On the pathophysiology of severe falciparum malaria with special reference to red cell deformability Dondorp, A.M.

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1 UvA-DARE (Digital Academic Repository) On the pathophysiology of severe falciparum malaria with special reference to red cell deformability Dondorp, A.M. Link to publication Citation for published version (APA): Dondorp, A. M. (1999). On the pathophysiology of severe falciparum malaria with special reference to red cell deformability General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam ( Download date: 23 Nov 2018

2 n zr &> CD The rheology of severe falciparum malaria -5 W A.M. Dondorp, P.A. Kager, J. Vreeken, N.J. White Department of Internal Medicine and Division of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Centre, Amsterdam, the Netherlands (A.M. Dondorp, MD, Prof. P.A. Kager, MD, PhD, Prof. J. Vreeken, MD, PhD). Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand (Prof. N.J. White, FRCP); Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford, United Kingdom (Prof. N.J. White). Submitted for publication

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4 The rheology of severe falciparum malaria Introduction Falciparum malaria is a potentially fatal infection. If left untreated in a non-immune subject the infection can develop quickly into severe disease, with multiple vital organ dysfunction, and a mortality of approximately 15-20% despite effective antimalarial drugs and optimal clinical care '. In high transmission areas anaemia in young children is the principle manifestation of severe malaria. In mesoendemic areas cerebral malaria and metabolic acidosis in a wider age range of children are the main complications seen 23. In hypoendemic (low transmission) areas, severe malaria occurs in all age groups. In this context acute renal failure, jaundice, and pulmonary oedema are more common in adult patients whereas anaemia, hypoglycaemia, and convulsions are more common in children, and cerebral malaria and metabolic acidosis occur at all ages 1 ' 4,5. Many pathophysiological aspects of severe malaria are still incompletely understood. Possible contributing mechanisms can be divided into those affecting microcirculatory flow and those involving the release of parasite derived products following erythrocyte rupture and the host reaction to these events. These mechanisms are not mutually exclusive, since impaired blood flow may focus the release of host or parasite derived toxins. In this review, a detailed description of the various contributors to the impaired rheology in falciparum malaria is given and the role of impaired microcirculatory flow in the pathogenesis of severe malaria will be discussed. Rheological changes in severe malaria According to the Poisseille-Hagen law, flow (Q) of a Newtonian fluid through a rigid tube depends on the pressure gradient (Ap) and the total resistance over the tube (R): Q = Ap / R. This resistance is in turn dependent on the diameter (r) and length (I) of the tube and the viscosity (ri) of the blood flowing through it: R= 8. I. r) / n. Ap. r 4. Because of the existence in the circulatory system of complex geometries, flow pulsatility, vessel distensibility and non-newtonian blood viscosity, the Poiseille-Hagen equation cannot be applied quantitatively to blood flow in vivo, but vascular resistance is still governed by the same parameters as in tube flow. Some of these parameters can be seriously affected in severe malaria (table 1). Blood pressure. Most patients with severe malaria are febrile with a high cardiac output, a low systemic vascular resistance, and a low to normal blood pressure that in itself should not limit normal tissue perfusion 6. In rare cases, patients may develop hypotension and shock in the absence of concomitant septicaemia, a condition called algid malaria 7. 27

5 Chapter 2 Arterioles Capillaries Postcapillary venules Reduction in flow - reduced red cell deformability (both infected and uninfected cells) - increased plasma viscosity - cytoadherence (sequestration) - reduced red cell deformability (both infected and uninfected cells) - cytoadherence (sequestration) - rosette formation - red cell aggregation - increased plasma viscosity Increase in flow - anaemia - anaemia - anaemia Table 1. Rheological factors affecting microcirculatory flow in falciparum malaria Vessel diameter Although there is some evidence of autonomic nervous system dysfunction in malaria 8, there is no evidence that abnormal neural control of vascular tone leads to impaired tissue perfusion. In cerebral malaria (coma in severe malaria) there seems to be a normal response of the cerebral resistance vessels (small arteries and arterioles) to changes in arterial pc The main pathological process appears to be mechanical obstruction to flow in the small blood vessels. The lumenal diameter of the capillaries and postcapillary venules, is reduced significantly by the sequestration of erythrocytes containing the more mature stages of the parasite, a phenomenon that has been recognized since the 19!h century 10. Blood viscosity Whole blood viscosity, or resistance to flow, depends on the local shear stress, and thus the section of the circulation that is considered, as blood behaves as a non-newtonian fluid with decreasing viscosity at higher shear rates. Whole blood viscosity in the larger vessels decreases exponentially with falling haematocrit,;, as in severe malaria, where anaemia is a common finding. This decrease in whole blood viscosity is counteracted by an increase in plasma viscosity through increased plasma fibrinogen levels, so that whole blood viscosity either does not change significantly or slightly increases 12 ' 13. Whole blood viscosity can also be increased by a reduction in red cell deformability. This is a determinant of viscosity mainly under high shear conditions. Despite lower flow rates, high shear stresses are encountered at the arteriolar end of the microcirculation (in vessels with a diameter below 250 urn), in which the red cells are elongated and migrate towards the axial centre of the vessel. This results in a lubricating plasma boundary layer along the vessel wall, which reduces viscosity considerably: the Fâhreus-Lindqvist phenomenon ". 28

6 The rheology of severe falciparum malaria Erythrocytes, with a normal average cell diameter of 7.5 u.m, must deform considerably in their normal passage through capillaries with a midpoint diameter of 3 to 7 u.m. The Fâhreus-Lindqvist phenomenon still occurs in capillaries with lumenal diameters down to 5 ^m. Below a vessel diameter of 5 u.m, there is a sudden sharp increase in the resistance to flow, known as inversion of the Fâhreus-Lindqvist phenomenon 14. The sequestration of parasitised red cells in the microcirculation is important in reducing the lumenal diameter of capillaries and small venules below this critical threshold. In this context red cell deformability becomes extremely important as a slight decrease in deformability may result in a hundred to thousand fold decrease in capillary flow rate ' 5. In severe malaria red cell deformability is reduced not only in the parasitised erythrocytes 16 ' 17, but also in the uninfected erythrocytes 18. Indeed it is the reduction in uninfected red cell deformability that has a strong association with outcome 1S19. Finally, on the venular side of the microcirculation, where low shear stresses are encountered, blood viscosity can be increased by red cell aggregation. The role of red cell aggregation in the pathogenesis of severe malaria remains uncertain. Aggregability is likely to be increased because of the increased plasma fibrinogen levels. In all the human malarias, red cell aggregates can also be formed as a result of the adherence of uninfected erythrocytes to parasitised erythrocytes (rosetting) and also by agglutination of parasitised red cells. However, unlike cytoadherence, which occurs with all P. falciparum isolates in vivo, rosetting only occurs with some isolates. These phenomena and their pathophysiological implications will now be described in more detail. Sequestration Erythrocytes containing P. falciparum in the second half of its intraerythrocytic development (trophozoites and schizonts) adhere to vascular endothelium (cytoadherence). This leads to sequestration of parasitized red cells in the microcirculation (fig.1 ). Sequestration occurs only in capillaries and post-capillary-venules. Sequestered red cells are not distributed uniformly throughout the body: in fatal cerebral malaria sequestration is greatest in the brain, particularly the white matter, but it is also prominent in the liver, kidneys, intestines and adipose tissue 20. In other cases of fatal falciparum malaria without cerebral involvement, there is less cerebral sequestration. Cytoadherence involves the binding of ligands protruding on the membrane of the infected erythrocyte ('adhesins') to a corresponding receptor ligand or ligands on the vascular endothelial surface. On the red cell surface the high molecular mass variants of the strain specific P. falciparum erythrocyte membrane protein 1 or PfEMP, has a central role as adhesion molecule 21-2I. This parasite-derived variant antigen is expressed on the exterior of the parasitised red cell 29

7 Chapter 2 in the second half of the asexual life cycle and is anchored through the membrane to accretions of another parasite-derived protein: histidine rich protein (PfHRPI) 23. These focal points of potential vascular attachment can be seen on electron-microscopy as electrondense knobs and are the points of adhesion to the vessel 20. Other putative candidates for cytoadherence on the red cell membrane, of uncertain in vivo importance, are the PfEMP, related protein sequestrin 24, expression of normally cryptic regions of the cytoskeleton protein band 3, called pfalhesin 25 ' 26, a LFA-1-like epitope 27 and fibrillar strands containing immunoglobulins 2S. The putative vascular endothelial host receptors involved in the process of cytoadherence have been defined mainly by the binding of parasitised erythrocytes to epitopes on different in vitro cell lines, such as the amelanotic melanoma cell line C32 29, or by binding to purified proteins. At least eight potential vascular receptors have been identified: thrombospondin 30, the leukocyte differentiation antigen CD36 (which binds thrombospondin) 31, the intercellular adhesion molecule 1 (ICAM-1) 32, vascular cell adhesion molecule-1 (VCAM-1) 33, E-selectin (formerly known as ECAM-1) 33, aß 34 3, PECAM-1 (CD31) 35 and chondroitin sulphate which may be particularly important in the placenta 36. Of these ICAM-I may be the most important in the brain and CD36 in other organs 37. Nearly all parasites isolated from patients bind well to CD36 (>90%), but adhere less well or not at all to the other potential ligands. Interestingly ICAM-1 and VCAM-1 expression on different cell lines can be upregulated by a number of factors including tumour necrosis factor (TNF) 38-39, ischaemia 40 and increased expression of ICAM-1 on vascular endothelium has been shown in autopsy studies 4 '. This suggests that the inflammatory response can influence the organ distribution of sequestration but also raises the possibility that some ICAM-1 expression may follow, rather than precede, the adherence of parasitised red cells. The increased expression of ICAM-1 is accompanied by increased plasma levels of soluble ICAM-1 in falciparum malaria 42. Adhesion studies under flow conditions show that the different receptors may have complimentary roles in modulating cytoadherence in microvessels 43. Initial interaction at high wall shear stress may be of a rolling type, mediated by ICAM-1, P-selectin 44 or other receptors (of which PECAM1 has been identified recently 3S ) with immobilisation and stabilisation occurring via CD36. ICAM-1 may act synergistically with CD36 in increasing the stability of cytoadherence 45. Under laminar flow conditions erythrocytes containing the mature forms of the parasite adhere to monolayers of cultured cell lines at wall shear stresses up to 0.1 to 0.2 Pa These low shear stresses, are encountered in wvoonly at the venular site of the microcirculation (postcapillary venules) ". That adherence of these parasitised red cells can have haemodynamic consequences in vivo is also suggested in studies in rat mesoappendix vasculature, where peripheral vascular resistance is increased in flow-experiments using cytoadhering strains of P. falciparum

8 The rheology of severe falciparum malaria '/,/'- } (wor^^xp * ; >,r a v* - ** \v - ' * l> fv,'.«i j& Ç) *M >-,. o K" <î>- i ; V* -VJa&iVj Figure 1. Histology of a post-mortem brain biopsy in a case of cerebral malaria, showing a capillary clogged up with sequestered erythrocytes, containing the mature stages of the parasite. The remaining capillary lumen is considerably reduced.

9 Chapter 2 Clinical correlates In man, generally only P. falciparum causes lethal disease, and this parasite is also the only one of four species pathogenic to man that shows the phenomenon of sequestration. Autopsy studies of fatal falciparum malaria clearly show the sequestration of erythrocytes in the small blood vessels (fig. 1) and some studies show an association between the percentage of cerebral vessels containing infected erythrocytes, the density of packing, and cerebral malaria It has proved more difficult to show a correlation between the degree of cytoadherence in vitro and the severity or organ distribution of disease. A correlation with the severity of disease, but not with the cerebral symptoms has been reported by some 3751, not by others 52. A recent study in 150 Kenyan children with falciparum malaria showed that strains binding to ICAM-1 were more frequent in cerebral malaria patients 53. Red cell deformability Parasitised erythrocytes Red blood cells infected with P. falciparum parasites become progressively less deformable as the intra-erythrocytic parasites mature All parameters determining red cell deformability are affected in the parasitised red cell. Slight geometrical changes leading to a reduction in the cell surface/ volume ratio (increased sphericity) already occur in infected cells while parasites are in the ring stage 56. As the parasite matures to the trophozoite and schizont stage the presence of the large relatively non deformable parasite itself results in a large increase in the internal viscosity of the red cell leading to marked loss in deformability 55. The erythrocytes containing more mature parasites also show a marked increase in membrane rigidity 56. There are several factors that could contribute to this increase in membrane rigidity. The structure of the cell membrane of infected erythrocytes is altered by neoantigens produced by the parasite. Some of these neoantigens are bound to the cytoskeleton, like MESA to band and RESA to spectrin 58, which could influence red cell deformability. Moreover, the parasite exerts oxidative stress on the host erythrocyte 59. Oxidative stress can lead to loss of membrane flexibility 60. Finally, alteration of the membrane phospholipid organisation occurs with an associated decrease in the fluidity of the lipid bilayer 61. However, reduced fluidity of the membrane does not directly relate to reduced membrane deformability. Phospholipid flip-flop is accelerated and phosphatidylserine appears on the exterior, although this has been contested 52. Non-parasitised erythrocytes Early studies showed that the "filterability" of red cells in uncomplicated malaria was reduced, suggesting that uninfected red cells might also be less deformable 63 ' 64. We 32

10 The rheology of severe falciparum malaria recently showed that in severe falciparum malaria the non-parasitised erythrocytes become considerably more rigid 18. Since the changes in deformability could not be restored by manipulation of the internal viscosity or cell geometry by suspending the cells in hypo- or hyperosmolar media, and there were no changes in the morphology of the uninfected red cells under light microscopy, changes in the red cell membrane are the most likely cause of the loss of deformability of these erythrocytes i8 > 19. The mechanism underlying these membrane changes remain to be elucidated, but could involve both rigidifying substances released by the parasite, uptake of membrane particles derived from parasitised erythrocytes at schizont rupture by the membranes of uninfected erythrocytes, systemic host factors or endothelial cell malfunction. Nauman has identified a heat labile exoantigen produced by in-vitro cultures of P. falciparum which binds reversibly to normal red cells and reduces their deformability 65. This was not confirmed be Paulitschke and Nash 56. Uninfected red cells containing the RESA-antigen on their membrane surface indicating previous infection and subsequent removal of the parasite could also be a source of rigid red cells, although they are insufficient in number to contribute more than a small part of the effect 65. _ a 0.30 i_ n CD 0.26 E ' o 13 u x> O _0 -Q o CD 0.18 healthy uncomplicated severe controls fatal cases Figure 2. Mean red cell deformability (RCD) in relation to severity of disease in adult patients with falciparum malaria on the Thai-Burmese border. El= elongation index, which is proportional with the ellipticity of the deformed erythrocytes. SS= shear stress applied on the red cells (reprinted with permission from Am J Trop Med Hyg 1997; 57: ). 33

11 Chapter 2 Clinical correlates Cerebral malaria Decreased red cell deformability under shear stresses encountered in arterioles and capillaries with low flow velocity, proved a strong predictor of mortality in adult Thai patients with severe falciparum malaria (fig. 2) 18. Recently this was confirmed in a separate study in Kenyan children with severe falciparum malaria (fig. 3) 19. In the Thai study, logistic regression analysis showed that red cell deformability was the most important prognostic factor for mortality, more important than other well established parameters like plasma lactate concentrations and other measures of acid base status ia67 - «w Blood lactate concentrations are increased in severe malaria mainly due to increased anaerobic glycolysis, and not hypermetabolism as evidenced by consistently elevated lactate-pyruvate ratios 6 ' Decreased red cell deformability leads to reduced microcirculatory flow and oxygen delivery in the tissues, and thus to anaerobic glycolysis. Red cell deformability correlates significantly with plasma base excess and lactate levels with an adjusted r 2 of 0.19 and 0.12 in children and adults with severe malaria respectively 1Si 19. This may be compounded by decreased lactate clearance by the liver resulting from reduced liver blood flow, where altered haemorheological factors may also play a role 6 " * 0.8 o 0.6 -O O mean red cell deformability (El) at a shear stress of 1.7 Pa Figure 3. Probability of a fatal outcome in severe falciparum malaria in relation to admission red cell deformability (RCD) measured at a shear stress (SS) of 1.7 Pa. The measure of RCD is the elongation index (El), which is proportional to the ellipticity of the red cells at a certain shear stress. Data derive from pooled data on adults (n=23) and children (n=99) with severe falciparum malaria ia

12 The rheology of severe falciparum malaria The reduction in RCD of the infected red cells can lead to margination of the normally axial flow of the erythrocytes in the smallest blood vessels This in turn can effect plasma skimming and red cell screening at branching points in the microcirculation 76 ' 77. Normally the vessel branch with the lower volume flow will also receive reduced quantities of erythrocytes. Rigid parasitised red cells might escape from this, resulting in routing through capillaries with low flow favouring their cytoadherence. This could also be a factor in the observed inhomogenity of sequestered red cells in autopsy studies. There is a generalised small increase in systemic capillary permeability in severe malaria 7S, but studies of computerised tomography or magnetic resonance imaging have generally shown only slight brain swelling in cerebral malaria, which was attributed to increased intracerebral blood, but no evidence of cerebral deem In adult patients with cerebral malaria intracerebral lactate production and CSF lactate concentrations are increased 81. Although cerebral blood flow was within the normal range, as measured by a modified Kety-Schmidt technique using differences in cerebral arterial-venous tracer concentrations, it was low in comparison to the arterial oxygen content. Cerebral vascular resistance was increased, whereas cerebral oxygen extraction was diminished 9. These findings can be explained by a mechanical obstruction of the cerebral microcirculation, resulting from a combination of reduced red cell deformability and sequestration. The surprising reversibility of coma, which may last in some cases for several days, indicates that there is incomplete ischaemia in cerebral malaria, although measurable focal infarction can occur in rare cases and there could be more extensive micro-infarction, which is not obvious clinically 82. Reduced flow, increased wall tensions, and the accumulation of metabolic products would all be expected to impair cellular function in the vascular territory affected. This would also provoke the release of regulatory vasodilatory factors such as nitric oxide (from enos) 8B, in addition to induction of nitric oxide synthesis by inos induced by the high concentrations of pro-inflammatory cytokines such as TNFa and interleukin-1 M. Besides being a potent vasodilator, nitric oxide is also a neurotransmitter, and local overproduction leading to impaired neuronal functioning has been proposed as a possible cause of coma in cerebral malaria 85. However, there is no direct proof of this elusive hypothesis, and there are some studies pointing to either a protective rather than deleterious effect < 88 or no effect of increased nitric oxide production in severe malaria Nitric oxide does not seem to influence red cell deformability directly, since no reduction was observed in patients with septicaemia and high plasma NO levels 1S -,9. Increased shear forces caused by rigid erythrocytes can also increase the release of other endothelial factors such as prostacyclin (epoprostenol), a potent vasodilator which inhibits platelet aggregation. Damage to the endothelium leading to decreased nitric oxide or prostacyclin production could therefore further impair microcirculatory flow. 35

13 Chapter 2 Anaemia In addition to the strong relationship between red cell deformability and disease outcome, decreased red cell deformability is also an important cause of anaemia in falciparum malaria. Anaemia results from a combination of parasitised erythrocyte destruction at schizont rupture, accelerated removal of both parasitised and unparasitised red cells, and ineffective erythropoiesis In Thai adults with falciparum malaria, decreased red cell deformability at high shear stresses (similar to those encountered in the spleen) correlated strongly with the severity of anaemia 93. Under normal circumstances rigid red cells are removed by the spleen since they will have to squeeze through the small intercellular gaps in the sinusoids of the spleen (width of u,m) Cells which are not able to deform are left in the cords of Billroth and removed by the immunologic effector cells. This mechanism acts synergistically with the mechanisms for clearance of antibody coated red cells %. The thresholds for splenic clearance of antibody coated cells are lowered in acute malaria 97. Recently we showed the same correlation between red cell deformability and anaemia in Kenyan children as in Thai adults with falciparum malaria, but in these children the correlation was also present at lower shear stresses 19. This could be a consequence of the different histological structure of the spleen in children 98. A logical deduction from these data is that very young children with severe falciparum malaria, who usually present with profound anaemia, should be relatively protected from developing cerebral malaria as the most rigid erythrocytes have already been removed by the spleen. Rosetting Rosette formation is the phenomenon in which uninfected red blood cells adhere to erythrocytes containing the mature forms of the parasite. More than ten uninfected erythrocytes are sometimes found stuck to a single infected erythrocyte. However, whereas all erythrocytes containing mature P. falciparum parasites cytoadhere, not all rosette " 10 ' 101. Since the peripheral blood, in general, does not contain the more mature forms of the parasite (trophozoites and schizonts), the parasites have to be cultured in vitro to show rosetting. The frequency of rosette formation in wild strains differs greatly in the various studies: from 5%-10% in both severe and non-severe patients in a recent Kenyan study 102, 49% in a mixed study population from African and South-American strains i03, to 100% in non-severe patients in Papua New Guinea 104. These differences could be explained partly by differences in laboratory techniques. Rosetting in P. falciparum has been associated with surface expression of PfEMPI and also 22 kda or 28 kda parasite proteins, termed 'rosettins' ] On the host red blood cell receptor side the complement receptor CR1 (also known as CD35) and possibly CD36 are involved 107 ',08. Rosettes can be disrupted by monoclonal antibodies to PfHRPI 109 or 36

14 The rheology of severe falciparum malaria immune serum from diseased children " ". Various other substances such as divalent cation depletion, heparin '"or sulphated glycoconjugates n2 also influence their stability. Investigations under flow conditions suggest that rosettes are able to resist shear stresses up to 1.6 Pa, corresponding to shear stresses encountered at the arterial side of the circulation 113. In contrast our studies showed that very low shear stresses of Pa were able to disrupt rosettes in a medium free of dextrans that possibly strengthen the adherence properties. If serum was added to the suspension medium, more mimicking the in vivo situation, the adhesive forces became stronger (personal observations). It is unclear if rosetting per se does occur in vivo, but parasitised cells do become sticky, and these adhesive forces may further slow the vascular passage of uninfected erythrocytes. In a rat mesocaecum vascular bed, P. falciparum strains showing both knob formation with cytoadherence as well as rosetting increased peripheral vascular resistance more than strains that only expressed knobs, but did not form rosettes m. Clinical correlates The pathophysiologic significance of rosette formation is not clear. Some studies show a correlation between the occurrence of rosette forming strains and cerebral malaria "."5."6 ; with severity of disease other than cerebral symptoms ul, or with severe anaemia only 53. Other studies do not show a correlation between rosetting and severity 11S Moreover, rosette formation has also been shown in the benign malarias caused by P. vivax uo P. ovale n \ and P. malariae,22, which questions their clinical relevance, although it has to be realised that quantitatively rosette formation will be much less in the benign malarias because of the much lower parasite burdens. Clinical implications Measures to improve microcirculatory flow could be important as additional treatment in severe falciparum malaria. One target could be the prevention of sequestration. The artemisinin derivatives artesunate and artemether are more potent than quinine in inhibiting cytoadherence 123. To date however, there is no convincing evidence of a difference in mortality between patients treated with these different antimalarial drugs 124 >,25. Heparin as ancillary treatment in falciparum malaria has not shown to improve clinical outcome 126 and in fact might be harmful 127, although it causes disruption of rosettes in vitro in. Another target could be red cell deformability, and there are some clues that this could be beneficial. Pentoxifyllin, a drug designed to improve red cell deformability but that has also an inhibitory effect on TNF production, was suggested to have a beneficial effect on duration of coma and on survival in cerebral malaria 128. The better outcome in the pentoxyfillin group was associated with a decline in serum levels of tumour necrosis factor 37

15 Chapter 2 (TNF) in a few patients. However, a recent study testing Pentoxifyllin as supportive treatment in uncomplicated falciparum malaria did not show any beneficial effect of pentoxyfillin on the clinical course of the disease or on the serum TNF levels 129. Pentoxifyllin has not been tested in the treatment of severe malaria in relation to changes in red cell deformability. Exchange transfusion is used widely in the management of severe malaria in returned travellers, although it has never been clear how it might be of benefit 13. The parasitised red cells causing pathology, are seguestered and not available for exchange. However, removal of rigid unparasitised cells and their replacement by more deformable new erythrocytes would provide a plausible explanation for the apparent benefit from this treatment. Patients with severely reduced red cell deformability form a subgroup that might benefit from exchange transfusion. We have recently shown that in African children with severe falciparum malaria blood transfusion does restore mean red cell deformability in a predictable way 19. Although administration of blood transfusions was not randomised, the data suggest that blood transfusion not only improved disease outcome through its effect on severe anaemia, but also through improvement of red cell deformability. The amount of transfused donor red cells reguired to improve mean red cell deformability above a certain threshold value can be calculated. With this knowledge a randomised trial of (exchange) transfusion in patients with severe malaria and severely reduced red cell deformability would be warranted. Conclusions Impaired microcirculatory flow is important in the pathophysiology of severe falciparum malaria. Several factors combine to reduce microcirculatory flow (fig. 4). First, parasitised red cells adhere to vascular endothelium and these rigid adherent cells simply block the vascular lumen. Second, these sequestered cells may adhere to the erythrocytes that have to squeeze past, through the processes that cause rosetting and agglutination in vitro. Thirdly the uninfected red cells are less deformable. This lack of deformability may be particularly important in areas of intense sequestration where the lumen is reduced below a critical threshold of around 5 im (inversion of the Fâhreus-Lindqvist phenomenon). Healthy erythrocytes can pass through pores as small as 1 um, due to the fact that they behave in many ways like fluid drops of very low internal viscosity. However, in severe falciparum malaria as yet undefined changes in the uninfected red cell membrane occur, reducing deformability considerably. These rigid cells may obstruct microcirculatory flow and they are prone to be filtered out by the spleen and thus contribute to malarial anaemia. The beneficial effects of exchange transfusion in severe malaria can be interpreted as resulting from replacement of undeformable cells by deformable ones. Also the addition 38

16 The rheology of severe falciparum malaria of deformable erythrocytes by simple blood transfusion seems to be beneficial. Drugs aimed at improving red cell deformability in falciparum malaria are a promising subject for future research. Endothelial surface 5 [Jm Cytoadhesion Endothelial surface Figure 4. Factors combining to reduce blood flow through a capillary in falciparum malaria. Red cells containing the mature stages of the parasite stick to the endothelial surface (cytoadherence). This partly obstructs flow. As a consequence passing erythrocytes will have to deform considerably. This ability to deform can be severely disturbed in severe falciparum malaria adding considerable to the impairment in microcirculatory flow. In addition to this, the adhesive forces between parasitised and uninfected red cells involved in rosetting can further hinder capillary flow. References 1 World Health Organisation.(1990) Control of Tropical Diseases. Severe and complicated malaria, 2nd edition. Trans. R. Soc. Trop. Med. Hyg. 84 (suppl.2), Snow, R.etal. (1997) Relation between severe malaria morbidity in children and level of Plasmodium falciparum transmission in Africa. Lancet. 349, Modiano, D. etal. (1998) Severe malaria in Burkina Faso: influence of age and transmission level on clinical presentation. Am. J. Trop. Med. Hyg. 59, Phillips, R.E. ef a/. (1986) The importance of anaemia in cerebral and uncomplicated falciparum malaria: role of complications, dyserythropoiesis and iron sequestration. Q. J. Med. 58, Luxemburger, C. etal. (1996) Morbidity from malaria among a Karen population living on the Thai-Burmese border. Trans. R. Soc. Trop. Med. Hyg. 90, White, N.J. and Ho, M. (1992) The pathophysiology of malaria, in Advances in Parasitology 31 (Baker, J.R. and Muller, R., eds) pp , London: Academic Press 7 Gage, A. (1926) Algid malaria. Ther. Gaz. 50, Supanaranond, W. etal. (1993) Abnormal circulatory control in falciparum malaria: the effects of antimalarial drugs. Eur. J. Clin. Pharmac. 44, Warrell, D.A. etal. (1988) Cerebral anaerobic glycolysis and reduced cerebral oxygen transport in human cerebral malaria. Lancet i,

17 Chapter 2 10 Marchiafava, E. and Bignami, A. (1884) On summer-autumn malarial fevers (translated from the first Italian edition by J.H. Thompson) London: New Sydenham Society 11 Matrai, A., Whittington, R.B. and Skalak, R. (1987) Biophysics of blood, in Clinicalhemorheology (Chien, S. etal. eds), pp 9-73, Martinus N ij hoff Publishers, Dordrecht, The Netherlands 12 Richardson, S.G.N, et al. (1979) Coagulation activation and hyperviscosity in infection. Br. J. Haematol. 42, Sitprija, V. etal. (1977) Renal failure in malaria: a pathophysiologic study. Nephron 18, Dintenfass, L. (1967) Inversion of the Fâhreus Lindqvist phenomenon in blood flow through capillaries with diminishing radius. Nature 21 5, Dintenfass, L. (1981) Basic aspects of rheology. in Hyperviscosity in hypertension (Dintenfass, L. ed) Pergamon Press, Sydney, Australia. 16 Cranston, H.A. et al. (1983) Plasmodium falciparum maturation abolishes physiologic red cell deformability. Science 223, Nash, G.B. etal. (1989) Abnormalities in the mechanical properties of red blood cells caused by Plasmodium falciparum. Blood 74, Dondorp, A.M. et al. (1997) Prognostic significance of reduced red cell deformability in severe falciparum malaria. Am. J. Trop. Med. Hyg. 57, Dondorp, A.M. ef a/. Reduced red cell deformability in the pathogenesis of severe falciparum malaria and its restoration by blood transfusion. Submitted for publication. 20 Macpherson, G.G. etal. (1985) Human cerebral malaria. A quantitative ultrastructural analysis of parasitised erythrocyte sequestration. Am. J. Pathol. 119, Magowan, C. et al. (1988) Cytoadherence by Plasmodium falciparum infected erythrocytes is correlated with the expression of a family of variable proteins on infected erythrocytes. J. Exp. Med. 168, Baruch, D.I. etal. (1996) Plasmodium falciparum erythrocyte membrane protein 1 is a parasitised erythocyte receptor for adherence to CD36, trombospondin, and intercellular adhesion molecule 1. Proc. Natl. Acad. Sei. USA. 93, Leech, J.H. etal. (1984) Identification of a strain-specific malarial antigen exposed on the surface of Plasmodium falciparum infected erythrocytes. J. Exp. Med. 159, Ockenhouse, CF. etal. (1991 ) Sequestrin, a CD36 recognition protein on Plasmodium falciparum malariainfected erythrocytes identified by anti-idiotype antibodies. Proc. Natl. Acad. Sei. USA. 88, Crandall, I. and Sherman, I.W. (1994) Cytoadherence-related neoantigens on Plasmodium falciparum (human malaria)-infected human erythrocytes result from the exposure of normally cryptic regions if the band 3 protein. Parasit. 108, Guthrie, N. etal. (1995) Monoclonal antibodies which recognize human band 3 residues recognize conformations of this protein in uninfected and Plasmodium falciparum-miected erythrocytes. Mol. Cell. Bloch. 144, Tacchini-Cottier, F. etal. (1995) Detection of a LFA-1-like epitope on the surface of erythrocytes infected with a strain of Plasmodium falciparum. Immun. 85, Scholander, C. etal. (1996) Novel fibrillar structure confers adhesive property to malaria-infected erythrocytes. Nature Med. 2, Schmidt, J.A. etal. (1982) Plasmodium falciparum malaria. An amelanotic melanoma cell line bears receptors for the knob ligand on infected cells. J. Clin. Inv. 70, Roberts, D.D. et al. (1985) Thrombospondin binds falciparum malaria parasitised erythrocytes and may mediate cytoadherence. Nature 318, Oquendo, P. et al. (1989) CD 36 directly mediates cytoadherence of Plasmodium falciparum parasitised erythrocytes. Cell 58, Berendt, A.R. ef a/. ( 1989) Intercellular adhesion molecule-1 is an endothelial cell adhesion molecule for Plasmodium falciparum. Nature 341, Ockenhouse, CF., etal. (1992) Human vascular endothelial cell adhesion receptors for Plasmodium fa/c/parum-infected erythrocytes: roles for endothelial leukocyte adhesion molecule-1 and vascular cell adhesion molecule-1. J. Exp. Med. 176,

18 The rheology of severe falciparum malaria 34 Siano, J.P. era/. (1998) Plasmodium falciparum: cytoadherence to aavbb3 on human microvascular endothelial cells. Am. J. Trop. Med. Hyg. 59, Treutiger, C.J. ef a/. (1997) PECAM-1/ CD31, an endothelial receptor for binding Plasmodium falciparum-'*nfected erythrocytes. Nature. Med. 3, Fried, M. and Duffy, P.E. (1996) Adherence of Plasmodium falciparum to chondroitine sulphate A in the human placenta. Science 272, Ockenhouse, CF. et al. (1991) Molecular basis of sequestration in severe and uncomplicated Plasmodium falciparum malaria: differential adhesion of infected erythrocytes to CD36 and ICAM- 1. J. Inf. Dis. 164, Udeinya, I.J. and Akogyeram, CO. (1993) Induction of adhesiveness in human endothelial cells by Plasmodium fa/c/pa/x/m-i nfected erythrocytes. Am. J. Trop. Med. Hyg. 48, Pober, J.S. era/. (1986) Overlapping patterns of activation of human endothelial cells by interleukin 1, tumor necrosis factor, and immune interferon. J. Immunol. 137, DeGraba, T.J. (1998) The role of inflammation after acute stroke: utility of pursuing anti-adhesion molecule therapy. Neurology 51 (suppl 3 ), S Turner, G.D. era/. (1994) An immunohistochemical study of the pathology of fatal malaria. Evidence for wide spread endothelial activation and a potential role for ICAM-1 in cerebral sequestration. Am. J. Pathology 145, Jacobsen, P.H. era/. (1994) Increased plasma concentrations of slcam-1, svcam-1 and selam-1 in patients with Plasmodium falciparum or P. vivax malaria and association with disease severity. Immunol. 83, Cooke, B.M. era/. (1994) Rolling and stationary cytoadhesion of red blood cells parasitised by Plasmodium falciparum: separate roles for ICAM-1, CD36 and thrombospondin. Br. J. Haemat. 87, Ho, M. ef a/. (1998) Characterization of Plasmodium fa/c/parum-infected erythrocytes and P-selectin " interaction under flow conditions. Blood 91, Mc Cormick, C.J. ef a/. (1997) Intercellular adhesion molecules and CD36 synergize to mediate adherence of Plasmodium fa/c/parum-infected erythrocytes to cultured human microvascular endothelial cells. J. Clin. Invest. 100, Nash, G.B. ef a/. (1992) Rheological analysis of the adhesive interactions of red blood cells parasitised by Plasmodium falciparum. Blood 79, Wick, T.M. and Louis, V. (1991) Cytoadherence of Plasmodium fa/c/parum-infected erythrocytes to human umbilical vein and human dermal microvascular endothelial cells under shear conditions. Am. J. Trop. Med. Hyg. 45, Raventos-Suarez, C ef a/. (1985) Membrane knobs are required for the microcirculatory obstruction induced by Plasmodium fa/c/parum-infected erythrocytes. Proc. Natl. Acad. Sei. 82, Aikawa, M. ef a/. (1990) The pathology of human cerebral malaria. Am. J. Trop. Med. Hyg. 43, Pongponratn, E. ef a/. (1991) Microvascular sequestration of parasitised erythrocytes in human falciparum malaria: a pathological study. Am. J. Trop. Med. Hyg. 44, Ho, M. ef a/. (1991) Clinical correlates of in vitro Plasmodium falciparum cytoadherence. Inf. Immun. 59, Goldring, J.D. ef a/. (1992) Plasmodium falciparum: diversity of isolates from Malawi in their cytoadherence to melanoma cells and monocytes in vitro. Br. J. Haem. 81, Newbold, C. etal. (1997) Receptor-specific adhesion and clinical disease in Plasmodium falciparum. Am. J. Trop. Med. Hyg. 57, Cranston, H.A., ef a/. (1983) Plasmodium falciparum maturation abolishes physiologic red cell deformability. Science 223, Nash, G.B. etal. (1989) Abnormalities in the mechanical properties of red blood cells caused by Plasmodium falciparum. Blood 74, Paulitschke, M. and Nash, G.B. (1993) Membrane rigidity of red blood cells parasitised by different strains of Plasmodium falciparum. J. Lab. Clin. Med. 122,

19 Chapter 2 57 Coppel, R.L. (1992) Repeat structures in a Plasmodium falciparum protein that binds human erythrocyte protein 4.1. Mol. Biochem. Parasitol. 50, Foley, M. et al. (1991) The ring-infected erythrocyte surface antigen of Plasmodium falciparum associates with spectrin in the erythrocyte membrane. Mol. Bioch. Parasit. 46, Hunt, N.H. and Stocker, R. (1990) Oxidative stress and the redox status of maiaria-infected erythrocytes. Blood Cells 16, Saltman, P. (1989) Oxidative stress: a radical view. Semin. Hematol. 26, Joshi, P. and Gupta, CM. (1988) Abnormal membrane phospholipid organization in Plasmodium falciparum infected human erythrocytes. Br. J. Haematol. 68, Moll, G.M. et al. (1990) Phopholipid asymmetry in the plasma membrane of malaria infected erythrocytes. Biochem. Cell. Biol. 68, Lee, M.V. era/. (1992) Diminished red blood cell deformability in uncomplicated human malaria. A preliminary report. J. Med. 13, Areekul, S. and Yamarat, P. (1988) Alterations in the viscosity and deformability of red cells with Plasmodium falciparum. J. Med. Assoc. Thai. 71, Nauman, K.M. ef al. (1991) Plasmodium falciparum exo-antigen alters erythrocyte membrane deformability. FEBS letters 292, Angus, B.J. ef al. (1997) In vivo removal of malaria parasites from red blood cells without their destruction in acute falciparum malaria. Blood 90, Taylor, T.E., Borgstein, A. and Molyneux, M.E. (1993) Acid-base status in paediatric Plasmodium falciparum malaria. 0- J. Med. 86, Krishna, S. et al. (1994) Lactic acidosis and hypoglycaemia in children with severe malaria: pathophysiological and prognostic significance. Trans. R. Soc. Trop. Med. Hyg. 88, English, M. ef al. (1997) Acidosis in severe childhood malaria. Q. J. Med. 90, Marsh, K. et al. (1995) Indicators of life-threatening malaria in African children. N. Eng. J. Med. 332, Molyneux, M.E., ef al. (1989) Clinical features and prognostic indicators in paediatric cerebral malaria: a study of 131 comatose Malawian children. Q. J. Med. 265, Pukrittayakamee, S. etal. (1992) Hepatic blood flow and metabolism in severe falciparum malaria: clearance of intravenously administered galactose. Clin. Sei. 82, Molyneux, M.E. etal. (1989) Reduced hepatic flow and intestinal malabsorption in severe falciparum malaria. Am. J. Trop. Med. Hyg. 40, Schmidt-Schonbein, G.W., Zweifach, B.W. and Kovalcheck,S. (1977) The application of stereological principles to morphometry of the microcirculation in different tissues. Microvasc. Res. 14, Secomb, T.W., Hsu, R. and Pries, A.R. (1998) A model for red blood cell motion in glycocalyx lined capillaries. Am. J. Physiol. 27'4, Schmidt-Schonbein, G.W. ef al. (1980) Cell distribution in capillary networks. Microvasc. Res. 19, Cokelet, GR. (1992) Macroscopic rheology and tube flow of human blood, in Microcirculation (Vol.1) (Grayson, J. and Zingg, W. eds), pp 9-31, New York, Plenum Publ. Corp. 78 Davis, T.M. ef al. (1992) Measures of capillary permeability in acute falciparum malaria: relation to severity of infection and treatment. Clin. Inf. Dis. 15, Newton, CR., Peshnu, N. and Kendall, B. (1994) Brain swelling and ischaemia in cerebral malaria. Arch. Dis. Child. 70, Looareesuwan, S. etal. (1995) Magnetic resonance imaging of the brain in cerebral malaria. Clin. Infect. Dis. 21, White, N.J. ef al. (1985) Pathophysiological and prognostic significance of cerebrospinal-fluid lactate in cerebral malaria. Lancet i, Brewster, D., Kwiatkowski, D. and White, N.J. (1990) Neurological sequelae of cerebral malaria in childhood, tancer 336,

20 The rheology of severe falciparum malaria 83 Busse, R. and Fleming, I. (1995) Regulation and functional consequences of endothelial nitric oxide formation. Ann. Med. 27, Rocket, K.A. et al. (1992) In vivo induction of nitrite and nitrate by tumor necrosis factor, lymphotoxin, and interleukin-1 : possible role in malaria. Infect. Immun. 60, Clark, I.A., Rockett, K.A. and Cowden, W.B. (1991) Proposed link between cytokines, nitric oxide, and human cerebral malaria. Parasitol Today 7, Clark, I.A. etal. (1996) Does malarial tolerance, through nitric oxide, explain the low incidence of autoimmune disease in tropical Africa? Lancet 348, Anstey, M. et al. (1996) Nitric Oxide in Tanzanian Children with Malaria: Inverse Relationship between Malaria Severity and Nitric Oxide Production/ Nitric Oxide Synthase Type 2 Expression. J. Experiment. Med. 84, Kremsner, P.G. et al. (1996) High plasma levels of nitrogen oxides are associated with severe disease and correlate with rapid parasitological and clinical cure in Plasmodium falciparum malaria. Trans. R. Soc. Trop. Med. Hyg. 90, Agbenyega, T. et al. ( 1997) Comparison of plasma nitrogen oxides and blood lactate concentrations in Ghanaian children with malaria. Trans. R. Soc. Trop. Med. Hyg. 45, Dondorp, A.M. etal. (1998) Nitric oxides in plasma, urine and cerebrospinal fluid in patients with severe falciparum malaria. Am. J. Trop. Med. Hyg. 59, Phillips, R.E. and Pasvol, G. (1992) Anaemia of Plasmodium falciparum malaria. Bailliere's Clin. Haem. 5, Weatherall, D.J. and Abdalla, S. (1982) The anaemia of Plasmodium falciparum malaria. Brit. Med. Bull. 38, Dondorp, A.M. etal. (1999) Red cell deformability as a predictor of anaemia in falciparum malaria. Am. J. Trop. Med. Hyg. in press 94 Rifkind, R.A. (1965) Heinz body anemia: a ultrastructural study II: red cell sequestration and " destruction. Blood 26, Rosse, W.F. (1987) The spleen as a filter. N. Eng. J. Med. 317, Ho, M. etal. (1990) Splenic Fc receptor function in host defense and anemia in acute Plasmodium falciparum malaria. J. Inf. Dis. 161, Looaresuwan, S. ef a/. (1987) Dynamic alteration in splenic function during acute falciparum malaria. N. Eng. J. Med. 317, Brozman, M. (1984) Precapillary spongy sheats in the human spleen. An immunohistological study. Acta Anat. 118, David, P.H. etal. (1988) Rosetting: a newcytoadherence property of malaria-infected erythrocytes. Am. J. Trop. Med. Hyg. 38, Handunetti, P.H. et al. (1989) Uninfected erythrocytes form 'rosettes' around Plasmodium falciparum infectes erythrocytes. Am. J. Trop. Med. Hyg. 40, Udonsangpetch, R. etal. (1989) Plasmodium falciparum infected erythrocytes form spontaneous erythrocyte rosettes. J. Exp. Med. 169, Newbold, C. etal. (1997) Receptor-specific adhesion and clinical disease in Plasmodium falciparum. Am. J. Trop. Med. Hyg. 57, Wahlgren, M. etal. (1990) Geographical distribution of Plasmodium falciparum erythrocyte rosetting and frequency of rosetting antibodies in human sera. Am. J. Trop. Med. Hyg. 43, Reeder, J.C etal. (1994) Diversity of agglutinatins phenotype, cytoadherence, and rosette-forming characteristics of Plasmodium falciparum isolates from Papua New Guinean children. Am. J. Trop. Med. Hyg. 51, Helmby, H. etal. (1993) Rosetting Plasmodium falciparum-'mfected erythrocytes express unique strain-specific antigens on their surface. Inf. Immun. 61, Carlson, J. and Wahlgren, M. (1992) Plasmodium falciparum erythrocyte rosetting is mediated be promiscuous lectin-like interactions. J. Exp. Med. 176, Rowe, J.A. etal. (1997) P. falciparum rosetting mediated by a parasite-variant erythrocyte membrane protein and complement-receptor 1. Nature 388,

21 Chapter Handunetti, S.M. ef al. (1993) Involvement of CD36 on erythrocytes as a rosetting receptor for Plasmodium falciparum infected erythrocytes. Blood 80, Carlson, J. etal. (1990) Antibodies to a histidine-rich protein (PfHRPI) disrupt spontateously formed Plasmodium falciparum erythrocyte rosettes. Proc. Natl. Acad. Sei. USA. 87, Carlson, J. etal. (1990) Human cerebral malaria: association with erthrocyte rosetting and lack of anti-rosetting activity. Lancet 336, Carlson, J. ef a/. (1992) Disruption of Plasmodium falciparum erythrocyte rosettes by standard heparin and heparin devoid of anticoagulant activity. Am. J. Trop. Med. Hyg. 46, Rowe, A. (1994) Plasmodium falciparum: a family of sulphated glycoconjugates disrupts erythrocyte rosettes. Exp. Parasit. 79, Nash, G.B. etal. (1992) Rheological properties of rosettes formed by red blood cells parasitised by Plasmodium falciparum. Br. J. Haemat. 82, Kaul, D.K. etal. (1991) Rosetting of Plasmodium fa/c/parum-infected red blood cells with uninfected red blood cells enhances microvascular obstruction under flow conditions. Blood 78, Treutiger, C.J. et al. (1992) Rosette formation in Plasmodium falciparum isolates and anti-rosette activity of sera from gambians with cerebral or uncomplicated malaria. Am. J. Trop. Med. Hyg. 46, Ringwald, P. et al. (1993) Parasite virulence factors during falciparum malaria: rosetting, cytoadherence, and modulation of cytoadherencce by cytokines. Inf. Immun. 61, Rowe, A. ef a/. (1995) Plasmodium falciparum rosetting is associated with malaria severity in Kenya. Inf. Immun. 63, AI Yaman, F. ef al. (1995) Human cerebral malaria: lack of significant association between erythrocyte rosetting and disease severity. Trans. R. Soc. Trop. Med. Hyg. 89, Ho, M. et al. (1991) Rosette formation of Plasmodium falciparum infected erythrocytes from patients with acute malaria. Inf. Immun. 59, Udomsangpetch, R. ef a/. (1995) Rosette formation by Plasmodium vivax. Trans. R. Soc. Trop. Med. Hyg. 89, Angus, B.J. etal. (1996) Short report: rosette formation in Plasmodium ovale infection. Am. J. Trop. Med. Hyg. 55, Lowe, B.S., Mosobo, M. and Bull, P.C. (1998) All four species of human malaria parasites form rosettes. Trans. R. Soc Trop. Med. Hyg. 92, Udonsangpetch, R. ef a/. (1996) Antimalarial drugs reduce cytoadherence and rosetting of Plasmodium falciparum. J. Inf. Dis. 173, Boele van Hensbroek, M. et al. (1996) A trial of artemether or quinine in children with cerebral malaria. N. Eng. J. Med. 335, Thin Hien, T. et al. (1996) A controlled trial of artemether or quinine in Vietnamese adults with severe falciparum malaria. N. Eng. J. Med. 335, Hemmer, C.J. et al. (1991) Neither heparin nor actylsalicylic acid influence the clinical course in human Plasmodium falciparum malaria: a prospective randomized study. Am. J. Trop. Med. Hyg. 45, Reid, H.A. and Sucharit, P. (1972) Ancrod, heparin and epsilon-aminocaproicacid in simian malaria. Lancet ii, Di Perri, G. et al. (1995) Pentoxifyllin as a supportive agent in the treatment of cerebral malaria in children, ƒ Inf. Dis. 171, Hemmer, C.J. et al. (1997) Supportive pertoxifyllin in falciparum malaria: no effect on tumor necrosis factor alpha levels or clinical outcome: a preospective, randomized, placebo-controlled study. Am. J. Trop. Med. Hyg. 56, Editorial. (1990) Exchange transfusion in falciparum malaria. Lancet 335,