A high-throughput colorimetric assay for detection of Schistosoma mansoni viability based on the tetrazolium salt XTT

Abstract

Background Schistosoma mansoni is a trematode parasite that causes schistosomiasis, one of the most prevalent neglected tropical diseases, leading to the loss of 2.6 million disability-adjusted life years. Praziquantel is the only drug available, and new drugs are required. The most common strategy in schistosomiasis drug discovery is the use of the schistosomula larval-stage for a pre-screen in drug sensitivity assays. However, assessing schistosomula viability by microscopy has always been a limitation to the throughput of such assays. Hence, the development of validated, robust high-throughput in vitro assays for Schistosoma with simple readouts is needed. Here, we present a simple and affordable alternative to assess schistosomula viability. The method employed is based on the hydrosoluble tetrazolium salt XTT which has been widely used in other organisms but has never been used to drug screen in schistosomes.ResultsWe showed that schistosomula reduce XTT salt to a coloured formazan product and that absorbance levels reflected the viability and parasites number. This XTT viability assay was validated for high throughput screening of compounds in schistosomula, and dose-response curves of compounds could be reproduced.ConclusionsWe conclude that the XTT viability assay could be applied for the screening of large compounds collections in S. mansoni and accelerate the identification of novel antischistosomal compounds.

Multi-center screening of the Pathogen Box collection for schistosomiasis drug discovery

“…ATP and NAD(P) metabolic indicators, DNA intercalation agents such as propidium iodide, and visual- or automated image-based systems) and readouts (e.g. percentage death, EC 50 value or phenotypic score), have been developed such that the general interpretability of data may be constrained by the particular assay employed [9–11, 24, 28, 32–34].…”

“…XTT (sodium-2,3-bis-[2-methoxy-4-nitro-5-sulfophenyl]-2H–tetrazolium-5-carboxanilide) was used as an indicator of schistosomula metabolic activity by the reduction of the yellow tetrazolium salt XTT to an orange formazan product [28, 29]. XTT was dissolved in Medium 199 without FBS to prepare a 1 mg/ml solution and the electron coupling reagent, N-methyl dibenzopyrazine methyl sulfate (PMS; Cat.…”

Determining the viability of Schistosoma mansoni cercariae using fluorescence assays: An application for water treatment

“…Plate readers have a significant cost, and the additional requirement for black-sided microplates designed for fluorescence assays increases the overall cost of the assays. Some studies found that simpler, less advanced machines could not measure the fluorescence accurately enough when quantifying schistosomula viability [6,16]. The assays should therefore be verified for cercariae using less-advanced plate readers, e.g.…”

“…The change in the dye's intensity can be used to quantify the sample's viability (visually under a microscope, or automated using spectrophotometers, flow cytometers or plate readers) [8]. These methods have significant advantages in terms of standardization, accuracy, simplicity of experiments, and replicability [16]. Fluorescence and colorimetric assays for determining parasite viability have been reviewed by Keiser [15] and Peak and Hoffmann [8], and although some of these methods have been shown to work on part of the schistosome lifecycle (for example MTT formazan on adult worms [17,18] or methylene blue on schistosomula [19]), there is a need to develop high-throughput assays for detecting cercarial viability.…”

“…The FDA-PI assay has been shown to accurately determine schistosomula viability by Peak et al [20]. However, other researchers have struggled to replicate these results [6,16], and limitations of the assay have been found in other fields of research [24]. We therefore also test the FDA-H dual stain, since Hoechst has been used to determine cercarial viability [25].…”