Reduced microcirculatory flow in severe falciparum malaria: pathophysiology and electron-microscopic pathology

Reduced microcirculatory flow in severe falciparum malaria: pathophysiology and electron-microscopic pathology

Acta Tropica 89 (2004) 309–317 Reduced microcirculatory flow in severe falciparum malaria: pathophysiology and electron-microscopic pathology Arjen M...

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Acta Tropica 89 (2004) 309–317

Reduced microcirculatory flow in severe falciparum malaria: pathophysiology and electron-microscopic pathology Arjen M. Dondorp a,b,∗ , Emsri Pongponratn a , Nicholas J. White a,b a


Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok 10400, Thailand Nuffield Department of Clinical Medicine, Centre for Tropical Medicine, John Radcliffe Hospital, Headington, Oxford, UK

Abstract The pathophysiology of severe falciparum malaria is complex, but evidence is mounting that its central feature is the old concept of a mechanical microcirculatory obstruction. Autopsy studies, but also in vivo observations of the microcirculation, demonstrate variable obstruction of the microcirculation in severe malaria. The principal cause of this is cytoadherence to the vascular endothelium of erythrocytes containing the mature forms of the parasite, leading to sequestration and obstruction of small vessels. Besides, parasitized red cells become rigid, compromising their flow through capillaries whose lumen has been reduced by sequestered erythrocytes. Adhesive forces between infected red cells (auto-agglutination), between infected and uninfected red cells (rosetting) and between uninfected erythrocytes (aggregation) could further slow down microcirculatory flow. A more recent finding is that uninfected erythrocytes also become rigid in severe malaria. Reduction in the overall red cell deformability has a strong predictive value for a fatal outcome. Rigidity may be caused by oxidative damage to the red blood cell membrane by malaria pigment released at the moment of schizont rupture. Anti-oxidants, such as N-acetylcysteine can reverse this effect and are promising as adjunctive treatment in severe malaria. © 2003 Elsevier B.V. All rights reserved. Keywords: Severe malaria; Microcirculation; Red blood cell deformability; Oxidative stress; Haemozoin

1. Introduction Severe malaria, caused by the parasite Plasmodium falciparum, is a potentially fatal disease, with a case mortality rate of 15–20% despite adequate treatment and access to facilities for intensive treatment (WHO, 2000). Despite serious scientific efforts, the pathophysiology of severe falciparum malaria is still incompletely understood. A lot of research has been focussed on the immunopathogenesis of the disease, establishing the role of cytokines and other mediators ∗

Corresponding author. E-mail address: [email protected] (A.M. Dondorp).

like nitric oxide. This short review, however, will focus on the role of a compromised microcirculation in the pathogenesis of severe malaria. The two mechanisms are not mutually exclusive: for example coma in cerebral malaria is probably not simply a result of global cerebral hypoxia, but it may result from neurotransmitter abnormalities secondary to microvascular obstruction and the consequent abnormal metabolic milieu and local release of parasite-derived toxic material and host derived inflammatory mediators. Clinically severe malaria is characterised by multiple organ failure, with a variable mix of affected organs, which partly depends on the age of the patient. Severe anaemia, hypoglycaemia and convulsions are

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more common in children, whereas acute renal failure, jaundice and pulmonary oedema are more prevalent in adult patients. Cerebral malaria is a frequent feature of severe malaria in all ages. Most survivors of cerebral malaria make a good neurological recovery, but of children surviving cerebral malaria, around 10% have neurological deficit and two-thirds of these have obvious strokes (Newton et al., 1997; Brewster et al., 1990). Metabolic acidosis is a consistent feature of severe malaria (Taylor et al., 1993). The severity of the accompanying metabolic acidosis, mainly caused by lactic acid, is a strong prognostic factor in both adults and children with severe malaria (Day et al., 2000; Krishna et al., 1994; Marsh et al., 1995). The increase in lactate concentrations is mainly due to increased anaerobic glycolysis, and not hyper metabolism as evidenced by consistently elevated lactate-pyruvate ratios (Day et al., 2000). Lactate production by the parasite itself and reduced hepatic clearance of lactate accounts for only a relatively small proportion of the increased plasma lactate levels (Pukrittayakamee et al., 1992). The increase in anaerobic glycolysis is caused by decreased perfusion at the microcirculatory or tissue level. Evidence for mitochondrial dysfunction as a cause of lactic acidosis has not been described. We will discuss the different contributing factors in the derailment of microcirculatory flow.

pressure (the difference between blood pressure and intracranial pressure) in two children with cerebral malaria (Newton et al., 1996). Usually, cerebrovascular autoregulation in cerebral malaria remains intact (Newton et al., 1996; Warrell et al., 1988). Overall cerebral blood flow has been assessed in the past by a modification of the Kety–Schmidt method, which showed that cerebral blood flow in Thai adults with cerebral malaria were within the range expected in healthy subjects, although they were considered inappropriately low for the arterial oxygen content, as most of the patients were anaemic. Calculated total cerebral vascular resistance was increased. However, it is difficult to extrapolate the findings from macrovascular studies of flow to events occurring in the microvasculature. A technique that has recently become available, named ‘orthogonal polarizing spectral (OPS) imaging’ enables visualisation of microvasculatory flow in patients with severe malaria in vivo. We have recently been able to show that capillary flow in the rectal mucosa was severely disturbed in proportion to disease severity (manuscript in preparation). The disturbance was independent of the systemic blood pressure. Moreover the blocking of the microvasculature was quite heterogeneous, with hypoperfused adjacent to hyperperfused regions. This can explain why macrovascular measurements cannot pick up these abnormalities. The different mechanisms that cause obstruction of the microvasculature will now be briefly discussed.

2. Haemodynamics The cardiac index is high and systemic vascular resistance is low in severe malaria, as a consequence of the increased metabolic demands associated with fever, the systemic inflammatory response, and the malaria associated progressive anaemia (Day et al., 1996). Shock is not a common feature of severe malaria. Blood pressure is usually normal or slightly reduced, but should in itself not limit normal tissue perfusion (White and Ho, 1992). An exception is perfusion of the brain in sporadic cases where an increase in intracerebral pressure is combined with a low blood pressure. A Kenyan study combining transcranial Doppler flow measurements in middle cerebral arteries with continuous intracranial pressure monitoring, showed evidence of passive dependence of cerebral blood flow velocity on cerebral perfusion

3. Sequestration Shortly after discovery of the malaria parasite over a century ago, the great Italian pathologists Marchiafava and Bignami observed that the blood vessels in the brains of patients who died from falciparum malaria were packed with red blood cells containing mature stages of the parasite (Marchiafava and Bignami, 1894). The sequestration of parasitized erythrocytes in the microvasculature of vital organs is the pathological hallmark of severe falciparum malaria. Sequestration in post-capillary venules and capillaries results from the interaction between adhesive, parasite-derived molecules expressed on the surface of the infected red cells, and one or more of several receptors expressed on the surface of the vascular

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endothelium. The most important parasite ligands consist of a family of highly variant proteins with a MW of 240–260 kDa called P. falciparum erythrocyte protein 1 (PfEMP1), which are encoded by a family of var genes distributed throughout the parasite genome (Magowan et al., 1988; Howard and Gilladoga, 1989; Su et al., 1995). PfEMP1 is anchored to the cytoskeleton protein ankyrin inside the erythrocyte through its binding to a submembranous accretion of knob associated histidine rich protein (KHARP), stabilised by PfEMP3 (Magowan et al., 2000). This can be visualised electron-microscopically as “knobs”, which form the points of attachment to the vascular ligands (Fig. 1). More recently it has become apparent that not only erythrocytes containing the mature forms of the parasite sequester, but also ring stage infected cells, although to a much lesser extent (Silamut et al., 1999; Pouvelle et al., 2000). The adhesion ligand on these ring stage infected cells is not PfEMP1. However expression of PfEMP1 on the ring stage-infected erythrocytes is upregulated by fever which would be


expected to accelerate cytoadherence (Udomsangpetch et al., 2002). On the vascular side, at least twelve different molecules expressed on endothelial cells and binding to PfEMP1 have been described. Of these CD36 appears to be the most important receptor outside the brain, whereas ICAM 1 may be the most important on cerebral vascular endothelium (Turner et al., 1994). Chondroitin sulphate A (CSA) is the main receptor for cytoadherence in the placenta (Fried and Duffy, 1996). Autopsy studies show that sequestration is not distributed equally throughout the body and is greatest in the brain, but also prominent in the heart, eyes, liver, kidneys, intestines, and adipose tissue (MacPherson et al., 1985). Within the brain the extend of vascular obstruction varies from vessel to vessel, which corresponds to the in vivo observations of the microcirculation in the rectal mucosa described above. This could be the reflection of asynchronous expression of endothelial receptors even in closely adjacent vessels. The vast subject of cytoadherence under static and flow condition has been reviewed

Fig. 1. Sequestration of a parasitized red blood cell to an endothelial cell of a microvessel, is mediated by contacts between electron dense knobs on its surface and the endothelial cell.


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extensively elsewhere (Ho and White, 1999; Cooke et al., 2000).

4. Rosetting, auto-agglutination and aggregation Rosette formation is the in vitro phenomenon in which uninfected red blood cells adhere to erythrocytes containing the mature forms of the parasite. However, whereas all erythrocytes containing the mature parasite cytoadhere, not all rosette

(Udomsangpetch et al., 1989; David et al., 1998). Adhesion molecules on the infected red blood cell membrane include PfEMP1 and possibly rifins (Chen et al., 1998; Rowe et al., 1997). The counter receptors on the red blood cell surface that can bind PfEMP1 include complement receptor 1 (CR1) (Rowe et al., 1997), heparin sulphate or related glycosaminoglycans (Barragan et al., 2000a) and the ABO blood group antigens, particularly blood group A (Barragan et al., 2000b). In epidemiological studies rosette forming strains have been associated with severe disease

Fig. 2. A large caliber cerebral venule with a number of URBCs and PRBCs, including PRBC ghosts in the lumen. The vessel is not packed, mature PRBC form a layer along the endothelium (margination, arrows). A number of young PRBCs and URBCs are seen in the center of the lumen. PRBCs appear attached to other PRBCs. Note an aggregate of URBCs surrounding a PRBC in the lumen (arrowhead), which probably represents rosette formation. Insert: P. falciparum rosette formation in vitro, showing three URBCs surrounding a PRBC (courtesy of Prof. David Ferguson).

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(Carlson et al., 1990; Ringwald et al., 1993; Rowe et al., 1995), whereas other studies have not found this association (Al-Yaman et al., 1995). Whether rosettes play a pathophysiologic role will depend on their resistance to shear stresses encountered in the human circulation. Rosettes are quite resistant against pulling forces (Nash et al., 1992), but less against more physiologic shearing forces (Chotivanich et al., 2000). A confounding factor in these studies is the composition of the in vitro rosette suspension medium, which determines quite strongly the stability of rosettes. Rosettes are seldom found in autopsy studies (Fig. 2). But even if rosettes per se are not likely to block the microcirculation in vivo, the sticky forces between cytoadherent parasitized red blood cells (PRBCs) and passing erythrocytes could slow down microcirculatory flow. A more recently described adherence property of parasitized red blood cells is the aggregation of parasitized red blood cells,


which is mediated via platelet CD36. Presence of this phenotype has been associated with disease severity both in Kenya and in Thailand (Pain et al., 2001), and Chotivanich, personal communication). The rheological properties of these clumps of parasitized red blood cells have yet to be determined. Also uninfected red blood cell aggregation, which will be enhanced because of the presence of acute phase proteins like fibrinogen, could contribute to vascular obstruction.

5. Coagulation Disseminated intravascular coagulation (DIC) causing clot formation in the microvasculature of vital organs is an important pathophysiologic feature in septicaemia. In contrast DIC in severe malaria is rare. Intravascular fibrin strands have been described in

Fig. 3. Red blood cell deformability: An unparasitized red blood cells shows considerable elongation in order to pass a rigid parasitized red blood cell adhering to the endothelium of the capillary.


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autopsy studies, but are rare (Oo et al., 1987; Turner, 1997). However, as yet unpublished results from autopsy studies in Malawi would suggest that fibrin thrombi are more common in the microvasculature of brain and other organs in children with fatal cerebral malaria than have been reported in adults. Generally, falciparum malaria is associated with a ‘controlled procoagulant state’ with an accelerated

coagulation cascade activity. Fibrinogen turnover is accelerated, there is consumption of antithrombin III, reduced factor XIII and increased concentration of fibrin degradation products (Hemmer et al., 1991; Pukrittayakamee et al., 1989; Holst et al., 1999). There may be prolonged prothrombin and partial thromboplastin times in severe infections, but significant bleeding is rare.

Fig. 4. A parasitized red blood cell (PRBC) from in vitro culture showing a distorted shape, and two unparasitized red blood cells. Also visible are knobs on the PRBC membrane, which in vivo form focal junctions with the endothelial cell membrane and are responsible for sequestration.

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6. Red blood cell deformability Capillaries and post-capillary venules containing parasitized erythrocytes adherent to the vessel wall will often leave a small gap where passing erythrocytes can squeeze through (Fig. 3). Normal red blood cells with an average cell diameter of 7.5 ␮m possess an amazing ability to elongate, allowing them to pass through capillaries with a patent lumen much smaller than their own diameter. However, maturation of the parasite inside the red blood cell progressively abolishes its deformability (Cranston et al., 1984; Nash et al., 1989). This rigidification is due to changes in the red cell membrane, including the incorporation of KHARP (Glenister et al., 2002), increase of the internal viscosity of the red blood cell by the presence of the relatively undeformable parasite and an unfavourable, more spherical, surface to volume ratio (Cooke et al., 2000) (Fig. 4). Moreover, the unparasitized red blood cells (URBCs) can become rigid in severe malaria. 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 ␮m (inversion of the Fåhreus Lindqvist phenomenon). Mean red blood cell deformability, mainly representing URBCs, is severely reduced in patients with severe malaria. When measured on admission, a severe reduction in red cell deformability is a strong predictor for mortality, both in adults and children with severe malaria (Dondorp et al., 1997, 2002). Reduced deformability in the URBCs is caused by rigidity of the membrane presumably through oxidative damage. Increased oxidation of URBC membranes has been shown in children with severe falciparum malaria (Griffith et al., 2001). Also in vitro, the presence of parasitized red blood cells induces chemical measures of membrane oxidation in URBCs, similar to changes that can be seen during red blood cell senescence (Omodeo-Sale et al., 2003). The oxidative agent produced by the parasite is possibly the malaria pigment or haemozoin, which is produced in large quantities of several grams per parasite cycle during severe infection. ␤-haematin, the chemical equivalent of malaria pigment, is the crystallisation product of haem dimers made by the parasite after digestion of the host haemoglobin. This is released into the circulation at schizont rupture, is insoluble in water, and will bind to adjacent cell


membranes including those of passing URBCs. Steric hindrance makes the iron moiety of ␤-haematin less toxic than in haem itself, but ␤-haematin still exerts an important pro-oxidant activity on membranes (Omodeo-Sale et al., 2001). In vitro ␤-haematin reduces red blood cell deformability in a dose dependent way (unpublished data). 7. Conclusions and implications Pathological studies, in vivo observations (OPS imaging) and circumstantial evidence (prognostic significance of lactic acidosis), all suggest that a compromised microcirculation is a pivotal factor in the pathogenesis of severe malaria. This obstruction is caused by sequestration of parasitized red blood cells, but a decrease in red blood cell deformability and adherent forces between parasitized and unparasitized red blood cells in different combinations will all contribute. Rigidity of the unparasitized red blood cells is presumably caused by oxidative damage to the red cell membrane mediated by malaria pigment. This provides us with a new target for intervention. The anti-oxidant N-acetylcysteine is able to reverse the detrimental effects of ␤-haematin on red cell deformability in vitro (manuscript in preparation). N-acetylcysteine has been shown a promising adjunctive treatment in severe malaria. A pilot study in 30 patients with severe malaria on the Thai-Burmese border showed that N-acetylcysteine reduced plasma lactate clearance times by half (Watt et al., 2002). A larger study is currently under way. Acknowledgements This review was supported by the Wellcome Trust Mahidol University Oxford Tropical Medicine Research Programme funded by The Wellcome Trust of Great Britain. References Al-Yaman, F., Genton, B., Mokela, D., Raiko, A., Kati, S., Rogerson, S., Reeder, J., Alpers, M., 1995. Human cerebral malaria: lack of significant association between erythrocyte resetting and disease severity. Trans. R. Soc. Trop. Med. Hyg. 89, 55–58.


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