Label-free microfluidic enrichment of ring-stage Plasmodium falciparum-infected red blood cells using non-inertial hydrodynamic lift

<b>Background</b> Understanding of malaria pathogenesis caused by Plasmodium falciparum has been greatly deepened since the introduction of in vitro culture system, but the lack of a method to enrich ring-stage parasites remains a technical challenge. Here, a novel way to enrich red blood cells containing parasites in the early ring stage is described and demonstrated.<p></p> <b>Methods</b> A simple, straight polydimethylsiloxane microchannel connected to two syringe pumps for sample injection and two height reservoirs for sample collection is used to enrich red blood cells containing parasites in the early ring stage (8-10 h p.i.). The separation is based on the non-inertial hydrodynamic lift effect, a repulsive cell-wall interaction that enables continuous and label-free separation with deformability as intrinsic marker.<p></p> <b>Results</b> The possibility to enrich red blood cells containing P. falciparum parasites at ring stage with a throughput of ~12,000 cells per hour and an average enrichment factor of 4.3 ± 0.5 is demonstrated.<p></p> <b>Conclusion</b> The method allows for the enrichment of red blood cells early after the invasion by P. falciparumparasites continuously and without any need to label the cells. The approach promises new possibilities to increase the sensitivity of downstream analyses like genomic- or diagnostic tests. The device can be produced as a cheap, disposable chip with mass production technologies and works without expensive peripheral equipment. This makes the approach interesting for the development of new devices for field use in resource poor settings and environments, e.g. with the aim to increase the sensitivity of microscope malaria diagnosis.<p></p&gt

ARAM: an automated image analysis software to determine rosetting parameters and parasitaemia in Plasmodium samples

Additional file 3: Figure S3. Bland-Altman diagrams. Left Comparison of the cell detection by ARAM and an operator. Right Comparison of the determined rosette size by ARAM and an operator

var gene transcription and PfEMP1 expression in the rosetting and cytoadhesive Plasmodium falciparum clone FCR3S1.2

<p>Abstract</p> <p>Background</p> <p>The pathogenicity of <it>Plasmodium falciparum </it>is in part due to the ability of the parasitized red blood cell (pRBC) to adhere to intra-vascular host cell receptors and serum-proteins. Binding of the pRBC is mediated by <it>Plasmodium falciparum </it>erythrocyte membrane protein 1 (PfEMP1), a large multi-variant molecule encoded by a family of ≈60 <it>var </it>genes.</p> <p>Methods</p> <p>The study of <it>var </it>gene transcription in the parasite clone FCR3S1.2 was performed by semi-quantitative PCR and quantitative PCR (qPCR). The expression of the major PfEMP1 in FCR3S1.2 pRBC was analysed with polyclonal sera in rosette disruption assays and immunofluorecence.</p> <p>Results</p> <p>Transcripts from <it>var</it>1 (FCR3S1.2_<it>var</it>₁; IT4<it>var</it>21) and other <it>var </it>genes were detected by semi-quantitative PCR but results from qPCR showed that one <it>var </it>gene transcript dominated over the others (FCR3S1.2_<it>var</it>₂; IT4<it>var</it>60). Antibodies raised in rats to the recombinant NTS-DBL1α of <it>var</it>2 produced in <it>E. coli </it>completely and dose-dependently disrupted rosettes (≈95% at a dilution of 1/5). The sera reacted with the Maurer's clefts in trophozoite stages (IFA) and to the infected erythrocyte surface (FACS) indicating that FCR3S1.2_{<it>var2 </it>}encodes the dominant PfEMP1 expressed in this parasite.</p> <p>Conclusion</p> <p>The major transcript in the rosetting model parasite FCR3S1.2 is FCR3S1.2_<it>var</it>₂(IT4<it>var</it>60). The results suggest that this gene encodes the PfEMP1-species responsible for the rosetting phenotype of this parasite. The activity of previously raised antibodies to the NTS-DBL1α of FCR3S1.2_<it>var</it>₁is likely due to cross-reactivity with NTS-DBL1α of the <it>var</it>2 encoded PfEMP1.</p

Survival of Plasmodium falciparum infected Red Blood Cell Aggregates in Elongational Shear Flow

Rosetting, the formation of red blood cell aggregates, is a life-threatening condition in malaria tropica and not yet fully understood. We study rosette stability using a set of microfluidic stenotic channels, with varied narrowing angle and erythrocytes of blood groups O and A. We find reduced ability of a rosette to pass a stenosis without disruption, the longer the tapered part of the constriction and the narrower the stenosis is. In general, this ability increases with rosette size and is 5-15% higher in blood group A. The experimental results are substantiated by equivalent experiments using lectin-induced red blood cell aggregates and a simulation of the underlying protein binding kinetics.</p

Simultaneous transcription of duplicated var2csa gene copies in individual Plasmodium falciparum parasites

Duplicated var2csa genes in one strain of Plasmodium falciparum are simultaneously transcribed, challenging the dogma of mutual exclusive var gene transcriptio

B-Cell Epitopes in NTS-DBL1 alpha of PfEMP1 Recognized by Human Antibodies in Rosetting Plasmodium falciparum

Plasmodium falciparum is the most lethal of the human malaria parasites. the virulence is associated with the capacity of the infected red blood cell (iRBC) to sequester inside the deep microvasculature where it may cause obstruction of the blood-flow when binding is excessive. Rosetting, the adherence of the iRBC to uninfected erythrocytes, has been found associated with severe malaria and found to be mediated by the NTS-DBL1 alpha-domain of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1). Here we show that the reactivity of plasma of Cameroonian children with the surface of the FCR3S1.2-iRBC correlated with the capacity to disrupt rosettes and with the antibody reactivity with a recombinant PfEMP1 (NTS-DBL1 alpha of IT4(var60)) expressed by parasite FCR3S1.2. the plasma-reactivity in a microarray, consisting of 96 overlapping 15-mer long peptides covering the NTS-DBL1 alpha domain from IT4var60 sequence, was compared with their capacity to disrupt rosettes and we identified five peptides where the reactivity were correlated. Three of the peptides were localized in subdomain-1 and 2. the other two peptide-sequences were localized in the NTS-domain and in subdomain-3. Further, principal component analysis and orthogonal partial least square analysis generated a model that supported these findings. in conclusion, human antibody reactivity with short linear-peptides of NTS-DBL1 alpha of PfEMP1 suggests subdomains 1 and 2 to hold anti-rosetting epitopes recognized by anti-rosetting antibodies. the data suggest rosetting to be mediated by the variable areas of PfEMP1 but also to involve structurally relatively conserved areas of the molecule that may induce biologically active antibodies.Swedish Research Council (VR)Swedish Academy of Sciences (KVA, Soderberg Foundation)Karolinska Institutet-DPAEU Network of Excellence EviMalarKarolinska Inst, Dept Microbiol Tumor & Cell Biol MTC, Stockholm, SwedenKarolinska Inst, Dept Lab Med, Therapeut Immunol TIM, Stockholm, SwedenKarolinska Univ Hosp, CAST, Huddinge, SwedenUniv Estadual Campinas, Dept Biochem, Campinas, SP, BrazilWeb of Scienc

A Sequence in Subdomain 2 of DBL1a of Plasmodium falciparum Erythrocyte Membrane Protein 1 Induces Strain Transcending Antibodies

Immunity to severe malaria is the first level of immunity acquired to Plasmodium falciparum. Antibodies to the variant antigen PfEMP1 (P. falciparum erythrocyte membrane protein 1) present at the surface of the parasitized red blood cell (pRBC) confer protection by blocking microvascular sequestration. Here we have generated antibodies to peptide sequences of subdomain 2 of PfEMP1-DBL1a previously identified to be associated with severe or mild malaria. A set of sera generated to the amino acid sequence KLQTLTLHQVREYWWALNRKEVWKA, containing the motif ALNRKE, stained the live pRBC. 50% of parasites tested (7/14) were positive both in flow cytometry and immunofluorescence assays with live pRBCs including both laboratory strains and in vitro adapted clinical isolates. Antibodies that reacted selectively with the sequence REYWWALNRKEVWKA in a 15-mer peptide array of DBL1a-domains were also found to react with the pRBC surface. By utilizing a peptide array to map the binding properties of the elicited anti-DBL1a antibodies, the amino acids WxxNRx were found essential for antibody binding. Complementary experiments using 135 degenerate RDSM peptide sequences obtained from 93 Ugandan patient-isolates showed that antibody binding occurred when the amino acids WxLNRKE/D were present in the peptide. The data suggests that the ALNRKE sequence motif, associated with severe malaria, induces strain-transcending antibodies that react with the pRBC surfac

Rosette-Disrupting Effect of an Anti-Plasmodial Compound for the Potential Treatment of Plasmodium falciparum Malaria Complications

The spread of artemisinin-resistant parasites could lead to higher incidence of patients with malaria complications. However, there are no current treatments that directly dislodge sequestered parasites from the microvasculature. We show that four common antiplasmodial drugs do not disperse rosettes (erythrocyte clusters formed by malaria parasites) and therefore develop a cell-based high-throughput assay to identify potential rosette-disrupting compounds. A pilot screen of 2693 compounds identified Malaria Box compound MMV006764 as a potential candidate. Although it reduced rosetting by a modest 20%, MMV006764 was validated to be similarly effective against both blood group O and A rosettes of three laboratory parasite lines. Coupled with its antiplasmodial activity and drug-likeness, MMV006764 represents the first small-molecule compound that disrupts rosetting and could potentially be used in a resource-limited setting to treat patients deteriorating rapidly from malaria complications. Such dual-action drugs that simultaneously restore microcirculation and reduce parasite load could significantly reduce malaria morbidity and mortality