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.

Transmission-blocking compound candidates against Plasmodium vivax using P. berghei as an initial screening.

BACKGROUND: Different strategies for improvement of malaria control and elimination are based on the blockage of malaria parasite transmission to the mosquito vector. These strategies include the drugs that target the plasmodial sexual stages in humans and the early developmental stages inside mosquitoes. OBJECTIVES: Here we tested Malaria Box compounds in order to evaluate their activity against male and female gametocytes in Plasmodium berghei, mosquito infection in P. vivax and ookinete formation in both species. METHODS/FINDINGS: The membrane feeding assay and the development of ookinetes by a 24 h ex vivo culture and the ookinete yield per 1000 erythrocytes were used to test transmission-blocking potential of the Malaria Box compounds in P. vivax. For P. berghei we used flow cytometry to evaluate male and female gametocyte time course and fluorescence microscopy to check the ookinete development. The two species used in this study showed similar results concerning the compounds' activity against gametocytes and ookinetes, which were different from those in P. falciparum. In addition, from the eight Malaria Box compounds tested in both species, compounds MMV665830, MMV665878 and MMV665941 were selected as a hit compounds due the high inhibition observed. CONCLUSION: Our results showed that P. berghei is suitable as an initial screening system to test compounds against P. vivax.

Transmission-blocking compound candidates against Plasmodium vivax using P. berghei as an initial screening

BACKGROUND Different strategies for improvement of malaria control and elimination are based on the blockage of malaria parasite transmission to the mosquito vector. These strategies include the drugs that target the plasmodial sexual stages in humans and the early developmental stages inside mosquitoes. OBJECTIVES Here we tested Malaria Box compounds in order to evaluate their activity against male and female gametocytes in Plasmodium berghei, mosquito infection in P. vivax and ookinete formation in both species. METHODS/FINDINGS The membrane feeding assay and the development of ookinetes by a 24 h ex vivo culture and the ookinete yield per 1000 erythrocytes were used to test transmission-blocking potential of the Malaria Box compounds in P. vivax. For P. berghei we used flow cytometry to evaluate male and female gametocyte time course and fluorescence microscopy to check the ookinete development. The two species used in this study showed similar results concerning the compounds' activity against gametocytes and ookinetes, which were different from those in P. falciparum. In addition, from the eight Malaria Box compounds tested in both species, compounds MMV665830, MMV665878 and MMV665941 were selected as a hit compounds due the high inhibition observed. CONCLUSION Our results showed that P. berghei is suitable as an initial screening system to test compounds against P. vivax.

Cardiovascular changes in patients with non-severe Plasmodium vivax malaria.

BACKGROUND: Cardiovascular system involvement in patients with Plasmodium vivax malaria has been poorly addressed. The aim of this study was to evaluate cardiac structures and function, and serum markers of cardiovascular injury in patients with the non-severe form of vivax malaria in Manaus, Amazonas State, Brazil. METHODS AND RESULTS: We prospectively evaluated 26 patients with vivax malaria in an outpatient referral hospital and compared results with a control group of 25 gender- and age-matched healthy individuals. Patients underwent clinical evaluation, laboratory tests, and transthoracic echocardiography at first evaluation (day zero, D0) and seven days (D7) after malaria diagnosis. At D0 echocardiography showed higher left ventricular (LV) systolic diameter (28.8 ± 2.82 vs 30.9 ± 4.03 mm; p = 0.037) and LV diastolic volume (82.4 ± 12.3 vs 93.8 ± 25.9 ml; p = 0.05), and lower LV ejection fraction (Teicholz method: 73.2 ± 6.59 vs 68.4 ± 4.87%; p = 0.004) in patients compared to controls. Right ventricle (RV) fractional area change (54.7 ± 5.11 vs 50.5 ± 6.71%; p = 0.014) was lower, and RV myocardial performance index (0.21 ± 0.07 vs 0.33 ± 0.19; p = 0.007), and pulmonary vascular resistance (1.13 ± 0.25 vs 1.32 ± 0.26 Woods unit; p = 0.012) were higher in patients than controls. Patients presented higher serum levels of unconjugated bilirubin (0.24 ± 0.15 vs 1.30 ± 0.89 mg/dL; p < 0.001), soluble vascular cell adhesion molecule-1 (sVCAM-1; 453 ± 143 vs 1983 ± 880 ng/mL; p < 0.001), N-terminal prohormone brain natriuretic peptide (0.59 ± 0.86 vs 1.08 ± 0.81 pg/mL; p = 0.045), and troponin T (861 ± 338 vs 1037 ± 264 pg/mL; p = 0.045), and lower levels of plasma nitrite (13.42 ± 8.15 vs 8.98 ± 3.97 µM; p = 0.016) than controls. Most alterations had reversed by D7. CONCLUSION: Patients with non-severe Plasmodium vivax malaria present subclinical reversible cardiovascular changes.

Influence of Plasmodium vivax malaria on the relations between the osmotic stability of human erythrocyte membrane and hematological and biochemical variables.

This study evaluated the influence of infection by Plasmodium vivax on the relations between hematological and biochemical variables and the osmotic stability of the erythrocyte membrane in a Brazilian Amazon population. A total of 72 patients with P. vivax malaria were included in the study and invited to return after 14 days, post-treatment with chloroquine and primaquine, for clinical and laboratorial reevaluations. The osmotic stability of the erythrocyte membrane was analyzed by nonlinear regression of the dependency of the absorbance of hemoglobin, released with hemolysis, as a function of the salt concentration, and it was represented by the inverse of the salt concentration at the midpoint of the curve (1/H 50) and by the variation of salt concentration, which promotes lysis (dX). Bivariate and multivariate methods were used in the analysis of the results. Prior to treatment of the disease, the erythrocytes showed greater stability, probably due to the natural selection of young and also more stable erythrocytes. The bivariate analysis showed that 1/H 50 was positively correlated with red cell distribution width (RDW), urea, triglycerides, and very low-density lipoprotein (VLDL)-cholesterol, but negatively associated with albumin, HDL-cholesterol, and indirect bilirubin, while dX was negatively associated with the mean corpuscular hemoglobin concentration. These associations were confirmed by canonical correlation analysis. Stepwise multiple linear regression showed that albumin, urea, triglycerides, and VLDL-cholesterol are the variables with the highest abilities of predicting erythrocyte stability. The bivariate analysis also showed that the hematological index RDW was related to elevated levels of bilirubin and decreased levels of albumin and urea, associated with liver damage resulting from malaria.

Lipid peroxidation and antioxidant enzymes activity in Plasmodium vivax malaria patients evolving with cholestatic jaundice.

BACKGROUND: Plasmodium vivax infection has been considered a benign and self-limiting disease, however, recent studies highlight the association between vivax malaria and life-threatening manifestations. Increase in reactive oxygen species has already been described in vivax malaria, as a result of the increased metabolic rate triggered by the multiplying parasite, and large quantities of toxic redox-active byproducts generated. The present study aimed to study the oxidative stress responses in patients infected with P. vivax, who developed jaundice (hyperbilirubinaemia) in the course of the disease, a common clinical complication related to this species. METHODS: An evaluation of the lipid peroxidation and antioxidant enzymes profile was performed in 28 healthy individuals and compared with P. vivax infected patients with jaundice, i.e., bilirubin < 51.3 µmol/L (8 patients) or without jaundice (34 patients), on day 1 (D1) and day 14 (D14) after anti-malarial therapy. RESULTS: Hyperbilirubinaemia was more frequent among women and patients experiencing their first malarial infection, and lower haemoglobin and higher lactate dehydrogenase levels were observed in this group. Malondialdehyde levels and activity of celuroplasmin and glutathione reductase were increased in the plasma from patients with P. vivax with jaundice compared to the control group on D1. However, the activity of thioredoxin reductase was decreased. The enzymes glutathione reductase, thioredoxin reductase, thiols and malondialdehyde also differed between jaundiced versus non-jaundiced patients. On D14 jaundice and parasitaemia had resolved and oxidative stress biomarkers were very similar to the control group. CONCLUSION: Cholestatic hyperbilirubinaemia in vivax malaria cannot be totally disassociated from malaria-related haemolysis. However, significant increase of lipid peroxidation markers and changes in antioxidant enzymes in patients with P. vivax-related jaundice was observed. These results suggest oxidative processes contributing to malaria pathogenesis, what may be useful information for future anti-oxidant therapeutical interventions in these patients.