title: Determination of schistosomiasis environmental contamination and microbial source tracking
Creators: name: Champion, Teteh Sian
Publisher: name: University of Glasgow
Year: 2022

description: Schistosomiasis and soil-transmitted helminths (STHs), cause major morbidity globally, predominantly in the world’s poorest populations, and exacerbate the cycle of poverty. Poor sanitation facilitates the transmission of the parasitic worms which cause these diseases. The transmission of the Schistosoma species, S. mansoni, can be interrupted by water, sanitation, and hygiene (WASH) interventions if they prevent the faecally deposited schistosome eggs from reaching water and infecting the parasite’s intermediate snail host. Adequate sanitation can also prevent STH ova being deposited into soils, and their ongoing transmission. The monitoring of schistosomiasis and the effect of WASH interventions has predominantly relied on epidemiological data, rather than direct examination of the parasite in the wider environment. In the last few years, environmental DNA (eDNA) techniques have been used to detect S. mansoni in water samples collected from known water contact sites in endemic areas. The sampling of the terrestrial environment has been neglected regarding environmental monitoring of schistosomiasis, despite the soil environment interfacing with sanitation practices such as open defecation and pit latrine usage. The overall aim of this thesis was to characterise the soil environment using eDNA techniques on soil samples that interface with sanitation facilities: sites of open defection and pit latrines in a community highly endemic for S. mansoni and co-endemic for STH. As the soil environment has not been previously investigated using eDNA based methods to detect S. mansoni, Chapter Two outlines the laboratory work carried out to apply existing eDNA-based techniques used on water samples, to the detection of S. mansoni in soil samples. An assay detecting the cytochrome oxidase I gene of S. mansoni was chosen and tested on soils spiked with varying numbers of S. mansoni eggs. It was tested to a lower limit of detection of a single Schistosoma egg extracted from 500 mg of soil. Although the qPCR assay could not reliably estimate the number of eggs in a sample, the assay was found to be highly sensitive. Therefore, this highly sensitive assay was then taken forward to be trialled in an absence/presence capacity on soil samples collected from a S. mansoni endemic area in Uganda in Chapter Three. Soil samples were collected from areas interfacing with sanitation (areas of open defecation and pit latrines) as well as a predicted human defecation free area (community football field). The collected soils were then tested for the detection of S. mansoni using the DNA extraction methods and qPCR-assay from Chapter Two. Although the qPCR assay performed as expected from the standard curve data generated, none of the soils collected had detectable amounts of S. mansoni DNA. Whilst this qPCR was not sensitive enough to detect S. mansoni eggs, or no eggs were present in these sanitation facilities-associated soils, that does not necessarily mean that faecal contamination of the soil environment was not occurring. Therefore, in Chapter Four, bacterial faecal markers were employed as a proxy for the presence of S. mansoni eggs, as they are transmitted into the environment through an infected individual’s faeces. To gain a broader insight into the soil environment four qPCR assays were used to detect and quantify all eukaryotic DNA (universal 18S), prokaryotic DNA (universal 16S), as well as indicators of faecal contamination using a general Bacteroides, a bacterial indicator of homeothermic (bird and mammal) faeces and a human specific Bacteroides marker. Faecal contamination was observed across all samples which could have been caused by the free roaming animals observed in this community. However, fewer samples from the predicted negative control site (the community football pitch) had human faecal markers detected, than soils from the sanitation-associated sites. As schistosomiasis is often co-endemic with STHs, including hookworm, additional studies were performed on these soil samples to assess if Chryseobacterium nematophagum (a nematode-eating bacteria) is found in areas endemic for hookworm. If found, this could indicate the potential for using these bacteria as a form of biological control for hookworm larvae in such an area. In the final experimental data chapter, Chapter Five, the soil samples collected from Uganda were therefore additionally tested for the presence of both hookworm and C. nematophagum. A single soil sample (out of 31) was positive for C. nematophagum. This soil sample was also positive for a hookworm species but as the technical replicate for this sample had CT values more than 0.5 cycles apart, they were considered too disparate from one another to be considered reliable data. Further work is therefore needed before any potential biological control intervention could be investigated further. The possible reasons for the variable results presented throughout the thesis, and recommendations for how to improve the assays, are discussed in the final chapter of the thesis, the General Discussion.