Nehad m. sayed (ain shams)1
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Nehad m. sayed (ain shams)1 Nehad m. sayed (ain shams)1 Presentation Transcript

  • بسم الله الرحمن الرحيم
  • ACKNOWLEDGMENT Prof. Dr. Mohamed Khairy Makled Professor of Parasitology Faculty 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-Sherbiny Assistant Professor of Zoology Faculty of Science, Cairo University   Dr. Gihan Mustafa Tawfeek Assistant Professor of Parasitology Faculty of Medicine, Ain Shams University
  • APPLICATION & ASSESSMENT OF IMMUNOCHROMATOGRAPHIC STRIPS & DIPSTICKS IN DIAGNOSIS OF SOME PARASITIC DISEASES
  • 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.
    • 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)..
    DIPSTICKS
    • Dipstick format of Dot-ELISA is a highly versatile solid-phase immunoassay for antibody or antigen detection.
  • APPLICATIONS OF DIPSTICKS IN PARASITOLOGY
    • Dipstick assay detected IgG, IgM and IgA to E/S antigen of Toxoplasma gondii in patients’ sera (Yamamoto et al. 1998)
    •  
    • 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) .
  • 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).
  • APPLICATIONS OF ICS IN PARASITOLOGY
    • ICS was used successfully in the detection of Entamoeba histolytica antigens in the stool samples ( Bhaskar, et al., 1996).
    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).
    • 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).
  • 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.
  • 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.
    •  
  • 4c 4b + Patients with 1. Ascarisis 2. Cysticercosi 3. Alveolar Echin. 4. Onchocerciasis 5. Filariasis (For dipsticks& ICS)   3c   3b + Patients with 1. Ascarisis 2. Cysticercosis 3. Alveolar Ech. 4. Onchocerciasis 5. Filariasis (For dipsticks &ICS)   1c   1b + Patients with 1. Ascarisis 2. Cysticercosis 3. Alveolar Ech. 4. Onchocerciasis 5. Filariasis (For dipsticks & ICS) Control Sera 4b Patients with 1.Hydatidodid 2. Toxoplasmosis 3. Schistosomiasis 4. Amoebiasis 5. Fascioliasis. (For EITB and FAST-ELISA) 3b Patients with 1.Hydatidosis 2. Trichinosis 3.Schistosomiasis 4. Amoebiasis 5. Fascioliasis. (For EITB and FAST-ELISA) Schistosomiasis Patients 1b   Patients with 1.Toxoplasmosis 2. Trichinosis 3.Schistosomiasis 4. Amoebiasis 5. Fascioliasis. (For EITB and FAST-ELISA) 4a Trichinosis patients 3a Toxoplasmosis Patients 1a Hydatidosis patients Group 5 Group 4 Group 3   Group 2   Group 1
    • 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]
    Experimental Study Clinical Study Evaluation of reactivity of these antigens to the collected Sera EITB FAST-ELISA EITB ELISA Dipsticks 1. Preparation 2. Assay ICS 1. Preparation 2. Assay
  • RESULTS
  • Reaction of Hydatidosis Patients, Normal Controls & Sera of other parasitic Diseases in EITB HYDATIDOSIS EITB
  • %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 HYDATIDOSIS EITB
  • Reaction of sera of hydatidosis patients, patients with other parasitic diseases and normal controls to crude HCF antigen by FAST-ELISA HYDATIDOSIS FAST-ELISA
  • Reactions of hydatidosis patients, normal controls and heterologous sera to crude HCF antigen in dipstick assay HYDATIDOSIS DIPSTICKS
    • Reactions of hydatidosis patients sera and sera of patients with other parasitic diseases to crude HCF antigen in dipstick assay
    HYDATIDOSIS DIPSTICKS
  • ICS using crude HCF antigen tested in hydatidosis patients, normal control sera, PBS and D.water HYDATIDOSIS ICS
  • HYDATIDOSIS   Tests   Sensitivity   Specificity   Efficiency Predictive value +ve PV -ve PV EITB     100%   91.4%   95.1%   89.7%   100%   FAST ELISA     96.2%   100%   98.4%   100%   97.2%   Dipsticks     100%   91.4%   95.1%   89.7%   100%
  • 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 SCHISTOSOMIASIS EITB
  • % positive reactions of sera of schistosomiasis patients and normal controls with different bands of MAMA and HAMA in EITB SCHISTOSOMIASIS EITB
  • SCHISTOSOMIASIS Number and percentage of positive reactions by FAST ELISA using MAMA FAST-ELISA   Groups   Total number of tested sera No. (%) Positive MAMA-FASTELISA   Schistosomiasis patients       30     29 (96.7)   Normal control     10   0 (0)
  • Reactions of Schistosomiasis patients and normal controls sera to S.mansoni Gp30 and S.haematobium Gp23 in dipsticks assay SCHISTOSOMIASIS DIPSTICKS
  • ICS using GP30 of S.mansoni SCHISTOSOMIASIS ICS using GP23 specific for S.haematobium ICS
  • SCHISTOSOMIASIS   Tests   Sensitivity   Specificity   Efficiency Predictive value (PV) +ve PV -ve PV   EITB     96.7%   100%   97.5 %   100 %   90.9%   FAST ELISA     96.7%   100 %   97 .5 %   100%   90.9%   Dipsticks   96.7%   100 %   97.5 %     100%   90.9%  
  • TOXOPLASMOSIS EITB analysis of antibody responses to crude tachyzoite antigen for sera from toxoplasmosis patient, controls, and sera of patients with other parasitic diseases EITB
  • % positive reactions of sera of toxoplasmosis patients, patients with other parasitic infections and normal controls with different bands of crude tachyzoite antigen TOXOPLASMOSIS EITB
  • Reaction of sera of toxoplasmosis patients, patients of other parasitic diseases and normal controls to crude tachyzoite antigen by FAST-ELISA TOXOPLASMOSIS FAST-ELISA
  • Reactions of toxoplasmosis patients’ sera, normal control and heterologous sera in dipstick assay TOXOPLASMOSIS DIPSTICKS
  • ICS using crude tachyzoites antigen of T.gondii TOXOPLASMOSIS ICS
  • TOXOPLASMOSIS Comparison between results of EITB, and FAST-ELISA in toxoplasmosis patients and controls   Tests   Sensitivity   Specificity   Efficiency Predictive value +ve PV -ve PV   EITB   100%   100%   100%   100 %   100%   FAST ELISA   95.5%   94.3%   94.7%   91.3%   97.1%
  • Reaction of trichinosis patients, normal controls, and sera of different parasitic diseases in EITB TRICHINOSIS EITB
  • %positive reactions of sera of trichinosis patients and normal controls with different bands of Trichinella antigen TRICHINOSIS EITB
  • Reaction of sera of trichinosis patients, patients of other parasitic diseases and normal controls to crude larval antigen of T.spiralis by FAST-ELISA   TRICHINOSIS FAST-ELISA
  • TRICHINOSIS Reactions of sera of trichinosis patients, normal controls, and heterologous sera to T.spiralis antigen in Dipstick DIPSTICKS
  • TRICHINOSIS % positive reactions of trichinosis patients’ sera, sera of patients with different parasitic diseases & normal control in dipstick assay. DIPSTICKS
  • TRICHINOSIS ICS using the crude T.spiralis larval antigen ICS
  • TRICHINOSIS   Tests   Sensitivity   Specificity   Efficiency Predictive value +ve PV -ve PV   EITB   100%   100%   100%   100%   100%   FAST ELISA   85.7%   85.7%   85.7%   54.5%   96.8%   Dipsticks   100%   100%   100%   100%   100%
  • TRICHINOSIS Reactions of experimentally infected mice sera at different time interval post infection to Trichinella antigen by EITB and dipsticks EXP. STUDY EITB DIPSTICKS
        • Dipstick assay is valuable immunodiagnostic test for diagnosis of hydatidosis, schistosomiasis, human and experimental trichinsosis, with high sensitivity and almost high specificity.
        •  
    • Dipstick assay
    CONCLUSION
    • Positive test can be visually identified
    • (color precipitate)
    • Doesn’t require any lab. Instruments
    • Field applicable
    • Very serum conservative
        • Very antigen conservative
      • Economic
        • Simple
        • 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.
    • Dipstick assay
    • 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.
        • 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.
    • 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.
    RECOMMENDATION
  • THANKS