Augmented plasma microparticles during acute Plasmodium vivax infection

Abstract

Background: In the last few years, the study of microparticles (MPs)--submicron vesicles released from cells upon activation or apoptosis--has gained growing interest in the field of inflammation and in infectious diseases. Their role in the human malaria parasite Plasmodium vivax remains unexplored. Because acute vivax malaria has been related to pro-inflammatory responses, the main hypothesis investigated in this study was that Plasmodium vivax infection is associated with elevated levels of circulating MPs, which may play a role during acute disease in non-immune patients.

Methods: Plasma MPs were analysed among thirty-seven uncomplicated P. vivax infections from an area of unstable malaria transmission in the Brazilian Amazon. The MP phenotype was analysed by flow cytometry using the classical MP marker, annexin, and fluorochrome-labeled monoclonal antibodies against specific cell surface markers. The frequencies of plasma MPs in P. vivax patients (n=37) were further compared to malaria-unexposed controls (n=15) and ovarian carcinoma patients (n=12), a known MPs-inducing disease non-related to malaria.

Results: The frequencies of plasma circulating MPs were markedly increased in P. vivax patients, as compared to healthy age-matched malaria-unexposed controls. Although platelets, erythrocytes and leukocytes were the main cellular sources of MPs during vivax malaria, platelet derived-MPs (PMPs) increased in a linear fashion with the presence of fever at the time of blood collection (β=0.06, p<0.0001) and length of acute symptoms (β=0.36, p<0.0001). Finally, the results suggest that plasma levels of PMPs diminish as patient experience more episodes of clinical malaria (β=0.07, p<0.003).

Conclusions: Abundant circulating MPs are present during acute P. vivax infection, and platelet derived-MPs may play a role on the acute inflammatory symptoms of malaria vivax.

Figure 1

Identification of plasma MPs. (A) MPs isolated from plasma were gated (R1) based on their forward (FSC) and side (SSC) scatter distribution as compared to 0.7 - 0.9 μm synthetic Nile Red microparticles (grey dots). (B) Events present in R1 were accessed for their annexin V positive staining using PE-conjugated mAbs. Mouse IgG PE-conjugated isotype control mAbs were used to place gates accurately (R2). (C) FITC immunolabeling for the cell markers CD41a (platelets), CD144 (endothelial cells), CD325a (erythrocytes), CD45 (leukocytes) and CD14 (monocytes) was further assessed within annexin V⁺ gated events (R2) and were compared in plasma samples from healthy donors, P. vivax malaria and ovary carcinoma patients (MPs-induced disease). Boxes represent median and interquartile interval, whiskers represent the 10 and 90 percentiles. Means were compared using the Mann-Whitney two-tailed test. A p value < 0.05 was considered significant.

Figure 2

Platelet derived MPs (PMPs) correlate with fever and length of symptoms of acute malaria and may be impacted by cumulative clinical malarial episodes. P. vivax malaria patients were categorized according to their body temperature (°C) (A); prolonged symptoms (B); cumulative malarial episodes (median 5, range 0 - 60) (C); and the time since last malarial episode (D). Boxes represent median and interquartile interval, whiskers represent the 10 and 90 percentiles. Data were transformed into neperian logarithm before applied to linear regression analysis. Positive linear trends were observed between the frequency of PMPs in plasma and presence of fever (above 37.5°C) at the time of blood collection (β = 0.06, p < 0.0001), length of malaria symptoms, estimated here by number of days with acute illness (β = 0.36, p < 0.0001), and previous clinical malaria episodes (β = 0.07, p < 0.003).

Figure 3

Plasma MPs levels decrease after anti-malarial chemotherapy. Levels of platelet (PMPs), erythrocyte (EMPs) and leukocyte derived-MPs (LMPs) were quantified in plasmas from five patients at the acute phase of P. vivax infection (0) and after 21 days of anti-malarial chemotherapy. MP levels were calculated and expressed as number/μl of plasma as detailed in Methods. Statistical analyses were performed using the two-tailed paired t-test. A p value < 0.05 was considered significant.