Study design and sample size determination for each objective
A cross-sectional study design was used to achieve the three study objectives, see STROBE checklist within supplemental materials. Based on previous epidemiological information , a sample size calculation with single population proportion formula (http://www.raosoft.com/samplesize.html) showed that a total sample size of 520 was sufficient to estimate overall prevalence of intestinal and urogenital schistosomiasis with < ±5% precision and 95% confidence.
Based on prevalence data provided by the authors of the May 2018 study , a Fisher’s exact test was used to show that sampling of 80 children from each of Samama and Mchoka schools in June 2019 was sufficient to detect a 25-percentage-point rise in prevalence of each Schistosoma species at each school (a<0.05, b<0.20) (objective 1). To ensure detection of S. mansoni if present at MOET and Koche schools (objective 2), 60 children were sampled from each. For objective 3, according to WHO recommendations for rapid mapping, 30 children per school were sampled per school . Random sampling was used at each school following stratification by age and gender. A study flow diagram is included, Figure 1.
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At each school, global position system (GPS) coordinates were taken using an Oregon 650 receiver (Garmin, Olathe, Kansas, USA). The GPS locations for each school in decimal degrees are as follows: Samama (-14.417465o, 35.217580 o), Mchoka (-14.439481 o, 35.220644 o), MOET (-14.320776 o, 35.131558 o), Koche (-14.330917 o, 35.146186 o), St Augustine II (-14.473926 o, 35.279613 o), Ndembo (-14.456385 o, 35.273794 o), Chikomwe (-14.422136 o, 35.265088 o), Chipeleka (-14.385387 o, 35.292935 o), Sungusya (-14.386472 o, 35.311398 o), St Martins (-14.351401 o, 35.294435 o), Makumba (-14.319806 o, 35.286104 o) and Mtengeza (-14.288932 o, 35.264073 o). A location map of the 12 schools is shown, Figure 2.
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Inclusion/exclusion criteria, diagnostics and praziquantel treatment of participants
The surveys took place during late May/June 2019; after obtaining written informed parental consent for each child, a total of 520 children, aged 6-15, of balanced gender, were enrolled. Children not attending school and acutely unwell children were excluded. Participants could withdraw consent at any point. On the appointed day of survey, each school child provided a mid-morning urine sample and when requested, a stool sample, alongside undertaking a brief interview by questionnaire documenting place of birth, recent travel, water-contact habits and praziquantel treatment history. If found infected, upon ova patent infection or ‘trace/positive’ urine CCA-dipstick test, each child was provided with praziquantel (IDA Foundation, Amsterdam, The Netherlands) at 40 mg/kg.
For detection of intestinal schistosomiasis, two drops of urine were applied to a CCA-dipstick (Rapid Medical Diagnostics, Pretoria, South Africa). Results were scored visually against a reference colour photograph as negative (-ve), trace (tr) or positive (+ve) and cross-checked . To augment urine CCA-dipsticks, on-site inspection of collected stool was performed with parasitological methods; at Mchoka, Samama, MOET and Koche schools, all children were asked to provide a stool sample with a total of 265 specimens obtained, Table 1. Following our rapid mapping protocol at 8 remaining schools, stool was only requested from urine CCA-dipstick +ve children, obtaining 70 specimens, Table 1.
To visualize helminth ova in stool, individual specimens were filtered across a 212 µm metal mesh then applied to produce duplicate thick (41.7 mg) Kato-Katz  smears as examined for lateral spine S. mansoni ova by microscopy (x100). Intensity of S. mansoni infection as eggs per gram (epg) was classified as: light (1 – 99 epg), medium (100 – 399 epg) and heavy (≥ 400 epg) according to WHO guidelines . To assess putative pathology associated with intestinal schistosomiasis , stools were screened for FOB using ALLTEST® cassettes (Access Diagnostic Tests UK Ltd, Aylsham, UK).
For detection of urogenital schistosomiasis, 10 ml of well-mixed urine was filtered by syringe across a circular nylon mesh of 1.5 cm diameter, with 20 µm pore size (Plastok® [Meshes and Filtration] Ltd, Birkenhead, UK). The mesh was stained with Lugol’s iodine, then inspected by microscopy (x100) to count terminal spine S. haematobium ova. Infection intensity was classified as light (< 50 ova per 10 ml) or heavy (≥ 50 ova per 10 ml) according to WHO guidelines . Putative pathology associated with urogenital schistosomiasis was assessed by Siemens Multistix® 10 SG reagent strips (Medisave UK Ltd, Weymouth, UK) for microhematuria .
During May/June 2019, all known locations where B. pfeifferi was found were re-surveyed, alongside several new locations as visited on the eastern shoreline of the lake, based upon convenience sampling from in-field observations of human water contact. At each site, two collectors searched, for 20 minutes, for B. pfeifferi by hand and with metal sieves. Global position system (GPS) coordinates, altitude and location photographs were taken with an Oregon 650 receiver (Garmin, Olathe, Kansas, USA). Water temperature (oC), pH and conductivity (µS) were recorded with a HI-98129 Pocket EC/TDS and pH Tester (Hanna Instruments Ltd, Leighton Buzzard, Bedfordshire, UK). Collected snails were kept for a week and screened daily for shedding S. mansoni cercariae by exposure to sunlight under a dissecting microscope (x20).
Demographic, questionnaire and diagnostic data were tabulated with statistical analysis carried out using IBM SPSS® Version 24 (IBM, Portsmouth, UK). Univariate analyses and Chi-square testing were first performed, then binary logistic regression undertaken, calculating adjusted odds ratios with generalised linear models, with stepwise subtraction of variables, to investigate (un)adjusted epidemiological associations.