Study design and population
The study was a purposive cross sectional study involving elementary school pupils who answered yes to whether or not they have any of the signs and symptoms of urinary schistosomiasis.
The S. haematobium species-specific MAb required for detection of the 29 kDa antigen was purified and the reactivity confirmed. Active MAb fractions were utilized for the urinary schistosomiasis MAb dipstick assay (USDA), microplate enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody test (IFAT). Urine samples for the study were collected from a total of 292 elementary school pupils from two villages, Kwashikumahman (n = 190) and Kojo Ashong (n = 102), hyperendemic for urinary schistosomiasis [11]. Aliquots of urine samples from subjects showing urinary schistosomiasis symptoms, haematuria and dysuria, were tested for S. haematobium antigens and eggs using USDA and microscopy respectively. Schistosome eggs were isolated from urine samples with >100 eggs/10 ml of urine for soluble egg antigen preparation, generation of parasite stages and for immunolocalization.
Study area
The study was conducted at Kojo Ashong and Kwashikumahman in the Greater Accra Region of Ghana. These villages are located on 5°43'N, 0°23.5'E and 5°43'N, 0°21.5'E, respectively. The vegetation along the banks of the slow flowing Densu River and Dobro stream, running at the outskirts of the villages, comprises mainly grassland and a few trees. The weedy river and stream banks contain decomposing plant leaves and twigs infested with urinary schistosomiasis vector snails, Bulinus globosus. The Densu River and the Dobro stream constitute the principal sources of water for domestic use and transmission of urinary schistosomiasis.
Ethical consideration
Study subjects were elementary school pupils assigned by the Ghana Health Service, Ministry of Education and Noguchi Memorial Institute for Medical Research for schistosomiasis examination and treatment. Subjects were recruited if they discharged urine with blood and/ or pain. Informed consent was obtained from Education Authorities and parents and/or guardians of pupils before recruitment. Infected children were treated with 40mg/kg body weight of praziquantel (Merck KGaA, Darmstadt, Germany) as recommended by WHO [12]. Approval for this study was given by the Noguchi Memorial Institute for Medical Research Institutional Review Board, referenced NMIMR-IRB CPN 042/06-07 rev 2008.
Collection and analysis of urine specimens
Field procedures
Collection and handling of urine samples
Twenty to 100ml of fresh clean catch urine was collected from each of 292 school pupils in the period between 11:00 and 14.00hours Greenwich Mean Time (GMT) into a 200ml urine container. The samples were then transported to the laboratory at the Noguchi Memorial Institute for Medical Research within 1hour on ice in ice-chest.
Laboratory procedures
Examination of urine samples for parasite antigens and eggs
Within 3hours after arrival at the laboratory urine samples were tested individually for S. haematobium antigen by MAb dipstick as described elsewhere [8,9,11]. Also, 10ml of the urine was filtered through a 25mm Nucleopore filter (12μm pore size) [11] to determine parasite density. The rest of the urine was centrifuged at 1,290 X g to isolate S. haematobium eggs.
Generation of S. haematobium parasite life-cycle stages
S. haematobium eggs were isolated by centrifugation and sedimentation as described by Bosompem and others [13] and then cleaned by density centrifugation through ficoll-paque™ (GE Healthcare Life Sciences, Buckinghamshire, UK). They were subsequently hatched into miracidia by exposure to clean aged tap water and light as described by Huyse and others [14]. Some of the miracidia were used to infect Bulinus snails (five miracidia/ snail) to generate cercariae as described elsewhere [15,16]. Some of the live cercariae were transformed into schostosomula by vortexing (Ikemoto Rikakogyo Co. Ltd., Japan) as described by Ramalho-Pinto and others [17] for 20min. Some cercariae were also used to infect BALB/c mice to generate adult worms [6,15]. Fractions of the eggs, miracidia, cercariae, schistosomula and adult worms were respectively homogenized by sonication [15] to prepare crude antigens or treated with fixatives for immunolocalization studies.
S. haematobium parasite stages and fixatives for immunolocalization
S. haematobium parasite stages were fixed for immunolocalization according to the method described elsewhere [18] with modification.
Washed S. haematobium eggshells, miracidia, cercariae, schistosomula and adult worms were suspended in PBS containing different concentrations of fixatives namely, paraformaldehyde, glutaraldehyde, Karnovsky’s fixative, methanol, ethanol and acetone, and incubated at 4°C for 5 min, 30 min, 60 min, 90 min and overnight (12 hrs). The concentrations of the various fixatives tested were, 0.1, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 10 and 100%. Fixed specimens were washed four times with PBS by centrifugation at 16,000 xg for 10 min at 4°C, stored in PBS at 4°C and analyzed by a preliminary IFAT to determine and select the best fixatives and conditions for immunolocalization studies.
Preparation of S. haematobium adult worms for (IFAT)
Adult worms were prepared for IFAT as described elsewhere [19-21] with modification. Fixed S. haematobium adult worms were incubated for 5mins in saturated picric acid to colour the specimen and enhance visualization. The worms were dehydrated stepwise by transferring them into ethanol (85% for 10 min, 95, 100 and 100%, each for 20 min) and then 100% chloroform (3 times, each for 10 min). The worms were transferred into molten paraffin wax at 56°C (2 times for 10 min each) to fill open cavities with wax, embedded in wax and then cut into 4 μm sections with a microtome (Yamato Koki Individuals. Co., Ltd., Tokyo, Japan.). Worm sections on glass microscope slides were deparaffinized by heating at 50°C for 2 hr and then washed twice in xylene (5 min/wash). The worms were then re-hydrated stepwise in 100, 100, 95 and 90% ethanol (5 min/step). Finally, the worm sections were rinsed, 5 min each, under running distilled water and in PBS.
Monoclonal antibody (MAb)
The IgG1MAb used in this study was generated by immunizing BALB/c mice with antigens extracted from S. haematobium infected human urine [6,7]. This antibody did not cross-react with Necator americanus (hookworm) egg antigens in micro-plate ELISA, and could bind ShSSA, from the eggs of both Ghanaian and Egyptian strains of S. haematobium and infected human urine [6-8]. The antibody in culture supernatant was concentrated ten-fold by Amicon filtration or by precipitation with 50% (v/v) ammonium sulphate [(NH4)2SO4] and then purified by ion-exchange chromatography. Microplate ELISA was used to determine the reactivity of this MAb to ShSSA in infected human urine and homogenates of parasite stages as described earlier [6].
IFAT procedure
IFAT was conducted on the prepared parasite stages as described elsewhere [19,22] with modification. Fixed eggshells, miracidia, cercariae and schistosomula in suspension were coated, 20–30 specimens/well, onto multi-well IFA slides by heating briefly over a Bunsen flame. Slides coated with worm sections or other parasite stages were immersed briefly in a destaining jar containing 0.2 M Phosphate Buffered Saline (PBS, pH 7.2) to wash off loosely bound materials. Excess PBS was blotted by touching the edge of the slides with filter paper after which the wells were incubated with Sh2/15.F, positive control (immune) sera or negative control (free medium) at 20 μl/well for 1 hr at room temperature. Primary antibodies, Sh2/15.F and immune sera were used at 1:40 and 1:50 dilution, respectively. The slides were washed twice (5mins per wash) with PBS and then incubated for 30 min (20 μl/well) with secondary antibody reagent [goat-anti-mouse IgG conjugated with fluorescein isothiocyanate (FITC) (Sigma Immuno Chemicals, St. Louis, MO) and 0.01% trypan blue (counter stain), all in PBS]. After incubation the slides were washed four times in PBS, blotted and then mounted in 50% glycerol (Sigma-Aldrich Co. Ltd.-Gillingham-Dorset, UK) in PBS. The specimens were observed using a flourescent microscope (Olympus Optical Co. Ltd., Japan) at x120 magnification.
