*2.7. DNA Extraction and Real Time PCR*

Parasite loads in heart tissue were quantified post mortem by qPCR. For this procedure, parasite DNA was extracted using a commercial genomic DNA extraction kit (Invitrogen Life Technologies—Thermo Scientific, Waltham, MA, USA), in accordance with the manufacturer's instructions. The obtained elute was quantified via Nanodrop 2000 (Thermo Scientific), and the purity and integrity of the DNA was verified in a 1.2% agarose gel. For parasite quantification in tissue by qPCR, the DNA extracted from cardiac tissue was amplified following the method described by Molina-Berríos [14], for identification of *T. cruzi* satellite DNA (GenBank accession number MH884804.1), employing Cruzi I: 5- -AST CGG CTG ATC GTT TTC GA-3 and Cruzi II: 5- AAT TCC TCC AAG CAG CGG ATA-3- , as forward and reverse primers, respectively; and Cruzi III: 6FAMCAC ACA CTG GAC ACC AAMGBNFQ, as aTaqMan probe (5- Fluorescent label 6-FAM and 3- Quencher MGB) acquired from Applied Biosystems (Applied Biosystems, Beverly, MA, USA). Cycle reactions were carried out in a Step One PlusTM 3 (Applied Biosystems, Beverly, MA, USA) under the following conditions: one initial cycle of denaturation at 2 min and 50 ◦C and 10 min at 95 ◦C, followed by 35 cycles of denaturalization (15 s at 95 ◦C), and annealing and extension (60 s at 50 ◦C).The borderline Ct (Cycle threshold) of positivity was defined by preparing a standard curve via serial dilutions of 1:10, starting from a pure culture of *T. cruzi* (strain Sylvio-X10/4 TcI), and taking DNA points at 0.1; 1.0; 10; 100; and 1000 parasites. All the experiments were carried out in duplicate and performed at least three times independently.
