Authors

R Barnes¹; J Reboud¹; JM Cooper¹;  ¹ University of Glasgow , UK

Discussion

The current diagnostic toolkit for schistosome infections does not offer the required sensitivity in low prevalence settings to support elimination campaigns. Nucleic acid amplification tests (NAATs) offer an opportunity to fill this requirement. However, current NAAT methods are too cumbersome and costly for high-throughput, point-of-care use required by schistosomiasis monitoring and evaluation campaigns. Two of the key barriers to current NAAT use in these settings are complex sample preparation, and the advanced equipment required for amplification and result readout.  Here we present a simple filter-based sample preparation method, and sensitive isothermal nucleic acid amplification assays with results presented on a lateral flow strip, to detect schistosome infection in urine. During infection, parasite DNA fragments are released into the circulation in extracellular vesicles and by dying parasites. This cell-free DNA (cfDNA) passes through the kidneys and into the urine, thus presenting a non-invasive diagnostic target that can be used to detect multiple schistosome species in a single sample. We leveraged the charge-switching properties of a chitosan-functionalised filter to rapidly enrich DNA in large volume samples, such as urine, to microlitre volumes that can be amplified by loop mediated isothermal amplification (LAMP).  We modified LAMP primers for S. haematobium and S. mansoni specific gene targets with labels that enable binding of the amplicons to gold nanoparticles, which are then immobilised on an antibody-coated test line of a lateral flow strip to indicate a positive result. The analytical sensitivity (LoD) of these assays for S. haematobium and S. mansoni was 4.9x10³ and 2.5x10³ target DNA copies per reaction, respectively. We will combine these assays onto a single lateral flow device, to be used alongside the cfDNA enrichment method, as a proof-of-concept that S. haematobium and S. mansoni can both be detected in a urine sample. Ultimately, a setup providing rapid, equipment-free DNA enrichment followed by an isothermal amplification with a lateral flow readout will move us one step closer to overcoming the logistical and cost challenges associated with getting NAATs to the point-of-care, where they are required.