The activity of methylene blue against asexual and sexual stages of Plasmodium vivax.

Methylene blue (MB) is an alternative for combating drug-resistant malaria parasites. Its transmission-blocking potential has been demonstrated in vivo in murine models, in vitro, and in clinical trials. MB shows high efficacy against Plasmodium vivax asexual stages; however, its efficacy in sexual stages is unknown. In this study, we evaluated the potential of MB against asexual and sexual forms of P. vivax isolated from the blood of patients residing in the Brazilian Amazon. An ex vivo schizont maturation assay, zygote to ookinete transformation assay, direct membrane feed assay (DMFA), and standard membrane feed assay (SMFA) using P. vivax gametocytes with MB exposure were performed. A cytotoxicity assay was also performed on freshly collected peripheral blood mononuclear cells (PBMCs) and the hepatocyte carcinoma cell line HepG2. MB inhibited the P. vivax schizont maturation and demonstrated an IC50 lower than that of chloroquine (control drug). In the sexual forms, the MB demonstrated a high level of inhibition in the transformation of the zygotes into ookinetes. In the DMFA, MB did not considerably affect the infection rate and showed low inhibition, but it demonstrated a slight decrease in the infection intensity in all tested concentrations. In contrast, in the SMFA, MB was able to completely block the transmission at the highest concentration (20 µM). MB demonstrated low cytotoxicity to fresh PBMCs but demonstrated higher cytotoxicity to the hepatocyte carcinoma cell line HepG2. These results show that MB may be a potential drug for vivax malaria treatment.

Rosette formation by Plasmodium vivax gametocytes favors the infection in Anopheles aquasalis.

Plasmodium vivax is a public health problem and the most common type of malaria outside sub-Saharan Africa. The capacity of cytoadhesion, rosetting, and liver latent phase development could impact treatment and disease control. Although the ability to P. vivax gametocyte develop rosetting is known, it is not yet clear which role it plays during the infection and transmission process to the mosquito. Here, we used ex vivo approaches for evaluate the rosetting P. vivax gametocytes capacity and we have investigated the effect of this adhesive phenotype on the infection process in the vector Anopheles aquasalis mosquito. Rosette assays were performed in 107 isolates, and we have observed an elevated frequency of cytoadhesive phenomena (77,6%). The isolates with more than 10% of rosettes have presented a higher infection rate in Anopheles aquasalis (p=0.0252). Moreover, we found a positive correlation between the frequency of parasites in rosetting with the infection rate (p=0.0017) and intensity (p=0.0387) in the mosquito. The disruption of P. vivax rosette formation through mechanical rupture assay confirmed the previously findings, since the paired comparison showed that isolates with disrupted rosettes have a lower infection rate (p<0.0001) and intensity (p=0.0003) compared to the control group (no disruption). Herein we have demonstrated for the first time a potential effect of the rosette phenomenon on the infection process in the mosquito vector An. aquasalis, favoring its capacity and intensity of infection, thus allowing the perpetuation of the parasite cycle life.