qPCR in heart tissue

The molecular technique of real time PCR was performed in cardiac tissue at 240 d.p.t. after necropsy of the animals according to the protocol of Caldas et al. (2012). The heart tissue previously stored at −80 °C was cut in fragments of 15–30 mg. DNA extraction was performed using the WizardTM Genomic DNA Purification kit (Promega) following the manufacturer's recommendations. For enzymatic digestion of the heart fragments, 30.0 µL of Proteinase K (Sigma-Aldrich^®, USA) at the concentration of 20.0 mg mL⁻¹ were added. To standardize the measure of parasite DNA detection in mouse heart fragments by qPCR, a standard curve was constructed in order to determine the number of T. cruzi DNA copies, which was used as reference. DNA dilutions were submitted to the same protocol of DNA extraction already described and were amplified as described: Samples were analysed in duplicate for amplification of T. cruzi DNA. For each qPCR reaction 3.0 µL of the diluted extracted sample containing 30.0 ng of genomic DNA, 5.0 µL of GoTaq^® qPCR Master Mix (Promega^®) and 10 µm of each primer were used: TCZ-F 5′-GCTCTTGCCCACAMGGGTGC-3′, wherein M = A or C, and TCZ-R 5′-CCAAGCAGCGGATAGTTCAGG-3′. In the same plaque, the reaction was also performed in unicate to assess the murine-specific tumour necrosis factor alpha (TNF-α), used as endogenous control, using the primers: TNF-5241 5′-TCCCTCTCATCAGTTCTATGGCCCA-3′, and TNF-5411 5′-CAGCAAGCATCATAGCACTTAGACCCC-3′ – R 5′ – CCAAGCAGCGGATAGTTCAGG-3′ according to Cummings and Tarleton (2003). All plates contained the test samples, negative and positive controls of the reaction. Each DNA sample was analysed in triplicate (duplicate for T. cruzi and unicate for TNF-α). PCR reactions were performed on 96 well plates – MicroAmp^®Optical 96 – Well Reaction Plate (Applied Biosystems by Life Technologies, USA), coated with Optical Adhesive Covers (Applied Biosystems by Life Technologies, USA) and processed in ABI Prism 7500 Sequence Detection System Thermal Cycler (Applied Biosystems, USA).

Survival rates: The survival rates of the animals were evaluated daily and expressed in cumulative percentage.

Cure criteria. The classical cure criterion according to II Brazilian Consensus on Chagas Disease, 2015 (Dias, 2016) was used. The susceptibility or resistance to treatments was defined according to the percentage of negative results obtained by the interpretation of the set of parasitological (FBE, HC and PCR) and serological (ELISA) methods used in the evaluation. Animals that were negative in all parasitological and serological tests were considered cured.

Additionally, the heart qPCR test of high sensibility and specificity (Caldas et al., 2012) was performed for better evaluation of the therapeutic activity.

Statistical analyses: Statistical analyses of data were carried out using Prism software v5.02 (GraphPad Software, San Diego, CA). Data were initially assessed by one-way analysis of variance (ANOVA). When interactions were significant, a Tukey test was used to determine specific differences between mean values. The Kolmogorov–Smirnov test was used to compare parasitaemia between infected groups that were either treated or not treated. One-way ANOVA or Mann–Whitney U tests were used to compare maximum peak values of parasitaemia among the different groups. The log-rank (Mantel–Cox) method was used to estimate the average survival for the different experimental groups. Values were expressed as means of the standard deviations. Differences in mean values were considered significant at P < 0.05.