Reduced red blood cell deformability in Plasmodium knowlesi malaria

Abstract

The simian parasite can cause severe and fatal human malaria. However, little is known about the pathogenesis of this disease. In falciparum malaria, reduced red blood cell deformability (RBC-D) contributes to microvascular obstruction and impaired organ perfusion. In infection, impaired microcirculatory flow has been observed in (rhesus macaques), unnatural hosts who develop severe and fatal disease. However, RBC-D has not been measured in human infection or in the natural host (long-tailed macaques). Using ektacytometry, we measured RBC-D in adults with severe and non-severe knowlesi and falciparum malaria and in healthy controls. In addition, we used micropipette aspiration to determine the relative stiffness of infected RBCs (iRBCs) and uninfected RBCs (uRBCs) in -infected humans and Ektacytometry demonstrated that RBC-D overall was reduced in human knowlesi malaria in proportion to disease severity, and in severe knowlesi malaria, it was comparable to that of severe falciparum malaria. RBC-D correlated inversely with parasitemia and lactate in knowlesi malaria and HRP2 in falciparum malaria, and it correlated with hemoglobin nadir in knowlesi malaria. Micropipette aspiration confirmed that in humans, infection increased stiffness of both iRBCs and uRBCs, with the latter mostly the result of echinocytosis. In contrast, in the natural host, echinocyte formation was not observed, and the RBC-D of uRBCs was unaffected. In unnatural primate hosts of , including humans, reduced deformability of iRBCs and uRBCs may represent a key pathogenic mechanism leading to microvascular accumulation, impaired organ perfusion, and anemia.

Methods to Investigate the Deformability of RBC During Malaria

“…These "storage-induced micro-spherocytes" are removed from the circulation (Roussel, 2019). A similar analysis of blood samples from three Plasmodium knowlesi-infected human subjects showed that sphericity was increased in iRBC compared with uninfected-RBC from the same patients (Barber et al, 2018). A reduction in the projected area of ring-infected RBC (P. falciparum culture in vitro) had also been observed using IFM and correlated with retention in microsphiltration (Safeukui et al, 2013).…”

“…Shear stresses of 30 Pa provide information on the RBC surface-tovolume ratio and may be a proxy for the ability of RBC to cross splenic slits (Barber et al, 2018). Reduced EI of circulating RBC, which contain a vast majority of uninfected RBC, in patients with malaria correlates with disease severity (Dondorp et al, 1999(Dondorp et al, , 2002Ishioka et al, 2016;Barber et al, 2018). A large-scale gene knockout strategy was combined with ektacytometry to determine the role of parasitic exported proteins in stiffness (Maier et al, 2008).…”

“…Micropipette aspiration requires training and it is a demanding single cell method ( Table 1). RBC infected with rings and mature stages require 1.5 and 4-6 times more pressure than normal RBC to enter into 3 µm-wide pipettes, respectively Cranston et al, 1984;Maier et al, 2008Dondorp et al, 1997, 1999Dondorp et al, 2002;Ishioka et al, 2016;Barber et al, 2018 Micropipette aspiration Nash et al, 1989;Glenister et al, 2002;Aingaran et al, 2012;Tiburcio et al, 2012;Shojaei-Baghini et al, 2013;Zhang et al, 2016 Microfluidics Live observation of RBC navigating along narrow channels or across slits in specifically designed PDMS biochips. Controled flow via micropumps/microvalves Ability of RBC to cross channels or slits, assessed by time of passage or sustained retention (quantitative).…”

“…Very informative but technically challenging. Qualitative and/or quantitative analysis Shelby et al, 2003;Antia et al, 2008;Handayani et al, 2009;Herricks et al, 2009Herricks et al, , 2012Hou et al, 2010;Imai et al, 2010;Bow et al, 2011;Huang et al, 2013;Wu and Feng, 2013;Picot et al, 2015;Guo et al, 2016Herricks et al, 2012 Mills et al, 2004Mills et al, , 2007Suresh et al, 2005;Bambardekar et al, 2008;Fedosov et al, 2011Fedosov et al, , 2014Hosseini and Feng, 2012;Ye et al, 2013 Imaging flow cytometry Barber et al, 2018 Frontiers in Physiology | www.frontiersin.org FIGURE 1 | Methods to investigate the deformability of RBC during malaria. Atomic force microscopy: Imaging mode reconstructs a 3D topography of the RBC surface using a cantiliver tip that scans in x and y directions.…”

Surface area-to-volume ratio, not cellular rigidity, determines red blood cell traversal through small capillaries

“…It is associated with high parasitemia infections and causes severe malaria in adult humans at a rate similar to P. falciparum (Cox-Singh et al, 2008, Barber et al, 2013. P. knowlesi does not express adhesins on the surface of the host RBC, but does exhibit increased the host cellular rigidity (Barber et al, 2018b). Despite the lack of adhesion, splenic rupture has been observed, suggesting blockage of sinusoidal vessels (Chang et al, 2018) and an autopsy of a fatal human knowlesi malaria case revealed brain capillaries congested with infected RBCs (Menezes et al, 2012).…”