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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060
EISSN: 1678-8060
Vol. 107, No. 5, 2012, pp. 637-643
Bioline Code: oc12109
Full paper language: English
Document type: Research Article
Document available free of charge

Memórias do Instituto Oswaldo Cruz, Vol. 107, No. 5, 2012, pp. 637-643

 en Validation of a modified fluorimetric assay for the screening of trichomonacidal drugs
Escribano, Alexandra Ibáñez; Marcel, Alfredo Meneses; Tugores, Yanetsy Machado; Ruiz, Juan José Nogal; Redó, Vicente J Arán; García-Trevijano, José Antonio Escario & Barrio, Alicia Gómez

Abstract

A fluorimetric microassay that uses a redox dye to determine the viability of the flagellate Trichomonas vaginalis check for this species in other resources 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.

Keywords
Trichomonas vaginalis - antitrichomonal agents - drug screening assay - resazurin - high-throughput screening assays

 
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