Fig 2. Phenotypic assays for Balamuthia mandrillaris.

(A) Assays performed: Schematic representing the workflow for each of the assays performed for Balamuthia. Red arrows represent addition of drug and outline arrows represent drug removal. (B) Speed of killing: Results from experiment to asses kinetics of drug action against Balamuthia trophozoites. Drugs were assayed at 2x the EC₅₀ as previously assessed [21, 44]: nitroxoline at 15.6μM and obatoclax at 3.2μM. Graph shows FDA signal as a percent of 0.5% DMSO control. Killing was assayed at 8, 24 and 48 hours, and three biological replicates were performed for each data point. (C) Cyst induction: Trophozoites were treated for 72h with obatoclax (3.2μM), nitroxoline (15.6μM) or DMSO (0.5%), in 24-well plates, then the numbers of trophozoites and cysts were counted. Nitroxoline induces substantial cyst formation, as previously observed [21], but obatoclax is similar to control. (D) Parasite recrudescence: Trophozoites were treated for 72h with obatoclax (3.2μM), nitroxoline (15.6μM) or DMSO (0.5%), in flasks, then drug was removed, and Balamuthia were seeded on a monolayer of mammalian cells. The monolayer was rapidly destroyed by trophozoites after obatoclax treatment, indicating that this concentration and/or duration of treatment was insufficient to prevent recrudescence.