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Schistosomiasis, caused by parasitic worms, is a neglected tropical disease affecting nearly 240 million people globally. The World Health Organization released a Target Product Profile in 2021 calling for the development of new diagnostics for monitoring and evaluation of Schistosomiasis, to move towards elimination. Traditional diagnostics using microscopy are reliant on laboratory infrastructure and technician skill. They also lack sufficient sensitivity and specificity. This thesis develops a loop-mediated isothermal amplification (LAMP) direct from urine Schistosomiasis assay for translation onto SnapDx platform, an electricity-free, point-of-care nucleic-acid diagnostics platform developed in the Prakash Lab for respiratory infections like Sars-CoV-2 detection. Here we demonstrate that the current version of the assay with genomic DNA from Schistosomiasis spiked in human urine in controlled lab settings displays a sensitivity of 77.50% at 1 ng/uL and 62.50% at 100 pg/uL and a specificity of 97.5% - all read via a simple colorimetric readout, in Eppendorf tubes. We further test the impact of various sample preparation methods on the assay. The lowest limit of detection reached on simulated urine samples is 1 pg/uL using samples purified with MidiPrep prior to the introduction of genomic DNA. We further find 11% of all the reactions run showing differing results between fluorescence and colorimetric readout. The mismatch is found to be greater at lower sample concentrations. Next we report on testing the functionality of the assay with wet bath inactivation and run, as required in the electricity-free SnapDx usage in field context. In future work, we intend to investigate different primer combinations, urine-based reaction inhibitors, and compact, low-cost sample preparation methods (such as urine egg concentration) are required to further optimize the assay. Next, reliability, sensitivity, specificity and limit of detection will need to be evaluated with real patient samples. Our work lays the foundation for the future implementation of this assay in field conditions without electricity, opening the door to molecular based surveillance approaches , which satisfies the usability and infrastructure requirements of the World Health Organization’s Target Product Profile. It also contributes to a body of work aiming to develop SnapDx applications for a wide range of infectious diseases, compatible with urine, saliva, blood, and other biological fluids. |
