Republic of Iraq
Ministry of Higher Education and Scientific Research
University of Wasit / College of Science
Department of Biology
The uses of molecular methods to detection of
genotyping to the Echinococcus granulosus
from human in Wasit province-Iraq
A Thesis
Submitted to the Council of the College of Science,
University of Wasit
By
Nour Al-Huda Saad Dawoud
Fourth year biology student
Supervised by
Assist. Prof. Dr. Abdulsadah Abdulabbas Rahi
March 2025A.D.
Gamadi al-khar1446A.H.
List of Contents
page
Contents
Summary
I
List of contents
II
List of Figures
V
List of tables
VI
List of appendixes
VI
List of abbreviation
VII
Chapter One: Introduction and Literatures Review
Number
Contents
page
1-1
Introduction
1
1-2
Literatures Review
4
1-2-1
Hydatidosis
4
1-2-2
Classification of parasite
5
1-2-3
Species of genus Echinococcus
6
1-2-4
General description of the parasite
7
1-2-5
Life cycle of the parasite
10
1-2-6
Pathogenesis & Clinical signs
12
1-2-7
Diagnosis
14
1-2-7-1
Staining method
15
1-2-7-2
Enzyme linked Immune sorbent Assays (ELISA)
1-2-9-3
Indirect Haem agglutination (IHA) Test
22
1-2-9-4
Indirect Immunofluorescent Antibody (IFA) Test
22
1-2-9-5
Complement Fixation (CFT) Test
23
1-2-9-6
Intradermal Casoni Test
23
1-2-9-7
Ultrasonography (US)
23
2
1-2-9-8
X- ray Investigation
24
1-2-9-9
Computerized Axial Tomography (CT) Test
24
1-2-9-10
Magnetic Resonance Imaging (MRI)
24
1-2-9-11
Polymerase Chain Reaction (PCR) Technique
24
1-2-9-12
DNA Sequencing Technique
25
1-2-10
Treatment
26
1-2-10-1
Surgery
26
1-2-10-2
Chemotherapy
The Puncture, Aspiration, Injection and
Reaspiration (PAIR) Technique
Prevention and Control
26
1-2-10-3
1-2-11
27
28
Conclusions & Recommendations
Conclusions
63
Recommendations
64
References
66
References
List of Figures
Num
ber
Subject
1-1
General morphological of parasite(cited from King & Hutchinson
, 2003).
E. granulosus eggs (Brower & Ghaffar, 2010).
life cycle of Echinococcus granulosus (Pedro &Peter,2009)
1-2
1-3
Figur
es
8
9
12
List of Tables
Numb
er
1-1
Subject
Characteristics for identification of Eshinococcosis species (Xiao
et al., 2006).
3
Tab
les
6
Chapterone
Introduction &Literatures Review
1-1 Introduction
Hydatidosis is a cyclozonotic disease of human and domesticated animals
caused by infection with the taeniidae metacestode {protoscolices(PSCs)}as a
larval stage of Echinococcus granulosus (Mathis et al., 2005). This disease
has serious impact on human and animal health (Snãbel et al.,2009). Is still a
major endemic, zoonotic and public health problem also is not yet an
organized national control program in Iraq (Athmar &Ben-Abbas, 2014).
Recognized as being one of the world major zoonotic parasites (Fadhil,
2008).The Hydatidosis has importance to public health level and at the level
countries due to migration of infected people and livestock, however have a
high fatality (Craige et al.,1996; Gonzales et al. ,2000).
This parasite requires the mammalian hosts to complete life cycle, intermediate
host (human, sheep, cattle, etc.), definitive host (dog or foxes) (Daniel et al.,
2014). Human serves as accidental or aberrant intermediate host and usually
consider the fate stage for the parasitic life cycle (Eckert et al., 2001; Rokni,
2008). This parasite has a worldwide prevalence but it has a particularly high
prevalence in southern Europe, East Africa, Australia, New Zealand and latin
America (Rokni ,2009). Outbreak of this disease have occurred in Asian
countries such as Lebanon, Jordan, Iraq, Saudi Arabia and Iran leading to
substantial health problems and economic losses (Abdi et al. ,2012). This
parasite have five medical important species of genus Echinococcus are E.
granulosus , E. multilocularis , E. vogeli , E. oligarthus and E. shiquicus ,
these species are morphologically distinction both adult and larval stages
(Abbasi et al., 2003). The E. oligarthrus and E. vogeli occur less frequently
than first two species (Bart et al., 2006a), until recently E. shiquicus had been
discovered only is a specific region of people republic of china (Buishi et al. ,
2005).
Human become infected when ingested contaminated food or water with
egg in material from infected animals ( Brunetti et al.,2010). The definitive
host becomes infected after ingestion of offal or organs infected with hydatid
cyst containing viable protoscolices from slaughtered livestock which be
considered intermediate host of parasite; there are several factors able to
stimulate invagination of protoscolices such as action of pepsin in stomach,
as well as increased temperature and bile secretions in the upper duodenum
(Thompson & McManus, 2001). Hydatid cyst can remain a symptomatic for
numerous years, until they cause compression on surrounding tissues
4
Chapterone
Introduction &Literatures Review
(Beyhan & Umurs ,2011).
The symptoms resemble those of slowly growing tumor; these symptoms of
cyst depend on the size, number and location of the metacestodes (Brunetti et
al., 2010). The clinical signs of cyst mass lesion, signs of cyst depend on the
size, number and the location of protoscoleces (Casulli et al.,2008). The cyst
appear to be usually only a single cyst, approximately ( 60-70% ) in the liver
and (20-25%) in the lungs (Foreyt et al., 2009). Also these cysts found in the
other organs of human including the bones , kidneys, spleen, muscles , central
nerve system (CNS) and behind the eyes (Goto et al., 2010). Development of
hydatid cyst causes pressure and necrosis to adjacent tissues and organs,
causing vital organs dysfunctions (Schipper et al., 2000).Further complication
are due to secondary bacterial infection and anaphylactic shock resulting from
rupture of cyst during surgical interference which may lead to sudden death
(Mirani et al.,2000).
In human hydatidosis , it is diagnosed mainly with imaging techniques such
as ultrasonic graph, radiology, radio wave energy or (T-scanning) computed
tomography scanning supported by serology tests (Dakkak ,2010).Serological
tests used in humans include enzyme linked immune-sorbent assays
(ELISAs),indirect immune fluorescence, indirect hemagglution, immune
blotting and latex agglutination, in the treatment of cyst using the Surgical
elimination is the unique treatment method of the hydatid cysts, since it is
hazardous method due to the possibility of cyst rupturing and spillage hydatid
fluid (Joshi et al., 2005).The Benzimidazole compounds (mebendazole,
flubendazole, albendazole and fenbendazole) have anticestodal activity
against larval stages of hydatid disease Albendazole (ABZ) and Mebendazole
(MBZ) compounds were the first line drugs for medical treatment of
hydatidosis (Mehlhorn et al.,2008). The control of hydatidosis include
education public, supervision of the slaughterhouse of livestock and disposal
of residue , control and reduction of stray dog population, also involved a
combination routine anthelmintic treatment of dogs.(Jenkins et al., 2005).
5
Chapterone
Introduction &Literatures Review
1-2 Literatures review
1-2-1 Hydatidosis
Echinococcus granulosus is hyper endemic and considered one of most
serious helminthes disease with important socio-economic problem, so far as it
affects both human and livestock (Saeed et al.,2000). It was the first
description of the disease hydatid cysts in 379-460 B.C.by Hippocrates as
described as cyst filled with water when observed in the liver of one of the
deceased, said the explosion in the abdomen and lead to the death of the
patient as to the disease for the first time in preventive medicine in the book of
the Razzie (Siracusano et al.,2012; Cox,2002).In the rural communities the
humans maintains contact with dogs (definitive host) and various domestic
animals that act as (intermediate host), therefore the disease is widely
distributed in rudimentary countries (Eckert et al., 2001).Also is affecting
agriculturally based countries (Wafaa ,2011), many years been a manifestation
of parasitic infection which can potentially lead even death (Stamatakos
,2009). Human hydatid disease is the most common presentation and probably
a accounts for (>95%) of the predestined (2-3) million cases in the globe
(Craig et al., 2007).
Hydatidosis was a continues to intrinsic cause of morbidity and mortality in
several parts of the countries, the major sources of morbidity are compression
influence from cyst size, location in a vital organ (brain, GTI, liver, lung,
reproductive tract, bone), cyst rupture with anaphylaxis and dissemination of
the infection; The most common affected organs are liver and lungs whereas
(90%) of hydatid cysts develop excepting; it is predilection sites for the larval
stage, the other organs of the host body are considered as unfamiliar sites of
location of hydatid disease (Mandal& Mandal ,2012).
6
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Introduction &Literatures Review
1-2-2 Classification of parasites
Classified the parasite Echinococcus granalosus site in classification
system (Karyakarte&Damel, 2002; Harder & Melhorn, 2008; Rokin, 2008).
as follows:
Kingdom: Animalia
Sub Kingdom: Metazoa
Phylum: Platyhelminthes
Class: Cestoda
Sub Class: Eucestoda
Order: Cyclophliidae
Sub Order: Taeniata
Family: Taeniidae
Sub Family: Echinococcina
Genus: Echinococcus
Species: granulosus
The present study emphasize on the first species, which have four sub species
of medical importance (WHO, 2003) ,which are as follow:
E. granulosus granulosus
E. granulosus equins
E. granulosus boreilis
E. granulosus Canadensis
7
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Introduction &Literatures Review
1-2-3 Species of genus Echinococcus
Species under genus Echinococcus are small tapeworms of carnivores with
larval (metacestode) stages known as hydatid cyst, a sexual reproduction in
various mammals including humans (xiao et al. ,2005).
There are four morphologically distinct species in this genus until, recently
when Echinococcus shiquicus was added to the previously known species:
E.granalosus, E.mulitlocularis , E.oligarthus , E.vogeli (Xiao et al.,2006a).
However only these four specie, only E.granulosus has medical and public
health importance in that they are widely prevalent and may cause severe
diseases in humans .(Makerro &Davies , 2001).
E.granulosus is a causative agent of cystic Echinococcosis (CE) or cystic
Hydatid Disease (CHD) and E.multilocalaris in human causes Alveolar
Echinococcosis (AE) or Alveolar Hydatid Disease (AHD) (Hofer et al.,
2000).The other two species are mainly restricted to sylvatic animals and
occur in some areas of central and South America. It rarely causes polycystic
Echinococcosis (Gemmell et al., 2001). A table (1-1) showing the properties
to determine the types of Hydatidosis in humans.
Table (1-1) characteristics for identification of Echinococcosis species (Xiao
et al., 2006a).
E.granulosus
E
.multilocularis
E.oligarthus
E.vogeli
E.shiquicus
Distribution
Cosmopolitan
Holarctic
region
Neotropical
region
Neotropical
region
Tibet plateau
Definitive
host
Dogs
Foxes
Wild felids
Bush dog
Tibetan fox
Intermediate
host
Ungulates
Microtine
rodents
Neotropical
rodents
Neotropical
rodents
Plateau pika
Body
length(mm)
2.0-11.0
1.2-4.5
2.2-2.9
3.9-5.5
1.3-1.7
Number of
segments
2-7
2-6
3
3
2-3
8
Chapterone
Introduction &Literatures Review
Length of
large
hooks(µm)
25.0-49.0
24.9-34.0
43.0-60.0
49.0-57.0
20.0-23.0
Length of
small
hooks(µm)
Number of
testes
17.0-31.0
20.4-31.0
28.0-45.0
30.0-47.0
16.0-17.0
25-80
16-35
15-46
50-67
12-20
a. Mature
segment
Near to middle
Anterior to
middle
Anterior to
middle
Posterior to
middle
Near to upper
edge
b. Gravid
segment
Posterior to
middle
Anterior to
middle
Near to
middle
Posterior to
middle
Anterior to
middle
Gravid uterus
Branching
laterally
Sac-like
Sac-like
Tubular
Sac-like
Metacestode
Unilocular cysts
in viscera
Multilocular
cysts in
viscera
Polycystic
cysts in
muscles
Polycystic
cysts in
viscera
Unilocular
cysts in
viscera
9
Chapterone
Introduction &Literatures Review
1-2-4 General description of the parasite
Echinococcus granulosus is a one of the smallest tapeworm, which ranges
in length between (2.5-9) mm and in width (0.5-1)mm ,and it has round
scolex (0.3)mm in diameter containing a rostellum surrounded by two rows of
hooks, their number is between (28-40),hooks length (22-42) micron as well
as also have four suckers having acetabulum shape , the scolex is connected
with cylindrical neck (Lawson & Gemmell,1983).
The neck region is the located of proglottid germination maturing proglottids
forming stroblia. The stroblia posterior to the neck region including of
reproductive units called segments (proglottids) that contain three to five of
proglottids, the proximal to the neck is immature proglottid and is followed by
maturing and finally gravid proglottid (contain mature eggs) (Rawson,1966).
The proglottids are range between (3-5) proglottids, they are different in
pattern of size and maturity, the first proglottid followed by neck is stumpy
and contain immature genital organs authority dark patch of cells called
immature proglottid (Thompson & Mcmanu , 2001).
The second proglottid called mature proglottid, because it contain mature
genital organs, also bigger than the first proglottid, rectangular in shape and
location, contain in the middle genital aperture .However, consist of (45-65)
pineapple testes spread on the front of the proglottid, female genital
consistency from one ovary located in the posterior with lobes a regular in
shape, also located ovary vitilline gland spherical shape (Tunaci et al., 1999).
The gravid proglottid is the largest proglottids where representing half the
length of the body nearly and where genital organ decay and remains uterus
characterized sac-like present (12-15) divaricate side-ways contain limits
(500-1000)eggs, separated from gravid porglottids for the worms body and
release embryonated egg in the feces(Muller et al., 2007). Figure (1-2).
10
Chapterone
Introduction &Literatures Review
Figure (1-1) General morphology of parasite (cited from King& Hutchinson, 2005).
The structure and function of the body covering have been of senior attention
to parasitology, which have used electron microscope to participate numerous
area of cestodology (Garcia, 2001). The tegument is the body covering
cestodes, it is known as a living tissue with high metabolic activity, the
cestode tegument is covered by minute microvilli which called microriches
that serve to increment the absorbent area of the tegument (Siracusano et al.,
2012). The microtriches are analogous in some regard to the microvilli found
on gut mucosal cells , they completely cover the worms surface including the
suckers ( Garcia,2001).The size of hydatid cysts ranged from (1.5 to 23cm) in
their greater diameter and the largest cyst was approximately (23*18cm);The
majority of the cysts (47.7%) were between (6-10cm) in their greater diameter
and only (9.9%) of those larger than (15cm) (Ahmadi& Bodi,2011).
11
Chapterone
Introduction &Literatures Review
Some cysts die spontaneously, while others persevere unchanged for years,
sometimes apart entire inner layers of the cysts calcify, which does not
necessarily mean cyst death (Goldsmith,2005).
The eggs contain inside on hexacanthembryo complete growth elliptical form in
diameter (25 micron), it contains three pairs of hooks lancet shape (Muller et
al.,2007.;Bouree,2001). Hexacanthembryo was surrounded by eight membranes
from the inside out Capsule, vi telline layer, outer embryophoiric membrane,
embryophore, basal membrane of granular layer, oncospheral membrane,
cimiting membrane, capsule assimilate pellicle disappears once set(Holcman &
Heath,1997) .
The eggs from definitive host cannot observed in feces samples, while
embryophore component from composing thick keratin in blocks, as features
high resistance ,it earns egg dark appearance planned (Imad, 2002; Thompson
& Mcmanus,2001; Markell et al.,1999). Figure (1-2).
Figure (1-2) E. granulosus eggs (Brower & Ghaffar, 2010).
12
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Introduction &Literatures Review
1-2-5 Life cycle of the parasite
The life cycle of Echinococcus was indirect employment dogs and
other canine as definitive host and many herbivorous, omnivorous species
including; wild life and domesticated livestock as intermediate hosts (Asma ,
2009). Human as accidental or aberrant intermediate host, they generally dead
end for the parasite infection. Adult E. granulosus worms have been detection
in various carnivorous such as; stray and farm dogs, red foxes and wolves
(Arbabi &Hooshyar ,2006).
Sexual maturity of adult E.granulosus occurs within (4-5) weeks after the host
ingests residue containing viable protoscolices in tissue of the intermediate
hosts (Mcmanus et al.,2003).Eggs remain viable for several weeks or months
in pastures or gardens and on fomite, they survive best under moist conditions
and in moderate temperatures, viable eggs have been found in water and damp
sand for three weeks. The eggs survive for only short periods of time if they
are exposed to direct sun light and dry conditions (Andresiu et al.,2009). Eggs
are highly resistant to bad environmental conditions such as sun light and
chlorine in tape water (Horton, 2003). Intermediate hosts get infection when;
ingestion of contaminate materials food or by direct contact with infected
dogs, especially children and veterinarian (Idris et al. ,1999).
Human become infected by ingestion of food or water contaminated with egg
in fecal material from infected canines (Heath et al.,1994).They are two
common sites for evolution liver and lung, but sometimes oncosphers
elopement into general systemic circulation and develop in to other organs
and tissues (Urquhart et al.,1996: Macpherson et al.,2000). The oncospher
penetrate the intestinal mucosa, passing in to lymphatic or mesenteric venues
and carried by the blood stream to various parts of the body. Also enters the
portal vein and lodges in the liver, reaching the lungs organs within (24hours)
after ingestion (Huddix et al.,1994 ; Gonzalez et al., 2002). The oncospher
metamorphosis have slow operation of growth in to a type of bladder worm
called unilocular hydatid cyst in about five months the hydatid develops a
thick outer creamy white laminated layer, non-cellular layer and an inner thin
nucleated germinal layer (Roberts & Janovy,2000).
The internal germinal layer produce many small vesicles or brood capsules in
about (5-6) months, after infection, each brood capsule contain up to (40)
11
13
Chapterone
Introduction &Literatures Review
scolices invaginated in to their neck portions and attached to the wall by
stalks. The brood capsules become discrete from the wall of the vesicle and
float freely in the vesicular fluid, giving rise to hydatid cyst (King &
Hutchinson,2005; Kul&Yildiz,2010)
If the oncospher is not destroyed by phagocytic cells, it develops in to hydatid
cyst (Huddixe et al .,1994;Gonzalez et al.,2002). The liver and lung are the
most organs to infected with hydatid cysts, these cysts grow slowly, generally
increases from less than (1cm to 5cm) in diameter each year (Kul & Yildiz
,2010). Although the larval stage is a fluid-filled bladder or hydatid cyst that
is unilocular, individual bladder may reach up to (30cm) in diameter and
occur most in the liver and lungs, but may develop in other internal organs,
the infection with this stage is referred to as hydatidosis ( Elayoubi &
craig,2004).
In the definitive host such as dogs and other canines, living viable protoscoleces
are evaginated and outspread in about (24) hours of ingestion (Macpherson et
al., 2000; Roberts& Janovy,2000). The successfully implantation of the parasite
requires physiochemical factors (e.g. gastric acidity, surface action of bile salts)
and gut enzymes (e.g. pepsin, trypsin and pancreatic) (Rafiei & Craig, 2002).
The worm grows in to adult in the small intestine of dogs in (6-7) weeks, when
mature the last segment of the tap worm may contain (1000) eggs.
A proglottid containing eggs is shed by the tap worm every (14) days, a new
segment filled with eggs then develops to take its places (King& Hutchinson,
2005). Gravid proglottids shed eggs in to feces; eggs have a sticky coat that
will adhere to an animal's surface .The eggs are shedding by the rhythmic
contraction from proglottids of adult worm in the final host widely on
pastures (Heath, 1971; Junghanss et al., 2008). Figure (1-3).
11
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Chapterone
Introduction &Literatures Review
Figure (1-3) life cycle of Echinococcus granulosus (Pedro &Peter,2009)
11
15
Chapterone
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1-2-6 Pathogenesis & Clinical signs
The initial phase of hydatid cysts development was asymptomatic; the size
of cysts being about (0.25-5 cm) this phase represented by formation of
capsule around the cyst by the infected organ, the incubation period varies
from several months to years (Craig & Larrieu, 2006; Dvorak et al., 2008).
After ingestion Echinococcus eggs hatch and release embryo in the small
intestine, penetration through the mucosa lead to blood borne distribution to
liver and other sites, when development to cysts begins (Pedro & Peter,
2009), whereas early signs appear when the cyst occupied in restricted and
vital organs such as brain, spinal cord and eyeball; hydatid cysts on the brain
mainly lead to death (Gutierrez, 2000; Marquardt et al., 2000).
In Iraq, the most cases of human hydatidosis occur in the liver (50%-75), lung
(25%) and (5%-10%) for other organs (Ahmed, 2011). The hydatid cysts cause
local inflammation of the surrounding tissues with formation of fibrous
capsulated membrane resulting from host's immune response against the parasite
that include fibrous layer derived by the host which mainly calcified alone
around the cyst and observed in the parenchymal tissue causing a pressure on the
infected organ as the infection progress (Rigano et al., 1997).
The remain cysts can be found anywhere in the body including; bones,
kidney, spleen, muscle, central nerve system (CNS) and behind the eye,
depending on the location, some cysts can become very large and may contain
up to several liters of fluid. Other climacteric location such as the brain is
symptomatic when they are still small (Schneider et al., 2010). Hydatid cysts
become a large size because of asexual reproduction and proliferation of the
most inner cyst layer (the germinal epithelium) (Mehlhorn et al., 2008).
The benign unilocular cyst may grow to great size without even producing
symptoms but detected early when it develops in areas like brain and bone
marrow; Hepatic cysts may cause several CE-no specific symptoms. It has
been reported to cause pain in the upper abdominal region, hepatomegaly,
cholestasis, biliary cirrhosis, portal hypertension, ascites and a verity of
other manifestation (Tortora et al., 2010). Hepatic cysts may rupture in the
peritoneal cavity, causing formation of secondary cyst or anaphylaxis
resulting from dissemination of protoscolices which can grow to larger
11
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Introduction &Literatures Review
cysts (Eckert & Deplazes, 2004), that hepatic cysts frequently cause
cholecystitis and pressure on liver which lead to enlargement and pain in
the upper right side of stomach, significant distraction of bile duct and
portal vein due to distension of cysts that lead to hepatic lobulation
(Nunnari et al.,2012).Renal cyst may reach a considerable size that lead to
rupture of cyst, then cyst fluid pass with urine, in some cases there is
development of membranous glomerulonephritis due to prolong release of
antigens from cysts with formation of immune complex which may become
deposited in kidneys, the growing cyst may cause a pressure necrosis on
neighboring tissues.
Hydatid cyst expansion depends on their location, oozing of hydatid fluid
from the cyst resulting in eosinophilia (Gelman et al., 2000). If bacterial
infection occur this will produce a number of gathering, depending on the
number of cysts (Gavidia et al., 2008). In the lungs (20%) of cases with
other organ embroilment for less than (10%) of cases (Pawlowiski et al.,
2001).
It may be linked with pulmonary tuberculosis or other lung infection,
complete or partial rupture of a hydatid cyst may lead to expectoration of
hydatid fluid and membrane, may be followed by bacterial over growth and
pulmonary abscess(Verastegui et al.,2003). Also the cysts cause respiratory
signs including; chronic cough, chest pain, dyspnea, hemoptysis and
pneumonic thorax can occur (Thompson, 2008).
Pulmonary cysts seepage or corrode in to a bronchus and rupture or become
large enough to blocking a bronchus causing chest pain, cough , dyspnea
and fever as the most common presenting symptoms in patients with intact
pulmonary hydatid cysts (Kuzucu et al., 2004; Darwish ,2006). The
growing cyst may cause a pressure necrosis on neighboring tissues. Hydatid
cyst expansion depends on their location, oozing of hydatid fluid from the
cyst resulting in eosinophilia. The severity of pulmonary hydatid cysts
when production of bloody-fluid mixture which may contain remarkable
hydatid tissue. As time goes results in gradual cure but secondary infection
lead to chronic lung abscess (John and Petri, 2006).
In the liver is the most common symptoms include; abdominal pain, nausea,
vomiting and indigestion (Zhao et al., 2009).If the cyst obstructs in the
biliary system, it can cause gallstones and pain or cholestasis jaundice,
anemia, pleural pain (Steta &Torre, 2009). Few cases were reported in the
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17
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Introduction &Literatures Review
pancreas, thyroid, breast, eye, bladder, tongue and salivary glands (Saez et
al.,2001). The cysts can visible in the brain and spinal cord, cyst in the
bones can destroy the cysts structure of bone and result in impulsive
fractures (Varcasia et al., 2007). In bones the state is atypical when it occurs
the invasion of marrow cavities and spongiosa is common and cause
extensive erosion of the bone, also bone cysts started as a small osteocytes
process (Moro & Schantz, 2009).
In the heart cyst presence of hydatid in the left ventricle of heart of a pure
holishtine cow from Al-Nasir farm swaira, south Baghdad ( Rahif, 2001).
Also, cause in pericardial effusion heart block or other arrhythmias and
sudden death (Varcasia et al ., 2008) .Even the infection may be acquired in
childhood , most cases of liver and lung cysts become symptomatic and
diagnosed in adult patients because of the slowly growing nature of hydatid
cyst (Jenkin et al ., 2005). While non – specific signs may include , anorexia ,
weight loss and littleness (OIE ,2008).Also can cause allergic reaction
including , shaking chills or fever , asthma , pruritus , urticarial and life
threatening anaphylaxis ( Xiao et al ., 2006b).
In the spleen, is the most common intra-abdominal organ to be affected by
hydatidosis, fever, abdominal pain and enlarged spleen are the most
frequently encountered symptoms of splenic cyst , but they are non- specific
(Garcia,2001; Raether& Hanel,2003). However, two cases of primary hydatid
cysts of adrenal glands with isolated abdominal pain was reported (Bedioui et
al., 2005).Also, a case of primary pelvic hydatid in a 36 years old female
presenting with a mass in the fallopian tubes of a young girl (Terek et al.,
2000). All body organs and tissues are considered to be exposed to hydatid
cyst infection expect the hair, nails and teeth (Muro et al., 2000).
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1-2-7 Diagnosis
1-2-7-1 Staining method
Staining method was used for parasite detection besides these, a number of
staining techniques using hydatid cysts from human infected liver from
hospital. The hydatid fluid of cysts aspirated by a 20 ml syringe and were
aseptically transferred into the glass cylinders and left for 30 min to allow the
protoscolices to settle to the bottom of the cylinder. Then, the supernatant was
removed. The viability of the protoscoleces was confirmed by 0.1% eosin
exclusive test as well as flame cell motility under a light microscope (Moazeni
et al., 2012).
1-2-7-2 Enzyme Linked Immune sorbent Assays (ELISA)
ELISA is a one important serological test used to disease diagnosis.
The test result rarely becomes negative even after many years and even when
it seems to cure disease (Sharma et al., 2004). ELISA has been extensively
used with completely good results, this test using for crude hydatid cyst fluid,
showed a high sensitivity, also the specificity was very low. Should be
montined that approximately (10-20%) of patient with hepatic cyst and about
(40%) with pulmonary cyst and with calcified cyst or cyst located in bones
and brain (More et al.,2000). ELISA used for diagnosis of cerebrospinal
hydatidosis , with giving higher sensitivity (Dekumyoy et al ., 2005).
1-2-7-3 Indirect Haemagglutination (IHA) Test
This test can also use to for diagnosis cystic hydatid disease, however
this test low sensitivity, but now has been replaced by the enzyme linked
immune assays (ELISA) for initial screening of sera (Pedro & Peter , 2009).
1-2-7-4 Indirect Immunofluorescent Antibody (IFA) Test
The test was diagnosed case of degree immune complex type of
membranous glomerulonephritis .In immunofluorescence (direct and indirect)
and immune peroxidase studies revealed glomerular deposits of hydatid
antigen and corresponding antibody. It is one of a great sensitivity in the
qualitative histological techniques (Tarp et al., 2000).
22
19
Chapterone
Introduction &Literatures Review
1-2-7-5 Complement Fixation (CFT) Test
In this test presence of complement fixing antibody in patients sera. It is
mainly associated with IgG antibodies and since these diminution rapidly
following surgical removal the cyst, It`s reported that have wide range from
variation in the sensitivity of CF test, complement fixation is now rarely used
(Matossian et al .,1992; Moreno et al ., 2002).
1-2-7-6 Intradermal Casoni Test
This test using fluid obtained from cysts of intermediate hosts origin, this
test requires special care in technique and in interpretation of reliable results
were to be obtained, the reagent in general use is pooled hydatid fluid derived
from uncomplicated cysts, fluid containing scolices being usually selected, the
fluid injected subcutaneously S/Q injection, the amount sufficient to raise a
white area approximately one centimeter in diameter.
The normal response to this test is stroke consists of red line confined to the
area of pressure followed within half minute by a spreading flush, also
prescence or absence generalized skin disease, pruritus, urticaria, high fever
and asthma, this test has been used to help in diagnosis. The specificity of this
test was in doubt because cross-reaction with schistosomasis and a false
positive reaction in casoni test occurred mainly in patients with other
helminthic infection (Poretti et al ., 1999) .
1-2-7-7 Ultrasonography (US)
Ultrasonography apparatus measure greater than other imaging
diagnostic methods for Hydatidosis, because provides real time results and
can be stored with high resolution and diagnostic precision (Macpherson et al
., 2003).
In liver cysts for example, US can clearly show natant membranes, daughter
cysts and hydatid grittiness macroscopic appearance when a cyst contains
thousands of protoscolices which are typical characteristic of hydatid cyst
(More et al., 2005).The specificity of US in diagnosis pulmonary hydatid
cysts using the wall sign, a double – layer border in univesicular and double –
23
20
Chapterone
Introduction &Literatures Review
layer in internal cysts (El Fortia et al., 2006).Also US developed for detected
hepatic cysts (WHO,2003).
1-2-7-8 X- ray Investigation
X-ray examination keep its diagnostic applicability in lung cystic
echinococcosis and sporadically in finding calcified lesions, it is diagnostic of
hydatid disease (Ben- Musa et al., 1990).
1-2-7-9 Computerized Axial Tomography (CT) Test
CT was superior than ultrasonography US by presentation the potential
of inspecting any organ and detecting smaller cysts , replaced that the
ultrasound technique which the primary diagnostic tool in the first period
(1985-1995) by CT for post- operative follow up in the second period (Tekin,
2008).
1-2-7-10 Magnetic Resonance Imaging (MRI)
MRI was not much superior than CT or US except for evaluation of
post- surgical residual lesions, recurrences and extra – hepatic infections such
as cardiac, vertebral and CNS infection (Chang & Han,1998).
MRI showed the exact anatomic location and the multivescular nature of
cardiac cyst and pulmonary cyst in hydatidosis patient and was useful in
planning surgical treatment (Pockros & Capozza,2005).
1-2-7-11 Polymerase Chain Reaction (PCR) Technique
The polymerase chain reaction took the world biological laboratories by
storm, and became the key technology in the field of molecular biology. The
generation of un limited amounts of specific PCR products made many new
methods of DNA analysis possible , such as downstream modifications (e.g.
cloning of the PCR product establish gene libraries)or comprehensive product
analysis techniques (e.g. gene expression or genotyping analysis) (Wojdacz et
al., 2008).
24
21
Chapterone
Introduction &Literatures Review
1-2-7-12 DNA Sequencing Technique
DNA sequencing technique denote to determine the order of nucleotide
bases adenine, guanine, cytosine and thymine in a molecule of DNA (
Sengupta & Cookson ,2010).
In studying biological processes for basic research the DNA sequencing
become knowing of gene and other parts of genome of organisms
indispensable on applied field such as diagnostic research (Schuster, 2008).
This technology using to determine the genetic characterization of
E.granulosus depends on the homology of the sequence of two mitochondrial
genes: cytochrome C oxidase subunit 1(CO1) and reduced nicotinamide
adenine dinucleotide subunit 1(ND1) (Mcmanus,2002; Lavikainen et
al.,2003; Snābel et al.,2009).
25
22
Chapterone
Introduction &Literatures Review
1-2-8 Treatment
1-2-8-1 Surgery
The first treat of hydatid cyst by a total surgical remove of cyst with
avoidance of the adverse consequences of spilling the contents (Schweiger,
2007). In liver cysts the preferred option is surgery when the cysts have size
are large (>10 cm) in diameter and located in certain organs (i.e. brain, lung
and kidney) (Kern et al., 2004). The best potential to remove cysts and
immediately complete treatment after surgical remove the infected cysts if
possible remnant cysts (Smego et al.,2003).
1-2-8-2 Chemotherapy
The Benzimidazole compounds (mebendazole, flubendazole , albendazole
and fenbendazole ) were spectrum anthelmintic activity against larval and
adult stages of worm. Albendazole(ABZ) and Mebendazole (MBZ)
compounds were the first line drugs for medical treatment of hydatidosis
(Mehlhorn et al.,2008).
The Benzimidazole compounds have experience chemotherapy and can be
recommended for many patients (Siqueira et al., 2007). In general relapses of
Benzimidazole compounds (Albendazole "ABZ" and Mebendazole "MBZ")
treatment were seen in up to (30%) cases ; however a high proportion of those
recurring cyst might be susceptible to a further cycle of chemotherapy
(Lorenzo et al .,2005).The detectable change for hepatic cysts is the secession
of the endocyst after (1-5) months from treatment followed by size reduction
and vanishing (3-13) months (Jimenez et al., 2002).Also the speed of liver
cyst retractility appears to be slow motion than cyst in other parts of the body
(Benito et al., 2006).
In the lung cysts changes appears when cyst rupture (10 days to 2 months)
after treatment initiation followed by evacuation , shrinkage , distortion and
vanishing within (5-9) months of the initiation of treatment .(Macpherson et
al.,2003).However rupture of cyst due to ABZ treatment has been observed in
many studies (Schantz et al., 2003).
26
23
Chapterone
Introduction &Literatures Review
Praziquantel (PZQ) has also been evaluation, both, alone and in combination
with (MBZ). PZQ appears to have a high efficacy at the beginning of cyst
differentiation (before 34days of infection) and zero efficacy (after 34 days)
in mature metacestode at least is an experimental mouse model (Dendurkuri
& Joseph , 2001).
1-2-8-3 The Puncture, Aspiration, Injection and Reaspiration
(PAIR) Technique
The treatment of hydatid cysts in the liver and some other locations
consist of percutaneous uncture using sonographic guidance, aspiration of
substantial amount of liquid contents, injection of a protosolicidal agent (95%
ethanol or hypertonic saline) for at least 15 minutes and re- aspiration (PAIR)
(Hûttner et al., 2008).
Using this technique when the hepatic cysts has size (≥ 5cm) in diameter ,
also this technique is using in children and pregnant women (Torgerson et
al.,2002). As well as in those with cysts communicating with biliary tree,
cysts in risky or inaccessible location in the liver , free cysts in the abdominal
cavity or cysts in the lungs , heart , brain and spinal cord (Budke et al., 2006).
The patients who refuse and cannot done the surgery and have single or
multiple cysts in liver, abdominal cavity, spleen, kidney and bones and
become treatment by PAIR; this technique is contraindicated for in accessible
specifically located liver cysts and for in active or calcified of cysts lesions
(Saez et al., 2001).
Application of a n effective vaccine to reduce hydatid infection in livestock
would be likely to have a substantial impact on threat of transmission disease
to humans (Chabalgoity et al., 2001). It was considered that vaccine
development a approach used in Taenia species such Teania ovis would also
be successful for Echinococcus granulosus using recombinant DNA
technology , an oncospher antigen vaccine EG95 was shown to be capable of
inducing a high protection against experimental challenge infection with
Echinococcus granulosus eggs in sheep (Kamiya ,2007).
Basic research on canine a mucosal immunology and Echinococcus infection
is required for progress (Varcusia et al., 2007) .
27
24
Chapterone
Introduction &Literatures Review
1-2-9 Prevention and Control
The control of Hydatidosis include education public, supervision of the
slaughterhouse of livestock and disposal of residue , control and reduction of
stray dog population, also involved a combination routine anthelmintic
treatment of dogs (Jenkins et al., 2002). Using Praziquantel as recommended
treatment for prepatent period of Echinococcus granulosus is approximately
6 weeks is the most effective anthelmintic viable for this purpose (Larrieu et
al ., 2002). Development of amended diagnostic tools for animal (definitive
and intermediate) hosts and human beings, therefore using effective vaccine
for livestock and dogs vaccines possibility against the worm E. granulosus,
educational programs and anti-parasitic effective oxfendazole for treatment
(Craig et al., 2007).
28
25
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