Maturation of the malarial phosphatidylserine decarboxylase is mediated by high affinity binding to anionic phospholipids.

Decarboxylation of phosphatidylserine (PS) to form phosphatidylethanolamine by PS decarboxylases (PSDs) is an essential process in most eukaryotes. Processing of a malarial PSD proenzyme into its active alpha and beta subunits is by an autoendoproteolytic mechanism regulated by anionic phospholipids, with PS serving as an activator and phosphatidylglycerol (PG), phosphatidylinositol, and phosphatidic acid acting as inhibitors. The biophysical mechanism underlying this regulation remains unknown. We used solid phase lipid binding, liposome-binding assays, and surface plasmon resonance to examine the binding specificity of a processing-deficient Plasmodium PSD (PkPSDS308A) mutant enzyme and demonstrated that the PSD proenzyme binds strongly to PS and PG but not to phosphatidylethanolamine and phosphatidylcholine. The equilibrium dissociation constants (Kd) of PkPSD with PS and PG were 80.4 nM and 66.4 nM, respectively. The interaction of PSD with PS is inhibited by calcium, suggesting that the binding mechanism involves ionic interactions. In vitro processing of WT PkPSD proenzyme was also inhibited by calcium, consistent with the conclusion that PS binding to PkPSD through ionic interactions is required for the proenzyme processing. Peptide mapping identified polybasic amino acid motifs in the proenzyme responsible for binding to PS. Altogether, the data demonstrate that malarial PSD maturation is regulated through a strong physical association between PkPSD proenzyme and anionic lipids. Inhibition of the specific interaction between the proenzyme and the lipids can provide a novel mechanism to disrupt PSD enzyme activity, which has been suggested as a target for antimicrobials, and anticancer therapies.

A simple polystyrene microfluidic device for sensitive and accurate SERS-based detection of infection by malaria parasites.

Early and accurate detection of infection by pathogenic microorganisms, such as Plasmodium, the causative agent of malaria, is critical for clinical diagnosis and ultimately determines the patient's outcome. We have combined a polystyrene-based microfluidic device with an immunoassay which utilises Surface-Enhanced Raman Spectroscopy (SERS) to detect malaria. The method can be easily translated to a point-of-care testing format and shows excellent sensitivity and specificity, when compared to the gold standard for laboratorial detection of Plasmodium infections. The device can be fabricated in less than 30 min by direct patterning on shrinkable polystyrene sheets of adaptable three-dimensional microfluidic chips. To validate the microfluidic system, samples of P. falciparum-infected red blood cell cultures were used. The SERS-based immunoassay enabled the detection of 0.0012 ± 0.0001% parasitaemia in a P. falciparum-infected red blood cell culture supernatant, an ∼7-fold higher sensitivity than that attained by most rapid diagnostic tests. Our approach successfully overcomes the main challenges of the current Plasmodium detection methods, including increased reproducibility, sensitivity, and specificity. Furthermore, our system can be easily adapted for detection of other pathogens and has excellent properties for early diagnosis of infectious diseases, a decisive step towards lowering their high burden on healthcare systems worldwide.

Choline and PEG dually modified artemether nano delivery system targeting intra-erythrocytic Plasmodium and its pharmacodynamics in vivo.

OBJECTIVE: The choline derivative (CD) and polyethylene-glycol (PEG) dually modified artemether (ARM) nanostructured lipid carriers (CD-PEG-ARM-NLC) have been designed to prolong the circulation of ARM in blood, as well as to develop targeting for new permeability pathways (NPPs) and erythrocyte choline carriers (ECCs) that are expressed on the Plasmodium-infected erythrocyte membrane. SIGNIFICANCE: The CD-PEG-ARM-NLC constructed in this study was found to be able to target endoerythrocytic Plasmodium by increasing the drug concentration and residence time in the infected erythrocytic microenvironment and minimizing toxicity and side effects. METHODS: CD-PEG-ARM-NLC was prepared using high-pressure homogenization followed by physicochemical characterization. The targeting ability of CD-PEG-NLC to infected erythrocytes probed by coumarin-6 was investigated by using fluorescence microscopy imaging. The SYBR Green I assay for parasite nucleic acid was adapted in order to assess the efficacy of inhibition against parasite growth in vitro. The antimalarial activity of ARM-loaded NLCs was evaluated by a Pearson four-day suppressive test in Pyy265BY-bearing mice. RESULTS: In vitro imaging indicated that the intracellular delivery of CD-PEG-ARM-NLC was efficiently taken up by the infected erythrocytes via ECCs and NPPs, which could be inhibited by addition of furosemide (an inhibitor of NPPs) and excessive choline (native substrate of ECCs). Moreover, in vitro and in vivo studies that evaluated antimalarial activity suggested that CD-PEG-ARM-NLC exhibited higher antimalarial activity in comparison to ARM-NLC and PEG-ARM-NLC. CONCLUSION: These findings suggested that choline and PEG dually modified NLC could be promising preparations for the production of hydrophobic antimalarial drugs, particularly for ARM.

Tailored environments to study motile cells and pathogens.

Motile cells and pathogens migrate in complex environments and yet are mostly studied on simple 2D substrates. In order to mimic the diverse environments of motile cells, a set of assays including substrates of defined elasticity, microfluidics, micropatterns, organotypic cultures, and 3D gels have been developed. We briefly introduce these and then focus on the use of micropatterned pillar arrays, which help to bridge the gap between 2D and 3D. These structures are made from polydimethylsiloxane, a moldable plastic, and their use has revealed new insights into mechanoperception in Caenorhabditis elegans, gliding motility of Plasmodium, swimming of trypanosomes, and nuclear stability in cancer cells. These studies contributed to our understanding of how the environment influences the respective cell and inform on how the cells adapt to their natural surroundings on a cellular and molecular level.

Development of an Ultrasensitive Impedimetric Immunosensor Platform for Detection of Plasmodium Lactate Dehydrogenase.

Elimination of malaria is a global health priority. Detecting an asymptomatic carrier of Plasmodium parasites to receive treatment is an important step in achieving this goal. Current available tools for detection of malaria parasites are either expensive, lacking in sensitivity for asymptomatic carriers, or low in throughput. We investigated the sensitivity of an impedimetric biosensor targeting the malaria biomarker Plasmodium lactate dehydrogenase (pLDH). Following optimization of the detection protocol, sensor performance was tested using phosphate-buffered saline (PBS), and then saliva samples spiked with pLDH at various concentrations. The presence of pLDH was determined by analyzing the sensor electrical properties before and after sample application. Through comparing percentage changes in impedance magnitude, the sensors distinguished pLDH-spiked PBS from non-spiked PBS at concentrations as low as 250 pg/mL (p = 0.0008). Percentage changes in impedance magnitude from saliva spiked with 2.5 ng/mL pLDH trended higher than those from non-spiked saliva. These results suggest that these biosensors have the potential to detect concentrations of pLDH up to two logs lower than currently available best-practice diagnostic tools. Successful optimization of this sensor platform would enable more efficient diagnosis of asymptomatic carriers, who can be targeted for treatment, contributing to the elimination of malaria.

Determinants of Gammaherpesvirus Shedding in Saliva Among Ugandan Children and Their Mothers.

Background: Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV) are transmitted via saliva, but factors associated with salivary shedding are unknown. Methods: We measured the DNA load of both viruses in saliva specimens collected from approximately 500 Ugandan mothers and their 6-year-old children, testing all participants for EBV and KSHV-seropositive individuals for KSHV. Results: EBV and KSHV were shed by 72% and 22% of mothers, respectively, and by 85% and 40% of children, respectively; boys were more likely than girls to shed KSHV (48% vs 30%) but were equally likely to shed EBV. Children shed more KSHV and EBV than mothers, but salivary loads of EBV and KSHV were similar. KSHV shedding increased with increasing anti-KSHV (K8.1) antibodies in mothers and with decreasing antimalarial antibodies both in mothers and children. Among mothers, 40% of KSHV shedders also shed EBV, compared with 75% of KSHV nonshedders; among children, EBV was shed by 65% and 83%, respectively. Conclusions: In summary, in this population, individuals were more likely to shed EBV than KSHV in saliva. We identified several factors, including child's sex, that influence KSHV shedding, and we detected an inverse relationship between EBV and KSHV shedding, suggesting a direct or indirect interaction between the two viruses.

Nanomedicines for Malaria Chemotherapy: Encapsulation vs. Polymer Therapeutics.

Malaria is one of the oldest infectious diseases that afflict humans and its history extends back for millennia. It was once prevalent throughout the globe but today it is mainly endemic to tropical regions like sub-Saharan Africa and South-east Asia. Ironically, treatment for malaria has existed for centuries yet it still exerts an enormous death toll. This contradiction is attributed in part to the rapid development of resistance by the malaria parasite to chemotherapeutic drugs. In turn, resistance has been fuelled by poor patient compliance to the relatively toxic antimalarial drugs. While drug toxicity and poor pharmacological potentials have been addressed or ameliorated with various nanomedicine drug delivery systems in diseases like cancer, no clinically significant success story has been reported for malaria. There have been several reviews on the application of nanomedicine technologies, especially drug encapsulation, to malaria treatment. Here we extend the scope of the collation of the nanomedicine research literature to polymer therapeutics technology. We first discuss the history of the disease and how a flurry of scientific breakthroughs in the latter part of the nineteenth century provided scientific understanding of the disease. This is followed by a review of the disease biology and the major antimalarial chemotherapy. The achievements of nanomedicine in cancer and other infectious diseases are discussed to draw parallels with malaria. A review of the current state of the research into malaria nanomedicines, both encapsulation and polymer therapeutics polymer-drug conjugation technologies, is covered and we conclude with a consideration of the opportunities and challenges offered by both technologies.

A paper and plastic device for the combined isothermal amplification and lateral flow detection of Plasmodium DNA.

BACKGROUND: Isothermal amplification techniques are emerging as a promising method for malaria diagnosis since they are capable of detecting extremely low concentrations of parasite target while mitigating the need for infrastructure and training required by other nucleic acid based tests. Recombinase polymerase amplification (RPA) is promising for further development since it operates in a short time frame (<30 min) and produces a product that can be visually detected on a lateral flow dipstick. A self-sealing paper and plastic system that performs both the amplification and detection of a malaria DNA sequence is presented. METHODS: Primers were designed using the NCBI nBLAST tools and screened using gel electrophoresis. Paper and plastic devices were prototyped using commercial design software and parts were cut using a laser cutter and assembled by hand. Synthetic copies of the Plasmodium 18S gene were spiked into solution and used as targets for the RPA reaction. To test the performance of the device the same samples spiked with synthetic target were run in parallel both in the paper and plastic devices and using conventional bench top methods. RESULTS: Novel RPA primers were developed that bind to sequences present in the four species of Plasmodium which infect humans. The paper and plastic devices were found to be capable of detecting as few as 5 copies/µL of synthetic Plasmodium DNA (50 copies total), comparable to the same reaction run on the bench top. The devices produce visual results in an hour, cost approximately $1, and are self-contained once the device is sealed. CONCLUSIONS: The device was capable of carrying out the RPA reaction and detecting meaningful amounts of synthetic Plasmodium DNA in a self-sealing and self-contained device. This device may be a step towards making nucleic acid tests more accessible for malaria detection.

Human biting activity, spatial-temporal distribution and malaria vector role of Anopheles calderoni in the southwest of Colombia.

BACKGROUND: Anopheles calderoni was first recognized in Colombia in 2010 as this species had been misidentified as Anopheles punctimacula due to morphological similarities. An. calderoni is considered a malaria vector in Peru and has been found naturally infected with Plasmodium falciparum in Colombia. However, its biting behaviour, population dynamics and epidemiological importance have not been well described for Colombia. METHODS: To assess the contribution of An. calderoni to malaria transmission and its human biting behaviour and spatial/temporal distribution in the southwest of Colombia, human landing catches (HLC) and larval collections were carried out in a cross-sectional, entomological study in 22 localities between 2011 and 2012, and a longitudinal study was performed in the Boca de Prieta locality in Olaya Herrera municipality between July 2012 and June 2013. All mosquitoes determined as An. calderoni were tested by ELISA to establish infection with Plasmodium spp. RESULTS: Larvae of An. calderoni were found in four localities in 12 out of 244 breeding sites inspected. An. calderoni adults were collected in 14 out of 22 localities during the cross-sectional study and represented 41.3% (459 of 1,111) of the collected adult specimens. Other species found were Anopheles albimanus (54.7%), Anopheles apicimacula (2.1%), Anopheles neivai (1.7%), and Anopheles argyritarsis (0.2%). In the localities that reported the highest malaria Annual Parasite Index (>10/1,000 inhabitants) during the year of sampling, An. calderoni was the predominant species (>90% of the specimens collected). In the longitudinal study, 1,528 An. calderoni were collected by HLC with highest biting rates in February, May and June 2013, periods of high precipitation. In general, the species showed a preference to bite outdoors (p < 0.001). In Boca de Prieta, two specimens of An. calderoni were ELISA positive for Plasmodium circumsporozoite protein: one for P. falciparum and one for Plasmodium vivax VK-210. This represents an overall sporozoite rate of 0.1% and an annual entomological inoculation rate of 2.84 infective bites/human/year. CONCLUSIONS: This study shows that An. calderoni is a primary malaria vector in the southwest of Colombia. Its observed preference for outdoor biting is a major challenge for malaria control.

Evaluation of Phosphatidylserine-Specific Peptide-Conjugated Liposomes Using a Model System of Malaria-Infected Erythrocytes.

Malaria is one of the most prevalent parasitic diseases and is most widespread in tropical regions. The malarial parasite grows and reproduces in erythrocytes during its life cycle, resulting in programmed erythrocyte death, termed eryptosis. Lipid scrambling, which occurs following the exposure of anionic lipids such as phosphatidylserine (PS) on the outer surface of erythrocytes, is a characteristic physical change that occurs early during eryptosis. Here, we prepared "PS specific peptide (PSP)"-conjugated liposomes (PSP-liposomes) and investigated whether PSP-liposomes hold promise as a novel strategy for actively targeting eryptosis. Eryptosis was induced by exposing red blood cells (RBCs) to ionomycin, a known calcium ionophore. When PSP liposomes were mixed with either RBCs or RBCs undergoing eryptosis (E-RBCs), the amount of PSP-liposome bound to E-RBCs was much higher than the amount bound to RBCs. However, the amount of PSP-liposome bound to E-RBCs was significantly inhibited by the presence of annexin V protein, which binds specifically to PS. These results suggest that PSP-liposomes could be an effective drug nanocarrier for treating E-RBCs and malaria-infected erythrocytes.

Mammalian and malaria parasite cyclase-associated proteins catalyze nucleotide exchange on G-actin through a conserved mechanism.

Cyclase-associated proteins (CAPs) are among the most highly conserved regulators of actin dynamics, being present in organisms from mammals to apicomplexan parasites. Yeast, plant, and mammalian CAPs are large multidomain proteins, which catalyze nucleotide exchange on actin monomers from ADP to ATP and recycle actin monomers from actin-depolymerizing factor (ADF)/cofilin for new rounds of filament assembly. However, the mechanism by which CAPs promote nucleotide exchange is not known. Furthermore, how apicomplexan CAPs, which lack many domains present in yeast and mammalian CAPs, contribute to actin dynamics is not understood. We show that, like yeast Srv2/CAP, mouse CAP1 interacts with ADF/cofilin and ADP-G-actin through its N-terminal α-helical and C-terminal ß-strand domains, respectively. However, in the variation to yeast Srv2/CAP, mouse CAP1 has two adjacent profilin-binding sites, and it interacts with ATP-actin monomers with high affinity through its WH2 domain. Importantly, we revealed that the C-terminal ß-sheet domain of mouse CAP1 is essential and sufficient for catalyzing nucleotide exchange on actin monomers, although the adjacent WH2 domain is not required for this function. Supporting these data, we show that the malaria parasite Plasmodium falciparum CAP, which is entirely composed of the ß-sheet domain, efficiently promotes nucleotide exchange on actin monomers. Collectively, this study provides evidence that catalyzing nucleotide exchange on actin monomers via the ß-sheet domain is the most highly conserved function of CAPs from mammals to apicomplexan parasites. Other functions, including interactions with profilin and ADF/cofilin, evolved in more complex organisms to adjust the specific role of CAPs in actin dynamics.

Plasmodium gametocyte carriage in humans and sporozoite rate in anopheline mosquitoes in Gondar zuria district, Northwest Ethiopia.

Although the overall burden of malaria is decreasing in Ethiopia, a recent report of an unpredictable increased incidence may be related to the presence of community-wide gametocyte-carrier individuals and a high proportion of infected vectors. This study aimed to reveal the current prevalence of gametocyte-carriage and the sporozoite infectivity rate of Anopheles vectors for Plasmodium parasites. A community-based cross-sectional study was conducted from May 01 to June 30/2019. A total of 53 households were selected using systematic random sampling and a 242 study participants were recruited. Additionally,515 adult female Anopheles mosquitoes were collected using Center for Diseases Control and Prevention (CDC) light traps and mouth aspirators. Parasite gametocytemia was determined using giemsa stain microscopy, while sporozoite infection was determined by giemsa staining microscopy and enzyme linked immunosorbent assay (ELISA). Among the total 242 study participants, 5.4% (95%, CI = 2.9-8.3) of them were positive for any of the Plasmodium species gametocyte. Furthermore, being female [AOR = 15.5(95%, CI = 1.71-140.39)], age group between 15-29 years old [AOR = 16.914 (95%, CI = 1.781-160.63)], no ITNs utilization [AOR = 16.7(95%, CI = 1.902 -146.727)], and high asexual parasite density [(95%, CI = 0.057-0.176, P = 0.001, F = 18.402)] were identified as statistically significant factors for gametocyte carriage. Whereas sporozoite infection rate was 11.6% (95%, CI = 8.2-15.5) and 12.7% (95%, CI = 9.6-16.3) by microscopy and ELISA, respectively. Overall, this study indicated that malaria remains to be an important public health problem in Gondar Zuria district where high gametocyte carriage rate and sporozoite infection rate could sustain its transmission and burden. Therefore, in Ethiopia, where malaria elimination program is underway, frequent, and active community-based surveillance of gametocytemia and sporozoite infection rate is important.

A colorimetric aptasensor for the diagnosis of malaria based on cationic polymers and gold nanoparticles.

Malaria, a major burden of disease caused by parasites of the genus Plasmodium, is widely spread in tropical and subtropical regions. Here, we have successfully developed a diagnostic technique for malaria. The proposed method is based on the interaction among the Plasmodium lactate dehydrogenase (pLDH), which is a biomarker for malaria, and pL1 aptamer against Plasmodium vivax lactate dehydrogenase (PvLDH) and Plasmodium falciparum lactate dehydrogenase (PfLDH). In addition, the cationic polymers, poly(diallyldimethylammonium chloride) (PDDA) and poly(allylamine hydrochloride) (PAH), aggregate gold nanoparticles (AuNPs) that should be possible to observe the change in color from red to blue, which depends on the concentration of pLDH. Using this aptasensor, pLDH proteins were successfully detected with low detection limits. Moreover, the specificity test proved that the aptasenor is very specific in targeting proteins over other interfering proteins. In addition, the pLDH from infected blood samples of the two main species of malaria were also detected. The limits of detection for P. vivax were determined as 80 parasites/µl for PDDA and 74 parasites/µl for PAH. The aptasenor has great advantages that can simply and rapidly diagnose malaria. Thus, the developed aptasensor for detection of pLDH can offer an effective and sensitive diagnosis of malaria.

Hemocyte differentiation to the megacyte lineage enhances mosquito immunity against Plasmodium.

Activation of Toll signaling in Anopheles gambiae by silencing Cactus, a suppressor of this pathway, enhances local release of hemocyte-derived microvesicles (HdMv), promoting activation of the mosquito complement-like system, which eliminates Plasmodium ookinetes. We uncovered the mechanism of this immune enhancement. Cactus silencing triggers a Rel1-mediated differentiation of granulocytes to the megacyte lineage, a new subpopulation of giant cells, resulting in a dramatic increase in the proportion of circulating megacytes. Megacytes are very plastic cells that are massively recruited to the basal midgut surface in response to Plasmodium infection. We show that Toll signaling modulates hemocyte differentiation and that megacyte recruitment to the midgut greatly enhances mosquito immunity against Plasmodium.

Malaria causes hundreds of thousands of deaths each year. This devastating disease is caused by Plasmodium parasites, which are transmitted to people through female Anopheles gambiae mosquitos. Mosquitos become infected with Plasmodium when they ingest blood containing these malaria-causing parasites. However, Plasmodium must avoid the mosquito immune system to survive and spread. The mosquito immune system is made up of several types of immune cells, including cells known as granulocytes. Granulocytes can further develop into additional cell subtypes, such as megacytes and antimicrobial granulocytes, but it is not clear how these types of cells work to protect mosquitos against infections. In the mosquitos that transmit malaria, a cell signaling pathway called Toll helps control immune responses to disease-causing microbes, such as Plasmodium. When Toll signaling is strongly triggered in mosquitos, Plasmodium infection is eliminated because immune cell responses are enhanced ­ which results in lower levels of transmission to humans. But what is the underlying mechanism through which high levels of Toll signaling eradicate Plasmodium infection? To find out, Barletta et al. collected cell samples from A. gambiae mosquitos and analyzed what happened when Toll signaling was strongly activated. They observed a large increase in the proportion of megacytes in these mosquitos (from 2% to 80% of all granulocytes). Toll signaling also caused megacytes to become bigger, cluster together, and have higher plasticity ­ meaning they could adopt different shapes. Barletta et al. used microscopy to show that these megacytes were releasing large mitochondria-like structures and membrane vesicles , which may be the trigger activating the mosquito's immune system. In live mosquitos, megacytes move towards the area of the Plasmodium infection and release microvesicles. These microvesicles are known to activate a part of the the mosquito's immune system called the complement-like system, destroying the parasites and preventing mosquito infection and disease transmission. These findings show how strong Toll signaling triggers the mosquito immune system to eliminate Plasmodium infections. Understanding how the mosquito immune system tackles Plasmodium infection may help reveal ways to reduce or block transmission.

Investigation of liposomal self-adjuvanting peptide epitopes derived from conserved blood-stage Plasmodium antigens.

Malaria is a vector born parasitic disease causing millions of deaths every year. Despite the high mortality rate, an effective vaccine against this mosquito-borne infectious disease is yet to be developed. Up to date, RTS,S/AS01 is the only vaccine available for malaria prevention; however, its efficacy is low. Among a variety of malaria antigens, merozoite surface protein-1(MSP-1) and ring-infected erythrocyte surface antigen (RESA) have been proposed as promising candidates for malaria vaccine development. We developed peptide-based Plasmodium falciparum vaccine candidates that incorporated three previously reported conserved epitopes from MSP-1 and RESA into highly effective liposomal polyleucine delivery system. Indeed, MSP-1 and RESA-derived epitopes conjugated to polyleucine and formulated into liposomes induced higher epitope specific antibody titres. However, immunized mice failed to demonstrate protection in a rodent malaria challenge study with Plasmodium yoelii. In addition, we found that the three reported P. falciparum epitopes did not to share conformational properties and high sequence similarity with P. yoelii MSP-1 and RESA proteins, despite the epitopes were reported to protect mice against P. yoelii challenge.

"Wonders unconceived": reflections on the birth of medical entomology.

Prior to Patrick Manson's discovery in 1877 that the mosquito Culex fatigans was the intermediate host of filariasis, the association of insects with disease and the nature of disease transmission was almost entirely speculation. Manson's work was incomplete, however, because it showed the manner in which the mosquito acquired the infection from humans, but failed to show the way in which the mosquito passed the infection to humans. That pathogens were transmitted by the bite of an infected female mosquito was later proven experimentally with bird malaria by Manson's protégé, Ronald Ross. In 1898 Ross demonstrated that the infective stage of the malarial parasite was injected into the host when the mosquito released saliva into the wound prior to injesting blood. Insects were suspected as carriers of disease for centuries, yet it was not until the late 1870s that the uncritical acceptance of folk beliefs was supplanted by research-based scientific medicine. Why did it take so long? The answer lies in the fact that early medicine itself was imprecise and could not have pursued the subject with any hope of useful results until the last quarter of the 19th century. A better understanding of the nature of the disease process (germ theory of disease) and improved technology (microscopes and oil-immersion lenses with greater resolving power, and synthetic tissue stains) were indispensable for revealing the nexus between those partners in crime: insects and parasites.

Extracellular vesicles derived from Plasmodium-infected and non-infected red blood cells as targeted drug delivery vehicles.

Among several factors behind drug resistance evolution in malaria is the challenge of administering overall doses that are not toxic for the patient but that, locally, are sufficiently high to rapidly kill the parasites. Thus, a crucial antimalarial strategy is the development of drug delivery systems capable of targeting antimalarial compounds to Plasmodium with high specificity. In the present study, extracellular vesicles (EVs) have been evaluated as a drug delivery system for the treatment of malaria. EVs derived from naive red blood cells (RBCs) and from Plasmodium falciparum-infected RBCs (pRBCs) were isolated by ultrafiltration followed by size exclusion chromatography. Lipidomic characterization showed that there were no significant qualitative differences between the lipidomic profiles of pRBC-derived EVs (pRBC-EVs) and RBC-derived EVs (RBC-EVs). Both EVs were taken up by RBCs and pRBCs, although pRBC-EVs were more efficiently internalized than RBC-EVs, which suggested their potential use as drug delivery vehicles for these cells. When loaded into pRBC-EVs, the antimalarial drugs atovaquone and tafenoquine inhibited in vitro P. falciparum growth more efficiently than their free drug counterparts, indicating that pRBC-EVs can potentially increase the efficacy of several small hydrophobic drugs used for the treatment of malaria.

K13, the Cytostome, and Artemisinin Resistance.

Artemisinins - the frontline antimalarial drug class - are compromised by emerging resistance, putting at risk the lives of hundreds of thousands of people each year. Resistance is associated with mutations in a malaria parasite protein, called Kelch 13 (K13). Recent work suggests that K13 is located at the cytostome (cell mouth) that the parasite uses to take up hemoglobin. Here we explore the proposal that K13 mutations confer artemisinin resistance by dampening hemoglobin endocytosis. This model suggests that the resultant decrease in hemoglobin-derived heme reduces artemisinin activation, which is sufficient to enable parasite survival in the early ring stage of infection. A fuller understanding of the resistance mechanism will underpin efforts to develop alternative antimalarial strategies.

Promising nanomaterials in the fight against malaria.

For more than one hundred years, several treatments against malaria have been proposed but they have systematically failed, mainly due to the occurrence of drug resistance in part resulting from the exposure of the parasite to low drug doses. Several factors are behind this problem, including (i) the formidable barrier imposed by the Plasmodium life cycle with intracellular localization of parasites in hepatocytes and red blood cells, (ii) the adverse fluidic conditions encountered in the blood circulation that affect the interaction of molecular components with target cells, and (iii) the unfavorable physicochemical characteristics of most antimalarial drugs, which have an amphiphilic character and can be widely distributed into body tissues after administration and rapidly metabolized in the liver. To surpass these drawbacks, rather than focusing all efforts on discovering new drugs whose efficacy is quickly decreased by the parasite's evolution of resistance, the development of effective drug delivery carriers is a promising strategy. Nanomaterials have been investigated for their capacity to effectively deliver antimalarial drugs at local doses sufficiently high to kill the parasites and avoid drug resistance evolution, while maintaining a low overall dose to prevent undesirable toxic side effects. In recent years, several nanostructured systems such as liposomes, polymeric nanoparticles or dendrimers have been shown to be capable of improving the efficacy of antimalarial therapies. In this respect, nanomaterials are a promising drug delivery vehicle and can be used in therapeutic strategies designed to fight the parasite both in humans and in the mosquito vector of the disease. The chemical analyses of these nanomaterials are essential for the proposal and development of effective anti-malaria therapies. This review is intended to analyze the application of nanomaterials to improve the drug efficacy on different stages of the malaria parasites in both the human and mosquito hosts.

Specific DNA probe for the diagnosis of Plasmodium falciparum malaria.

Malaria can be diagnosed either by direct microscopic examination of blood smears, which is time consuming and requires expertise, or by immunological techniques, which are effective but do not distinguish between past and present infections. In this study, a simple procedure was developed for spotting lysed blood from infected patients directly onto nitrocellulose paper and identifying the malaria species on the basis of hybridization of parasite DNA with a species-specific probe. A genomic DNA library of Plasmodium falciparum was screened to detect clones containing DNA sequences that are highly repeated within the parasite genome. Several such clones were further analyzed to identify those that hybridize specifically with P. falciparum DNA but not with DNA from humans, P. vivax, or P. cynomolgi. This technique appears to be sensitive enough to detect 10 picograms of purified P. falciparum DNA (equivalent to 100 parasites) and in field studies is able to detect approximately 40 parasites per microliter of blood.