The EDTA vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ) were used to collect 35 mL of whole blood from each infected patient, which were divided into two aliquots: a 15 mL sample for mosquito feeding and a 20 mL sample that was transported to the blood bank of the Valle del Lili Clinic for routine screening for a panel of common infectious agents (viral, bacterial, parasitic), including confirmation of Plasmodium species ( P. vivax, P. falciparum, P. malariae) by polymerase chain reaction (PCR) (Table 2).15 The rationale behind the blood screening and direct mosquito feeding on human volunteers was based on two premises: 1) standard blood bank screening is universally accepted as a safety procedure that ensures that no pathogens are transmitted from human-to-human during a blood transfusion. Therefore, blood samples determined to be negative for pathogens other than P. vivax by standard blood bank screening should be safe enough to be transferred from human-to-human through a mosquito bite. 2) Anopheles mosquitoes have not been reported to trans mit any pathogen other than Plasmodium. Before the study, we carried out an extensive bibliographic search and consulted with U.S. and Colombian experts on virology, parasitology, and entomology about potential pathogens that might be transmitted by Anopheles and could represent a threat for the volunteers exposed to challenge. There was a consensus that as yet no other pathogen had been reported to be transmitted by Anopheles mosquitoes, including those comprised in the blood bank screening. However, mosquitoes were discarded using a biosafety procedure if after feeding blood was confirmed to have any co-infection.