Parasite DNA extraction

1ml blood sample, for mutation molecular detection, was collected from each patient and stored in a labeled EDTA tube. A mixture of 1ml of Saponin (0.5%) (Sigma-Aldrich) and 150 µL of each blood sample was incubated overnight at 4°C, and then centrifuged at 6000 rpm speed. 1ml of phosphate-buffered saline (PBS) (Sigma-Aldrich) was added and the mixture was centrifuged, at 6000 speed, for four consecutive times. 70 µL of chelex (Bio-Rad) and 30 µL of water were added to the precipitant and the mixture heated at 95°C for 20 minutes in 4 rounds (5 min/round). After every round the mixture was vortexed for 1 minute. DNA was collected and stored at -20°C until a PCR reaction was conducted.