ACKNOWLEDGMENT

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ACKNOWLEDGMENT
Prof. Dr. Mohamed Khairy MakledProfessor of
ParasitologyFaculty of Medicine, Ain Shams
University
Prof. Dr. Mohamed Al-Hussieny Fayad Professor of
Parasitology Faculty of Medicine, Ain Shams
University
Prof. Dr. Abd-Al Magid Mohamed Kamal Professor of
Parasitology Faculty of Medicine, Ain Shams
University
Dr. Magid Mustafa Al-SherbinyAssistant Professor
of ZoologyFaculty of Science, Cairo University 
Dr. Gihan Mustafa TawfeekAssistant Professor of
ParasitologyFaculty of Medicine, Ain Shams
University
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APPLICATION ASSESSMENT OF IMMUNOCHROMATOGRAPHIC
STRIPS DIPSTICKS IN DIAGNOSIS OF SOME
PARASITIC DISEASES
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INTRODUCTION 

• One of the most pronounced problems in
  controlling the morbidity and mortality
  caused by different parasites is limited
  access to early, rapid and effective diagnosis in
  order to provide proper treatment (Tsang
  Wilkins, 1991).
• Different serological tests based on antibody
  detection are widely used but the need for
  special kits, the technical problems for the
  adequate preparation and reading of results and
  the time consuming incubation steps are several
  disadvantages of these tests. Dipstick format of
  Dot-ELISA and Immunochromatographic strips
  (ICS) are two simple immunodiagnostic tests
  that recently developed for diagnosis of
  parasitic diseases.

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DIPSTICKS

• Dipstick format of Dot-ELISA is a highly
  versatile solid-phase immunoassay for antibody or
  antigen detection.

• The assay uses minute amounts of antigen
  dotted onto solid surface. After
  incubation with antigen-specific antibody
  and enzyme-conjugated anti-antibody, the
  addition of a chromogenic substrate causes the
  formation of a colored dot on the solid phase,
  which is visually read (Pappas, 1988)..

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APPLICATIONS OF DIPSTICKS IN PARASITOLOGY

• The dipstick was both sensitive in detecting
  kala-azar in sera from patients with confirmed
  infections and specific in excluding healthy
  geographically matched controls (Pappas, 1988).

• Using the sandwich style of antigen detection,
  dipstick assay for detection of Taenia species
  coproantigens in the stool was developed (Allan,
  et al., 1992).

• Dipsticks were used as a simplified antigen
  detection assay to detect Schistosome antigen in
  urine (Van Etten et al. 1994). This assay was
  modified to detect antibodies specific to
  Schistosoma species in serum (Al-Sherbiny,
  1996).

• Dipstick assay detected IgG, IgM and IgA to E/S
  antigen of Toxoplasma gondii in patients sera
  (Yamamoto et al. 1998)
•  

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ICS

• Immunochromatographic assay is widely used
  for the detection of various analytics such as
  hormones, antigens, antibodies, other proteins,
  and drugs.

• The assay is performed on a nitrocellulose
  membrane strip and the result is determined by a
  visual read out of colored colloidal gold without
  using conjugates or substrates.

• Physicians and medical technicians use these
  assays for rapid diagnosis and therapeutic
  monitoring of a variety of conditions and
  disorders, due to the simplicity of the
  procedures and the rapidity of the result (Shin,
  et al., 2001).

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APPLICATIONS OF ICS IN PARASITOLOGY
1. ANTIGEN DETECTION ICS

• ICS was developed as a rapid diagnostic test for
  Malaria based on an antigen capture (Shiff, et
  al., 1993).

• For detection of Bancroftian filariasis, ICS was
  employed using specific polyclonal and
  monoclonal antibodies to Wuchereria bancrofti
  antigen (Bhumiratana, et al., 1999).

• ICS was used successfully in the detection
  of Entamoeba histolytica antigens in the stool
  samples ( Bhaskar, et al., 1996).

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• 2. ANTIBODY DETECTION ICS
•  

• Antibodies detection ICS was not optimized in
  diagnosis of parasitic infections, despite its
  successful application in diagnosis of bacterial
  infection with Mycobacterium tuberculosis
  (Grobusch, et al., 1998).

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AIM OF THE WORK

• Apply dipstick format of Dot-ELISA and
  Immunochromatographoic strips (ICS) as
  immunodiagnostic tests in the diagnosis of human
  schistosomiasis, hydatidosis, toxoplasmosis, and
  trichinosis using the crude antigen prepared for
  each of them.
• Evaluate the dipstick assay and ICS in diagnosis
  of these diseases in comparison to the enzyme
  immmunoelectrotransfer blot (EITB) FAST-ELISA
• Apply and evaluate the dipstick assay in
  diagnosis of trichinosis in experimentally
  infected mice in comparison to EITB and ELISA.

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MATERIALS METHODS

• A. Clinical Study
• Sera were collected from patients of
  hydatidosis, schistosomiasis, toxoplasmosis
  trichinosis in addition to sera of normal healthy
  individuals as control samples
• B. Experimental Study
• Experimental infection of mice with
  Trichinella spiralis larvae and collection
  of sera were done.
•  

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• For both studies the following were done
• Preparation of
• Crude HCF antigen
• Crude T. gondii tachyzoite antigen
• Crude T.spiralis antigen
• MAMA HAMA were supplied by ERDC

Evaluation of reactivity of these antigens to the
collected Sera
Experimental Study
Clinical Study
EITB ELISA
EITB FAST-ELISA
Dipsticks 1. Preparation 2. Assay
ICS 1. Preparation 2. Assay
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RESULTS
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EITB
HYDATIDOSIS
Reaction of Hydatidosis Patients, Normal Controls
Sera of other parasitic Diseases in EITB
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positive reactions of sera of hydatidosis
patients, patients with other parasitic
infections and normal controls with different
bands of crude HCF antigen bands in EITB
EITB
HYDATIDOSIS
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FAST-ELISA
HYDATIDOSIS
Reaction of sera of hydatidosis patients,
patients with other parasitic diseases and
normal controls to crude HCF antigen by
FAST-ELISA
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DIPSTICKS
HYDATIDOSIS
Reactions of hydatidosis patients,
normal controls and heterologous sera to crude
HCF antigen in dipstick assay
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DIPSTICKS
HYDATIDOSIS

• Reactions of hydatidosis patients sera and sera
  of patients with other parasitic diseases to
  crude HCF antigen in dipstick assay

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ICS using crude HCF antigen tested in hydatidosis
patients, normal control sera, PBS and D.water
ICS
HYDATIDOSIS
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HYDATIDOSIS
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EITB
SCHISTOSOMIASIS
Reactions of Schistosomiasis patient and normal
control sera with S.mansoni specific fraction in
EITB
Reactions of Schistosomiasis patient and normal
control sera with S.haematobium specific fraction
in EITB
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positive reactions of sera of schistosomiasis
patients and normal controls with different bands
of MAMA and HAMA in EITB
EITB
SCHISTOSOMIASIS
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FAST-ELISA
SCHISTOSOMIASIS
Number and percentage of positive reactions by
FAST ELISA using MAMA
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DIPSTICKS
SCHISTOSOMIASIS
Reactions of Schistosomiasis patients and normal
controls sera to S.mansoni Gp30 and S.haematobium
Gp23 in dipsticks assay
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ICS using GP30 of S.mansoni
ICS
SCHISTOSOMIASIS
ICS using GP23 specific for S.haematobium
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SCHISTOSOMIASIS
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EITB
TOXOPLASMOSIS
EITB analysis of antibody responses to crude
tachyzoite antigen for sera from toxoplasmosis
patient, controls, and sera of patients with
other parasitic diseases
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positive reactions of sera of toxoplasmosis
patients, patients with other parasitic
infections and normal controls with different
bands of crude tachyzoite antigen
EITB
TOXOPLASMOSIS
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Reaction of sera of toxoplasmosis patients,
patients of other parasitic diseases and normal
controls to crude tachyzoite antigen by
FAST-ELISA
FAST-ELISA
TOXOPLASMOSIS
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Reactions of toxoplasmosis patients sera, normal
control and heterologous sera in dipstick assay
DIPSTICKS
TOXOPLASMOSIS
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ICS using crude tachyzoites antigen of T.gondii
ICS
TOXOPLASMOSIS
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TOXOPLASMOSIS
Comparison between results of EITB, and
FAST-ELISA in toxoplasmosis patients and controls
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Reaction of trichinosis patients, normal
controls, and sera of different parasitic
diseases in EITB
EITB
TRICHINOSIS
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positive reactions of sera of trichinosis
patients and normal controls with different bands
of Trichinella antigen
EITB
TRICHINOSIS
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Reaction of sera of trichinosis patients,
patients of other parasitic diseases and normal
controls to crude larval antigen of T.spiralis by
FAST-ELISA 
FAST-ELISA
TRICHINOSIS
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DIPSTICKS
TRICHINOSIS
Reactions of sera of trichinosis patients,
normal controls, and heterologous sera to
T.spiralis antigen in Dipstick
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DIPSTICKS
TRICHINOSIS
positive reactions of trichinosis patients
sera, sera of patients with different parasitic
diseases normal control in dipstick assay.
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ICS
TRICHINOSIS
ICS using the crude T.spiralis larval antigen
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TRICHINOSIS
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EXP. STUDY
EITB
DIPSTICKS
TRICHINOSIS
Reactions of experimentally infected mice sera at
different time interval post infection to
Trichinella antigen by EITB and dipsticks
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CONCLUSION

• Dipstick assay is valuable immunodiagnostic test
  for diagnosis of hydatidosis, schistosomiasis,
  human and experimental trichinsosis, with high
  sensitivity and almost high specificity.
•  

• Dipstick assay

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• Dipstick assay

• Can be used as a qualitative test to screen
  large numbers of samples or as a quantitative
  assay to determine endpoint titration of
  individual sera.

• Further improvement may make the dipsticks assay
  suitable for wide scale use in field studies and
  in rural areas were equipped laboratories are not
  available.

• In schistosomiasis the dipstick assay has the
  advantage of the capability of speciation of
  two Schistosomes S. mansoni and S.
  haematobium on the same strip.

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RECOMMENDATION

• Apply Dipstick assay for diagnosis of
  Hydatidosis, Schistosomiasis, and Trichinosis in
  field studies in endemic areas.

• Apply Dipstick assay in diagnosis of Trichinosis
  in swine in slaughter houses in control programs

• Develop ICS for diagnosis of hydatidosis,
  schistosomiasis, toxoplasmosis, and trichinosis
  using highly purified antigens and amplifying
  signals that hindered us in developing this test.
  

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THANKS