Drug sensitivity assays and IC50 calculations

We evaluated the viability of the worms after 24, 48, and 72 hours of continuous drug exposure by means of visual microscopy. The worms’ viability was evaluated assigning a score ranging from 0 to 3, with 0.25 unit intervals. A score of 0 was given to dead worms, while a score of 3 was assigned to fully vital worms. The criteria used to score the viability were movement, frequency of body contractions, granularity and transparency and tegument morphology.

The activity of six drugs, namely auranofin (Enzo Life Sciences AG, Lausen, Switzerland), mefloquine (Sigma-Aldrich, Buchs, Switzerland), nicardipine (Sigma-Aldrich, Buchs, Switzerland), oxamniquine (kindly donated by Pfizer, NY, USA), oxethazaine (Sigma-Aldrich, Buchs, Switzerland) and praziquantel (Sigma-Aldrich, Buchs, Switzerland) was evaluated against both in vitro and in vivo developed S. mansoni by 21 days of development. For praziquantel, we additionally compared the drug sensitivity of worms developed for 7, 14, 28 and 35 days in vitro and in vivo. The concentrations used to calculate the IC₅₀ values were: for praziquantel: 2.5, 1.25 and 0.625 μM, for oxamniquine: 50, 25, 12.5 μM, for auranofin, mefloquine, nicardipine and oxethazaine: 10, 5, 2.5, 1.25 μM. The selection of the dose range was based on previous published studies describing the sensitivity of different stages of development to these drugs [13,18–20]. The experiments were performed in duplicate using at least three worms per well and were repeated once. The drug effect was calculated by normalizing the viability of the worms exposed to the drugs, to the viability score of the control wells. The control wells consisted of worms incubated in the same culture media as the test conditions, and had the amount of DMSO corresponding to highest concentration of the assay (0.5% V/V). IC₅₀ values were calculated using the software Graph Pad Prism V8.0 (San Diego, CA, USA).