ACKNOWLEDGMENT

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ACKNOWLEDGMENT
Prof. Dr./ Magda El-Sayed Azab
Prof. Dr./ Sawsan Abdel-Hamid Bishara
Prof. Dr./ Reda Rashad Ramzy
Dr./ Nihad Mohamed Oteifa
Dr./ Laila Mohamed El-Hoseiny
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Prof.Dr./ Nabila El-Sheikh
Prof.Dr./ Laila El-Okby
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Human Leucocytic Antigen SystemandParasite
Strainas Determinants of Human Susceptibility to
Unilocular Cystic Echinococcosis
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Introduction
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• Cystic echinococcosis (CE) is a major public
  health problem in many countries around the
  world, concentrated in the major sheep-raising
  and pastoral areas (Flisser, 1998). In Egypt, the
  distribution of E. granulosus is endemic but of
  focal or limited occurrence (Shambesh, 1997).
• There is an apparent variability in
  susceptibility of people to E. granulosus
  infection (Lightowlers et al., 1993). Individuals
  who contract the infection can be categorized
  into a group who develop CE (susceptible to
  disease) and a group in whom CE cannot be
  detected (resistant to disease) (Craig et al.,
  1986).

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• Reasons explaining the functional or baseline
  differences between resistance and susceptibility
  may be due to
• Host-related factors among the numerous
  host-related factors, Immunogenetics is the most
  important (i.e. dependent upon specific human
  leucocytic antigens (HLA) types).
• Parasite related factors parasite strain may
  also affect markedly the apparent spectrum of
  resistance/susceptibility exhibited by hosts.

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• Numerous studies have provided evidence that E.
  granulosus exists as a complex of different
  strains, which differ in a wide variety of
  criteria that impact on the epidemiology,
  pathology, and control of cystic hydatid disease
  (CHD).
• Furthermore there is evidence to suggest that
  some strains are more infective to humans than
  others but this question warrants further studies
  (Eckert and Thompson, 1997).

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Objective of the Work
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• Demonstrate the potential immunogenetic
  predisposition for susceptibility and resistance
  to unilocular echinococcosis (by HLA typing)
• Compare the parasite-specific humoral immune
  response in patients showing different clinical
  manifestations and cyst characters (by IgG and
  IgG1 ELISA)
• Detect genetic variability in Egyptian human and
  non-human isolates of Echinococcus granulosus (by
  RAPD-PCR)

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HLA TYPING
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• HLA antigens are membrane glycoproteins that are
  encoded by a closely linked set of genes
  collectively known as the major
  histocompatibility complex (MHC).
• The MHC is located on the short arm of chromosome
  6. It consists of 3 regions namely, class I, II,
  and III, which are arranged as centromere-class
  II-, class III- class I (Mackay and Rosen, 2000).
• HLA-DRB1 gene is the most polymorphic of the
  human class II genes, making it a powerful marker
  for individual identification (Bodmer et al.,
  1995).

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• The role of MHC in regulation of the immune
  response to Echinococcus is suggested because
  antigens of the extracellular parasite
  Echinococcus are presented by MHC molecules in a
  restricted way through the MHC class II molecules
  (Godot et al., 2000).
• In spite of the good evidence for the role of
  HLA-DRB1 genes in determining susceptibility and
  severity of alveolar echinococcosis (AE)
  (Gottstein and Bettens, 1994), studies concerning
  the correlation between CE and HLA were mostly on
  class I HLA (Shcherbakov and Monje-Barredo, 1989)
  without any reports concerning class II HLA.

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Subjects

• 35 patients with confirmed CE
• 100 apparently healthy individuals as a control
  group (C1)

Samples
Whole blood sample (from patients only)
Steps

• DNA extraction
• HLA-DRB1 amplification (biotinylated primers)
• Molecular typing of HLA-DRB1 alleles

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Results
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HS
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HS
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Distribution of HLA-DR3 and HLA-DR11 antigens in
the controls and the CE patients relative to the
site of the cysts
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Distribution of HLA-DR3 and HLA-DR11 antigens in
the controls and the CE patients relative to the
number of the cysts
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Distribution of HLA-DR3 and HLA-DR11 antigens in
the controls and the CE patients relative to the
size of the cysts
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Distribution of HLA-DR3 and HLA-DR11 antigens in
the controls and the CE patients relative to the
state of the cysts
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Distribution of HLA-DR3 and HLA-DR11 antigens in
the controls and the CE patients relative to the
radiological picture of the cysts
HS
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Compilation of Data
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Distribution of HLA-DR3 and HLA-DR11 antigens in
the controls and the CE patients relative to the
parasite-specific humoral immune response (using
commercial ELISA)
S
S
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Distribution of HLA-DR3 and HLA-DR11 antigens in
the controls and the CE patients relative to the
parasite-specific humoral immune response (using
local ELISA for total IgG)
HS
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Distribution of HLA-DR3 and HLA-DR11 antigens in
the controls and the CE patients relative to the
parasite-specific humoral immune response (using
local ELISA for IgG1)
S
S
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Compilation of data regarding the distribution
of HLA-DR3 and HLA-DR11 antigens amongst patients
and controls (C1) in relation to the
parasite-specific humoral immune response
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ELISA
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Subjects

• 35 patients with confirmed CE
• 30 apparently healthy individuals as a control
  group

Samples
Serum (from patients and controls)
Methods
Commercial kit (Novum Diagnostica) Total
IgG Purified bovine antigen
Locally devised ELISA (RTC) Total IgG and
IgG1 Crude camel antigen
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Results
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Sensitivity
Commercial kit
Locally devised ELISA
48.6
IgG1
Total IgG
94.3
88.6
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Mean Echinococcus antibody units for total IgG
and IgG1 in sera from CE patients and controls
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Mean Echinococcus antibody units for total IgG
and IgG1 in sera from male and female patients
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Mean Echinococcus antibody units for total IgG
and IgG1 in sera from CE patients of different
age groups
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Comparison of the mean levels of IgG and IgG1
antibody units in groups of patients identified
according to detailed medical history
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Mean Echinococcus antibody units for total IgG
and IgG1 in sera from patients with complicated
and non-complicated CE
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Comparison of the mean levels of IgG and IgG1
antibody units in groups of patients identified
according to cyst characters
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RAPD-PCR
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Steps

• Processing of hydatid cyst fluid (to prepare
  protoscolices) and host tissues
• DNA extraction from protoscolices, host tissues,
  and human blood
• RAPD-PCR (using Kit H from Operon Technologies)
  OPH-03, OPH-05, OPH-12, OPH-15, OPH-18
• Gel electrophoresis

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Principle

• RAPD-PCR technique is based on using a single
  oligonucleotide primer of known, but arbitrary
  nucleotide sequence (targets specific but unknown
  sites in the genome)
• It does not require previous sequence knowledge
• Amplification products are analyzed by gel
  electrophoresis. A particular DNA fragment that
  is generated for one individual but not for
  another represents a DNA polymorphism

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Results
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RAPD-PCR
Strain Variation
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RAPD-PCR
Strain Variation
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Similarity Coefficients (Strain Variation)
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RAPD-PCR
Individual variation (3 pig isolates)
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RAPD-PCR
Individual variation (3 camel isolates)
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RAPD-PCR
Individual variation (3 human isolates)
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RAPD-PCR
Individual variation ( additional 3 human
isolates)
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Similarity Coefficients (Individual variation)
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Conclusion
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Conclusion

• HLA-DR3 and HLA-DR11 correlated positively with
  the occurrence of CE and some of its clinical
  presentations in Egyptian patients
• Patients with CE are more liable to complications
  if they are carriers of some HLA antigens as DR3
• Therefore the present study may contribute to the
  identification of groups of individuals at higher
  risk for the development of CE early enough so
  that appropriate preventive measures can be
  applied. so a great care in treatment should be
  applied to those patients

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• Locally devised IgG1 ELISA using crude camel
  antigen is the most useful diagnostic system with
  94.3 sensitivity
• RAPD-PCR technique using randomly chosen primers
  was capable of discriminating among Egyptian
  human, camel and pig E. granulosus isolates.
  Primer OPH-03 was the most effective for
  discrimination
• Human hydatidosis in Egypt is suggested to be of
  camel/dog strain, so camels are important hosts
  for transmission of the disease
• Even though individual variation has been
  detected, the result is considered valid because
  there are many more differences between strains
  than between individuals

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Recommendations
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Recommendations

• This is the first study dealing with the
  correlation of HLA antigens and CE in Egyptian
  patients. So, it is recommended to perform more
  studies using the same and/or different class of
  HLA antigens in the Egyptian population
• The distribution of the HLA-DR types in the
  Egyptian community is not identified. So further
  extended studies in the Egyptian population are
  recommended to establish the prevalent HLA-types
  in the community

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Recommendations

• It is recommended to use IgG1 ELISA as screening
  diagnostic test of CE using crude camel antigen
• Epidemiological studies and adequate measures
  must be taken for hydatid control and public
  health in Egypt considering that camel is an
  important source for transmission of human
  hydatidosis in Egypt
• It is recommended to investigate larger number of
  human samples to confirm the individual variation
  detected
• Before detection of strain variation it is
  recommended to start with investigating
  individual variation as a preceding step

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THANK YOU