title: Validation of a modified fluorimetric assay for the screening of trichomonacidal drugs.
abstract: A fluorimetric microassay that uses a redox dye to determine the viability of the flagellate Trichomonas vaginalis has been optimised to provide a more sensitive method to evaluate potential trichomonacidal compounds. Resazurin has been used in recent years to test drugs against different parasites, including trichomonadid protozoa; however, the reproducibility of these resazurin-based methods in our laboratory has been limited because the flagellate culture medium spontaneously reduces the resazurin. The objective of this work was to refine the fluorimetric microassay method previously developed by other research groups to reduce the fluorescence background generated by the media and increase the sensitivity of the screening assay. The experimental conditions, time of incubation, resazurin concentration and media used in the microtitre plates were adjusted. Different drug sensitivity studies against T. vaginalis were developed using the 5-nitroimidazole reference drugs, new 5-nitroindazolinones and 5-nitroindazole synthetic derivatives. Haemocytometer count results were compared with the resazurin assay using a 10% solution of 3 mM resazurin dissolved in phosphate buffered saline with glucose (1 mg/mL). The fluorimetric assay and the haemocytometer counts resulted in similar percentages of trichomonacidal activity in all the experiments, demonstrating that the fluorimetric microtitre assay has the necessary accuracy for high-throughput screening of new drugs against T. vaginalis.
authors.affiliation: Departamento de Parasitologia, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, España. alexandraibanez@pdi.ucm.es
authors.forename: Alexandra Ibáñez
authors.initials: AI
authors.lastname: Escribano
authors.forename: Alfredo Meneses
authors.initials: AM
authors.lastname: Marcel
authors.forename: Yanetsy Machado
authors.initials: YM
authors.lastname: Tugores
authors.forename: Juan José Nogal
authors.initials: JJ
authors.lastname: Ruiz
authors.forename: Vicente J Arán
authors.initials: VJ
authors.lastname: Redó
authors.forename: José Antonio Escario
authors.initials: JA
authors.lastname: García-Trevijano
authors.forename: Alicia Gómez
authors.initials: AG
authors.lastname: Barrio
mesh_terms: D000994:Antitrichomonal Agents / Q000494:pharmacology*; D003022:Clotrimazole / Q000494:pharmacology*; D005470:Fluorometry; D057166:High-Throughput Screening Assays; D008795:Metronidazole / Q000031:analogs & derivatives* / Q000494:pharmacology*; D010078:Oxazines; D021261:Parasitic Sensitivity Tests; D012680:Sensitivity and Specificity; D014246:Trichomonas vaginalis / Q000187:drug effects*; D014966:Xanthenes
chemical_list: D000994:Antitrichomonal Agents; D010078:Oxazines; D014966:Xanthenes; D008795:Metronidazole; C005843:resazurin; D003022:Clotrimazole; C016724:secnidazole