Design

Quarterly assessments of inhabitants in the region where the first malaria case detected in 2010 occurred were scheduled, resulting in a total of eight assessments over 21 months. Before the first assessment the participants signed a voluntary informed-consent form. During each assessment, a questionnaire was filled out covering demographic data and any instances since the previous collection of febrile illnesses, as well as thick-smear tests for malaria and trips outside the area where the participant lived. A total of 5 mL of blood was then collected in Vacutainer tubes containing EDTA, thick and thin peripheral blood smears were prepared, and abdominal palpation for splenic enlargement was performed.

The blood collected was centrifuged at 300 g for 10 min in the Protozoology Laboratory at the Tropical Medicine Unit, Federal University of Espírito Santo, to separate the plasma from the red blood cells. The latter were then aliquoted and stored at -20 °C in Eppendorf tubes, which were sent to the Protozoology Laboratory at the São Paulo Institute of Tropical Medicine, where DNA was extracted and PCR was performed.