A Dual-Reporter Fluorescence–Luminescence Assay for Drug Screening in Promastigote and Intracellular Amastigote Stages of Leishmania

Leishmaniasis is a Neglected Tropical Disease caused by protozoa of the Leishmania genus. No human vaccine is available, and current treatments are limited by toxicity and drug resistance. Thus, the identification of new active molecules remains an urgent priority. However, drug discovery is severely hampered by the lack of fast, robust, and high-throughput screening assays, particularly for the clinically relevant intracellular amastigote stage. Here, a transgenic Leishmania infantum line stably co-expressing the bioluminescent marker PpyRE9H fused to the fluorescent reporter tdTomato was generated and used to develop a novel dual-reporter microplate-based platform to identify compounds active against promastigotes or intracellular parasite stages. The engineered strain exhibited comparable growth and infectivity to the wild-type strain, validating its suitability for drug screening assays. The activity of reference drugs was evaluated, and results were compared to those obtained using standard methods, such as MTT assay for promastigotes and Giemsa staining with microscopic quantification for amastigotes. Comparable IC50 values were obtained using fluorescent/bioluminescent assays and conventional methods. These proposed assays provide a sensitive and reproducible alternative to conventional methods. The microplate format enables higher throughput screening, while the dual bioluminescence and fluorescence readouts enable cross-validation of results and reduce the risk of compound-specific signal interference.