Characterization of exosome-like vesicles from chloroquine-resistant Plasmodium vivax patients

Background: Chloroquine resistance in Plasmodium vivax (CQRPv) has become an obstacle to elimination strategies based on the use of antimalarials. Exosome-like vesicles, which play an important role on intercellular communication in malaria, are not well characterized in P. vivax infection and may promote the transfer of resistance carrying gene expression regulation factors. This study proposes to investigate and characterize exosome-like vesicles in malaria patients with CQRPv. Materials and Methods: Patient recruitment was carried out at Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, in Manaus, Brazilian Amazon. P. vivax resistant and sensitive to CQ samples from clinical trials were included. These patients received treatment with CQ and primaquine accordingly the Brazilian Ministry of Health, with 42-days follow-up. Aliquots of 300µL of plasma collected at admission (D0) and at the recrudescence day (DR) were used to exosome isolation by size exclusion chromatography (SEC). Resulting fractions were labeled with CD5 and CD71 antibodies in a bead based assay and further analyzed by flow cytometry (FC) to confirm exosome presence and to estimate the vesicles amount, using MFI (immunofluorescence median) values. The resulting pools of fractions richer in exosomes were analyzed by mass spectrometry (LTQ Orbitrap Fusion apparatus). Data analysis will search differences in the proteic content of exosomes from CQ sensitive and resistant P. vivax. Results: Exosome characterization by FC was performed from 22 samples, with 4 CQ sensitive, 7 resistant (D0 and DR samples) and 4 negative controls. Exosome quantity was greater in the P. vivax positive samples compared to negative controls, using anti-CD71 as marker. The mean of MFI of CQ resistant samples at D0 (1,739.71 [CI95%: 822.91-2656.52]) was greater when compared to the malaria negative group (335.88 [CI95%: 66.81-604.94]) (p=0.02, unpaired t test). There was no significant difference in exosome quantity between the MFI means of sensitive and resistant samples. There was no correlation between parasitemia and exosomes quantity (p=0.47, linear regression). Conclusions: Exosome characterization by FC showed more quantity of exosomes during P. vivax infection. Proteomic analysis will allow to know the exosomes content and to confirm the carriage of parasite antigens related to CQ resistance.