These observations prompt a modification of the current paradigms of the pathogenesis of malaria and clear the way to investigate the pathophysiology of P. vivax infections.

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BackgroundAlthough thrombocytopenia is a hematological disorder commonly reported in malarial patients, its mechanisms are still poorly understood, with only a few studies focusing on the role of platelets phagocytosis.Methods and FindingsThirty-five malaria vivax patients and eight healthy volunteers (HV) were enrolled in the study. Among vivax malaria patients, thrombocytopenia (<150,000 platelets/µL) was found in 62.9% (22/35). Mean platelet volume (MPV) was higher in thrombocytopenic patients as compared to non- thrombocytopenic patients (p = 0.017) and a negative correlation was found between platelet count and MPV (r = −0.483; p = 0.003). Platelets from HV or patients were labeled with 5-chloromethyl fluorescein diacetate (CMFDA), incubated with human monocytic cell line (THP-1) and platelet phagocytosis index was analyzed by flow cytometry. The phagocytosis index was higher in thrombocytopenic patients compared to non-thrombocytopenic patients (p = 0.042) and HV (p = 0.048). A negative correlation was observed between platelet count and phagocytosis index (r = −0.402; p = 0.016). Platelet activation was assessed measuring the expression of P-selectin (CD62-P) in platelets’ surface by flow cytometry. No significant difference was found in the expression of P-selectin between thrombocytopenic patients and HV (p = 0.092). After evaluating the cytokine profile (IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and IL-17) in the patients’ sera, levels of IL-6, IL-10 and IFN-γ were elevated in malaria patients compared to HV. Moreover, IL-6 and IL-10 values were higher in thrombocytopenic patients than non-thrombocytopenic ones (p = 0.044 and p = 0.017, respectively. In contrast, TNF-α levels were not different between the three groups, but a positive correlation was found between TNF-α and phagocytosis index (r = −0.305; p = 0.037).Conclusion/SignificanceCollectively, our findings indicate that platelet phagocytosis may contribute to thrombocytopenia found in vivax malaria. Finally, we believe that this study opens new avenues to explore the mechanisms involved in platelet dysfunction, commonly found in vivax malaria patients.

Abstractobjective To investigate risk factors associated with the acquisition of antibodies against Plasmodium vivax Duffy binding protein (PvDBP) -a leading malaria vaccine candidate -in a well-consolidated agricultural settlement of the Brazilian Amazon Region and to determine the sequence diversity of the PvDBP ligand domain (DBP II ) within the local malaria parasite population.methods Demographic, epidemiological and clinical data were collected from 541 volunteers using a structured questionnaire. Malaria parasites were detected by conventional microscopy and PCR, and blood collection was used for antibody assays and molecular characterisation of DBP II .results The frequency of malaria infection was 7% (6% for P. vivax and 1% for P. falciparum), with malaria cases clustered near mosquito breeding sites. Nearly 50% of settlers had anti-PvDBP IgG antibodies, as detected by enzyme-linked immunosorbent assay (ELISA) with subject's age being the only strong predictor of seropositivity to PvDBP. Unexpectedly, low levels of DBP II diversity were found within the local malaria parasites, suggesting the existence of low gene flow between P. vivax populations, probably due to the relative isolation of the studied settlement.conclusion The recognition of PvDBP by a significant proportion of the community, associated with low levels of DBP II diversity among local P. vivax, reinforces the variety of malaria transmission patterns in communities from frontier settlements. Such studies should provide baseline information for antimalarial vaccines now in development.

Violacein is a violet pigment extracted from the gram-negative bacterium Chromobacterium violaceum. It presents bactericidal, tumoricidal, trypanocidal, and antileishmanial activities. We show that micromolar concentrations efficiently killed chloroquine-sensitive and -resistant Plasmodium falciparum strains in vitro; inhibited parasitemia in vivo, even after parasite establishment; and protected Plasmodium chabaudi chabaudiinfected mice from a lethal challenge.Violacein is a violet pigment isolated from Chromobacterium violaceum, a gram-negative betaproteobacterium found in the Amazon River in Brazil. It has been reported to kill bacteria (4) and induces apoptosis in various types of cancer cells (1,5,7,8,10,11). Moderate activity against Trypanosoma cruzi and Leishmania amazonensis promastigotes has also been observed (3, 9). Due to the widespread presence of drug resistance in the malaria parasite, resulting in dramatically decreased efficacy of available antimalarial drugs (15), and the fact that immunoprotection achieved by the most successful malaria vaccine is only partial and short-lived (14), we evaluated the in vitro and in vivo effects of violacein on human and murine blood stage forms of Plasmodium parasites.Isolation and purification of violacein, (Fig. 1), from C. violaceum (CCT3496) were performed as previously described (12). Toxicity was measured as the concentration-dependent lysis of normal erythrocytes (NE) by counting red blood cells per milliliter with the aid of a Neubauer chamber. After 48 h of exposure to various concentrations of violacein, the percent red blood cell density (RBCD) relative to that of the control (without violacein) was monitored and calculated according to the formula (RBCD per milliliter in the presence of violacein/RBCD per milliliter without violacein) ϫ 100. As shown in Fig. 2A, a slight reduction in the RBCD percentage at violacein concentrations of Ͼ8.0 M was observed. Significant (Mann-Whitney U test, P Ͻ 0.05) toxicity to NE occurred at a concentration of 14.0 M.Next, we performed dose-response assays to obtain the 50% inhibitory concentrations (IC 50 s) of violacein against erythrocytes infected with chloroquine-sensitive or -resistant strains of P. falciparum (3D7 [16] or S20 [2], respectively) at 1% parasitemia and a 2% final hematocrit. We used [ 3 H]hypoxanthine (Amersham Biosciences, Amersham, United Kingdom) incorporation to assess parasite growth according to a protocol described elsewhere (13). Violacein was tested in triplicate at least three times with different batches and cells, and parasite growth was compared to that in nontreated infected erythrocytes (IE), which represented 100% parasite growth. Percent parasite growth inhibition was calculated according to the formula [1 Ϫ (cpm of treated IE Ϫ cpm of NE/cpm of nontreated IE Ϫ cpm of NE)] ϫ 100. After a 48-h incubation, violacein inhibited parasite development even at the lowest tested concentration of 0.06 M and completely abrogated parasite viability at concentrations of Ͼ1.0 M (Fig. 2B).The ...

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BackgroundPlasmodium vivax is the causative agent of human malaria of large geographic distribution, with 35 million cases annually. In Brazil, it is the most prevalent species, being responsible by around 70 % of the malaria cases.MethodsA cross-sectional study was performed in Manaus (Amazonas, Brazil), including 36 adult patients with primary malaria, 19 with recurrent malaria, and 20 endemic controls. The ex vivo phenotypic features of circulating leukocyte subsets (CD4+ T-cells, CD8+ T-cells, NK, NKT, B, B1 and Treg cells) as well as the plasmatic cytokine profile (IL-2, IL-4, IL-6, IL-10, TNF and IFN-γ) were assessed, aiming at establishing patterns of immune response characteristic of primary malaria vs recurrent malaria as compared to endemic controls.ResultsThe proportion of subjects with high levels of WBC was reduced in malaria patients as compared to the endemic control. Monocytes were diminished particularly in patients with primary malaria. The proportion of subjects with high levels of all lymphocyte subsets was decreased in all malaria groups, regardless their clinical status. Decreased proportion of subjects with high levels of CD4+ and CD8+ T-cells was found especially in the group of patients with recurrent malaria. Data analysis indicated significant increase in the proportion of the subjects with high plasmatic cytokine levels in both malaria groups, characterizing a typical cytokine storm. Recurrent malaria patients displayed the highest plasmatic IL-10 levels, that correlated directly with the CD4+/CD8+ T-cells ratio and the number of malaria episodes.ConclusionThe findings confirm that the infection by the P. vivax causes a decrease in peripheral blood lymphocyte subsets, which is intensified in the cases of “recurrent malaria”. The unbalanced CD4+/CD8+ T-cells ratio, as well as increased IL-10 levels were correlated with the number of recurrent malaria episodes. These results suggest that the gradual remodelling of the immune response is dependent on the repeated exposure to the parasite, which involves a strict control of the immune response mediated by the CD4+/CD8+ T-cell unbalance and exacerbated IL-10 secretion.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1501-5) contains supplementary material, which is available to authorized users.

The Plasmodium vivax Duffy binding protein (PvDBP) and its erythrocytic receptor, the Duffy antigen receptor for chemokines (DARC), are involved in the major P. vivax erythrocyte invasion pathway. An open cohort study to analyze DARC genotypes and their relationship to PvDBP immune responses was carried out in 620 volunteers in an agricultural settlement of the Brazilian Amazon. Three cross-sectional surveys were conducted at 6-month intervals, comprising 395, 410, and 407 subjects, respectively. The incidence rates of P. vivax infection was 2.32 malaria episodes per 100 person-months under survey (95% confidence interval [CI] of 1.92-2.80/100 person-month) and, of P. falciparum, 0.04 per 100 person-months (95% CI of 0.007–0.14/100 person-month). The distribution of DARC genotypes was consistent with the heterogeneous ethnic origins of the Amazon population, with a predominance of non-silent DARC alleles: FY*A > FY*B. The 12-month follow-up study demonstrated no association between DARC genotypes and total IgG antibodies as measured by ELISA targeting PvDBP (region II, DBPII or regions II–IV, DBPII-IV). The naturally acquired DBPII specific binding inhibitory antibodies (BIAbs) tended to be more frequent in heterozygous individuals carrying a DARC-silent allele (FY*BES). These results provide evidence that DARC polymorphisms may influence the naturally acquired inhibitory anti-Duffy binding protein II immunity.