Study setting
North-western Tanzania comprises Mwanza, Mara, Kigoma, Kagera, Simiyu and Shinyanga regions (Fig. 1). Mara is located on the eastern, Simiyu on the south-eastern, Mwanza on the southern and Kagera on the south-western shoreline of Lake Victoria. Shinyanga is located to the south of Lake Victoria, and does not border the lake. Kigoma is located south-west of Lake Victoria and is located on the north-eastern shoreline of Lake Tanganyika (Fig. 1). All of these regions are traditionally endemic for STH, S. mansoni, STH and S. haematobium [1]. The districts bordering the southern shoreline of Lake Victoria are endemic for S. mansoni, whereas mixed infections of S. haematobium and S. mansoni have been reported for more southerly districts outside the basin, with S. haematobium being the predominant species [1]. STH are widely distributed in the region of Lake Victoria, with hookworm being common along the shoreline and in dry areas, while T. trichiura and A. lumbricoides are typically found in wet areas on the western side of the lake [3, 5]. Pre-MDA schistosomiasis mapping between 2004 and 2005 categorized the selected districts as having low to moderate endemicity, with prevalence ranging from 10 to 49.9% [5]. That survey included 5201 SAC from 86 schools selected from the same districts as those included in the present study, and the overall prevalence of S. haematobium was 20.7% (1075/5201) [5]. The prevalences of S. mansoni, A. lumbricoides, T. trichiura and hookworm were 0.8%, 0.28%, 0.13% and 31.8%, respectively [5].
The study reported here was undertaken in 29 districts of Mwanza, Mara, Kagera, Kigoma, Shinyanga and Simiyu regions. The ecology of these regions is characterized as tropical, and the rainfall is bimodal, with the long rainy season starting in February/March and lasting until the end of May and the short rainy season starting in October and lasting until December. The area receives between 1100 mm to 1600 mm of rainfall annually and the average temperature is 25–28 °C. The area is characterized by a flat landscape, an extension of the Serengeti plains, to the south of Lake Victoria, and V-shaped hills and valleys to the west of Lake Victoria. Seasonal and permanent rivers flow down the plains to Lake Victoria and Lake Tanganyika. The regions’ predominant livelihoods are livestock keeping, rice farming, and cotton and maize cultivation. The major tribe is the Wasukuma, a Bantu group, and the other, smaller, tribes in the area are the Waha, Wanyatunzu, Wakurya, Wakerewe, Wajita and Wazinza.
Study population and inclusion and exclusion criteria
Primary school children aged 9–13 years, who attended the selected schools, were targeted for the study as they comprise the high-risk age group for STH and schistosomiasis in endemic areas [16, 17]. Inclusion criteria for the children were as follows: 9–13 years of age, permanent residents of the selected areas, no history of anthelminthic treatment in the past 6 months, submitted a single urine and stool sample, and provided signed parental consent for participation in the study.
Sampling and sample size
A ward(s) within a defined ecological zone was used as a sampling unit. An ecological zone was defined as areas located within a similar distance from bodies of water (e.g. rivers, lakes, streams, ponds, dams, irrigation channels) within a locality [9, 12, 13]. An interactive mapping process was used to identify and map all the water bodies in the selected wards. All of the schools in each ward within the selected district were mapped. For the selection of the wards, each district was sequentially subdivided into lower administrative levels such as wards and villages. Then the district was divided into one, two or three different ecological zones based on its proximity to water bodies (near, < 1 km; medium, 1-5 km; and far, > 5 km) [12, 13]. All the wards and schools within a particular ecological zone were identified according to WHO guidelines [9]. From five to 15 schools were sampled from each ecological zone. It is worth noting that the selected schools met the criteria of the possible mapping scenarios (known, unknown and suspected) as described in the WHO guidelines; at least five schools were selected from each category. To ensure representation of each ward/cluster, selected schools in different ecological zones were mutually exclusive, thus a school was only included for one administrative level/ward/cluster. In the case where several wards/clusters were located in the same ecological zone (where the transmission of schistosomiasis was likely to be similar), the wards/clusters were combined into a single mapping unit and less than 15 primary schools were selected from this mapping unit, while ensuring representation of each ward/cluster [9]. After selecting the schools, a simple random sampling technique was used to select 60 SAC (30 boys and 30 girls) between the ages of 9 and 13 years, using the lottery method (drawing of numbers in a random fashion) [13, 18]. The WHO recommends the inclusion of 50 SAC from each school in a study [13]. To take into consideration a 16% rate of non-response/dropout of participants during a study, an additional 10 SAC were included in addition to the number recommended by the WHO.
Recording of demographic information of participants
Age, sex, village/ward name, school name and school identification number were recorded in the master enrolment list. The barcode of a sticker with the specific number of each participant was used as a unique identification number on the biodata form for each study participant.
Parasitological examination of stool and urine samples for schistosomiasis
Stool and urine sample collection
On the day of sample collection, the signed informed consent forms were collected from all the eligible children, who also gave their assent for inclusion in the study. All the participants were assigned an individual study identification number, which was used as an identifier for the sample collection containers, microscopic slides, register, enrolment forms, written informed consent forms and their assent. Each study participant then received an empty, clean, labelled container and tissue paper and was instructed on how to hygienically collect urine and stool samples. After sample collection, the participants were instructed to wash their hands with soap (liquid medicated soap provided by the study team).
Examination of S. mansoni eggs using the KK thick technique
A single fresh stool sample was collected from each participating child using a stool container, and duplicate KK thick smears were prepared using a slide template of 41.7 mg per thick smear [19]. The KK smears were examined within 1 h of preparation for the presence of hookworm eggs, and after 24 h, the slides were examined for the presence of eggs of S. mansoni and other STH (T. trichiura, A. lumbricoides) by two independent laboratory technicians. For quality assurance, 20% of all the positive and negative slides were re-examined by a third examiner blinded with respect to the results of the first two examiners.
Examination of S. haematobium infection using a urine filtration technique
A single urine sample was collected from all study participants between 10.00 a.m. and 2.00 p.m. within the school environment. The urine samples underwent gross examination for the presence of macrohaematuria and a urine dipstick/urinalysis reagent strip (Mission Expert; ACON, USA) was used to test for the presence of microhaematuria. The microhaematuria results were recorded as per the manufacturer’s instructions (negative, trace +, ++ and +++). After urine filtration, light microscopy was used to examine the urine filters stained with Lugol’s iodine or povidone iodine for the presence of S. haematobium eggs [20]. Eggs were counted and the number recorded. For the purpose of quality assurance, a third technician re-examined 20% of all the positive and negative urine filters at the field sites.
Geographical distribution of infections
To determine the geographical distribution of schistosomiasis and STH prevalence, a Geographic Information Systems/Global Positioning System was employed to map their spatial patterns. Maps were generated using R statistical software (version 2022.02.2+461) via the package ggplot2 to plot point prevalence and choropleth maps based on WHO infection categories. Sub-county- and ward-level shapefiles were imported with the support of the Ministry of Health, and site/village geographical coordinates were recorded via Kobo Collect throughout the data collection process.
Ethical approval and permission to conduct the study
Ethical approval for this study was sought from the National Ethical Committee, National Institute for Medical Research Tanzania (NIMR/HQ/R.8a/Vol. IX/3481). Further permission was sought from the regional and district administrative authorities of the regions involved in the study. Two days before participating in the study, the children received an informed consent form translated into Kiswahili to give to their parent(s)/guardian(s), who were invited to the school on the day of screening to give their consent. An assent form was developed for children aged 9–13 years that the children could read to understand the study procedures and objectives. The purpose of the study, participation and withdrawal rights, and risks and benefits were explained fully to the participants and their parents/guardians.
On the day of sample collection, only the assenting children whose consent forms had been signed by their parents/guardians participated in the study. Participation in the study was voluntary and the children were free to withdraw from the study at any time. For confidentiality purposes, unique codes were assigned to each participant, which were used to identify them for the purposes of the study instead of their names. All the study records were kept at a secured local store by the principal investigator (PI). All the children with schistosomiasis or STH were treated using PZQ (40 mg/kg) and/or ALB [21]. Any side-effects related to the PZQ treatment were monitored and managed by health professionals (nurses, medical doctors) who were part of the team.
Data analysis
Data were double entered into a Microsoft Excel sheet, cleaned, and exported to Stata version 15 (Stata statistical software 2017; StataCorp, College Station, TX). The focus of the analysis was to determine the prevalence of STH, S. haematobium and S. mansoni based on the KK technique and urine filtration technique variables summarized as proportions, median or mean ± SD. Chi-square or Fisher’s exact tests were used to compare frequencies/proportions/categorical variables, whereas continuous variables were compared using a t-test. For S. mansoni and STH eggs, arithmetic mean egg counts were calculated from the counts of two KK smears and multiplied by 24 to obtain an individual’s number of eggs per gram of faeces. The mean egg counts for S. mansoni and STH were compared between sex and age groups using either t-test (two groups) or ANOVA (for more than two groups). The intensity of infection was categorized according to WHO criteria as low, moderate or heavy as follows: for S. mansoni, 1–99 eggs per gram of faeces (epg), 100–399 epg and ≥ 400 epg, respectively; for hookworm, 1–1999, 2000–3999 and ≥ 4000 epg, respectively; for T. trichiura, 1–999, 1000–9999 and ≥ 10,000, respectively; for A. lumbricoides, 1–4999, 5000–49,999 and ≥ 50,000, respectively [21]. For S. haematobium, infection intensities were classified into two categories as per WHO recommendations [22]: light infection (< 50 eggs/10 ml of urine) and heavy infection (≥ 50 eggs/10 ml of urine). The geometric mean egg output for each helminth species included in the study was estimated based on infected children only.
The observed combined prevalence (results from all wards/schools involved in the study to calculate the prevalence of schistosomiasis) of S. haematobium and S. mansoni infection was used to categorize implementation units (wards) for treatment strategies based on WHO prevalence thresholds as follows: high risk area (prevalence ≥ 50%), moderate risk area (prevalence ≥ 10% and < 50%), and low risk area (prevalence 1% and < 10%)[23]. The combined prevalence of STH was also used to categorize implementation units for treatment strategies as follows: low risk areas (> 20% and < 50%) and high-risk area (prevalence ≥ 50%) [10].