QUANTIFICATION OF TRYPANOSOMA CRUZI EPIMASTIGOTES: A COMPARATIVE STUDY OF FLUOROMETRIC METHODS

LUCIA R. FERNANDEZ, DIANA BERNAL, GABRIEL FERRI, MARTIN M. EDREIRA, DANIEL MUSIKANT

Congreso; REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS; 2017

Cell counting with Neubauer chamber is a traditional tool of greatutility and precision in the quantification of parasitic forms of T. cruzi.However, this method has some limitations, including long-operatingtimes and subjectivity in counts, which makes it impractical fortesting antiparasitic compounds at medium or large scale. With theobjective of validating a technique that allows us to test a libraryof compounds, two alternative fluorescent methods of quantificationof parasites were tested: transgenic GFP epimastigotes and a resazurin(Rz) reduction-based assay. GFP transgenic and wild typeepimastigotes of the Y strain were grown under standard cultureconditions and treated with the reference drugs (Benznidazole-BZandNifurtimox -NFX-). Fluorescence was recorded on a plate fluorometer(BMG Fluostar Optima). Neubauer chamber count wasperformed in parallel. We confirmed a linear correlation (r2 = 0.99)between the number of parasites and the fluorescence signal, withboth Rz and GFP. The linearity, sensitivity, limit of detection, limitof quantification and range for both methods were analyzed. Theaccuracy and precision of the two techniques were evaluated usingthe EJCR (Elliptic Confidence Region) test. Although both methodsshowed good correlation, GFP showed a better correlation with thestandard method.