Parasite Burden of Trypanosoma cruzi in Whole Blood and Buffy Coat Determined by Real-Time PCR in Individuals with Chronic Chagas Disease

Abstract

The objective of this study was to compare, by qPCR, the circulating blood parasite load of Trypanosoma cruzi in the buffy coat, and in whole blood mixed with boiled and unboiled guanidine hydrochloride-EDTA buffer, of individuals with chronic ChD. The concentration and purity of DNA were evaluated in a Nanodrop Denovix DS-11FX Series Spectrophotometer (DeNovix Inc., Wilmington, NC, USA). The parasite load was determined with the Taqman® qPCR system using a Stratagene Mx3000P thermocycler (Agilent Technologies, Santa Clara, CA, USA) with Cruzi 1 and Cruzi 2 satellite primers. Student’s t-test with Bonferroni correction, Chi-squared (χ2) tests and Spearman’s correlation coefficient were applied. The concentration and purity of DNA were higher in the buffy coat. Parasite DNA was detected and quantifiable in the three types of samples in seven patients, without statistically significant differences in the parasite load obtained. Higher correlations were found between the total DNA concentrations and the parasite loads obtained in the samples of the buffy coat.