الفهرس 
Historical background of Echinococcus granulosus
Life Cycle and Morphology of E. granulosusOnly 14 pages are availabe for public view
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Abstract
Cystic echinococcosis (CE) caused by the larval stage
of Echinococcus granulosus is a re-emerging problem with
great economic and medical impact in developing countries
including Egypt and it was listed by WHO as one of the
neglected tropical diseases.
E. granulosus has a number of intraspecific variants
or strains which have been characterized by morphological,
biochemical, biological, developmental and molecular
approaches. They were identified and classified by
mitochondrial and nuclear markers into ten genotypes (G1 to
G10) namely E. granulosus sensu stricto (s.s.) (G1–G3),
Echinococcus equinus (G4), Echinococcus ortleppi (G5) and
Echinococcus canadensis (G6–G10) and the lion strain
(Echinococcus felidis). These strains shows differences in
geographical distribution, host specificity, ecology,
transmission dynamics, development rate, infectivity to
human, antigenicity and sensitivity to chemotherapeutic
agents.
Camels have been involved in the transmission cycle
and the epidemiology of E. granulosus especially in rural
communities where dogs (definitive host) are infected by
ingestion of infected camel carcasses containing the hydatid
cysts. However the most common strain infecting human
was known to be G1, recent molecular characterization of
human and animal isolates demonstrated the involvement of
the camel G6 strain in causing human infection. To detect
the specific strain in any endemic locality is a must to apply
the most suitable control programmes.
CE is diagnosed by different methods; immunological
and serological tests, radiological and by molecular techniques as PCR which showed high sensitivity and
specificity.
Detection of circulating E. granulosus antibodies in
patients’ sera remains the method of choice and showed
greater sensitivity than detection of circulating antigens in
diagnosis of CE. Standardization of techniques of antigen
preparation and characterization of new antigens still needed
in order to improve the immunodiagnosis of CE.
The present work aimed to study and compare the
protein profile of HCF obtained from different Egyptian CE
patients as well as different livestock reservoir animals with
identification of antigenic proteins. Moreover, probing HCF
and patients’ sera for the detection of the G1 or G6 genotype.
This aims to identify new antigens, which may provide
information about parasite survival strategies in the host and
thus improving CE diagnosis, treatment and control.
The study was conducted on 90 subjects of both sexes;
70 patients and 20 healthy individuals after taking informed
consent as well as animal isolates after permission.
Human subjects were classified into two groups:
group I (CE patients):
-group I-A: including cases of hepatic CE
-group I-B: including cases of pulmonary CE.
-group I-C: including cases of CE with multiple organs
affection.
-group I-D: including cases of CE after medical and/or
surgical treatment.
They were selected from Abdominal
Ultrasonographic Unit of Tropical Medicine Department,Kasr El-Aini Hospital, Department of Cardiothoracic
Surgery, Faculty of Medicine, Ain Shams University and
Diagnostic and Research Laboratory of Parasitic Diseases in
the Department of Medical Parasitology, Faculty of
Medicine, Ain Shams University.
group II (Control group):
-group II-A: including cases of other parasitic infections;
schistosomiasis, fascioliasis, taeniasis and amoebic liver
abscess.
-group II-B: including cases of patients with other mass
occupying lesions; hepatocellular carcinoma, simple liver
cyst and bronchogenic carcinoma.
-group II-C: including cases of apparently healthy
individuals with no history of parasitic infections, negative
urine and stool examination and negative IHAT for hydatid
disease.
They were selected from Department of Tropical
Medicine, Chest Medicine (pulmonary diseases and
tuberculosis), Faculty of Medicine, Ain Shams University
and the Diagnostic and Research Laboratory of Parasitic
Diseases in the Department of Medical Parasitology, Faculty
of Medicine, Ain Shams University.
Animal samples (isolates):
Cyst from animals (camel and sheep) were obtained
from Cairo abattoirs and the fluid was examined for
protoscolices. No cysts from Pigs were available.
The present study had two parts; molecular and
immunological.The Descriptive analysis of the CE cases showed that
CE was more common among females (65%) and most of
the cases represented in the middle age (the mean is 33.9
years).
The molecular part was carried out by purification of
HCF of human and animal’s isolates. Deposited
protoscolices underwent DNA extraction with subsequent
PCR using specific primers for detection of either G6 or G1
genotype. Then DNA sequencing and comparing the results
on GenBank was done.
The human cases’ HCF were collected by
percutanuous aspiration injection reaspiration (PAIR)
technique. Using conventional PCR, all the CE cases under
the study; hepatic, pulmonary and multiple organ, were of
G6 genotype (camel strain); showed band at 254 bp.
Regarding the animal isolates, the examined camel
cysts were fertile and they were all of the G6 genotype (band
at 254 using G6 primers). All examined sheep cysts (30 were
negative either microscopically (no protoscolices) and by
PCR using G6 or G1 primers. This suggests that the camels
are the main animal reservoir of CE in Egypt and the cause
of the presented human infections.
Also sera of the studied human groups were collected
to detect the G1 or G6 genotype, 27 out of 30 samples were
G6 genotype showed characteristic band at 254 bp using G6
primers and were all negative using G1.
Results of PCR in sera was compared to microscopic
examination of the HCF, showed relatively low sensitivity
90% against 100%, however it still represent a reliable tool
for CE diagnosis more safe than PAIR technique and
microscopic examination of HCF. Moreover, PCR for G6and G1 of CE was negative in all control subjects, giving it
a 100% specificity.
The results of PCR were confirmed and verified by
bidirectional DNA sequencing. The tested isolates showed
100% homology with G6 Japanese strain (Camel) by Nakao
et al. (2007) in the GenBank. Also, alignment of the isolates
with the Egyptian G6 strain, showed 100% identities and (0)
gabs which indicate homology.
The immunological part of the study, crude HCF
antigens were prepared from the supernatant of HCF of
human and animal isolates. Their protein profiles were
analyzed by using 12% SDS-PAGE under reducing
conditions. After Standardization of protein concentration,
25 µg protein showed the best band resolution.
The protein profiles of human and camel isolates were
similar composed of 11 bands (164, 130, 110, 80, 75, 48, 35,
25, 20, 18 and 12 KDa) with some differences at the high
molecular weight bands; the164, 130 KDa were absent in all
camel isolates.
Using human crude HCF antigen pool, EITB revealed
that anti E. granulosus total IgG of the hyperimmune rat
serum reacted with 9 antigenic bands, varying in molecular
weight from 110 kDa to 12 kDa (110, 80, 75, 65, 56, 48, 35,
25 and 12).
Statistical analysis of these molecular weights bands
recognized by total IgG of the studied groups’ sera showed
that the 48-kDa and 12-kDa protein bands reacted against 30
(100%) of the patient’s sera IgG. None of the sera from
control groups (GpΠ) recognized the 12 KDa band
indicating a highly significant difference but the 48 KDaband was only recognized by 2 sera of control group of other
parasitic infections.
Also, 35 kDa bands reacted against 20 (66.7%) of CE
cases versus 2 (4%) of schistosomal sera (GpΠ-A).
Moreover, 75, 56 and 25 KDa bands reacted with 7 (23.3%),
3(10%) and 10 (33.3%) of the CE patients’ sera, respectively
and none of the control sera recognized these bands which
indicating a highly significant difference (p<0.01). On
contrast, 110, 80 and 65 KDa bands were not recognized by
both CE cases and control groups.
Also, EITB was used in the present study to follow
up patients after medical or surgical treatment, sera of 10 CE
cases 6-12 months after treatment were blotted against
human crude HCF pool antigen. It showed absence of all
antigenic bands except 35 KDa band which was recognized
by 4 (40%) patients (P value: <0.01) indicating a highly
significant difference.
On statistical analysis of the recognition of each
protein band by anti E. granulosus IgG using human crude
antigen pool showed that 12 kDa gave the highest sensitivity
(100%) and specificity (100%). Moreover, it wasn’t detected
in any sera of patient after treatment making it sensitive to
be used in the assessment and follow up of treatment.
On conclusion, G6 genotype was detected in all
human CE cases as well as camel isolates. Moreover, EITB
was used to identify antigenic proteins of human crude HCF
to be used in diagnosis and post- treatment follow up of CE
patient. The test showed high sensitivity and specificity in
the identification of potent antigens. The 48 and 12 KDa
antigenic proteins were detected in 100% of CE cases’ sera
and disappeared 6-12 months after treatment making them sensitive for the use in diagnosis and post treatment follow
up.