This was a nested-Case Control study conducted among children and adults of ages 6-30 years from the urban Asokwa District in Kumasi, Ghana (see plate 1). This was part of a larger study to assess plasmodium transmission in persons infected with Schistosomiasis (NCT02769013). Ethical approval was obtained from the Committee for Human Research Publication and Ethics, School of Medical Sciences, Kwame Nkrumah University of Science and Technology, Ghana. All participants were required to sign an informed consent and an assent form in case of minors. Cases were respondents diagnosed to have S. haematobium by routine microscopic examination of urine samples. Controls from the same communities, without laboratory or clinical detection of urinary schistosomiasis infection were age and sex matched with cases.
Apromase, Deduako, Emena and Kokoben were the study communities in the urban Asokwa District with a population of 140,161 inhabitants in 36, 183 households . These communities are located between latitude 6°30’ and 7°00’ North and longitude 1°30’ and 2°00 West of Kumasi, the capital city of the Ashanti Region of Ghana. The four communities have Saman (Kokoben and Apromase), Oda (Deduako) and Subin (Emena) as names of three rivers running through it.
Climatic conditions are tropical with temperatures varying from 20.2 °C to 37.1 °C. Rainfall pattern is seasonally bimodal with major rains extending from late April to August with a minor one from September to October . The average annual rainfall for the area is 6.25mm with peaks of 214.3mm and 16.2mm in June and September respectively. The dry season (harmattan) is from November to March with humidity ranging between 53% and 93%.
Screening and enrolment
A census of the selected communities was conducted with the ages and number of inhabitants per building collected along with corresponding GPS coordinates using Personal Digital Assistants (PDAs). Households within the buildings were selected and their members asked for written informed consent to be screened in the study. Twenty millilitres (20 ml) of urine samples were collected once, from consenting participant into well-labelled 30 ml urine containers. The urine samples were collected within the hours of 6:00 am and 12 noon. Subsequently, the samples were kept in cold boxes at temperature of 4-6 oC until transported to the laboratory at Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR) about 15-20 minute drive from the study sites.
Asymptomatic schistosomiasis positive (SP) cases and schistosomiasis-negative (SN) controls based on screening results were invited to participate in the study.
2.1 Sampling procedures
A total of 1258 participants were screened for schistosomiasis out of which 104 were positive. All 104 schistosome positive participants (Figure 1) were placed on PZQ60 treatment. Controls were selected from the same communities and matched in a 2: 1 ratio with cases by sex and age. Out of the 104 positive cases, 32 started the treatment phase with 28 successfully completing the course with samples analyzed. On the other hand, 53 controls had all samples analyzed.
2.2 Design of Experiment
Biochemical parameters and schistosome counts were analysed before and after treatment for both cases and controls (Figure 3). In between pre- and post-treatment, the biochemical and schistosome counts were monitored before the 2nd and 3rd dosages of PZQ60 for cases.
2.3 Laboratory Processes
2.3.1 Processing of Urine and S. haematobium quantification
Freshly voided urine was collected between 6:00am and 12:00pm in a sterile wide-mouthed screw-top plastic container (30 ml) and transported to the laboratory on ice at 4oC to 8 oC. Urine processing and quantification were done as described by Cheesebrough . Briefly, blunt-ended forceps were utilized to place a polycarbonate membrane filter of pore size of 12.0μm (Whatmann Nuclepore) on the filter-support of a filter-holder (Swinnex 25mm support chamber). The filter holder was re-assembled and attached to a 10ml syringe filled with well-mixed urine which was filtered with the aid of the plunger. The filter was carefully removed and transferred with the face upwards to a clean glass-slide. A drop of physiological saline was added, covered with a cover-slip, and examined by two independent expert microscopists using the 10X objective (Carl Zeiss Microscope) with the condenser iris closed sufficiently to give good contrast. The entire filter was examined systematically for the presence of S. haematobium eggs. The number of the eggs counted per 10ml of urine was recorded and the average of the two counts was calculated. A slide was declared negative when no parasites were detected.
2.3.2 Blood sampling and processing
Blood samples were collected from the antecubital vein with the aid of a tourniquet and the puncture site cleaned with 70% alcohol prep. Blood drawn into separator gel tubes (5 ml) were centrifuged at 1780 x g for 10 minutes at 4oC to obtain the sera which were subsequently stored at -80oC.
2.3.3 Biochemical Analysis
Assays for the liver function; albumin, globulin, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and gamma glutamyl transferase (GGT) and renal function (urea and creatinine), were conducted using a chemistry analyzer (HumaStar 200, Human, Germany). Elevated levels of AST, ALT and GGT are known indications of liver damage which may be caused by drug metabolism, infections and alcohol consumption. Increase in the levels of globulin is also found to correlate with infection or an inflammatory state. Aliquots of the serum were dispensed into cuvettes after thawing and placed at pre-programmed positions in the auto-analyzer and analysis done in batches. Due to the limited strength of serum urea and creatinine as markers of renal function, renal insufficiency based on estimated glomerular filtration was used as a more robust indicator. Renal insufficiency was assessed based on the 4-variable Modification of Diet in Renal Disease (4v-MDRD) and the Chronic Kidney Disease Epidemiology Program (CKDEPI) equations . The 4v-MDRD estimates the glomerular filtration rate based on serum creatinine concentration, age, sex and race whiles the CKDEPI uses variations of an equation based on cut-offs for serum creatinine concentrations and sex.
2.4 Statistical analysis
Data were entered into excel and analyzed with Stata V.12 (StataCorp, USA). Continuous variables were reported as median with interquartile ranges and categorical variables as proportions. The Wilcoxon signed rank test was used to assess differences in continuous variables between groups with statistical significance set at p< 0.05.