Anti- compounds can be screened using spp. expressing red fluorescence ().

The main challenges associated with leishmaniasis chemotherapy are drug toxicity, the possible emergence of resistant parasites, and a limited choice of therapeutic agents. Therefore, new drugs and assays to screen and detect novel active compounds against leishmaniasis are urgently needed. We thus validated (Lb) and (Li) that constitutively express the tandem tomato red fluorescent protein () as a model for large-scale screens of anti- compounds. Confocal microscopy of and revealed red fluorescence distributed throughout the entire parasite, including the flagellum, and flow cytometry confirmed that the parasites emitted intense fluorescence. We evaluated the infectivity of cloned promastigotes and amastigotes constitutively expressing , their growth profiles in THP-1 macrophages, and susceptibility to trivalent antimony, amphotericin, and miltefosine . The phenotypes of mutant and wild-type parasites were similar, indicating that the constitutive expression of did not interfere with the evaluated parameters. We applied our validated model to a repositioning strategy and assessed the susceptibility of the parasites to eight commercially available drugs. We also screened 32 natural plant and fungal extracts and 10 pure substances to reveal new active compounds. The infectivity and Glucantime treatment efficacy of BALB/c mice and golden hamsters infected with and mutant lines, respectively, were very similar compared to animals infected with wild-type parasites. Standardizing our methodology would offer more rapid, less expensive, and easier assays to screen of compounds against and and . Our method could also enhance the discovery of active compounds for treating leishmaniasis.