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بسم الله الرحمن الرحيم
ACKNOWLEDGMENT
Prof. Dr. Mohamed Khairy MakledProfessor of Parasitology
Faculty of Medicine, Ain Shams University
Prof. Dr. Mohamed Al-Hussieny FayadProfessor of Parasitology
Faculty of Medicine, Ain Shams UniversityProf. Dr. Abd-Al Magid Mohamed Kamal
Professor of Parasitology
Faculty of Medicine, Ain Shams UniversityDr. Magid Mustafa Al-Sherbiny
Assistant Professor of ZoologyFaculty of Science, Cairo University
Dr. Gihan Mustafa Tawfeek
Assistant Professor of ParasitologyFaculty 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.
Group 1 Group 2 Group 3 Group 4 Group 5
1a
Hydatidosis patients
3a
Toxoplasmosis Patients
4a
Trichinosis patients
1b Patients with
1.Toxoplasmosis
2. Trichinosis 3.Schistosomiasis
4. Amoebiasis
5. Fascioliasis.
(For EITB and FAST-
ELISA)
Schistosomiasis Patients
3bPatients with 1.Hydatidosis
2. Trichinosis 3.Schistosomiasis
4. Amoebiasis
5. Fascioliasis.
(For EITB and FAST-ELISA)
4bPatients with 1.Hydatidodid
2. Toxoplasmosis
3. Schistosomiasis
4. Amoebiasis
5. Fascioliasis.
(For EITB and FAST-ELISA)
Control Sera
1c 1b + Patients with
1. Ascarisis
2. Cysticercosis
3. Alveolar Ech.
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)
4c4b + Patients with
1. Ascarisis
2. Cysticercosi
3. Alveolar Echin.
4. Onchocerciasis
5. Filariasis
(For dipsticks& ICS)
For both studies the following were done:Preparation of
Crude HCF antigenCrude T. gondii tachyzoite antigen
Crude T.spiralis antigen[ MAMA & HAMA were supplied by ERDC]
Experimental StudyClinical Study
Evaluation of reactivity of these antigens to the collected Sera
EITB FAST-ELISA EITB ELISA
Dipsticks1. Preparation2. Assay
ICS1. Preparation2. Assay
Reaction of Hydatidosis Patients, Normal Controls & Sera of other parasitic Diseases in EITB
0
10
20
30
40
50
60
70
80
90
100
%
200 138 56 45-50 38 27.5 23.5 16.5 12
Crude HCF antigen (KDa)
Hydatidosis patients
P atients wuth otherparasitic diseases
Normal control
%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
0
10
20
30
40
50
60
70
80
90
100 %
po
sit
ive
Hydatidosispatients
Patients withother parasitic
diseases
Normal control
Groups
Reaction of sera of hydatidosis patients, patients with other parasitic diseases and normal controls to crude HCF antigen by FAST-ELISA
Reactions of hydatidosis patients, normal controls and heterologous sera to crude HCF antigen in dipstick assay
Reactions of hydatidosis patients sera and sera of patients with other parasitic diseases to crude HCF antigen in dipstick assay
0
10
20
30
40
50
60
70
80
90
100
Groups
%positive
ICS using crude HCF antigen tested in hydatidosis patients, normal control sera, PBS and D.water
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
+ve -ve +ve -ve +ve -ve
Hydatid Control
EITB Dipsticks ELISA
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
0
10
20
30
40
50
60
70
80
90
100
%
MAMA (30-32KDa) HAMA (23KDa)
Patients
control
% positive reactions of sera of schistosomiasis patients and normal controls with different bands of MAMA and
HAMA in EITB
Groups
Total number of tested sera
No. (%) PositiveMAMA-
FASTELISA
Schistosomiasis patients
30
29 (96.7)
Normal control
10
0 (0)
Number and percentage of positive reactions by FAST ELISA using MAMA
Reactions of Schistosomiasis patients and normal controls sera to S.mansoni Gp30 and S.haematobium Gp23 in dipsticks assay
ICS using GP30 of S.mansoni
ICS using GP23 specific for S.haematobium
Tests
Sensitivity
Specificity
EfficiencyPredictive 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%
0
10
20
30
40
50
60
70
80
90
100
%
+ve -ve +ve -ve +ve -ve
Patients
Control
EITB Dipstick FAST-ELISA
EITB analysis of antibody responses to crude tachyzoite antigen for sera from toxoplasmosis patient, controls, and sera of patients with other parasitic diseases
0
10
20
30
40
50
60
70
80
90
100
%
30 23
KDa
Toxoplasmosis control
% positive reactions of sera of toxoplasmosis patients, patients with other parasitic infections and normal controls with different bands of
crude tachyzoite antigen
0
10
20
30
40
50
60
70
80
90
100
%
Toxoplasmosis Patients with otherparasitic diseases
Normal control
Groups
% positive
Reaction of sera of toxoplasmosis patients, patients of other parasitic diseases and normal controls to crude tachyzoite antigen by FAST-
ELISA
Reactions of toxoplasmosis patients’ sera, normal control and heterologous sera in dipstick assay
ICS using crude tachyzoites antigen of T.gondii
0
10
20
30
40
50
60
70
80
90
100
%
+ve -ve +ve -ve
EITB FAST ELISA
Patients
Control
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
0
10
20
30
40
50
60
70
80
90
100
%
87 52 45 40
KDa
Trichimosis
Controls
%positive reactions of sera of trichinosis patients and normal
controls with different bands of Trichinella antigen
0
10
20
30
40
50
60
70
80
90
%
Trichinosis patients Patients w ith otherparasitic diseases
Normal control
Groups
Reaction of sera of trichinosis patients, patients of other parasitic diseases and normal controls to crude larval antigen of T.spiralis by
FAST-ELISA
Reactions of sera of trichinosis patients, normal controls, and heterologous sera to T.spiralis antigen in Dipstick
0
10
20
30
40
50
60
70
80
90
100
%
Trichinosis patients Patients w ith otherparasitic infection
Normal control
Groups
% positive reactions of trichinosis patients’ sera, sera of patients with different parasitic diseases & normal control
in dipstick assay.
ICS using the crude T.spiralis larval antigen
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%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
+ve -ve +ve -ve +ve -ve
Trichinella (Human) Control
EITB Dipsticks ELISA
Reactions of experimentally infected mice sera at different time interval post infection to Trichinella antigen by EITB and dipsticks
Dipstick assay is valuable immunodiagnostic test for diagnosis of hydatidosis, schistosomiasis, human and experimental trichinsosis, with high sensitivity and almost high specificity.
Simple Economic
Very antigen conservative Very serum conservative
Doesn’t require any lab. Instruments
Field applicable
Positive test can be visually identified
(color precipitate)
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.
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.
