title: Validation of novel fluorescence assays for the routine screening of drug susceptibilities of Trichomonas vaginalis.
abstract: OBJECTIVE
  To evaluate existing protocols, based on Alamar blue (resazurin), for the routine determination of drug susceptibility in trichomonads, develop new ones and validate these by screening small antiprotozoal libraries.
  
  
  METHODS
  The resazurin-based assay was evaluated by determining fluorescence development in Trichomonas media with various cell densities after various intervals and in the presence of metronidazole. Similar investigations were performed with the alternative fluorophores propidium iodide (PI) and resorufin. The optimized protocols were used to screen for new antitrichomonal compounds.
  
  
  RESULTS
  Anaerobic cultures of Trichomonas vaginalis rapidly reduced blue resazurin to red, fluorescent resorufin. However, the ascorbic acid in the culture medium produced similar effects, even in the absence of cells, causing high background fluorescence and variability. Moreover, T. vaginalis rapidly metabolized resorufin to the non-fluorescent and colourless metabolite dihydroresorufin, making the fluorescent signal transient. In contrast, resorufin proved to be an excellent viability probe for Trichomonas due to its chemical stability in media and rapid metabolism by the parasite. We also show that staining with PI after cell permeabilization similarly constitutes a reliable measurement of trophozoite numbers. Using the PI and resorufin assays we determined reproducible EC(50) values and identified potent antitrichomonal compounds from a limited screen of phosphodiesterase inhibitors and phosphonium salts.
  
  
  CONCLUSIONS
  The resorufin- and PI-based assays are suitable for routine and high-throughput drug screening, whereas resazurin-based assays are not. These assays constitute a major advance in the current protocols as demonstrated by a successful screen for new antitrichomonal lead compounds.
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA,  UK.
authors.forename: Manal J
authors.initials: MJ
authors.lastname: Natto
authors.forename: Francesca
authors.initials: F
authors.lastname: Savioli
authors.forename: Neils B
authors.initials: NB
authors.lastname: Quashie
authors.forename: Christophe
authors.initials: C
authors.lastname: Dardonville
authors.forename: Boris
authors.initials: B
authors.lastname: Rodenko
authors.forename: Harry P
authors.initials: HP
authors.lastname: de Koning
mesh_terms: D000981:Antiprotozoal Agents / Q000494:pharmacology*; D005260:Female; D005453:Fluorescence; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D008297:Male; D010078:Oxazines / Q000378:metabolism; D021261:Parasitic Sensitivity Tests / Q000379:methods*; D011419:Propidium / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods; D014246:Trichomonas vaginalis / Q000187:drug effects* / Q000254:growth & development; D014966:Xanthenes / Q000378:metabolism
chemical_list: D000981:Antiprotozoal Agents; D010078:Oxazines; D014966:Xanthenes; C005843:resazurin; D011419:Propidium; C014180:resorufin