Abstract. A safe and reproducible Plasmodium vivax infectious challenge method is required to evaluate the efficacy of malaria vaccine candidates. Seventeen healthy Duffy (+) and five Duffy (−) subjects were randomly allocated into three (A-C) groups and were exposed to the bites of 2-4 Anopheles albimanus mosquitoes infected with Plasmodium vivax derived from three donors. Duffy (−) subjects were included as controls for each group. Clinical manifestations of malaria and parasitemia were monitored beginning 7 days post-challenge. All Duffy (+) volunteers developed patent malaria infection within 16 days after challenge. Prepatent period determined by thick smear, was longer for Group A (median 14.5 d) than for Groups B and C (median 10 d/each). Infected volunteers recovered rapidly after treatment with no serious adverse events. The bite of as low as two P. vivax -infected mosquitoes provides safe and reliable infections in malaria-naive volunteers, suitable for assessing antimalarial and vaccine efficacy trials.

Abstract. Vaccine development for Plasmodium vivax malaria is underway. A model to assess the protective efficacy of vaccine candidates in humans is urgently needed. Given the lack of continuous P. vivax cultures, we developed a system to infect Anopheles albimanus mosquitoes using blood from P. vivax-infected patients and determined parameters for challenge of malaria-naive volunteers by mosquito bite. Absence of co-infections in parasitized blood was confirmed by tests consistent with blood bank screening. A total of 119 experiments were conducted using batches of 900-4,500 mosquitoes fed by an artificial membrane feeding method. Optimal conditions for mosquito probing and infection were determined. Presence of oocyst and sporozoites were assessed on Days 7-8 and 14-15, respectively, and conditions to choose batches of infected mosquitoes for sporozoite challenge were established. Procedures to infect volunteers took a 2-hour period including verification of inoculum dose. Anopheles albimanus mosquitoes represent a valuable resource for P. vivax sporozoite challenge of volunteers.

Plasmodium vivax transmission-blocking activity was assessed in sera from acutely infected patients from a malaria-endemic area in Colombia. We measured reduction in the number of oocysts that developed in the midguts of Anopheles albimanus mosquitoes artificially fed with blood from these patients. Of 88 mosquito batches that developed infections when parasites were mixed with normal AB human serum, one-third (36.4%) showed full transmission-blocking activity (>or= 90% inhibition) when mixed with autologous sera, 29.6% showed partial activity (50-89%), 17.0% did not block transmission (0-50%), and 17% did not enhance transmission. Transmission-blocking activity correlated with antibody titer by an immunofluorescent antibody test and decreased with the serial dilution of the sera. This activity disappeared at a 1:4 dilution in most sera tested. Afro-Colombian individuals showed lower activity than other ethnic groups and febrile patients produced stronger inhibition than those without fever.

Artificial membrane feeding (AMF) assays are used to determine malaria transmission-blocking activity in Anopheles. The purpose of this study was to determine the effect of the most widely used anticoagulants, EDTA and heparin, on development of the Plasmodium vivax sporogonic cycle. Blood samples collected from 60 patients carrying P. vivax infections were used to feed An. albimanus using AMF. Seven days after feeding, mosquitoes were dissected to assess mosquito infection. Mosquitoes fed with blood containing EDTA showed a lower mean oocyst number as compared with those fed blood with heparin. However, this effect was minimized upon reduction of EDTA concentrations in the serum. This result may be explained by the fact that microgametocytes require Ca(2+), Mn(2+), and Mg(+2) to activate enzymes important for exflagellation process and for motility of ookinetes. We therefore recommend that heparin be used as the anticoagulant of choice for blood used in AMF assays.