Oguttu, D. W. | Search bioRxiv

bioRxiv2025

Improving eDNA filtration and purification for qPCR detection of Schistosoma mansoni in lakes Albert and Victoria

BackgroundSchistosomiasis, a parasitic disease caused by Schistosoma trematodes, remains a significant public health burden in sub-Saharan Africa, particularly in regions with limited access to clean water, sanitation and hygiene. Effective disease control strategies rely on large-scale surveillance to accurately identify and target high-risk populations. However, traditional methods such as malacological surveys and stool/urine microscopy in humans often lack sensitivity and scalability. Environmental DNA (eDNA) is emerging as a promising tool for non-invasive surveillance of aquatic pathogens, offering enhanced sensitivity and feasibility for large-scale monitoring. MethodsThis study evaluated the efficacy of eDNA-based detection of Schistosoma mansoni in water samples from Lake Albert and Lake Victoria, Uganda. Three eDNA filtration techniques: open membrane, Waterra capsule and Sylphium capsule filters, were compared for DNA yield and detection efficiency. SYBR Green quantitative polymerase chain reaction (qPCR) targeting the mitochondrial cytochrome oxidase subunit I (COI) gene was performed to quantify S. mansoni eDNA, following in silico and in vitro optimisation of primers. Conventional malacological surveys were conducted in parallel to validate eDNA findings, and statistical analyses examined the influence of environmental factors (turbidity and total dissolved solids (TDS)) on eDNA yield and detection rates. ResultsThe SYBR Green S. mansoni qPCR assay had a practical limit of detection (LOD), defined as amplification in >95% of 33 technical replicates, of 100 copies per reaction. The assay amplified in 82% of reactions with 10 DNA copies and 76% with a single copy. The theoretical LOD, determined via amplification probabilities in RStudio, was 83 copies per reaction, which was also the calculated limit of quantification (LOQ). Schistosoma mansoni eDNA was detected in 58.1% (25/43) of filters from Lake Albert and 19.2% (10/52) from Lake Victoria. The Waterra capsule filter yielded the highest eDNA concentrations, while the Sylphium-coupled capsule filter exhibited comparable detection efficiency. Among the DNA purification methods tested, the Sylphium precipitation-based protocol produced significantly higher eDNA yield than column-based kits (DNeasy Blood and Tissue and Zymo Research). Despite significant variation in eDNA recovery across filtration methods, qPCR detection rates were consistent. No significant correlation was observed between turbidity and S. mansoni eDNA detectability. ConclusionOur findings highlight the potential of eDNA as a sensitive, scalable tool for schistosomiasis surveillance. While Waterra filters and Sylphium extraction maximised DNA yield, lower-yield filtration methods still enabled S. mansoni detection in high-transmission settings. These findings support the adaptability of eDNA approaches across varying resource contexts. Future work should prioritise protocol standardisation, ecological validation, and development of field-ready diagnostics such as LAMP to enable broader implementation in endemic regions.

molecular biology↗

bioRxiv2022

Identification of the onchocerciasis vector in the Kakoi-Koda focus of the Democratic Republic of Congo

BackgroundThe objective of this study was to characterise the vector in a small hyper-endemic focus of onchocerciasis (the Kakoi-Koda focus) which has recently been discovered on the western slopes of the rift valley above Lake Albert. Methodology/Principal FindingsAquatic stages of blackflies were collected by hand from streams and rivers, and anthropophilic adult females were collected by human landing catches. Using a combination of morphotaxonomy and DNA barcoding, the blackflies collected biting humans within the focus were identified as Simulium dentulosum and S. vorax, which were also found breeding in local streams and rivers. Simulium damnosum s.l., S. neavei and S. albivirgulatum were not found (except for a single site in 2009 where crabs were carrying S. neavei). Anthropophilic specimens from the focus were screened for Onchocerca DNA using discriminant qualitative real-time triplex PCR. One specimen of S. vorax was positive for Onchocerca volvulus in the body, and out of 155 S. dentulosum, 30% and 11% were infected and infective (respectively). Conclusions/SignificanceSimulium dentulosum currently appears to be the main vector of human onchocerciasis within the Kakoi-Koda focus, and S. vorax may be a secondary vector. It remains possible that S. neavei was the main (or only) vector in the past having now become rare as a result of the removal of tree-cover and land-use changes. Simulium vorax has previously been shown to support the development of O. volvulus in the laboratory, but this is the first time that S. dentulosum has been implicated as a vector of onchocerciasis, and this raises the possibility that other blackfly species which are not generally considered to be anthropophilic vectors might become vectors under suitable conditions. Because S. dentulosum is not a vector in endemic areas surrounding the Kakoi-Koda focus, it is probable that the Kakoi-Koda focus is significantly isolated. AUTHOR SUMMARYRiver blindness (=onchocerciasis) is a severely debilitating disease caused by the nematode parasite Onchocerca volvulus, and in Africa it is known to be transmitted from person to person by blood-sucking blackflies (Diptera: Simuliidae) of the Simulium damnosum complex, the S. neavei group and (rarely) by S. albivirgulatum. Using classical morphological characteristics and DNA analysis we have unexpectedly identified the vector blackfly as S. dentulosum (and possibly S. vorax) in a small endemic area (the Kakoi-Koda focus) which has been recently discovered on the western slopes of the rift valley in the Democratic Republic of Congo above Lake Albert. In the surrounding endemic areas, the vectors are S. damnosum complex and/or S. neavei (as normally expected), and because S. dentulosum is not a vector in these surrounding areas, it follows that this focus is entomologically isolated from immigrant blackfly species which might otherwise have carried new infections into the Kakoi-Koda focus (and vice-versa). This is of local importance, because it makes elimination of the parasite easier, but our findings have wider significance across Africa, because they raise the possibility that under the right conditions, other common and widely-distributed blackfly species might unexpectedly become man-biters and significant vectors.

ecology↗