**2. Materials and methods, results and discussion**

#### **2.1 Parasites**

The isolates were obtained from specimens of *Triatoma vitticeps* captured in the locality of Triunfo whose latitude is 22° 02′52″S and longitude 41° 56′ 32″W, in the Santa Maria Madalena and Conceição Macabu cities, both the State of Rio de Janeiro. In this study, we used a total of 14 samples obtained from triatomines collected in three distinct regions for this study called A, B and C. Six of them (SMM1, SMM10, SMM51, SMM57, SMM88 and SMM98) were isolated from area A, area deforested to make way for banana cultivation, being located at an altitude of 250 m and 3.5 km away from the village, seven samples (SMM9, SMM30, SMM34, SMM36, SMM39, SMM82 and SMM89) from area B, located in a valley with vegetation preserved around thinking to 130 m above sea level and 4 km far from the village. These two areas are 2 km far from each other and separated by mountainous area with altitudes between 400 and 900 m, belonging to the locality of Triunfo, Municipality of Santa Maria Madalena. One sample (SMM106) were isolated from D area, a preserved area of 10 km far from area C.

#### **2.2 Growth of parasite**

The samples were maintained in tubes containing NNN medium plus LIT (Liver Infusion Tryptose), as the liquid phase, supplemented 30% fetal calf serum. The tubes were incubated in an oven of the BOD (FANEM) to 27.3°C, subcultured regularly at intervals of 14 days for maintenance of the samples.

#### **2.3 DNA extraction**

Cultures of *T. cruzi* (10 ml) in the exponential growth phase were washed three times by centrifugation in PBS (Phosphate Buffered Saline) at 2,000 rpm for 10 minutes. After removal of the supernatant, the DNA was extracted by DNAzol (Invitrogen) following the manufactured instructions.
