Nehad m. sayed (ain shams)1Presentation 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
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)..
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) .
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:
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
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
ICS using crude HCF antigen tested in hydatidosis patients, normal control sera, PBS and D.water HYDATIDOSIS ICS
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