Microwaves can kill malaria parasites non-thermally.

Malaria, which infected more than 240 million people and killed around six hundred thousand only in 2021, has reclaimed territory after the SARS-CoV-2 pandemic. Together with parasite resistance and a not-yet-optimal vaccine, the need for new approaches has become critical. While earlier, limited, studies have suggested that malaria parasites are affected by electromagnetic energy, the outcomes of this affectation vary and there has not been a study that looks into the mechanism of action behind these responses. In this study, through development and implementation of custom applicators for in vitro experimentation, conditions were generated in which microwave energy (MW) killed more than 90% of the parasites, not by a thermal effect but via a MW energy-induced programmed cell death that does not seem to affect mammalian cell lines. Transmission electron microscopy points to the involvement of the haemozoin-containing food vacuole, which becomes destroyed; while several other experimental approaches demonstrate the involvement of calcium signaling pathways in the resulting effects of exposure to MW. Furthermore, parasites were protected from the effects of MW by calcium channel blockers calmodulin and phosphoinositol. The findings presented here offer a molecular insight into the elusive interactions of oscillating electromagnetic fields with P. falciparum, prove that they are not related to temperature, and present an alternative technology to combat this devastating disease.

Treatment of Plasmodium falciparum merozoites with the protease inhibitor E64 and mechanical filtration increases their susceptibility to complement activation.

Plasmodium falciparum malaria killed 451,000 people in 2017. Merozoites, the stage of the parasite that invades RBCs, are a logical target for vaccine development. Treatment with the protease inhibitor E64 followed by filtration through a 1.2 µm filter is being used to purify merozoites for immunologic assays. However, there have been no studies to determine the effect of these treatments on the susceptibility of merozoites to complement or antibodies. To address this gap, we purified merozoites with or without E64 followed by filtration through either a 1.2 or 2.7 µm filter, or no filtration. Merozoites were then incubated in either 10% fresh or heat-inactivated serum followed by surface staining and flow cytometry with monoclonal antibodies against the complement effector molecules C3b or C5b9. To determine the effect of anti-merozoite antibodies, we incubated merozoites with MAb5.2, a mouse monoclonal antibody that targets the merozoite surface protein 1. We used an amine-reactive fluorescent dye to measure membrane integrity. Treatment with E64 resulted in an insignificant increase in the proportion of merozoites that were C3b positive but in a significant increase in the proportion that were C5b9 positive. Filtration increased the proportion of merozoites that were either C3b or C5b9-positive. The combination of filtration and E64 treatment resulted in marked deposition of C3b and C5b9. MAb5.2 induced greater complement deposition than serum alone or an IgG2b isotype control. The combination of E64 treatment, filtration, and MAb5.2 resulted in very rapid and significant deposition of C5b9. Filtration through the 1.2 µm filter selected a population of merozoites with greater membrane integrity, but their integrity deteriorated rapidly upon exposure to serum. We conclude that E64 treatment and filtration increase the susceptibility of merozoites to complement and antibody. Filtered or E64-treated merozoites are not suitable for immunologic studies that address the efficacy of antibodies in vitro.

Plasmodium falciparum strains spontaneously switch invasion phenotype in suspension culture.

The extensive redundancy in the use of invasion ligands by Plasmodium falciparum, and its unique ability to switch between invasion pathways have hampered vaccine development. P. falciparum strains Dd2 and W2mef have been shown to change from sialic acid (SA)-dependent to SA-independent phenotypes when selected on neuraminidase-treated erythrocytes. Following an observation of increasing ability of Dd2 to invade neuraminidase-treated cells when cultured for several weeks, we systematically investigated this phenomenon by comparing invasion phenotypes of Dd2, W2mef and 3D7 strains of P. falciparum that were cultured with gentle shaking (Suspended) or under static (Static) conditions. While Static Dd2 and W2mef remained SA-dependent for the entire duration of the investigation, Suspended parasites spontaneously and progressively switched to SA-independent phenotype from week 2 onwards. Furthermore, returning Suspended cultures to Static conditions led to a gradual reversal to SA-dependent phenotype. The switch to SA-independent phenotype was accompanied by upregulation of the key invasion ligand, reticulocyte-binding homologue 4 (RH4), and the increased invasion was inhibited by antibodies to the RH4 receptor, CR1. Our data demonstrates a novel mechanism for inducing the switching of invasion pathways in P. falciparum parasites and may provide clues for understanding the mechanisms involved.

A Murine Model to Study Epilepsy and SUDEP Induced by Malaria Infection.

One of the largest single sources of epilepsy in the world is produced as a neurological sequela in survivors of cerebral malaria. Nevertheless, the pathophysiological mechanisms of such epileptogenesis remain unknown and no adjunctive therapy during cerebral malaria has been shown to reduce the rate of subsequent epilepsy. There is no existing animal model of postmalarial epilepsy. In this technical report we demonstrate the first such animal models. These models were created from multiple mouse and parasite strain combinations, so that the epilepsy observed retained universality with respect to genetic background. We also discovered spontaneous sudden unexpected death in epilepsy (SUDEP) in two of our strain combinations. These models offer a platform to enable new preclinical research into mechanisms and prevention of epilepsy and SUDEP.

Blood Stage Plasmodium falciparum Exhibits Biological Responses to Direct Current Electric Fields.

The development of resistance to insecticides by the vector of malaria and the increasingly faster appearance of resistance to antimalarial drugs by the parasite can dangerously hamper efforts to control and eradicate the disease. Alternative ways to treat this disease are urgently needed. Here we evaluate the in vitro effect of direct current (DC) capacitive coupling electrical stimulation on the biology and viability of Plasmodium falciparum. We designed a system that exposes infected erythrocytes to different capacitively coupled electric fields in order to evaluate their effect on P. falciparum. The effect on growth of the parasite, replication of DNA, mitochondrial membrane potential and level of reactive oxygen species after exposure to electric fields demonstrate that the parasite is biologically able to respond to stimuli from DC electric fields involving calcium signaling pathways.

Complement and Antibody-mediated Enhancement of Red Blood Cell Invasion and Growth of Malaria Parasites.

Plasmodium falciparum malaria is a deadly pathogen. The invasion of red blood cells (RBCs) by merozoites is a target for vaccine development. Although anti-merozoite antibodies can block invasion in vitro, there is no efficacy in vivo. To explain this discrepancy we hypothesized that complement activation could enhance RBC invasion by binding to the complement receptor 1 (CR1). Here we show that a monoclonal antibody directed against the merozoite and human polyclonal IgG from merozoite vaccine recipients enhanced RBC invasion in a complement-dependent manner and that soluble CR1 inhibited this enhancement. Sialic acid-independent strains, that presumably are able to bind to CR1 via a native ligand, showed less complement-dependent enhancement of RBC invasion than sialic acid-dependent strains that do not utilize native CR1 ligands. Confocal fluorescent microscopy revealed that complement-dependent invasion resulted in aggregation of CR1 at the RBC surface in contact with the merozoite. Finally, total anti-P. berghei IgG enhanced parasite growth and C3 deficiency decreased parasite growth in mice. These results demonstrate, contrary to current views, that complement activation in conjunction with antibodies can paradoxically aid parasites invade RBCs and should be considered in future design and testing of merozoite vaccines.

The HFE genotype and a formulated diet controlling for iron status attenuate experimental cerebral malaria in mice.

Plasmodium falciparum infects approximately 500million individuals each year. A small but significant number of infections lead to complications such as cerebral malaria. Cerebral malaria is associated with myelin damage and neurological deficits in survivors, and iron status is thought to impact the outcome of infection. We evaluated whether a mouse model of experimental cerebral malaria with Plasmodium berghei ANKA strain was altered by dietary iron deficiency or genetic iron overload (H67D HFE). We found that H67D mice had increased survival over H67H (wild type) mice. Moreover, a specifically designed formulation diet increased survival regardless of whether the diet was iron deficient or iron adequate. To determine potential mechanisms underlying demyelination in experimental cerebral malaria, we measured Semaphorin4A (Sema4A) protein levels in the brain because we found it is cytotoxic to oligodendrocytes. Sema4A was increased in wild type mice that developed experimental cerebral malaria while consuming standard rodent chow, consistent with a decrease in myelin basic protein, an indicator of myelin integrity. The brains of iron deficient and H67D mice had lower levels of Sema4A. Myelin basic protein was decreased in brains of mice fed the iron deficient diet as has been previously reported. We also examined erythropoietin, which is under consideration for treatment of cerebral malaria, and IL-6, which is known to increase during infection. We found that plasma erythropoietin was elevated and IL-6 was low in H67D mice and in the mice fed the formulation diets. These data reveal a paradigm-shifting concept that maintaining iron status may not increase the mortality associated with malaria and provide a dietary strategy for further examination. Moreover, the data provide clues for exploring the mechanism to limit the co-morbidity associated with experimental cerebral malaria that appears to include decreased Sema4A in brain as well as elevated erythropoietin and lower IL-6 in plasma.

Analysis of Erythrocyte Invasion Mechanisms of Plasmodium falciparum Clinical Isolates Across 3 Malaria-Endemic Areas in Ghana.

BACKGROUND: Plasmodium falciparum invades human erythrocytes by using an array of ligands that interact with several receptors, including sialic acid (SA), complement receptor 1 (CR1), and basigin. We hypothesized that in malaria-endemic areas, parasites vary invasion pathways under immune pressure. Therefore, invasion mechanisms of clinical isolates collected from 3 zones of Ghana with different levels of endemicity (from lowest to highest, Accra, Navrongo, and Kintampo) were compared using standardized methods. METHODS: Blood samples were collected from children aged 2-14 years in whom malaria was diagnosed, and erythrocyte invasion phenotypes were determined using the enzymes neuraminidase, chymotrypsin, and trypsin, which differentially cleave receptors from the erythrocyte surface. In addition, antibodies against CR1 and basigin were used to determine the contributions of these receptors to invasion. Gene expression levels of P. falciparum invasion ligands were also examined. RESULTS: The parasites generally expressed SA-independent invasion phenotypes across the malaria-endemic areas, with parasites from Kintampo showing the highest invasion rates in neuraminidase-treated erythrocytes. CR1 was a major mediator of SA-independent invasion, while basigin was essential for both SA-dependent and SA-independent invasion mechanisms. Furthermore, expression of the basigin ligand PfRh5 was the best predictor of donor parasitemia. CONCLUSIONS: Erythrocyte invasion phenotypes expressed by P. falciparum are influenced by endemicity levels, and the PfRh5-basigin pathway is a potential vaccine target.

Use of mosquito preventive measures is associated with increased RBC CR1 levels in a malaria holoendemic area of western Kenya.

Malaria is responsible for close to 1 million deaths each year, mostly among African children. Red blood cells (RBCs) of children with severe malarial anemia show loss of complement regulatory proteins such as complement receptor 1 (CR1). We carried out this study to identify socio-economic, environmental, and biological factors associated with the loss of RBC CR1. A cross-sectional study was conducted in a malaria holoendemic area of western Kenya. Twelve socioeconomic, environmental, and biological factors were examined for a relationship with RBC CR1 level using bivariate linear regression followed by creation of a multivariate linear regression model. A significant positive relationship between RBC CR1 level and use of mosquito countermeasures was found. However, there was no evidence of a significant relationship between RBC CR1 level and malaria infection or parasitemia level. Reducing mosquito exposure may aid in the prevention of severe malarial anemia by reducing the number of infections and thus preserving RBC CR1.

Facile synthesis of antimalarial 1,2-disubstituted 4-quinolones from 1,3-bisaryl-monothio-1,3-diketones.

A new strategy was developed to synthesize 1,2-disubstituted 4-quinolones in good yield starting from 1,3-bisaryl-monothio-1,3-diketone substrates. The synthesized compounds were evaluated for antimalarial activity using Plasmodium falciparum strains. All compounds, except for two, showed good activity. Of these, seven compounds exhibited an excellent antimalarial activity (IC50, <2 µM). More importantly, all seven compounds were equally effective in inhibiting the growth of both chloroquine-sensitive and chloroquine-resistant strains. The cytotoxicity assessment using carcinoma and non-carcinoma human cell lines revealed that almost all synthesized compounds were minimally cytotoxic (IC50, >50 µM).

Complement activation in malaria: friend or foe?

Complement is activated during malaria infection, but there is little evidence that it benefits the host. On the contrary, growing evidence points to the central role of complement activation in the pathogenesis of complicated malaria. Recent evidence suggests a critical role for C5a and the membrane attack complex in the pathogenesis of cerebral malaria, and for C5a in the pathogenesis of placental malaria. In addition, erythrocytes of children with severe malarial anemia have increased deposition of C3b and decreased capacity to regulate complement activation, that probably increase their susceptibility to destruction by liver and splenic macrophages. These observations justify further investigation of the role of complement in malaria and the testing of complement inhibitors as adjunctive treatment for severe malaria.

Use of telemedicine to diagnose tinea in Kenyan schoolchildren.

OBJECTIVE: Internet-based telemedicine has the potential to alleviate the problem of limited access to healthcare in developing countries. The Mashavu project aims to deploy kiosks that transmit health data and pictures from patients in underdeveloped countries who have no immediate access to healthcare to clinics for analysis by trained personnel. To test this principle, we investigated whether dermatophytic fungal infections (tinea) could be diagnosed by Kenyan clinicians solely from pictures of the lesions. SUBJECTS AND METHODS: Six physicians, five physician assistants, and five nurses from Nyeri Provincial Hospital took a test consisting of 15 pictures of potassium hydroxide (KOH) prep-confirmed tinea lesions and 15 pictures of KOH prep-negative lesions obtained from local children. RESULTS: The mean (standard deviation) sensitivity and specificity for the whole group were 73% (19%) and 83% (11%), respectively. The physicians had the highest sensitivity and specificity, although only sensitivity reached statistical significance when compared with physician assistants. CONCLUSIONS: These results suggest that telemedicine can be used to diagnose simple skin conditions in a low resource setting with reasonable sensitivity and specificity.

Sequential Plasmodium chabaudi and Plasmodium berghei infections provide a novel model of severe malarial anemia.

Lack of an adequate animal model of Plasmodium falciparum severe malarial anemia (SMA) has hampered the understanding of this highly lethal condition. We developed a model of SMA by infecting C57BL/6 mice with P. chabaudi followed after recovery by P. berghei infection. P. chabaudi/P. berghei-infected mice had an initial 9- to 10-day phase of relatively low parasitemia and severe anemia, followed by a second phase of hyperparasitemia, more profound anemia, reticulocytosis, and death 14 to 21 days after infection. P. chabaudi/P. berghei-infected animals had more intense splenic hematopoiesis, higher interleukin-10 (IL-10)/tumor necrosis factor alpha and IL-12/gamma interferon (IFN-γ) ratios, and higher antibody levels against P. berghei and P. chabaudi antigens than P. berghei-infected or P. chabaudi-recovered animals. Early treatment with chloroquine or artesunate did not prevent the anemia, suggesting that the bulk of red cell destruction was not due to the parasite. Red cells from P. chabaudi/P. berghei-infected animals had increased surface IgG and C3 by flow cytometry. However, C3(-/-) mice still developed anemia. Tracking of red cells labeled ex vivo and in vivo and analysis of frozen tissue sections by immunofluorescence microscopy showed that red cells from P. chabaudi/P. berghei-infected animals were removed at an accelerated rate in the liver by erythrophagocytosis. This model is practical and reproducible, and its similarities with P. falciparum SMA in humans makes it an appealing system with which to study the pathogenesis of this condition and explore potential immunomodulatory interventions.

A case of congenital plasmodium vivax malaria from a temperate region in Central China.

In February 2011, a rare case of congenital Plasmodium vivax malaria was diagnosed in a temperate region of Central China. An infant developed intermittent fever 20 days after delivery. Since this occurred during the non-transmission winter season in a low malaria endemic region and the infant's mother did not have a clear malaria history or showed malaria symptoms at the time of the delivery, malaria infection was not suspected at the beginning. Later, on suspicion of potential malignant haematological illness due to persistence of the fever, bone marrow smear was examined, which revealed infection by P. vivax parasite. This rare case of congenital vivax malaria underlines that malaria diagnosis might need to be included in the healthcare of neonates born in vivax-endemic areas.

Complement receptor 1 and malaria.

Plasmodium falciparum malaria is an intracellular parasite that is transmitted by Anopheles mosquitoes. It is responsible for approximately 1 million deaths per year. Most deaths occur as a result of complications such as severe anaemia or cerebral malaria (coma). The complement receptor 1 is a key complement regulator found on the surface of red cells and most leucocytes. A growing body of evidence suggests that this molecule plays a critical role in the pathogenesis of P. falciparum malaria. Initial reports showed that CR1 enables the binding of infected red cells to uninfected red cells to form rosettes, which can potentially obstruct small capillaries. However, further evidence suggests that CR1 is also important in the control of complement activation and immune complex formation during malaria infection. Most recently, CR1 has also been shown to be a receptor for the invasion of red cells by the parasite. Its polymorphic nature almost certainly has allowed the selection of variants that have helped humankind survive the scurge of malaria. The finding of conflicting reports about the exact role of these variants in severe disease underlies the complexity of the parasite-host interactions and highlights the need for further studies.

Plasmodium falciparum field isolates use complement receptor 1 (CR1) as a receptor for invasion of erythrocytes.

A majority of Plasmodium falciparum strains invade erythrocytes through interactions with sialic acid (SA) on glycophorins. However, we recently reported that complement receptor 1 (CR1) is a SA-independent invasion receptor of many laboratory strains of P. falciparum. To determine the role of CR1 in erythrocyte invasion among P. falciparum field isolates, we tested eight isolates obtained from children in Kenya. All the parasites examined were capable of invading in a SA-independent manner, and invasion of neuraminidase-treated erythrocytes was nearly completely blocked by anti-CR1 and soluble CR1 (sCR1). In addition, anti-CR1 and sCR1 partially inhibited invasion of intact erythrocytes in a majority of isolates tested. Sequencing of the hypervariable region of P. falciparum AMA-1 showed considerable diversity among all the isolates. These data demonstrate that CR1 mediates SA-independent erythrocyte invasion in P. falciparum field isolates.

Complement receptor 1 is a sialic acid-independent erythrocyte receptor of Plasmodium falciparum.

Plasmodium falciparum is a highly lethal malaria parasite of humans. A major portion of its life cycle is dedicated to invading and multiplying inside erythrocytes. The molecular mechanisms of erythrocyte invasion are incompletely understood. P. falciparum depends heavily on sialic acid present on glycophorins to invade erythrocytes. However, a significant proportion of laboratory and field isolates are also able to invade erythrocytes in a sialic acid-independent manner. The identity of the erythrocyte sialic acid-independent receptor has been a mystery for decades. We report here that the complement receptor 1 (CR1) is a sialic acid-independent receptor for the invasion of erythrocytes by P. falciparum. We show that soluble CR1 (sCR1) as well as polyclonal and monoclonal antibodies against CR1 inhibit sialic acid-independent invasion in a variety of laboratory strains and wild isolates, and that merozoites interact directly with CR1 on the erythrocyte surface and with sCR1-coated microspheres. Also, the invasion of neuraminidase-treated erythrocytes correlates with the level of CR1 expression. Finally, both sialic acid-independent and dependent strains invade CR1 transgenic mouse erythrocytes preferentially over wild-type erythrocytes but invasion by the latter is more sensitive to neuraminidase. These results suggest that both sialic acid-dependent and independent strains interact with CR1 in the normal red cell during the invasion process. However, only sialic acid-independent strains can do so without the presence of glycophorin sialic acid. Our results close a longstanding and important gap in the understanding of the mechanism of erythrocyte invasion by P. falciparum that will eventually make possible the development of an effective blood stage vaccine.

The levels of CD16/Fc gamma receptor IIIA on CD14+ CD16+ monocytes are higher in children with severe Plasmodium falciparum anemia than in children with cerebral or uncomplicated malaria.

Fc gamma receptor IIIA (CD16/Fc gamma RIIIA) on monocytes/macrophages may play an important role in the pathogenesis of severe malarial anemia (SMA) by promoting phagocytosis of IgG-coated uninfected red cells and by allowing the production of tumor necrosis factor alpha (TNF-alpha) upon cross-linking by immune complexes (ICs). However, not much is known about the differential expression of this receptor on monocytes of children with severe malaria and uncomplicated malaria. Therefore, we investigated the expression of CD16/Fc gamma RIIIA on monocytes of children with SMA, cerebral malaria (CM), and their age-matched uncomplicated malaria controls by flow cytometry. Since CD14 low (CD14(+)) monocytes are considered more mature and macrophage-like than CD14 high (CD14(++)) monocytes, we also compared the level of expression of CD16/Fc gamma RIIIA according to the CD14 level and studied the relationship between CD16/Fc gamma RIIIA expression and intracellular TNF-alpha production upon stimulation by ICs. CD16/Fc gamma RIIIA expression was the highest overall on CD14(+) CD16(+) monocytes of children with SMA at enrollment. At convalescence, SMA children were the only ones to show a significant decline in the same parameter. In contrast, there were no significant differences among groups in the expression of CD16/Fc gamma RIIIA on CD14(++) CD16(+) monocytes. A greater percentage of CD14(+) CD16(+) monocytes produced TNF-alpha upon stimulation than any other monocyte subset, and the amount of intracellular TNF-alpha correlated positively with CD16/Fc gamma RIIIA expression. Furthermore, there was an inverse correlation between hemoglobin levels and CD16/Fc gamma RIIIA expression in children with SMA and their controls. These data suggest that monocytes of children with SMA respond differently to Plasmodium falciparum infection by overexpressing CD16/Fc gamma RIIIA as they mature, which could enhance erythrophagocytosis and TNF-alpha production.