are single reports which
provided evidence for an overlap between these assemblages and suspected hosts [42].
Other reports proposed that these assemblages (C-H) can also infect humans; however
more investigation is required to illustrate the above finding [60].
The following two tables below summarises Giardia species and G. duodenalis
assemblages with their potential hosts [16].
01
Table 2.2: Different Giardia species and its potential hosts. G. duodenalis is only
species that can infect humans as well as other mammals. Source:[16].
Species Major Host
G .agilis Amphibians
G. ardeae Birds
G. muris, Muskrats and Voles
G. microti Rodents
G. psittaci Birds
G. duodenalis
Mammals and Humans
G.varani Lizards
Table 2.3: G.duodenalis assemblages and their suspected host. Assemblage A and B
only can infect humans, assemblages C- H appeared to be host specific. Source: [16]
Assemblage Major Host / hosts
A Human, nonhuman primates, domestic and wild ruminants,
alpacas, pigs, horses, domestic and wild canines, cats,
rodents, marsupials
B Humans, non-human primates, cattle, dogs, horses, rabbits,
beavers, muskrats
C Domestic and wild canines
D Domestic and wild canines
E Domestic ruminants, pigs
F Cats
G Mice and rats
H Seals
05
2.10 Diagnosis:
Diagnosis of G. duodenalis can be performed by various methods starting from simple
microscopic examination by wet mount preparation to more sensitive immunoassay
techniques and more specific discrimination tools using different molecular based assays
(PCR) [61, 62].
210.1 Microscopic examination:
Microscopic examination is the original basic method which is routinely used in Giardia
diagnosis worldwide, although it has many problems and drawbacks such as the low
number of skilled medical technologists who are trained and able to identify the parasite in
wet mount preparations, and recurrent faecal samples collection for several days to
compensate for the low sensitivity that result from the intermittent shedding of the
Giardial cysts [63].
Some modification is introduced to the microscopic examination by using stains such as
methylene blue or iodine that might improve the sensitivity of microscopic detection;
however this technique still suffers from low sensitivity which ranges from 65% to 75%
according to some studies [64]. The use of fluorescent microscope can improve the
specificity of microscopic detection by using specific fluorescent dye or fluorescent
antibody against Giardial antigen which gives it a distinctive colour to be clearly visualized
[65, 66].
2.10.2: Immune assays
There are many immunological tests which were used for Giardia diagnosis which have
been developed to overcome the drawbacks of microscopic examination through the
specific detection of G. duodenalis antigens or antibodies such as direct fluorescent-
antibody (DFA) test, immunochromatographic card test (copro antigen rapid test), enzyme
immunoassays (EIA) and enzyme linked immunosorbent assay (ELISA) [53]. The
immunological tests are easy to perform, give precise results, and preserve time and effort.
Among the immunological tests which is regarded as the most accurate and applicable
assay is ELISA since it possess high sensitivity and specificity (100% and 91%
respectively) to detect Giardia even if it has low concentration in the sample, and it can be
06
performed on a large number of samples at once [53]. Therefore recently FDA proposed
ELISA as an impressive and favourable method to detect Giardia antigen from stool
specimens [67]. Immunochromatographic card test (copro antigen rapid test) is the fastest
and easiest immune assay; however it has lower sensitivity than other immunological
assays. The average sensitivity and specificity of all these assays is about 95±5 % and
96±4 % respectively. All immunological tests can be regarded as good alternative methods
for the microscopic examination [68].
2.10.3: Molecular analysis:
Molecular and genetic analysis especially polymerase chain reaction (PCR) is regarded to
be a distinctive method for G. duodenalis detection and a great tool for its assemblages and
sub-assemblages identification, which is highly useful for studying the epidemiology and
population genetics of G. duodenalis [69]. DNA extraction is considered a primary and
essential step which is required for proper PCR analysis. Therefore, it should be carefully
done especially with parasitic faecal samples as for Giardia detection; given the
complexity of faecal sample content and stuffiness of Giardia cyst wall that enclose the
DNA. The efficiency of PCR analysis depends on the use of primers that target the
amplification of specific gene loci such as glutamate dehydrogenase (GDH), beta–giardin
(BG), trios phosphate isomerase (tpi), small sub-unit ribosomal RNA(SSU-rRNA) and
elongation factor 1 alpha (elf-a) [70]. The sequence of the amplified PCR product which
target one of the above mentioned genetic loci is perfectly used to determine the
assemblage and sometimes sub- assemblages of G. duodenalis [71]. Although all pervious
genetic loci are used in PCR, but recently amplification of beta giardin is commonly used
due to its sensitivity and ability to assemblages and sub-assemblages identification through
sequencing or RFLP easily after amplification using this gene locus [72]. Regarding many
studies which were conducted to investigate the sensitivity of PCR as a diagnostic method
of giardiasis diagnosis, it was proposed that different types of PCR targeting different G.
duodenalis gene loci is the most accurate detection method with high sensitivity and
specificity [71, 72].
2.10.4: Other diagnostic techniques:
07
Loop-Mediated Isothermal Amplification (LAMP):
This newly developed technique provides a simple, sensitive, fast and cheap tool to
amplify specific G. duodenalis genetic loci through the use of isothermal circumstances
and Bacillus stearothermophilus DNA Polymerase (Bst) polymerase instead of regular Taq
polymerase which forces the DNA strand self cycling by its displacement activity. This
ambitious method overcomes the high cost and slow procedures of ordinary PCR in
addition to improvement in sensitivity [73].
2.11 Pathogenesis:
When the Giardia cyst enters the human body with contaminated food or drink, it
transforms into a trophozoite stage which attaches itself to the epithelial cell of the small
intestine by its adhesive disks and begins to multiply without invading the intestinal
mucosa [74]. However, this attachment may enhance enterocyte apoptosis through certain
genetic modification such as alteration in genes responsible for apoptotic proteins
formation and inactivation of anti-apoptotic Bcl-2 molecules, in addition to encouraging
free radical nitric oxide building that accelerates the death of cells [75]. This remarkable
elevation in the number of apoptotic cells especially in chronic infection leads to the
increased intestinal epithelial permeability, where extensive infiltration of immune cells
can be observed clearly around the Giardia colonization sites [76]. The most adverse effect
of giardiasis usually results from shortened or damaged intestinal microvilli which causes
malabsorption of many elements and substances such as glucose, iron, zinc and sodium
which may be occur due to alteration in some permeability regulator proteins like
zonulaoccludens-1 (ZO-1) [77]. Although many studies were conducted to understand the
pathogenesis mechanisms of Giardia but still none of them is fully understood and require
further investigations [77].
2.12 Immunity against Giardia:
Human immune response is specifically activated against Giardia trophozoite antigens
like giardin and tubulin which are normally expressed on trophozoite surface upon its
attachment to the intestinal wall. Moreover, some studies propose many isolates that
undergo antigenic variation phenomenon through genetic alteration of these cysteine rich
08
antigens which frequently represent certain molecules with different molecular mass [77].
Various innate immunity mechanisms can be stimulated in response to giardiasis where
cells such as neutrophils which is mainly responsible for inflammatory reaction usually
recruited in acute giardiasis. However, in chronic cases of giardiasis humoral and cellular
acquired immunity have major role in the defence against giardiasis which is driven by
IgA ,CD4 Tcell, IL6, and interferon gamma (IFNγ) [78]. Furthermore, nitric oxide is
supposed to have an effect in anti-giardiasis defence especially of trophozoite damage to
the villi [79]. Recently some studies have proposed non -immune substances that possess a
destroying effect to Giardia such as unsaturated fats and conjugated bile of human milk
[78, 79].
2.13 Virulence:
The impact of G.duodenalis virulence is usually associated with several virulence factors
including enterotoxin, ease of transmission, high replication rate, and low infective dose
(only 10-100 cyst can induce the disease) and antigenic variation [79, 80]. Normally
Giardia enterotoxin is responsible for fluid accumulation and elements in intestinal lumen
to be utilized later by the trophozoite [81]. The most important virulence factor of G.
duodenalis is related to the antigenic variation; through continuous switching of antigens
all the time which leads to the weakening of the immune response against it. Other
virulence factors that also influence giardiasis are the high proliferation rate of Giardia
trophozoite and the low Giardia infectivity dose. The quick dissemination of Giardia
cysts is considered as a powerful virulence tool that increases its infectivity. Some studies
consider the adhesive (sucking disks) as an important virulence factor since it facilitates
the tight attachment of Giardia to the intestinal wall which help Giardia trophozoite
proliferation and multiplication [82].
2.14 Metabolism:
The metabolism of Giardia is quite different from other eukaryotes,for example its
carbohydrate metabolism does not include oxidative phosphorelation that usually takes
place in the mitochondria, also glucose is not completely catabolised, it is converted to
acetate ethanol, alanine, and carbon dioxide, instead of being completely converted into
09
water and carbon dioxide in order to produce energy as what happens in the normal state
[83]. Amino-acids metabolism is considered as an important process for Giardia since it
has a major role in producing energy from the metabolism of aspartate, alanine, or arginine
through arginine dihydrolase pathway and aspartate transaminase [58]. Other benefit of
amino-acid metabolism is the production of alanine and cysteine which are responsible for
osmotic condition regulation and axenic growth of Giardia trophozoite respectively, other
amino- acids as serine, glycine and threonine, glutamine and asparagine are usually
introduced from the intestinal external environment since it can't be synthesized inside
trophozoite [84]. Lipid metabolism usually takes place during axentic development of
Giardia trophozoite growth through using cholesterol, phospholipids and fatty acids
present in the host’s bile [83]. Synthesis of lipid in trophozoite is quite limited to some
small fatty acids. This may be attributed to the fact that fatty acids cause toxicity to
trophozoite , therefore several studies have suggested breast milk as a lethal tool to Giardia
trophozoite upon lipolysis. In the other hand nucleic acid metabolism depend on salvage
and obtain purines and pyrmidines from the host in order to use it in metabolism [58, 84].
2.15 Giardiasis treatment:
The main medication prescribed to treat giardiasis is 5-Nitroimidazoles and its derivates as
metronidazole, tinidazole, secnidazole. However, other medication can also be used in
giardiasis therapy such as furazolidone, paromomycin, nitazoxanide , bacitracin zinc, and
chloroquine, these have been used in cases where undesirable side effect as nausea
anorexia are reported, or failure to respond due to G.duodenalis resistance to
nitroimidazoles group [85].
One promising study which was conducted on albino rats propose ginger and cinnamon to
be used for the treatment of giardiasis as an alternative natural house recipes since it
contain effective ingredients that possess anti-inflammatory, anti-diarrhea, spasmolytic
action, antioxidant characteristic such as alkaloids, polyphenolic, glycosides, and
flavonoids [86]. In some cases giardiasis doesn't require any type of therapy since it can be
regarded as a self limiting disease [86, 87].
Chapter 3
Materials and methods
3.1 Sample collection:
A total of 96 microscopy-positive Giardia human faecal samples were collected from
patients who suffer from diarrhoea, vomiting and abdominal cramps and referred to
clinical laboratories of major hospitals in 5 regions of Jordan (Amman, Irbid, Jordan
Valley, Zarqa and Maan) from November 2014 to October 2015. Demographic data
regarding age, gender, residency, medical history, duration of symptoms, and common life
style habits were obtained upon filling a designed questionnaire for this study by the
patient himself or their guardian in case of children patients (Appendix A).
3.2 Ethics statement:
Ethical issues were approved by the Institutional Review Board at Hashemite University
(Appendix B) (ethics permit number 1401254/32). Written consent forms were assigned by
each patient or guardian (Appendix C).
3.3 Microscopic examination:
Routine direct examination was performed in the clinical labs of hospitals on fresh faecal
sample by taking a significant quantity of the sample which was then mixed with one drop
of 0.9% normal saline or distilled water in order to exam it under 10X ,40X and 100X
light microscope objective lenses to confirmed the presence G.duodenalis cysts or
trophozoites. Modified acid fast stain was also used on some samples to easily recognize
Giardia trophozoite and cyst morphology. Briefly; the staining procedures followed the
manufacture procedures which started by making a faecal smear on a clean glass slide and
allowing it to dry, then fixing it with methanol for five minutes. After that carbolfuchion
was flooded over the slide for three minutes then rinsed with tap water. The next step was
performed through dipping the slides in 5% acid alcohols for 20 seconds and rinsed with
tap water, finally slides were immersed in methylene blue for one minute and rinsed with
tap water and examined under oil immersion lens [88].
20
3.4 ELISA:
All samples were prepared for ELISA test using RIDASCREEN® Giardia- Germany kit.
This test employs specific antibodies in a sandwich-type method against specific antigen (
65000 molecular weight glycoprotein (65 GSA) of Giardia duodenalis cysts and
trophozoite coated to the 96 micro wells plate. Each stool sample has been diluted 1:11
with sample dilution buffer, and samples were mixed thoroughly by vortex before
beginning the test. Controls and samples were pipetted into wells and mixed with
biotinylated anti-Giardia antibodies (conjugate buffer 1) before being incubated for 30
minute at room temperature. The wells were washed to remove unbound antibodies. After
the washing step streptavidin poly-peroxidase conjugate (Conjugate 2) was added and
wells were incubated at room temperature. The wells were washed again and hydrogen
perioxide substrate solution was added to the wells. A blue colour develops after
incubation in darkness for 15 minutes in case the sample was positive for Giardia. The
addition of the stopping reagent changed the colour from blue to yellow.
The intensity of colour is directly proportional to the amount of Giardia antigen present in
samples. The extinction of colour was measured by microplate (ELISA) reader at 450 nm,
which is related to the concentration of giardial antigen, and the concentration was
expressed as micro international units per ml (μIU/mL).
3.5 DNA extraction:
Total DNA was extracted from the faecal samples using QIAamp DNA Stool Mini Kit
(Qiagen, Germany ). This kit protocol is designed for purification of total DNA from fresh,
fixed or frozen samples. A suitable amount of each sample was mixed with inhibition EX
buffer in order to inhibit enzymatic reaction and prevent DNA degradation. This mixture
was subjected to four rounds of freezing thawing cycles using liquid nitrogen and 95°C
water bath. After centrifugation all samples were placed in QIAamp spin columns to
adsorb and trap DNA onto QIAamp membrane. Finally concentrated DNA was eluted
from the QIAamp column in 50μI of the elution buffer.
22
3.6 PCR:
For the molecular work nested PCR technique is done to amplify beta- giardin gene locus
using forward primer G7 (5-AAGCCCGACGACCTCACCCGCAGTGC-3) and reverse
primer G759 (5- AGGCCGCCCTGGATCTTCAGACGAC-3) for first PCR reaction and
forward primer BGf (5-GAACGAACGAGATCGAGGTCCG-3) and reverse primer BGr
(5-CTCGACGAGCTTCGTGTT-3) [80], to produce a 753 and 511 bp amplicon size for
both first and second reactions respectively. All reactions were prepared with a total
volume of 25 μI which consists of 12.5 µl of the master mix: 1 µl of MgCl2 , 0.5 µl of
forward primer , 0.5 µl of reverse primer , 5.5 µl of nuclease free water and 5 µl of DNA
in first reaction and 3 µl of product of first reaction for second PCR reaction. The two PCR
reactions were performed using ESCO thermal cycler (USA) under the following
conditions 94ºC for 5min for initialization step, 94 ºC for 20 seconds for denaturation step,
53ºC for 45 seconds and 51ºC for 45 seconds for first and second reactions annealing step
respectively, 72 ºC for 50 seconds for extension step, and finally 72ºC for 10min for final
extension step [56]. The PCR product checked by applying 5µl of samples on 1.5% agrose
gel and visualization by ethidium bromide.
:analysis Statistical3.7
Data analysis was performed using SPSS Version 20 software package. Tests were applied
to compare three diagnostic tests through calculating sensitivity for all diagnostic tests
since these measures are global based.
Chapter 4
Results
During the study, a total of 96 faecal samples were collected from patients suffering from
diarrhoea and abdominal pain and confirmed to have G. duodenalis trophozoites or cysts
upon microscopic examination.
4.1 Association between gender and giardiasis:
The infection rate of giardiasis appeared to be higher in male than in female, since out of
96 patients who suffered from intestinal giardiasis, 56 cases were males and 40 were
females (Table 4.1). Univariate analysis proposed that males were more likely to be
infected more with giardiasis since the odd ratio (OR ) is above one (OR = 1.6667 95%
CI=0.7066-3.9311) .
4.2 Association between age and giardiasis:
The age group of giardiasis patients ranged between 5 months to 57 years. .The highest
numbers of positive samples were observed in the young groups. (Table 4.1) illustrates the
distribution of patients into different age groups
4.3 Association between regions and giardiasis:
According to our data, higher infection of giardiasis was observed in persons originated
from rural areas of Jordan as 51 samples from patients who confirmed to be giardiasis
positive by bg nested PCR came from regions such as Irbid villages, Camps and Jordan
valley while only 45 samples of the 96 positive were collected from urban cities (Table
4.1) .
The results for the infected patients who are diagnosed with giardiasis by bg nested PCR
according to age groups, gender and region illustrated in (Table 4.1)
21
Table 4.1: Distribution and number of G. duodenalis positive samples which were
diagnosed by microscopy according to different age groups, gender and regions.
Demographic
data
Number of
Positive
samples
tested by
Microscopy
Percentage of
Positivity
Gender
Male 56 58.33%
Female 40 41.67%
Age groups
5 month -14
years
59 61.46%
15-29 years 17 17.71%
30- 44 years 15 15.62%
45-60 years 5 5.21%
Region
Urban 45 46.88%
Rural 51 53.12%
25
4.4 Microscopy, ELISA and bg nested PCR results:
During the present study, all 96 faecal samples which were collected from the clinical
medical labs were confirmed to be positive for G. duodenalis cysts or trophozoite s upon
microscopic examination of the wet mount preparations. However based on ELISA
technique, only 60 out of the 96 samples were positive for the detection of G. duodenalis
antigen using cut off value, 0.346 0.316 for plate A and plate B, respectively (Fig 4.4 )
(Appendix D ,E) . For bg nested PCR, 64 out of the 96 samples were positive for G.
duodenalis and show amplification on gel electrophoresis (Fig 4.5). All these result are
illustrated in table 4.2 below.
Table 4.2 Result of each sample after analysed by three diagnostic tests (Microscopic
examination, ELISA, and beta giardin) .
26
Sample
number
Microscopy ELISA PCR
1 + + -
2 + + -
3 + - -
4 + + -
5 + + +
6 + + +
7 + + +
8 + - -
9 + + +
10 + + +
11 + + +
12 + + +
13 + - -
14 + + +
15 + - -
16 + - -
17 + + -
18 + + +
19 + + +
20 + - -
21 + + +
22 + - -
23 + - -
24 + - -
25 + + +
26 + + +
27 + + -
28 + - -
29 + - -
30 + + -
31 + - +
32 + - -
33 + - -
34 + + +
35 + - +
36 + - +
37 + - -
38 + - -
39 + - -
40 + - -
41 + + +
42 + - -
43 + + +
44 + + +
45 + + +
46 + - +
47 + + +
48 + + +
49 + + +
50 + + +
Sample
number
Microscopy ELISA PCR
51 + + +
52 + - +
53 + - +
54 + + +
55 + + +
56 + + +
57 + + +
58 + + +
59 + - +
60 + - -
61 + - -
62 + + -
63 + + +
64 + + +
65 + + +
66 + + +
67 + + +
68 + - -
69 + + +
70 + + +
71 + - -
72 + + +
73 + + +
74 + - +
75 + + +
76 + + +
77 + + +
78 + - -
79 + + +
80 + + +
81 + + +
82 + - +
83 + + +
84 + + +
85 + + +
86 + + +
87 + - -
88 + - -
89 + + +
90 + + +
91 + + +
92 + - +
93 + - +
94 + + +
95 + + +
96 + + +
27
Fig4.1: ELISA results for the 96 stool samples on A and B micro-plates. Plate A
contain faecal samples from 1-48, plate B contain faecal samples from 49-96.
A B C D E F G H I J A.
Fig 4.2 : Beta giardin nested PCR result on agrose gel with band size=511bp .A-
Ladder, B- Negative control, C-Positive control, D-J -Tested clinical samples (selected
samples).
4.5. Comparison between ELISA sensitivity with Microscopic Examination:
Based on Microscopy outcome, sensitivity of ELISA was 62.5%. With ELISA 36 cases
were recorded as false negative results since they were positive by microscopic
examination as illustrated in Table 4.3.
28
Table 4.3: Comparison between ELISA and Microscopy result. Sensitivity = 60/ 96
(62.5%), Specificity ,FN and TN couldn't be calculated. TP (true positive), FP (false
positive), FN (false negative), TN (true negative).
Microscopy
positive samples
Microscopy
negative samples
ELISA positive 60 (TP) --------- (FP)
ELISA negative 36 (FN) ---------- (TN)
Total 96 -----------
4.6 Comparison of bg neated PCR sensitivity with Microscopic examination:
Based on Microscopy outcome, sensitivity of bg nested PCR was 66.67%. With bg neated
PCR 32 cases were recorded as false negative results since they were positive by
microscopic examination as illustrated in Table 4.4.
Table 4.4 : Comparison between bg nested PCR and Microscopy result. Sensitivity =
64/96 (66.67%), Specificity ,FN and TN couldn't be calculated. TP (true positive),
FP (false positive), FN (false negative), TN (true negative).
Microscopy
positive samples
Microscopy
negative samples
Bg nested PCR
positive
64 (TP) --------- (FP)
Bg nested PCR
negative
32 (FN) ---------- (TN)
Total 96 -----------
29
4.7 Comparison between ELISA and bg nested PCR results :
Upon comparison of ELISA results to bg nested PCR results, 60 cases were positive by
ELISA while 64 samples were positive for G.duodenalis .11 cases were recorded as
negative results by ELISA although they showed an obvious amplification by bg nested
PCR analysis. On the other hand 7 cases were recorded as a negative by bg nested PCR
despite they were positive by ELISA. Table 4.5 summarize the sensitivity and number of
positive samples for three tests.
Table 4.5: Comparison of microscopy and ELISA with bg nested PCR results for
their ability to diagnose G. duodenalis Numbers of positive samples and sensitivity.
Microscopy ELISA PCR
Number of positive
sample
96 60 64
Sensitivity 100% 62.5% 66.67%
Chapter 5
Discussion
Giardia. duodenalis is regarded as an important parasite which is usually associated with
diarrhea, malabsorption and gastroenteritis worldwide. Giardiasis show a higher
prevalence rate in developing countries compared to the developed ones [16, 79]. In the
Middle East several studies in many countries showed an elevation in the prevalence of
giardiasis where the Egypt and United Arab Emirates were recorded the highest
prevalence 80% and 60% respectively, while the lowest prevalence was recorded in Iran
4.7% [47, 49, 51]. The possible cause of this increase prevalence in Egypt may refer to
poor hygiene and low income however the highest prevalence in UAE might be associated
to variety of nationalities that comes to UAE for working as those comes from poor health
countries as India and Pakistan. Relatively little is known about the prevalence of
giardiasis among humans in Jordan with previous studies using microscopy reporting large
differences in the disease prevalence (0.8% to 44.8), with the highest being reported in
rural areas and among children aged between 6-9 years [8, 9, 45]. Therefore, knowledge of
the burden of giardiasis and its association with gastroenteritis is still not fully understood
in this country.
In agreement with other previous studies which were conducted in Jordan [8, 9, 45], the
present data showed that the higher number of positive samples giardiasis was mostly
observed in children and among males compared to females. Moreover, the higher
number of infected patients was recovered from rural area compared with urban areas.
5.1 Relationship between gender and giardiasis:
In the present study, number of infected males were higher than female Table 4.1. The
possible explanation for this finding might be the fact that men have more outdoors
activities than females, also they might be employed in jobs in farms and ranches with
greater contact with animals which was consistent with a previous observation in two
studies which were conducted in Sicilian farmers in Argentine [89]. Other study in
30
Germany reported that male also have higher prevalence rate of giardiasis than in female
in all age groups [90]. Contradictory to the above mentioned studies and our present study,
two studies one in Pakistan and the other in Bergen in Norway indicated that the
prevalence of giardiasis was higher in females compared to males [91, 92].
5.2 Relationship between age and giardiasis:
Upon inspecting if there was any correlation between giardiasis and age, the present results
. Higher number of giardiasis cases (59) were observed in the young age groups which
ranged from 5 month to14 years Table 4.1. This result was consistent with a previous result
from a study which was conducted in Bangladesh which found that giardiasis was higher
in the young age group ( 4 ‐ 5 years) [94]. Other study in Cuba showed an elevation of
giardiasis cases in children below 12 years old compared to adults [94]. More investigation
in developed countries such as the one which was conducted in Auckland where giardiasis
was recorded as a gastroenteritis threat to children less than 5 years old [95]. Also in
Jordan two studies conducted in 1994 and 2004 were proposed that young children had the
higher prevalence of giardiasis .The possible explanation why children are at high risk to
suffer from giardiasis compared to adults may be related to many factors such as the poor
hygiene practices especially among children in schools and in day care centres [96, 97].
5.3 Relationship between regions and giardiasis:
During the present study giardasis was observed among rural and urban areas of Jordan
although the majority of positive cases came from rural area (Table 4.1). This might be
attributed to many factors such as the low education level, low income, and improper
drainage for sewage as well as poor sanitation [40]. These results are consistent with a
Malaysian study which showed a predominance of intestinal protozoa including G.
duodenalis cases among rural areas and claimed that the reason was the poor hygiene and
lower health education among the community of these areas. This observation applied also
to the developed countries where a study in Ontario, Canada reported a high prevalence of
giardiasis cases in rural areas [16, 98]. However some studies indicated that the elevation
of giardiasis cases in urban area might be due to the increase in the number of children
32
who attend day care centres which become very popular in urban areas due to the increase
in the percentage of working women [97, 99].
5.4 Comparison between diagnostic tests:
In order to compare between three diagnostic test ( microscopy, ELISA, and bg nested
PCR) statistical analysis were applied in order to measure and evaluate the sensitivity for
the three diagnostic tests. Usually sensitivity reflect the ability of the test to detect the true
positive samples and overcome any false negatives on the other hand specificity express
capability to highlight the negative samples without misleading with positive ones [100].
The percentage of sensitivity would be closely agreed with sensitivity which can be
conducted on same types of diagnostic test worldwide, because both tests don't related to
characteristics and population prevalence criteria. Therefore usually sensitivity taken in
consideration in order to decide which is the most appropriate diagnostic method [100,
101].
The present study was the first study that investigate the sensitivity of bg nested PCR
molecular tool and ELISA as a method for the diagnosis of G.duodenalis in Jordan.
Microscopic examination was used as a basic reference to all calculations in this study
with sensitivity were considered to be 100% .The results of the present study comes in
agreement with other previous studies were conducted in different countries regional and
worldwide as ( Iraq [102], India [62] and Germany [103] ) .which were supposed that
microscopic examination was an accurate technique with high sensitivity and specificity
can be used for G.duodenalis diagnosis and could be considered as a basic standard. In the
present study both bg nested PCR and ELISA were regarded as good sensitive technique
used for giardiasis diagnosis depending on microscopic results .However both techniques
(ELISA and bg nested PCR) were recorded a large number of false negative results. In
present study unfortunately specificity couldn't be calculate for ELISA and bg nested
PCR since this required recovering large number of stool samples ( nearly 100
samples) that should be first confirmed to be negative regarding to microscopic
examination and then screen them by ELISA and bg nested PCR to account true negative
33
and false positive cases which required for specificity calculation, however this couldn’t be
performed during this study since it is out of research budget.
5.4.1 Comparison between ELISA and microscopy results:
During the present study and relaying on ELISA results it can be indicated that this
technique is a sensitive one which can be used for the diagnosis of giardiasis which
reached up 62.5% (Table 4.3). our data was consistent with Many studies that conducted
in different countries including Bangladesh [104], Georgia [105] and Turkey [53], which
proposed ELISA as a valuable sensitive diagnostic test for giardiasis diagnosis with
sensitivity ranged from 89% to 100 %. However upon comparing ELISA with
microscopy, 36 cases were reported as false negative cases. This large number of false
positive cases might be related to low concentration of Giardia cyst or trophozoite
antigens in these samples which can lead to the decrease in the optical density to below
the detection limits (behind the borderline of OD ELISA reader ) [105]. These findings
were consistent with other previous studies such as one which conducted in Germany
which recorded an 82% sensitivity for Giardia Rida ELISA as it detect 37 cases out of
45confirmed positive samples by microscopy [102].
5.4.2 Comparison between bg nested PCR and Microscopy results:
During the present study and relaying on bg nested PCR it can also be indicated as a good
technique for giardiasis detection since it recorded a relatively good sensitivity percentage
which was 67.67% . Several previous studies were indicated that PCR own a high
sensitivity for giardiasis diagnosis such as study conducted in Germany which proposed
85.4 % sensitivity of PCR (Table 4.4) [106]. In the present study upon applying bg nested
PCR, 32 cases were reported as a false negative cases which might be referred to lower or
DNA concentration in samples below the detection limits of PCR amplification that may
resulting from presentence of DNA inhibitors or DNA extraction problems [107].
5.4.3 Comparison between ELISA and PCR:
Upon comparing ELISA with bg nested PCR results, this study show that ELISA still less
sensitive than bg nested PCR, although both percentage of sensitivity were relatively
31
close which were recorded 62.5% and 66.67% for ELISA and bg nested PCR receptively.
Therefore they could be used as an alternative test for each other in case of any one of
them are not available in the clinical diagnostic labs (Table 4.5). According to present
data 11 cases were negative by ELISA although these cases were reported to be positive
regarding bg nested PCR . In the other hand 7 cases were recorded as a negative cases by
bg nested PCR although these cases were reported to be positive using ELISA. the possible
explanation of both false negativity was illustrated previously in the text.
In Jordan, almost all clinical and medical labs use microscopic examination as a gold
routine test for the diagnosis of G.duodenalis in patient’s faecal samples which is
considered as a cheap test to be performed while the ELISA and PCR might be more
expensive and require sophisticated machines, equipments, chemicals and kits. However it
was recommended after the completion of this study that PCR or ELISA also can be
considered as an sensitive method for giardia diagnosis that could be used to confirm
giardiasis diagnosis in addition to Microscopy especially in case the medical technologist
in labs might be unskilled or of low experience to discriminate G.duodenalis cyst from
white blood cells or parasitic cysts such as Entameoba cysts in order to have an accurate
final diagnosis[108].
Chapter 6
Conclusion
Giardia .duodenalis is a major waterborne parasite which is frequently associated with
diarrhoea and gastroenteritis among Jordanian patients since it easily transmitted through
contaminated food or water..
Although Microscopic examination is the most common method which is routinely used
used for G.duodenalis detection in most clinical labs in Jordan, ELISA and bg nested
PCR also show a good sensitivity for giardiasis diagnosis. Therefore it can be use as an
additional conformation of microscopic examination in order to achieve an final accurate
diagnosis for G. duodenalis.
36
Future trends
1- Determine the accurate prevalence of giardiasis in Jordan by conducting a study on
larger number of samples which should be collected from different areas.
2- Perform a molecular characterisation study to identify the different G. duodenalis
assemblages in Jordan in order to tract any potential zoonotic transmission in case of out
breaks occurrence.
3- Spread awareness and health education about giardiasis especially in day care centers
and kinder-gardens through lectures and posters in order to protect the children mainly
from getting the infection
37
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17
ppendix AA
Questionnaire Form
المقارنة بين استخدام المجهر والفحص المناعي المعتمد على االنزيم وتفاعل البلمرة المتسلسل في
.تشخيص االصابة بالجيارديا ديودينالس في األردن
:المعلومات الشخصية
------------------------- : عمرلا -----------------------------:اإلسم
------------------------- :الوظيفة --------------------------:الجنس
--------------------- :مكان السكن
:المعلومات الطبية من األعراض التالية؟ هل لديك أي -1
براز دموي مع مخاط. ألم في البطن د. انتفاخ ج. إسهال ب. أ
األعراض؟ هذه من تعاني وأنت متى منذ -2
واحد أسبوع من أكثر. د أيام 7-5. جأيام 4-3. ب أيام 2-1. أ
ال. ب نعم. أ األعراض؟ هذهتصاب فيها ب التي األولى المرة هي هذه هل -3
ال. ب نعم. أ األعراض؟ هذه نفس من عائلتك أفراد من فرد أييعاني هل -4
ال.نعم ب.أ؟ .....(م،اغنواأل ب،والكال ،طالقط) مثل الحيوانات مع تعامل مباشر أي لديك هل -5
ال. نعم ب.أ المطاعم؟ في أو البيت فيفي العادة هل تأكل -6
ما هو مصدر مياه الشرب التي تشربها؟ -7
المياة المعبئه بالزجاجات البالستيكية. المياه المقطرة في المنزل ج. مياه الحنفية ب. أ
ال. ب نعم. أ ؟ أمراض أي تعاني من هل -8
:إذا كانت إجابتك نعم أذكرها
...............................................................................................................
ال. نعم ب. هل تأخذ أي نوع من أنواع األدوية؟ أ -9
:إذا كانت اجابتك نعم أذكرها
..............................................................................................................
:النتائج المخبرية -11
........................التحليل الجزيئي....................... الفحص المجهري
18
Appendix B
IRP approval
19
.Appendix C
Constent Form
ةـــــرار مــوافقــة تطـوعيـقإ
:ةالدراسعنوان المقارنة بين استخدام المجهر والفحص المناعي المعتمد على االنزيم وتفاعل البلمرة المتسلسل في
.بالجيارديا ديودينالس في األردنة تشخيص االصاب
وصف الدراسةللمرضى المراجعين لمستشفى األمير ديودونالسالجيارديا لالدراسه على بحث انتشار طفيتقوم هذه
الفحص )التشخيص في في األردن، ومقارنة األساليب الثالثة المستخدمة ( منطقة عمان)حمزة
ديودونالس الجيارديا لكشف أنواع طفي باإلضافة الى. (زيئيالمجهري، الفحص المناعي والتحليل الج
.المنتشرة في منطقة عمان في األردن
:المدة الزمنية المتطلبة إلكمال هذا البحث شهر 02
:الدراسةالمخاطر المحتملة من هذه
.ال يوجد أي مخاطر محتملة لهذه الدراسة
:ما سيجنيه المتطوع من المشاركة في هذه الدر اسةالمعلومات المكتسبة من إجراء هذا البحث ال يوجد فائدة مباشرة للمشاركة في هذه الدراسة،ولكن
ديودونالس في عمان وتحديد الطريقة الجيارديا لسيكون لها دور كبير في تقييم مدى انتشار طفي
.األكثر دقة لتشخيص الصحيح
: لهويتك ومعلوماتك البحثية( الخصوصية)الحفاظ على السرية لمنع اآلخرين من . اسمك ورقم التعريف بكأحد غير أولئك الذين يجرون هذه الدر اسه سيعرف ال
الباحث الرئيسي فقط سيحتفظ برموز . معرفة اسمك ، سيستخدم رمز تعريف للعينة بدال لالسم الحقيقي
.التعريف في حجرة مكتبه
: الظروف التي ستوقف مشاركتك في هذه الدر اسه بدون اخذ موافقتكول على موافقة مسبقة منك، إذا تبين أن كنت ال يجوز إنهاء مشاركتكم في هذه الدراسة دون الحص
.تتناسب مع معايير االختيار، أو أن هناك ظروف تستثنيك من هذه الدراسة
: تمويل هذه الدراسة تمويل هذه الدر اسه من منحة الماجستير فى الجامعة الهاشمية
: االسئلةنسخة من هذه . الرجاء عدم التردد في طرح أي اسئله من شأنها إن تبين لك بوضوح هذه الدر اسه
.االستمارة ألموقعه ستقدم لكم
51
وأن لك كامل األهلية لتعطي الموافقة وتتطوع سنة 08بالتوقيع أدناه تقر أن عمرك ال يقل عن
ة قد تم شرحها لك من قبل أعضاء فريق والمضايقات والمخاطر المتوقعلالشتراك في الدراسة اعالة
.البحث
وتمت اإلجابة علي كافة أسئلتك الرجاء التوقيع أدناه إذا كنت مستعدا للمشاركة بهذه الدر اسه
.اسةتنشأ اسئله أخرى بشأن مشاركتكم في هذه الدرقد . بشكل تام وكامل المتعلقة بهذه الدراسة
التاريخ توقيع المتطوع
اشهد إن هذا النموذج قد تم توضحيه وقرأته للشخص أعاله وان أية اسئله حول هذه الدر اسه قد تم
االجابه عليها
التاريخ توقيع الباحث
50
Appendix D
ELISA Result
Plate A
52
Appendix E
ELISA result
Plate B
53
الملخص
عن المسوؤلة رئيسيةال ديودينالس هي أحد االسباب الجياردياما اذا كانت دراسة إلىالبحث دف هذه يه: مقدمة
مثل العمر العوامل الديموغرافيةفي االردن باالضافه الى دراسة تأثير بعض واألمعاءاإلسهال والتهاب المعدة
المقارنة بين بعض الفحوصات ة إلى كما وتهدف هذه الدراس .والجنس والمنطقة السكنية على انتشار هذا الطفيل
تفاعل البلمرة ، تمد على االنزيم و فحص المناعة المع،المجهر ) .ديودينالس الجيارديا تشخيصالمخبرية التي تستخدم ل
.إلختيار الطريقة االنسب للتشخيص( المتسلسل
الفئات مختلف وينتمون الى تم جمع ستة وتسعين عينة براز من مرضى يعانون من االسهال : المواد والطريقة
و ،المجهر ) الثالثة طرق الفحص باستخدامواختبارها جميع العينات كما تم فحص . من كال الجنسين العمرية و
(.تفاعل البلمرة المتسلسل، فحص المناعة المعتمد على االنزيم
وقد أظهرت. ( 40/96)من اإلناث ( 56/96)الذكور عند أكثر انتشار طفيل الجيارديا ديودينالس لوحظ : النتائج
تم تسجيل أعلى نسبة انتشار بين أطفال تتراوح اذ نخفض مع التقدم في السن يمعدل اإلصابة هذه الدراسة ايضا ان
كما لوحظ ايضا من خالل هذه الدراسه ان المناطق الريفية كانت أكثر المناطق . عاما 01أشهر و 5أعمارهم بين
كما وقد تم حساب كل من . حالة 51عرضة ألنتشار هذا الطفيل أذ بلغت الحاالت التي جمعت من تلك المناطق
بناء على نتائج الفحص المجهري اذ ( تفاعل البلمرة المتسلسلو، المناعة المعتمد على االنزيم )ي الحساسية لفحص
% 011على التوالي بينما تم اعتبار الحساسية للفحص المجهري % 66.7و % 62.5 بلغت الحساسية للفحصين
لخصوصية فلم يتم حسابها ألي من أما بالنسبة ل ،وذلك بسبب اعتبار هذا الفحص هو الفحص االساسي للمقارنة
.الفحصين
، االردن في إلسهال المعدي المعويبالهي واحدة من األسباب الرئيسية المرتبطة ديودينالس الجيارديا: الخالصة
وحتى نستطيع تأكيد االصابة بهذا الطفيل فأننا يمكن ان نستخدم اي . واألجناس مختلف األعمار تصيبوالتي يمكن أن
الطرق الثالثة في التشخيص وذلك لحساسيتها العالية كما ويمكن استخدام طريقتين للفحص لحصول على تشخيص من
.نهائي دقيق جدا
