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.

Biospeckle laser digital image processing for quantitative and statistical evaluation of the activity of Ciprofloxacin onEscherichia coliK-12

“…The experimental setup was the same as previously described [16] and is shown in Fig 1. Briefly, the setup rests on top of an anti-vibration table.…”

“…Ciprofloxacin is a broad-spectrum antimicrobial carboxyfluoroquinoline which selectively poisons preferentially the GyrA subunit of gyrase in Gram-negative bacteria and the ParC subunit of Topo IV in Gram-positive bacteria, with generation of chromosome breaks, which culminate in cell death [14,15]. An assay using VDRL plates that had already been used for testing Benznidazol on Trypanosoma cruzi [16], was adapted to E . coli .…”

Evaluation of Maize Seeds Treated with Trichodermil® through Biospeckle

“…Grassi et al [13] used the biospeckle to evaluate the protozoan Trypanosoma cruzi. Sendra et al [14] used biospeckle to detect bacterial activity.…”

When noise became information: State-of-the-art in biospeckle laser

“…Otherwise, we can cite some applications in medicine using offline techniques such as identification of cancer adjacent to normal tissue in animal samples (Braga et al, 2012) or even changes in muscle tissues (Maksymenko;Muravsky;Berezyuk, 2015). Offline methods can also be applied to identify parasites (Pomarico et al, 2004;Grassi et al, 2016) or even the effect of drugs in cancer cells (González-Peña et al, 2014).…”