Abstract
BackgroundSchistosomiasis, caused by infection with the blood fluke Schistosoma, is responsible for greater than 200,000 human deaths per annum. Objective high-throughput screens for detecting novel anti-schistosomal targets will drive ‘genome to drug’ lead translational science at an unprecedented rate. Current methods for detecting schistosome viability rely on qualitative microscopic criteria, which require an understanding of parasite morphology, and most importantly, must be subjectively interpreted. These limitations, in the current state of the art, have significantly impeded progress into whole schistosome screening for next generation chemotherapies.Methodology/Principal FindingsWe present here a microtiter plate-based method for reproducibly detecting schistosomula viability that takes advantage of the differential uptake of fluorophores (propidium iodide and fluorescein diacetate) by living organisms. We validate this high-throughput system in detecting schistosomula viability using auranofin (a known inhibitor of thioredoxin glutathione reductase), praziquantel and a range of small compounds with previously-described (gambogic acid, sodium salinomycin, ethinyl estradiol, fluoxetidine hydrochloride, miconazole nitrate, chlorpromazine hydrochloride, amphotericin b, niclosamide) or suggested (bepridil, ciclopirox, rescinnamine, flucytosine, vinblastine and carbidopa) anti-schistosomal activities. This developed method is sensitive (200 schistosomula/well can be assayed), relevant to industrial (384-well microtiter plate compatibility) and academic (96-well microtiter plate compatibility) settings, translatable to functional genomics screens and drug assays, does not require a priori knowledge of schistosome biology and is quantitative.Conclusions/SignificanceThe wide-scale application of this fluorescence-based bioassay will greatly accelerate the objective identification of novel therapeutic lead targets/compounds to combat schistosomiasis. Adapting this bioassay for use with other parasitic worm species further offers an opportunity for great strides to be made against additional neglected tropical diseases of biomedical and veterinary importance.
Highlights
Infection with the parasitic trematode Schistosoma mansoni causes a wide range of quantifiable clinical pathologies [1], which collectively lead to the death of approximately 200,000 individuals/annum [2]
In our continued effort to identify novel drug and vaccine targets, we detail a dual-fluorescence bioassay that can objectively be used for assessing Schistosoma mansoni schistosomula viability in a medium or high- throughput manner to suit either academic or industrial settings
To first determine whether propidium iodide (PI) and fluorescein diacetate (FDA) could be used individually to detect with corresponding polarized bright field microscopy imaging of the same schistosomula samples providing morphological confirmation of death (i.e. parasites that were uniform in shape and size and displayed no movement (Fig. 1B))
Summary
Infection with the parasitic trematode Schistosoma mansoni causes a wide range of quantifiable clinical pathologies [1], which collectively lead to the death of approximately 200,000 individuals/annum [2]. All involve microscopic techniques where the experimenter manipulates the parasite in vitro and assesses the effect of such manipulation by brightfield examination of morphology This technique has been employed in immunological studies [8], RNA interference (RNAi) assays [9], drug screening protocols [9,10] and general manipulations of parasite development [11]. Current methods for detecting schistosome viability rely on qualitative microscopic criteria, which require an understanding of parasite morphology, and most importantly, must be subjectively interpreted. These limitations, in the current state of the art, have significantly impeded progress into whole schistosome screening for generation chemotherapies
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