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We report on the application of the biospeckle laser method (BLM) to test drug effects on Trypanosoma cruzi (T. cruzi) parasites. The method, enabled to assess the activity of parasites under different drug concentrations, thus demonstrating the effectiveness of the proposed methodology. The spatial-temporal correlation and speckle grain size were measured in order to assess the immediate action of the drug on parasites. The achieved results allowed the validation of the methodology as a fast and non-invasive for testing the effectiveness of Epirubicin on T. cruzi parasites. The proposed methodology was also validated by other well-known digital processing approaches.

Antibiotic susceptibility testing is a necessary step prior to the treatment of clinical infections. A major concern is the time required to obtain a fast and reliable result. The aim of this work is to use Biospeckle laser in a 15min assay for an antimicrobial susceptibility test of Ciprofloxacin in serial two-fold dilutions onEscherichia coliK-12 using Venereal Disease Research Laboratory (VDRL) plates. Analysis of images by video edition is performed on a quantitatively selected region of interest, and processed with ImageJ-ImageDP; and by the construction of time series and analysis with either statistical diagnostics tests or Autoregressive Integrated Moving Average (ARIMA) models. Antimicrobial susceptibility tests are also performed for the purpose of quantitative comparison, showing a profile that is comparable to the result obtained with ImageJ-ImageDP processing after 15min of antibiotic action. Only the time series of the least affected bacteria (low Ciprofloxacin concentration) behaves in an expected manner, being non-independent and mainly non-linear, non-normal, and heteroscedastic. The most affected bacteria (higher Ciprofloxacin concentration) are non-independent and tend to be linear, normal and heteroscedastic. Adjustment to a linear regression identifies both, the culture medium without bacteria and the most affected bacteria, normality identifies the most affected bacteria and heteroscedasticity-homoscedasticity distinguishes the presence-absence of bacteria, respectively. ARIMA models (1,1,1)(1,0,1)11and (4,1,1)(1,1,1)11fit the time series of the most affected bacteria while the latter also fits the culture medium without bacteria. The time series of the least affected bacteria are identified by a (7,1,2)(1,0,1)11model. The non-linear, non-normal and heteroscedastic behavior of this group is probably responsible for its adjustment to a model with a relatively high parameter. The four methods: diagnostic statistical tests, fitting of ARIMA models, ImageJ-ImageDP and antimicrobial susceptibility tests, show similar results, being able to distinguish among the groups of assays with bacteria and Ciprofloxacin below and above the Minimal Inhibitory Concentration.Author SummaryBiospeckle laser patterns occur when a dynamic surface is illuminated. This research describes its application to the activity ofEscherichia colibacteria and the effect of 18 different concentrations of an antibiotic (Ciprofloxacin) in a 15min assay, using VDRL plates where the sample has a relatively small volume and is flat shaped. The assay is performed on an anti-vibration table in a dark room with a laser that sequentially illuminates each of the wells of the plate. A camera takes short 30sec videos with approximately 750 frames and sends them to a computer where image processing takes place. In order to select a segment of 80 successive frames to analyze, the region with the higher variation was identified, punched out and edited as a “flip-book animation” with a program named ImageJ and processed with another program named ImageDP that takes the difference between successive frames and is able to describe the speed with which the Biospeckle dots move, expecting to show that the antibiotic affects the bacteria by changing the speed with which they move. Also in each video, within the region of higher differential activity, a pixel was selected to construct a time series which is the successive value of that pixel in 253 frames, representing a recording of 8sec. This was analyzed with two statistical methods: diagnostic statistics tests and ARIMA models, both of which try to demonstrate how the results are organized. All the results, the speed of the dots in the “flip-book animation” and the structure of the data of the time series, were comparable to those obtained with traditional antimicrobial susceptibility tests with the same bacteria and antibiotic.

This paper reports on the biospeckle processing of biological activity using a visualization scheme based upon the digital imaging information technology. Activity relative to bacterial growth in agar plates and to parasites affected by a drug is monitored via the speckle patterns generated by a coherent source incident on the microorganisms. We present experimental results to demonstrate the potential application of this methodology for following the activity in time. The digital imaging information technology is an alternative visualization enabling the study of speckle dynamics, which is correlated to the activity of bacteria and parasites. In this method, the changes in Red-Green-Blue (RGB) color component density are considered as markers of the growth of bacteria and parasites motility in presence of a drug. The RGB data was used to generate a two-dimensional surface plot allowing an analysis of color distribution on the speckle images. The proposed visualization is compared to the outcomes of the generalized differences and the temporal difference. A quantification of the activity is performed using a parameterization of the temporal difference method. The adopted digital image processing technique has been found suitable to monitor motility and morphological changes in the bacterial population over time and to detect and distinguish a short term drug action on parasites.

Antibiotic susceptibility testing is a necessary step prior to the treatment of clinical infections. A major concern is the time required to obtain a fast and reliable result. The aim of this work is to use Biospeckle laser in a 15min assay for an antimicrobial susceptibility test of Ciprofloxacin in serial two-fold dilutions on Escherichia coli K-12 using Venereal Disease Research Laboratory (VDRL) plates. Analysis of images by video edition is performed on a quantitatively selected region of interest, and processed with ImageJ-ImageDP; and by the construction of time series and analysis with either statistical diagnostics tests or Autoregressive Integrated Moving Average (ARIMA) models. Antimicrobial susceptibility tests are also performed for the purpose of quantitative comparison, showing a profile that is comparable to the result obtained with ImageJ-ImageDP processing after 15min of antibiotic action. Only the time series of the least affected bacteria (low Ciprofloxacin concentration) behaves in an expected manner, being non-independent and mainly non-linear, non-normal, and heteroscedastic. The most affected bacteria (higher Ciprofloxacin concentration) are non-independent and tend to be linear, normal and heteroscedastic. Adjustment to a linear regression identifies both, the culture medium without bacteria and the most affected bacteria, normality identifies the most affected bacteria and heteroscedasticity-homoscedasticity distinguishes the presence-absence of bacteria, respectively. ARIMA models (1,1,1)(1,0,1)11 and (4,1,1)(1,1,1)11 fit the time series of the most affected bacteria while the latter also fits the culture medium without bacteria. The time series of the least affected bacteria are identified by a (7,1,2)(1,0,1)11 model. The non-linear, non-normal and heteroscedastic behavior of this group is probably responsible for its adjustment to a model with a relatively high parameter. The four methods: diagnostic statistical tests, fitting of ARIMA models, ImageJ-ImageDP and antimicrobial susceptibility tests, show similar results, being able to distinguish among the groups of assays with bacteria and Ciprofloxacin below and above the Minimal Inhibitory Concentration.

This paper reports on the application of the motion history image (MHI) method on dynamic laser speckle processing as a result of a specific drug action on Trypanosoma cruzi parasites. The MHI procedure is based on human action recognition, and unlike other methods which use a sequence consisting of several frames for recognition, this method uses only an MHI per action sequence for recognition. MHI method avoids the complexity as well as the large computation in sequence matching-based methods and detects a change in the speckle pattern. Experimental results of MHI on real-time monitoring of activity (motility) under the influence of the drug demonstrate the effectiveness of the proposed method. The MHI showed an online result without loss of resolution and definition if we compare with routine LASCA method. The obtained results highlight the advantage of the MHI analysis over traditional qualitative image intensity-based methods and demonstrate the potential of measuring the activity of parasites via dynamic laser speckle analysis. The data was further numerically analyzed in the time domain, and the results presented the ability of the technique to monitor the action of the drug, particularly Epirubicin (100 μg/ml).

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., Author Summary Biospeckle refers to a pattern that occurs when a laser beam illuminates a dynamic surface, such as a liquid that contains microorganisms. The movement or the roughness of its surface causes the wave fronts to interfere and produce a pattern of moving dots that resemble boiling water. This research describes the application of Biospeckle to Trypanosoma cruzi, the parasite that causes Chagas disease. The purpose was to observe the movement of the Biospeckle dots and to detect differences depending on the presence of the parasite, the quantity of the parasite and the conditions of the parasites when they are affected by a drug. We designed a method using VDRL plates where the sample has a relatively small volume and is flat shaped, a laser, a camera and a lens. The Biospeckle pattern is recorded in a video in a computer and shows the Biospeckle dots which move rapidly as the concentration of parasites increases and less rapidly as the concentration decreases or as the parasites are affected by a drug such as benznidazole. We designed algorithms which take the difference between successive frames and expressed them in a program in Java, in a script in R Commander and SAGA and in ImageJ. Thus we obtained a quantitative description of the movement of T. cruzi.

In this paper, we report on a biospeckle laser method used to monitor a specific drug action on T. cruzi parasites. Experimental results from fast biospeckle monitoring of the parasites' activity under the influence of the drug demonstrate the effectiveness of the proposed method. We measure the speckle parameters such as spatiotemporal correlation and speckle grain size to assess the immediate action of the drug on the parasites during a very short incubation period. From a practical point of view, this aproach allows us to validate biospeckle as a fast, non-invasive and alternative method to test candidate drugs on T. cruzi parasites.