|Year : 2011 | Volume
| Issue : 2 | Page : 99-103
Urinary schistosomiasis transmission in Epe, an urban community of Southwest Nigeria
OP Akinwale1, VN Akpunonu2, MB Ajayi1, DO Akande1, MA Adeleke1, PV Gyang1, MO Adebayo1, AA Dike1
1 Molecular Parasitology Research Laboratory, Public Health Division, Nigerian Institute of Medical Research, Lagos State, Nigeria
2 Department of Medical Microbiology and Parasitology, School of Medical Laboratory Sciences, Lagos University Teaching Hospital, Idi Araba, Lagos State, Nigeria
|Date of Web Publication||31-Oct-2011|
O P Akinwale
Molecular Parasitology Research Laboratory, Public Health Division, Nigerian Institute of Medical Research, PMB 2013, Yaba, Lagos State
| Abstract|| |
Background: A survey of Schistosoma haematobium infection in Epe, an urban community in Lagos State, Southwest Nigeria, was carried out to ascertain the possibility that schistosomiasis, otherwise considered a rural disease, could reach urban populations. Materials and Methods: About 100 ml of voided urine samples from 200 pupils aged 6-13 years [109 (54.5%) males and 91 (45.5%) females], attending an Anglican primary school, Ebute Afuye, and a community primary school, Erepoto, were examined parasitologically for hematuria and S. haematobium ova following informed consent obtained from their parents/guardians. All samples were screened using polymerase chain reaction (PCR) amplification of the schistosome Dra1 gene. Fourteen Bulinus snails collected from the two sites, Ebute Afuye (6) and Erepoto (8), were screened for schistosome infection by the PCR amplification of the schistosome Dra1 gene. PCR-RFLP of the snails' its region was analyzed for species identification and a subregion of the cox1 gene from four infected snails (two from each site) was amplified and sequenced. Results: In the Anglican primary school, Ebute Afuye, and community primary school, Erepoto, 16% and 29% were positive for hematuria, and 16% and 17% had schistosome ova, respectively. PCR analysis showed that 57% and 40% were positive for the infection in Anglican primary school, Ebute Afuye, and community primary school, Erepoto, respectively. PCR screening of the snails confirmed that four from Ebute Afuye and three from Erepoto were infected with schistosomes. PCR-RFLP showed that all the 14 snails were Bulinus truncatus while phylogenetic analysis of the sequenced partial cox1 gene corroborated the PCR-RFLP results. Conclusions: There was a high prevalence of S. haematobium infection among the participants detected by PCR, which was able to detect infection in cases otherwise shown to be negative by hematuria. We also observed that B. truncatus is one of the snail species responsible for the transmission of urinary schistosomiasis in the Epe community. For national control programs, it is very important that trends in the prevalence and intensity of schistosomiasis in urban cities be monitored.
Keywords: Molecular, survey, Nigeria, urinary schistosomiasis
|How to cite this article:|
Akinwale O P, Akpunonu V N, Ajayi M B, Akande D O, Adeleke M A, Gyang P V, Adebayo M O, Dike A A. Urinary schistosomiasis transmission in Epe, an urban community of Southwest Nigeria. Trop Parasitol 2011;1:99-103
|How to cite this URL:|
Akinwale O P, Akpunonu V N, Ajayi M B, Akande D O, Adeleke M A, Gyang P V, Adebayo M O, Dike A A. Urinary schistosomiasis transmission in Epe, an urban community of Southwest Nigeria. Trop Parasitol [serial online] 2011 [cited 2020 Jul 10];1:99-103. Available from: http://www.tropicalparasitology.org/text.asp?2011/1/2/99/86944
| Introduction|| |
Schistosomiasis is the second most prevalent tropical disease after malaria, and a leading cause of severe morbidity in many parts of the world.  The disease is caused by the parasitic helminth of the genus Schistosoma and transmitted through fresh water snail intermediate hosts. The disease poses a threat to 600 million people in more than 76 countries including Nigeria.  Urinary schistosomiasis occurs in all 36 states of Nigeria including the federal capital territory.  The disease is associated with water resource development projects such as dams and irrigation schemes, and slow-flowing and stagnant water which support the breeding and survival of the snail intermediate host. The residents of the communities around such water development projects or inland water whose style of life promote regular contact with the contaminated water are at the great risk of the disease, most importantly, the school-age children. ,
One of the banes still confronting the effective control of schistosomiasis in Nigeria is the paucity of information on the extent of distribution of the disease and the total number of affected people. There is therefore a need for renewed interest in documenting the prevalence of the disease in different parts of the country with the view of planning effective control strategies and the treatment of the affected population. Until now, the diagnosis of urinary schistosomiasis had relied mostly on the presence of blood in urine (hematuria), detection of eggs in urine, and detection of adult worm antigens in urine and sera of infected individuals (serology). Although these methods are useful for the detection of heavy infections, their low sensitivity to detect light infections has great epidemiological implications as many endemic communities could be underestimated and many infected people could be left untreated. 
As efforts to enhance schistosomiasis diagnosis lingered on, the use of polymerase chain reaction (PCR) was adapted for the detection of urinary schistosomiasis. Recently, a tandemly repeated DNA sequence termed Dra1 was identified in the genome of Schistosoma haematobium.  The present study therefore utilized both molecular and parasitological methods to diagnose urinary schistosomiasis among school-age children in two public schools in Epe, Lagos State, Southwest Nigeria, with a view to generating more reliable data on the current status of urinary schistosomiasis in this urban community.
| Materials and Methods|| |
The study area
The study was conducted in two public primary schools: Anglican primary school, Ebute Afuye, and Community primary school, Erepoto, in Epe local government area of Lagos State, Southwest Nigeria. The two schools were selected because of their proximity to the fresh water habitats which are frequently visited by the residents for fishing, recreational, and domestic purposes.
Participant selection (inclusion/exclusion criteria)
One hundred pupils were randomly selected from each school thereby bringing the total number of participating pupils to 200. However, pupils under the age of five and any girl menstruating at any point of urine collection were not selected. At the end of the selection, the study participants from the two schools were made up of 109 (54.5%) males and 91 (45.5%) females [Table 1].
The study was approved by the Lagos State Ministry of Health and Epe local government area authority. Permission to carry out the study was taken from the authorities of the two schools selected for the study and informed consent was obtained from the parents/guardians of the participating pupils, prior to sample collections.
Demographic data including the name, surname, age, sex, and weight were recorded for each pupil while a unique study code of three digits was given to each of them for proper identification. Sterile wide mouth specimen containers bearing the study code were distributed among the corresponding participants for urine collection. About 200 ml of voided mid-stream urine was collected between 10:00 am and 2:00 pm from each participant.
A total of 14 snails, 6 from Ebute Afuye and 8 from Erepoto, were collected from identified human-water contact sites in the fresh water habitats using a standard snail scoop. The contents were washed and the snails picked manually. Recovered snails were transported to the laboratory in prelabeled plastic containers, rinsed, sorted, and counted. Each snail was identified based on its morphological characteristics using a field guide to African freshwater snails. 
The urine samples were examined immediately for hematuria using commercially prepared reagent strips (Hemastix; Biehringer Mannheim, Germany) while the samples were transported thereafter to the laboratory without delay. The presence of S. haematobium eggs in each urine sample was confirmed using the sedimentation-by-gravity method. 
Molecular examination for schistosome infection
Urine cell pellet preparation
Each urine sample was allowed to sediment; the supernatant was decanted and the sediment was centrifuged at 5000 g for 10 min. The supernatant was decantedand cell pellets were washed three times with 25 ml PBS (0.8% NaCl, 2.7 mM KCl, 1.8 mM KH 2 PO 4 , 8 mM Na 2 HPO 4 , pH 7.4). The cell pellets were stored immediately at −80°C until use.
Extraction of genomic DNA from urine pellets
Urine cell pellets were digested with 1% SDS and 50 μg/ml proteinase K (Roche Diagnostics, Mannheim, Germany) at 48°C overnight. Genomic DNA (gDNA) was extracted from the solution by adding an equal volume of chloroform/isoamyl alcohol (24:1) to each tube. The organic and aqueous layers were gently mixed for 5 min and spun at 13,000 rpm for 20 min. The upper aqueous layer was removed into another sterile Eppendorf tube and an equal volume of 100% ethanol was added, mixed, and was incubated at −20°C overnight in order to enhance DNA precipitation. The solution was spun at 13,000 rpm for 20 min and the pellet was washed with 70% ethanol and was spun for another 20 min. The supernatant was removed and the pellet was dried at room temperature. When completely dry, the pellet was resuspended in 25 μl of water and stored at 4°C until use.
Genomic DNA extraction from snails
Genomic DNA was extracted from each snail using a CTAB extraction buffer containing 2-mercaptoethanol, hexadecyltrimethylammonium bromide (CTAB; solid), tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid, disodium salt solution (EDTA), and sodium chloride. Each snail was removed from the 70% ethanol and soaked in TE (10 mM Tris HCl and 1 mM EDTA) overnight so as to get rid of the remaining ethanol. Tissue from each of the snails was placed in a sterile 1.5 ml Eppendorf tube; 500 μl of the CTAB solution was added and the tissue was grounded followed by the addition of 10 μl of the proteinase K solution (20 mg/ml) and incubated at 55°C for 1 h, with occasional gentle mixing. gDNA was extracted from the CTAB buffer by adding an equal volume of chloroform/isoamyl alcohol (24:1) to each tube. The organic and aqueous layers were gently mixed for 5 min and spun at 13,000 rpm for 20 min. The upper aqueous layer was removed to another sterile Eppendorf tube and an equal volume of 100% ethanol was added, mixed, and was incubated at −20°C overnight in order to enhance DNA precipitation. The solution was spun at 13,000 rpm for 20 min and the pellet was washed with 70% ethanol and was spun for another 20 min. The supernatant was removed and the pellet was dried at room temperature. When completely dry, the pellet was resuspended in 25 μl of water and stored at 4°C until use.
PCR screening of pupils and snails for schistosome infection
Following a previous method,  the extracted gDNA was subjected to the PCR amplification of the schistosome Dra1 repeat using forward primers 5′-GATCTCACCTATCAGACGAAAC-3′ and reverse primers 5′-TCACAACGATACGACCAAC-3. All the PCR amplifications were performed with the thermal cycler (Hybaid, OmniGene, Pegasus Scientific Inc., Rockville, MD, USA) and the amplified products were visualized on 1.5% agarose gel. Photo documentation was performed with the gel documentation and analysis system (Clinx Science Instruments, Shanghai, China).
Data were analyzed using Statistical Package for Social Sciences (Windows version 16.0; SPSS Inc, Chicago, IL, US) with the application of a chi-square test. Descriptive analysis was used to express gender occurrence while the prevalence of infection was expressed in percentage. The difference in the prevalence of infection between the gender groups was determined using a chi-square test.
| Results|| |
Overall results obtained from parasitological examinations of 200 pupils from the two schools showed that 45 (22.5%) were positive for hematuria; 33 (16.5%) had S. haemtatobium eggs in their urine while 97 (48.5%) were positive for the infection as shown by the PCR amplification of the schitosome Dra1 repeat [Table 2]. It was observed in the results of the three diagnostic methods that males were more infected than females; however, the difference in prevalence was not statistically significant (P>0.05; [Table 3]). PCR screening of the snails for the schistosome Dra1 repeat confirmed that 4 from Ebute Afuye and 3 from Erepoto were infected while the PCR-RFLP analysis showed that all the 14 snails were Bulinus truncatus. Phylogenetic analysis of their sequenced partial cox1 genes corroborated the PCR-RFLP results while this result has been reported earlier in another publication. 
|Table 3: The prevalence of urinary schistosomiasis among male and female pupils examined |
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| Discussion|| |
The prevalence of infection in almost half of the participants (48.5%) as indicated by the PCR amplification of the schistosome Dra1 repeat showed that the study area was endemic with urinary schistosomiasis. The high prevalence may possibly be a reflection of intense water contact activities in the area. The relatively low sensitivity of hematuria and egg count at detecting schistosome infection as compared with the PCR technique confirmed earlier observations that the PCR technique is more sensitive than any other method in diagnosing schistosome infections. , Therefore, the reliance on hematuria and egg count as methods of determining the prevalence of schistosomiasis could be misleading and give inaccurate data on the occurrence of the infection in such localities. The nonsignificant differences observed in the prevalence of infection in both sexes possibly indicate that both school-age males and females are equally exposed to water activities. Researchers have posited that the school-age children are the most vulnerable group to urinary schistosomiasis because of frequent engagement with water activities such as swimming, fishing, and washing of clothes and plates.  Thus, the estimation of the prevalence of the infection in school-age children can be used as an index for assessing community prevalence. 
| Conclusion|| |
The present study has shown the high prevalence of urinary schistosomiasis among the school children in Epe local government area of Lagos State. We recommend that the study should be extended to cover both out-of-school children and their parents/guardians in the two communities for effective disease control, and further studies covering other parts of Lagos State should be carried out so as to document the current status of urinary schistosomiasis in the state. This will help to provide reliable data that could reveal areas of high risk as well as quantifying the population at risk of schistosomiasis in the state. We further recommend that the government should provide health education to the people in the study area, for a better understanding of the transmission chain of schistosomiasis and the health hazards of improper waste disposal. Pipe-borne water should also be provided to the people so as to reduce the frequency of visits to the rivers. Implementing these measures would have a more permanent effect on the control of schistosomiasis and would also result in other benefits to the population. Lastly, these findings could be used as a basis for the development of well-designed strategies for effective mass drug delivery that will benefit the communities and the state.
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[Table 1], [Table 2], [Table 3]
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