Cultured Plasmodium Falciparum Research Articles

Simple supplementation of serum-free medium produces gametocytes of Plasmodium falciparum that transmit to mosquitoes

BackgroundHuman serum is a major component of Plasmodium falciparum culture medium, and can be replaced with AlbuMAX™ II, a lipid-rich bovine serum albumin, for asexual cultures. However, gametocytes produced without serum are poorly infective to mosquitoes. Serum suffers from high cost, limited availability, and variability in quality.MethodsSeveral commercially-available media supplements were tested for their ability to support parasite growth and production of P. falciparum (3D7) gametocytes in standard RPMI1640 medium containing 0.5% AlbuMAX. The impact on asexual growth and gametocyte production with each supplement was assessed and compared to standard RPMI1640 medium containing 10% human serum, as well as to medium containing 0.5% AlbuMAX alone. The infectivity of gametocytes produced with one supplement to Anopheles gambiae sensu stricto was assessed by standard membrane feeding assay and measuring both prevalence of infection and oocyst intensity.ResultsSupplementation of medium containing 0.5% AlbuMAX with five supplements did not affect asexual growth of P. falciparum, and four of the five supplements supported early gametocyte production. The supplement producing the highest number of gametocytes, ITS-X, was further investigated and was found to support the production of mature gametocytes. Infection prevalence and oocyst intensity did not differ significantly between mosquitoes given a membrane feed containing gametocytes grown in medium with 0.5% AlbuMAX + ITS-X and those grown in medium with 10% human serum. Infection prevalence and oocyst intensity was significantly higher in case of ITS–X supplementation when compared to AlbuMAX alone. Infectious gametocytes were also produced from two field clones using ITS–X supplementation.ConclusionsSerum-free medium supplemented with ITS-X was able to support the growth of gametocytes of P. falciparum that were as infectious to An. gambiae as those grown in medium with 10% serum. This is the first fully serum-free culture system able to produce highly infectious gametocytes, thereby removing the requirement for access to serum for transmission assays.

Breath biomarkers of pediatric malaria: reproducibility and response to antimalarial therapy.

Many insect-borne pathogens appear to manipulate the odors of their hosts in ways that influence vector behaviors. In our prior work, we identified characteristic changes in volatile emissions of cultured Plasmodium falciparum parasites in vitro and during natural human falciparum malaria. In the current study, we prospectively evaluate the reproducibility of these findings in an independent cohort of children in Blantyre, Malawi. We enrolled febrile children under evaluation for malaria and collected breath from children with and without malaria, as well as healthy controls. Using gas-chromatography/mass spectrometry, we characterized breath volatiles associated with malaria. By repeated sampling of children with malaria before and after antimalarial use, we determined how breath profiles respond to treatment. In addition, we investigated the stage-specificity of biomarkers through correlation with asexual and sexual stage parasitemia. Our data provide robust evidence that P. falciparum infection leads to specific, reproducible changes in breath compounds. While no individual compound served as adequate classifier in isolation, selected volatiles together yielded high sensitivity for diagnosis of malaria. Overall, the results of our predictive models suggest the presence of volatile signatures that reproducibly predict malaria infection status and determine response to therapy, even in cases of low parasitemia.

AlbuMAX supplemented media induces the formation of transmission-competent P. falciparum gametocytes

Asexual blood stage culture of Plasmodium falciparum is routinely performed but reproducibly inducing commitment to and maturation of viable gametocytes remains difficult. Culture media can be supplemented with human serum substitutes to induce commitment but these generally only allow for long-term culture of asexual parasites and not transmission-competent gametocytes due to their different lipid composition. Recent insights demonstrated the important roles lipids play in sexual commitment; elaborating on this we exposed ring stage parasites (20–24 hours hpi) for one day to AlbuMAX supplemented media to trigger induction to gametocytogenesis. We observed a significant increase in gametocytes after AlbuMAX induction compared to serum. We also tested the transmission potential of AlbuMAX inducted gametocytes and found a significant higher oocyst intensity compared to serum. We conclude that AlbuMAX supplemented media induces commitment, allows a more stable and predictable production of transmittable gametocytes than serum alone.

Green synthesized silver nanoparticles of Terminalia bellirica leaves extract: synthesis, characterization, in-silico studies, and antimalarial activity

Malaria is a mosquito-borne infectious disease that is caused by the Plasmodium parasite. Most of the available medication are losing their efficacy. Therefore, it is crucial to create fresh leads to combat malaria. Green silver nanoparticles (AgNPs) have recently attracted a lot of attention in biomedical research. As a result, green mediated AgNPs from leaves of Terminalia bellirica, a medicinal plant with purported antimalarial effects, were used in this investigation. Initially, cysteine-rich proteins from Plasmodium species were studied in silico as potential therapeutic targets. With docking scores between −9.93 and −11.25 kcal/mol, four leaf constituents of Terminalia bellirica were identified. The green mediated silver nanoparticles were afterward produced using leaf extract and were further examined using UV-vis spectrophotometer, DLS, Zeta potential, FTIR, XRD, and FESEM. The size of synthesized TBL-AgNPs was validated by the FESEM results; the average size of TBL-AgNPs was around 44.05 nm. The zeta potential study also supported green mediated AgNPs stability. Additionally, Plasmodium falciparum (3D7) cultures were used to assess the antimalarial efficacy, and green mediated AgNPs could effectively inhibit the parasitized red blood cells (pRBCs). In conclusion, this novel class of AgNPs may be used as a potential therapeutic replacement for the treatment of malaria.

Amino acid supplementation confers protection to red blood cells before Plasmodium falciparum bystander stress

AbstractMalaria is a highly oxidative parasitic disease in which anemia is the most common clinical symptom. A major contributor to the malarial anemia pathogenesis is the destruction of bystander, uninfected red blood cells (RBCs). Metabolic fluctuations are known to occur in the plasma of individuals with acute malaria, emphasizing the role of metabolic changes in disease progression and severity. Here, we report conditioned medium from Plasmodium falciparum culture induces oxidative stress in uninfected, catalase-depleted RBCs. As cell-permeable precursors to glutathione, we demonstrate the benefit of pre-exposure to exogenous glutamine, cysteine, and glycine amino acids for RBCs. Importantly, this pretreatment intrinsically prepares RBCs to mitigate oxidative stress.

Rapid and non-invasive detection of malaria parasites using near-infrared spectroscopy and machine learning.

Novel and highly sensitive point-of-care malaria diagnostic and surveillance tools that are rapid and affordable are urgently needed to support malaria control and elimination. We demonstrated the potential of near-infrared spectroscopy (NIRS) technique to detect malaria parasites both, in vitro, using dilutions of infected red blood cells obtained from Plasmodium falciparum cultures and in vivo, in mice infected with P. berghei using blood spotted on slides and non-invasively, by simply scanning various body areas (e.g., feet, groin and ears). The spectra were analysed using machine learning to develop predictive models for infection. Using NIRS spectra of in vitro cultures and machine learning algorithms, we successfully detected low densities (<10-7 parasites/μL) of P. falciparum parasites with a sensitivity of 96% (n = 1041), a specificity of 93% (n = 130) and an accuracy of 96% (n = 1171) and differentiated ring, trophozoite and schizont stages with an accuracy of 98% (n = 820). Furthermore, when the feet of mice infected with P. berghei with parasitaemia ≥3% were scanned non-invasively, the sensitivity and specificity of NIRS were 94% (n = 66) and 86% (n = 342), respectively. These data highlights the potential of NIRS technique as rapid, non-invasive and affordable tool for surveillance of malaria cases. Further work to determine the potential of NIRS to detect malaria in symptomatic and asymptomatic malaria cases in the field is recommended including its capacity to guide current malaria elimination strategies.

Chemoproteomics-based profiling reveals potential antimalarial mechanism of Celastrol by disrupting spermidine and protein synthesis

BackgroundMalaria remains a global health burden, and the emergence and increasing spread of drug resistance to current antimalarials poses a major challenge to malaria control. There is an urgent need to find new drugs or strategies to alleviate this predicament. Celastrol (Cel) is an extensively studied natural bioactive compound that has shown potentially promising antimalarial activity, but its antimalarial mechanism remains largely elusive.MethodsWe first established the Plasmodium berghei ANKA-infected C57BL/6 mouse model and systematically evaluated the antimalarial effects of Cel in conjunction with in vitro culture of Plasmodium falciparum. The potential antimalarial targets of Cel were then identified using a Cel activity probe based on the activity-based protein profiling (ABPP) technology. Subsequently, the antimalarial mechanism was analyzed by integrating with proteomics and transcriptomics. The binding of Cel to the identified key target proteins was verified by a series of biochemical experiments and functional assays.ResultsThe results of the pharmacodynamic assay showed that Cel has favorable antimalarial activity both in vivo and in vitro. The ABPP-based target profiling showed that Cel can bind to a number of proteins in the parasite. Among the 31 identified potential target proteins of Cel, PfSpdsyn and PfEGF1-α were verified to be two critical target proteins, suggesting the role of Cel in interfering with the de novo synthesis of spermidine and proteins of the parasite, thus exerting its antimalarial effects.ConclusionsIn conclusion, this study reports for the first time the potential antimalarial targets and mechanism of action of Cel using the ABPP strategy. Our work not only support the expansion of Cel as a potential antimalarial agent or adjuvant, but also establishes the necessary theoretical basis for the development of potential antimalarial drugs with pentacyclic triterpenoid structures, as represented by Cel.5LH6oqsCtKBdVDis1L_PeKVideo

Cultivation of Asexual Intraerythrocytic Stages of Plasmodium falciparum.

Successfully developed in 1976, the continuous in vitro culture of Plasmodium falciparum has many applications in the field of malaria research. It has become an important experimental model that directly uses a human pathogen responsible for a high prevalence of morbidity and mortality in many parts of the world and is a major source of biological material for immunological, biochemical, molecular, and pharmacological studies. Until present, the basic techniques described by Trager and Jensen and Haynes et al. remain unchanged in many malaria research laboratories. Nonetheless, different factors, including culture media, buffers, serum substitutes and supplements, sources of erythrocytes, and conditions of incubation (especially oxygen concentration), have been modified by different investigators to adapt the original technique in their laboratories or enhance the in vitro growth of the parasites. The possible effects and benefits of these modifications for the continuous cultivation of asexual intraerythrocytic stages of P. falciparum, as well as future challenges in developing a serum-free cultivation system and axenic cultures, are discussed.

Culture and transfection: Two major bottlenecks in understanding Plasmodium vivax biology.

The long term in vitro culture of Plasmodium falciparum was successfully established by Trager and Jensen in 1976; however it largely remains unachieved for P. vivax. The major obstacle associated with Plasmodium vivax in vitro culture is its predilection for invading younger reticulocytes and the complex remodelling of invaded reticulocytes. There are many factors under exploration for this predilection and host-parasite interactions between merozoites and invaded reticulocytes. These include various factors related to parasite, host and environment such as compromised reticulocyte osmotic stability after invasion, abundance of iron in the reticulocytes which makes them favourable for P. vivax growth and propagation and role of a hypoxic environment in P. vivax in vitro growth. P. vivax blood stage transfection represents another major hurdle towards understanding this parasite's complex biology. Efforts in making this parasite amenable for molecular investigation by genetic modification are limited. Newer approaches in sustaining a longer in vitro culture and thereby help advancing transfection technologies in P. vivax are urgently needed that can be explored to understand the unique biology of this parasite.

Uncovering the antimalarial potential of toad venoms through a bioassay-guided fractionation process

Malaria remains to date one of the most devastating parasitic diseases worldwide. The fight against this disease is rendered more difficult by the emergence and spread of drug-resistant strains. The need for new therapeutic candidates is now greater than ever. In this study, we investigated the antiplasmodial potential of toad venoms. The wide array of bioactive compounds present in Bufonidae venoms has allowed researchers to consider many potential therapeutic applications, especially for cancers and infectious diseases. We focused on small molecules, namely bufadienolides, found in the venom of Rhinella marina (L.). The developed bio-guided fractionation process includes a four solvent-system extraction followed by fractionation using flash chromatography. Sub-fractions were obtained through preparative TLC. All samples were characterized using chromatographic and spectrometric techniques and then underwent testing on in vitro Plasmodium falciparum cultures. Two strains were considered: 3D7 (chloroquine-sensitive) and W2 (chloroquine-resistant). This strategy highlighted a promising activity for one compound named resibufogenin. With IC50 values of (29 ± 8) μg/mL and (23 ± 1) μg/mL for 3D7 and W2 respectively, this makes it an interesting candidate for further investigation. A molecular modelling approach proposed a potential binding mode of resibufogenin to Plasmodium falciparum adenine-triphosphate 4 pump as antimalarial drug target.

Phytochemical screening, cytotoxicity assessment and evaluation of in vitro antiplasmodial and in vivo antimalarial activities of Mentha spicata L. methanolic leaf extract

Ethnopharmacological relevanceMalaria causes extensive morbidity and mortality, and the decreasing efficacy of artemisinin and its partner drugs has posed a serious concern. Therefore, it is important to identify new antimalarials, and the natural compounds from plants provide a promising platform. Mentha spicata L. representing the Lamiaceae family has been used in traditional medicine for various diseases including malaria. Aim of the studyThis study was aimed at evaluating the antiplasmodial activity of M. spicata methanolic leaf extract using Plasmodium falciparum (Pf) cultures (Pf3D7 and artemisinin (ART)-resistant PfCam3.IR539T strains) and antimalarial activity using Plasmodium berghei (Pb)-infected mice. Dry leaf powder and methanolic leaf extract were examined for in vivo antimalarial activity and the efficacy of oral versus parenteral administration was compared. Materials and methodsLeaves of M. spicata were collected and extracted using 70% methanol in water (v/v). [3H]-hypoxanthine incorporation assays and Giemsa-stained smears were used to assess the in vitro antiplasmodial activity of M. spicata methanolic extract against Pf3D7 and ART-resistant PfCam3.IR539T strains. Cytotoxicity was evaluated in HeLa and HEK-293T cell lines using MTT assays. Hemolysis assays were performed using red blood cells (RBCs). In vivo antimalarial activities of M. spicata dry leaf powder and methanolic leaf extract were examined in P. berghei-infected mice by Rane's curative test and Peters' 4-day suppressive test. ResultsPhytochemical screening of M. spicata methanolic leaf extract indicated the presence of reducing sugars, phenolic compounds, flavonoids, glycosides, sterols, saponins, alkaloids, coumarins, tannins, carbohydrates, and proteins. In vitro studies carried out using Pf cultures showed that M. spicata methanolic leaf extract had significant antiplasmodial activity against Pf3D7 cultures with a 50% inhibitory concentration (IC50) of 57.99 ± 2.82 μg/ml. The extract was also effective against ART-resistant PfCam3.IR539T strain with an IC50 of 71.23 ± 3.85 μg/ml. The extract did not show significant in vitro cytotoxicity, hemolysis, and in vivo toxicity. In vivo studies performed using Pb-infected mice treated with M. spicata dry leaf powder and methanolic leaf extract showed ∼50% inhibition in parasite growth at 1500 mg/kg and 1000 mg/kg doses, respectively. There was also a significant delay in the mortality of treated mice. Parenteral administration was found to be appropriate for the in vivo treatment. ConclusionsOur in vitro and in vivo findings from Pf and Pb parasites suggested the therapeutic potential of M. spicata leaf extract as an antimalarial. M. spicata leaf extract could also inhibit the growth of ART-resistant Pf strain. Further studies on fractionation and active component analysis of M. spicata leaf extract would be required to identify the bioactive phytochemicals having pharmaceutical and therapeutic values. Such efforts would help us in developing new antimalarials to combat malaria.

Safety, tolerability, and Plasmodium falciparum transmission-reducing activity of monoclonal antibody TB31F: a single-centre, open-label, first-in-human, dose-escalation, phase 1 trial in healthy malaria-naive adults

Malaria elimination requires interruption of the highly efficient transmission of Plasmodium parasites by mosquitoes. TB31F is a humanised monoclonal antibody that binds the gamete surface protein Pfs48/45 and inhibits fertilisation, thereby preventing further parasite development in the mosquito midgut and onward transmission. We aimed to evaluate the safety and efficacy of TB31F in malaria-naive participants. In this open-label, first-in-human, dose-escalation, phase 1 clinical trial, healthy, malaria-naive, adult participants were administered a single intravenous dose of 0·1, 1, 3, or 10 mg/kg TB31F or a subcutaneous dose of 100 mg TB31F, and monitored until day 84 after administration at a single centre in the Netherlands. The primary outcome was the frequency and magnitude of adverse events. Additionally, TB31F serum concentrations were measured by ELISA. Transmission-reducing activity (TRA) of participant sera was assessed by standard membrane feeding assays with Anopheles stephensi mosquitoes and cultured Plasmodium falciparum gametocytes. The trial is registered with Clinicaltrials.gov, NCT04238689. Between Feb 17 and Dec 10, 2020, 25 participants were enrolled and sequentially assigned to each dose (n=5 per group). No serious or severe adverse events occurred. In total, 33 grade 1 and six grade 2 related adverse events occurred in 20 (80%) of 25 participants across all groups. Serum of all participants administered 1 mg/kg, 3 mg/kg, or 10 mg/kg TB31F intravenously had more than 80% TRA for 28 days or more, 56 days or more, and 84 days or more, respectively. The TB31F serum concentration reaching 80% TRA was 2·1 μg/mL (95% CI 1·9-2·3). Extrapolating the duration of TRA from antibody kinetics suggests more than 80% TRA is maintained for 160 days (95% CI 136-193) following a single intravenous 10 mg/kg dose. TB31F is a well tolerated and highly potent monoclonal antibody capable of completely blocking transmission of P falciparum parasites from humans to mosquitoes. In areas of seasonal transmission, a single dose might cover an entire malaria season. PATH's Malaria Vaccine Initiative.

Homeostasis of extracellular ATP in uninfected RBCs from a Plasmodium falciparum culture and derived microparticles

Plasmodium falciparum, a dangerous parasitic agent causing malaria, invades human red blood cells (RBCs), causing hemolysis and microvascular obstruction. These and other pathological processes of malaria patients are due to metabolic and structural changes occurring in uninfected RBCs. In addition, infection activates the production of microparticles (MPs).ATP and byproducts are important extracellular ligands modulating purinergic signaling within the intravascular space.Here, we analyzed the contribution of uninfected RBCs and MPs to the regulation of extracellular ATP (eATP) of RBCs, which depends on the balance between ATP release by specific transporters and eATP hydrolysis by ectonucleotidases.RBCs were cultured with P. falciparum for 24–48 h prior to experiments, from which uninfected RBCs and MPs were purified. On-line luminometry was used to quantify the kinetics of ATP release. Luminometry, colorimetry and radioactive methods were used to assess the rate of eATP hydrolysis by ectonucleotidases. Rates of ATP release and eATP hydrolysis were also evaluated in MPs.Uninfected RBCs challenged by different stimuli displayed a strong and transient activation of ATP release, together with an elevated rate of eATP hydrolysis. MPs contained ATP in their lumen, which was released upon vesicle rupture, and were able to hydrolyze eATP.Results suggest that uninfected RBCs and MPs can act as important determinants of eATP regulation of RBCs during malaria.The comparison of eATP homeostasis in infected RBCs, ui-RBCs, and MPs allowed us to speculate on the impact of P. falciparum infection on intravascular purinergic signaling and the control of the vascular caliber by RBCs.

Presence of Phylloquinone in the Intraerythrocytic Stages of Plasmodium falciparum.

Malaria is one of the most widespread parasitic diseases, especially in Africa, Southeast Asia and South America. One of the greatest problems for control of the disease is the emergence of drug resistance, which leads to a need for the development of new antimalarial compounds. The biosynthesis of isoprenoids has been investigated as part of a strategy to identify new targets to obtain new antimalarial drugs. Several isoprenoid quinones, including menaquinone-4 (MK-4/vitamin K2), α- and γ-tocopherol and ubiquinone (UQ) homologs UQ-8 and UQ-9, were previously detected in in vitro cultures of Plasmodium falciparum in asexual stages. Herein, we described for the first time the presence of phylloquinone (PK/vitamin K1) in P. falciparum and discuss the possible origins of this prenylquinone. While our results in metabolic labeling experiments suggest a biosynthesis of PK prenylation via phytyl pyrophosphate (phytyl-PP) with phytol being phosphorylated, on the other hand, exogenous PK attenuated atovaquone effects on parasitic growth and respiration, showing that this metabolite can be transported from extracellular environment and that the mitochondrial electron transport system (ETS) of P. falciparum is capable to interact with PK. Although the natural role and origin of PK remains elusive, this work highlights the PK importance in plasmodial metabolism and future studies will be important to elucidate in seeking new targets for antimalarial drugs.

Fast detection and quantification of Plasmodium species infected erythrocytes in a non-endemic region by using the Sysmex XN-31 analyzer

BackgroundDue to increased travel from endemic countries, malaria occurs more frequently in non-endemic regions. It is a challenge for diagnostic laboratories in non-endemic countries to provide reliable results, as experience of staff is often limited to only a few cases per year. This study evaluated the diagnostic accuracy of the fully automated Sysmex XN-31 malaria analyzer in a routine diagnostic setting in a non-endemic region was evaluated.MethodsSamples from 112 patients suspected for malaria were examined by the Sysmex XN-31 analyzer to determine the absolute count of malaria-infected red blood cells count (MI-RBC/µL). Microscopic examination of both Quantitative Buffy Coat capillary tubes and thick and thin blood films were used as reference methods. Limits of blank (LoB), detection (LoD) and quantification (LoQ) were investigated using an in vitro Plasmodium falciparum culture. Nine hundred twenty samples of patients with RBC abnormalities were included to determine which RBC abnormalities trigger indeterminate or false positive results.ResultsNo false positive nor false negative results were obtained for the examined patient samples suspected for malaria. For 3% of samples an indeterminate result by the XN-31 was obtained. The Passing-Bablok regression line for diagnostic accuracy of the parasitaemia was y = 39.75 + 0.7892 × showing a positive bias of about 21% when comparing the MI-RBC results to microscopy. The LoB, LoD and LoQ were calculated to be 4.7, 5.9, and 19.0 infected RBC/μL, respectively. From the 920 abnormal RBC samples collected, 4.6% resulted in a false positive MI-RBC result and almost half of the samples produced indeterminate results. These results were related to increases in nucleated red blood cells, reticulocytes and other abnormal RBC morphologies such as sickle cells.ConclusionsBased on the results, the XN-31 is a fast and reliable screening method in the detection and quantification of Plasmodium species in patients However, if an abnormal red blood cell morphology is present, the results of the XN-31 should be interpreted with caution as false positive results can be caused by interfering abnormal erythrocytes.

Biotransformation of artemisinin to a novel derivative via ring rearrangement by Aspergillus niger

Artemisinin is a component part of current frontline medicines for the treatment of malaria. The aim of this study is to make analogues of artemisinin using microbial transformation and evaluate their in vitro antimalarial activity. A panel of microorganisms were screened for biotransformation of artemisinin (1). The biotransformation products were extracted, purified and isolated using silica gel column chromatography and semi-preparative HPLC. Spectroscopic methods including LC-HRMS, GC–MS, FT-IR, 1D and 2D NMR were used to elucidate the structure of the artemisinin metabolites.1H NMR spectroscopy was further used to study the time-course biotransformation. The antiplasmodial activity (IC50) of the biotransformation products of 1 against intraerythrocytic cultures of Plasmodium falciparum were determined using bioluminescence assays. A filamentous fungus Aspergillus niger CICC 2487 was found to possess the best efficiency to convert artemisinin (1) to a novel derivative, 4-methoxy-9,10-dimethyloctahydrofuro-(3,2-i)-isochromen-11(4H)-one (2) via ring rearrangement and further degradation, along with three known derivatives, compound (3), deoxyartemisinin (4) and 3-hydroxy-deoxyartemisinin (5). Kinetic study of the biotransformation of artemisinin indicated the formation of artemisinin G as a key intermediate which could be hydrolyzed and methylated to form the new compound 2. Our study shows that the anti-plasmodial potency of compounds 2, 3, 4 and 5 were ablated compared to 1, which attributed to the loss of the unique peroxide bridge in artemisinin (1). This is the first report of microbial degradation and ring rearrangement of artemisinin with subsequent hydrolysis and methoxylation by A.niger.Key points• Aspergillus niger CICC 2487 was found to be efficient for biotransformation of artemisinin• A novel and unusual artemisinin derivative was isolated and elucidated• The peroxide bridge in artemisinin is crucial for its high antimalarial potency• The pathway of biotransformation involves the formation of artemisinin G as a key intermediate

Tenfold difference in DNA recovery rate: systematic comparison of whole blood vs. dried blood spot sample collection for malaria molecular surveillance

BackgroundMolecular and genomic surveillance is becoming increasingly used to track malaria control and elimination efforts. Blood samples can be collected as whole blood and stored at − 20 °C until DNA extraction, or as dried blood spots (DBS), circumventing the need for a cold chain. Despite the wide use of either method, systematic comparisons of how the method of blood sample preservation affects the limit of detection (LOD) of molecular diagnosis and the proportion of DNA recovered for downstream applications are lacking.MethodsExtractions based on spin columns, magnetic beads, Tween-Chelex, and direct PCR without prior extraction were compared for whole blood and dried blood spots (DBS) using dilution series of Plasmodium falciparum culture samples. Extracted DNA was quantified by qPCR and droplet digital PCR (ddPCR).ResultsDNA recovery was 5- to 10-fold higher for whole blood compared to DBS, resulting in a 2- to 3-fold lower LOD for both extraction methods compared to DBS. For whole blood, a magnetic bead-based method resulted in a DNA recovery rate of 88–98% when extracting from whole blood compared to 17–33% for a spin-column based method. For extractions from DBS, the magnetic bead-based method resulted in 8–20% DNA recovery, while the spin-column based method resulted in only 2% DNA recovery. The Tween-Chelex method was superior to other methods with 15–21% DNA recovery, and even more sensitive than extractions from whole blood samples. The direct PCR method was found to have the lowest LOD overall for both, whole blood and DBS.ConclusionsPronounced differences in LOD and DNA yield need to be considered when comparing prevalence estimates based on molecular methods and when selecting sampling protocols for other molecular surveillance applications.

Preparation, characterization, and evaluation of eosin B-loaded nano-liposomes for growth inhibition of Plasmodium falciparum.

Malaria is a deadly disease in humans caused by the Plasmodium parasite. High prevalence of malaria and resistance of malaria parasite to currently proposed drugs have increased the need to introduce and use new and effective antimalarial agents. In this study, eosin B was used as an effective antimalarial agent, the efficacy of which has already been confirmed by in vitro models. Also, for efficacy and safety improvement of eosin B, liposomal nanocarrier was used because of diversity and adaptability in controlled drug delivery and targeting. Eosin B was trapped inside liposomal nanocarriers by thin layer hydration method and its optimization was performed based on size, polydispersity index, and drug entrapment efficiency. Finally, the eosin B-loaded liposomes were tested on Plasmodium falciparum in culture to evaluate its anti-plasmodial effect. According to the results, the formulation with DSPC:cholesterol 8:1 (molar ratio) and drug concentration of 3 mg/ml was selected as the optimal form. The optimal nano-liposomes showed a size of 163.3 nm, a polydispersity index of 0.250, and an encapsulation efficiency of 69.94%. The process of drug release from nanocarriers was also obtained about 63% at the end of 72 h. Stability studies over 2 months at 25 °C and 4 °C on the optimum sample showed that the samples stored in the refrigerator were more stable in terms of size characteristics, polydispersity index, and drug entrapment efficiency. The results indicate a greater effect of liposomal-formulated eosin B on inhibiting parasite growth compared to the free eosin B.

Single-Cell Sorting of Plasmodium falciparum-Infected Erythrocytes Expressing Particular PfEMP1 Variants.

Cultures of Plasmodium falciparum often contain a heterogeneous parasite population. However, several studies require analysis of single infected erythrocytes (IEs) or a clonal parasite population derived from a single parasite. This protocol describes an efficient method for cloning by using fluorescence-activated cell sorting (FACS). For this, an antibody for a particular IEs surface protein it is added to the cell mixture to separate positive and negative IEs for that marker. After the separation, the viable homogeneous population can be used to grow in culture or for molecular analysis.

Genotyping of Plasmodium falciparum to Assess Clone Composition in Parasite Cultures.

Defining clone composition in Plasmodium falciparum cultures is key to verify that in vitro experiments are performed on the parasite line of interest. Genotyping of the highly polymorphic merozoite surface protein 2 gene(msp2) is a widely established method to define P. falciparum clones. Specific size variants from the two msp2 families (IC and FC27) can be used as "fingerprints" to identify individual clones in parasite mixtures. Size variant genotyping of msp2 using fluorescent nested PCR followed by fragment analysis by capillary electrophoresis (CE) provides accurate information about the presence of one or multiple parasite clones. Here, we describe an adaptation of this approach to assess the integrity and purity of P. falciparum lines kept in in vitro culture. In addition, we describe the use of synthetic mock parasite mixtures with the msp2 sequences from the parasite lines kept in culture that can provide a good estimate of the assay sensitivity, specificity, and reproducibility. We suggest that genotyping of P. falciparum lines should be performed ona regular basis as part ofthe standard procedures of in vitro parasite culture, as a way to secure that the parasite lines of interest are cultivated, and to monitor any cross-contamination and/or recombination events.