Red blood cell trans-dispersion revealing biophysical signatures in malaria parasitism

Red blood cell (RBC) deformability plays an important role in the pathogenesis of Plasmodium falciparum malaria, and therefore could potentially enable simple, rapid, and reagent-free biophysical assays. A key challenge, however, is that pathological cells often only represent a small fraction of the sample, which requires testing a large number of individual cells to enable their detection. Additionally, it is often desirable to perform multiple assays simultaneously, which require technologies capable of parallelized analysis. Traditional technologies for analyzing RBC deformability are limited by their experimental difficulty, extensive instrumentation requirements, as well as their lack of throughput and parallelizability. Here, a new microfluidic mechanism called trans-dispersion is developed to address these issues, enabling a high-throughput and parallelized analysis of RBC deformability. The trans-dispersion mechanism transports single RBCs through a series of constrictions in a microfluidic channel, where their transit speed is a function of their deformability. This process is analogous to gel-electrophoresis, where the migration speed of molecules depends on their length. To ensure a sensitive and consistent measurement, the geometry of the constriction is sized such that the transiting cell forms a temporary seal with each constriction while supporting microchannels ensure consistent forces are applied to each deformation channel. After undergoing repeated deformations, the final position of each RBC, indicating its deformability, is determined using simple bright-field microscopy and automated image processing, and thereby resulting in a repeatable, high-throughput and parallelized process. The performance of this mechanism was evaluated by detecting changes in RBC deformability resulting from chemical degradation, malaria parasitism and exposure to anti-malarial drugs. This device can distinguish variation in RBC deformability following chemical degradation using small concentrations (0.0005%) of glutaraldehyde (GTA). P. falciparum-infected RBCs (iRBCs) show distinct deformability curves compared to the uninfected controls. The linear correlation between the parasitemia and the percentage of non-transiting cells could potentially be used to infer the parasitemia of clinical specimen. Furthermore, this device was able to simultaneously assess the efficacy of several antimalarial compounds; showing that rigidification of P. falciparum-iRBCs can potentially be used to evaluate antimalarial drug efficacy, as well as serve as a functional screen for new antimalarials.Applied Science, Faculty ofMechanical Engineering, Department ofGraduat

REDUCED DEFORMABILITY OF PARASITIZED RED BLOOD CELLS AS A BIOMARKER FOR ANTIMALARIAL DRUG EFFICACY

Background Malaria remains a challenging and fatal infectious disease throughout the developing world. Malaria progressively induces structural and functional changes causing rigidification (loss of deformability) of infected red blood cells. Antimalarials may accelerate this process, thereby allowing the infected erythrocyte to be removed from the circulation earlier. The rapid spread of antimalarial drug resistance increases the urgency for the development of new drugs. Many biomarkers, including malaria specific genes and parasite synthesized proteins and metabolites, have been developed to evaluate drug efficacy as well as to screen new drugs. Methods Recently, we developed a microfluidic mechanism, called the multiplexed fluidic plunger that provides sensitive and rapid measurement of single red blood cell deformability. Here, we systematically evaluated the deformability changes of late stage trophozoite infected-RBCs after treatment with various well known as well as unknown antimalarials. Results We show a concentration and time - dependent response relationship that exists between chloroquine treatment and iRBCs deformability change. We determined that rigidification of trophozoite-infected RBCs is a universal property of almost all clinical antimalarial drug treatments. Spiroindolone compounds (+)-SJ733 and NITD246, inhibitors to a Plasmodium falciparum cation-transporting ATPase ATP4, induced the highest rigidified trophozoite-infected RBCs. Collectively, these results suggest that changes in the deformability of iRBCs could be used as a biomarker for antimalarial drug treatments. Therefore, as a proof-of-principle, we tested a group of bisindole alkaloids. The results revealed that cladoniamide A, which has a rare scaffold and lower IC50 value than that of chloroquine may be a promising antimalarial drug candidate. Conclusions Our results demonstrate that rigidification of infected-RBCs may be used as a biomarker for antimalarial drug efficacy as well as for new drug screening. As a proof of principle, we successfully discovered a potential antimalarial drug

Cell-­phoresis o fRed Blood Cells Revealing Biophysical Signatures in Falciparum Malaria

We describe the cell-phoresis mechanism for massively parallel analysis of red blood cell (RBC) deformability by transporting single cells through microstructures to measure their spatial dispersion. Analogous to gel electrophoresis, which transport molecules through nanostructures to measure their length, the spatial dispersion of RBCs within microstructures indicate their deformability. Similar to gel electrophoresis, cell-phoresis require minimal instrumentation, provide a simple image-based readout, and could be performed simultaneously on multiple samples as part of a biophysical assay. We applied the cell-phoresis mechanism to study the biophysical signatures of falciparum malaria where we demonstrate label-­‐free and calibration-­‐free detection of ring-­‐stage infection, as well as in vitro assessment of antimalarial drug efficacy. We show that all clinical antimalarial drugs rigidify RBCs infected P. falciparum and that recently discovered PfATP4 inhibitors show a distinct biophysical signature. We anticipate cell-phoresis to be a functional assay for screening new antimalarials and adjunctive agents, as well as for validating their mechanisms of action