title: Quantitative Laser Biospeckle Method for the Evaluation of the Activity of Trypanosoma cruzi Using VDRL Plates and Digital Analysis.
abstract: In this paper we report a quantitative laser Biospeckle method using VDRL plates to monitor the activity of Trypanosoma cruzi and the calibration conditions including three image processing algorithms and three programs (ImageJ and two programs designed in this work). Benznidazole was used as a test drug. Variable volume (constant density) and variable density (constant volume) were used for the quantitative evaluation of parasite activity in calibrated wells of the VDRL plate. The desiccation process within the well was monitored as a function of volume and of the activity of the Biospeckle pattern of the parasites as well as the quantitative effect of the surface parasite quantity (proportion of the object's plane). A statistical analysis was performed with ANOVA, Tukey post hoc and Descriptive Statistics using R and R Commander. Conditions of volume (100μl) and parasite density (2-4x104 parasites/well, in exponential growth phase), assay time (up to 204min), frame number (11 frames), algorithm and program (RCommander/SAGA) for image processing were selected to test the effect of variable concentrations of benznidazole (0.0195 to 20μg/mL / 0.075 to 76.8μM) at various times (1, 61, 128 and 204min) on the activity of the Biospeckle pattern. The flat wells of the VDRL plate were found to be suitable for the quantitative calibration of the activity of Trypanosoma cruzi using the appropriate algorithm and program. Under these conditions, benznidazole produces at 1min an instantaneous effect on the activity of the Biospeckle pattern of T. cruzi, which remains with a similar profile up to 1 hour. A second effect which is dependent on concentrations above 1.25μg/mL and is statistically different from the effect at lower concentrations causes a decrease in the activity of the Biospeckle pattern. This effect is better detected after 1 hour of drug action. This behavior may be explained by an instantaneous effect on a membrane protein of Trypanosoma cruzi that could mediate the translocation of benznidazole. At longer times the effect may possibly be explained by the required transformation of the pro-drug into the active drug.
authors.affiliation: Facultad de Farmacia y Bionálisis, Universidad de Los Andes, Mérida, Venezuela.
authors.forename: Hilda Cristina
authors.initials: HC
authors.lastname: Grassi
authors.affiliation: Facultad de Farmacia y Bionálisis, Universidad de Los Andes, Mérida, Venezuela.
authors.forename: Lisbette C
authors.initials: LC
authors.lastname: García
authors.affiliation: Cátedra de Farmacología y Terapéutica, Departamento de Biopatología, Facultad de Odontología, Universidad de Los Andes, Mérida, Venezuela.
authors.forename: María Lorena
authors.initials: ML
authors.lastname: Lobo-Sulbarán
authors.affiliation: Centro Multidisciplinario de Ciencias, Instituto Venezolano de Investigaciones Científicas, Mérida, Venezuela.
authors.forename: Ana
authors.initials: A
authors.lastname: Velásquez
authors.affiliation: Ogangi Corporation, Miami, Florida, United States of America.
authors.forename: Francisco A
authors.initials: FA
authors.lastname: Andrades-Grassi
authors.affiliation: Centro Multidisciplinario de Ciencias, Instituto Venezolano de Investigaciones Científicas, Mérida, Venezuela.|SPIE-ICTP Anchor Research in Optics Program Laboratory, International Centre for Theoretical Physics, Trieste, Italy.
authors.forename: Humberto
authors.initials: H
authors.lastname: Cabrera
authors.affiliation: Facultad de Ciencias Forestales y Ambientales, Departamento de Ordenación de Cuencas, Universidad de Los Andes Mérida, Venezuela.
authors.forename: Jesús E
authors.initials: JE
authors.lastname: Andrades-Grassi
authors.affiliation: Facultad de Farmacia y Bionálisis, Universidad de Los Andes, Mérida, Venezuela.
authors.forename: Efrén D J
authors.initials: ED
authors.lastname: Andrades
mesh_terms: D000465:Algorithms; D000818:Animals; D014355:Chagas Disease; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000379:methods*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D015151:Immunoblotting / Q000379:methods*; D007834:Lasers; D009038:Motion; D009593:Nitroimidazoles / Q000378:metabolism / Q000494:pharmacology; D010783:Photometry / Q000295:instrumentation / Q000379:methods; D011933:Reagent Kits, Diagnostic; D014344:Trypanocidal Agents / Q000378:metabolism / Q000494:pharmacology; D014349:Trypanosoma cruzi / Q000187:drug effects / Q000302:isolation & purification / Q000502:physiology*
chemical_list: D009593:Nitroimidazoles; D011933:Reagent Kits, Diagnostic; D014344:Trypanocidal Agents; C009999:benzonidazole