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.

Results: We 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.

Conclusions: We 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.

Keywords: Drug screening; High - throughput screening; Schistosoma mansoni; XTT.

Fig. 1

Absorbance levels of formazan product for different parasites concentration over time. Mechanically transformed schistosomula were distributed in 96-well plates at the concentrations of 100, 200, 300 and 400 parasites per well. Schistosomula were cultured for 48 h before addition of XTT labeling mixture. Parasites were incubated with XTT labelling mixture for 4, 6 and 24 h before absorbance measurements. Linear regression r ² values are indicated on the graph

Fig. 2

Correlation of absorbance levels to parasites concentration in 96-well (a) and 384-well (b) plates formats after 24 h incubation with XTT labeling mixture. Linear regression r ² values are indicated on the graph and statistical analysis using Two-way ANOVA with Bonferroni post-hoc test between each replicate (n = 6). Significance values are represented by asterisks: **P < 0.01; ***P < 0.001

Fig. 3

Correlation of absorbance levels to viable parasites concentration in 96-well (a) and 384-well (b) plates formats. Schistosomula viability was calculated from absorbance measured on 96-wells plates (c) and on 384-wells plates (d). Linear regression r ² values are indicated on the graph

Fig. 4

Plate uniformity study performed with different parasites preparations: DMSO treated parasites (Live); heat killed parasites (Dead). Parasites were distributed in three plates following the interleaved signal format in two independent experiments (templates available at https://www.ncbi.nlm.nih.gov/books/NBK83783/). The Z’ factor for each plate is represented in the graph

Fig. 5

Dose-response curves of schistosomula exposed to compounds with reported activity against S. mansoni (a; mefloquine, ivermectin, JQ1, praziquantel) generated by the XTT viability assay and microscopy assessment. Microscopic counts and XTT viability assay started 48 h post-treatment. (b) Comparison of the readouts of the XTT viability assays of compounds that present yellow coloration (curcumin and amphotericin) performed without or with a washing step