• View in gallery

    A, A cerebral microvessel from a case with non-cerebral malaria showing loosely packed parasitized red blood cells (PRBCs) (PR) and a PRBC ghost containing parasite debris (arrow) adhering to an endothelial cells (EC) (bar = 2 μm). B, A congested cerebral venule from a case with cerebral malaria showing margination of the PRBCs (PR) with uninfected red blood cells (R) in the center of the lumen. Note that many of the parasites are at a similar stage of development. There is vacuolation of the pericyte (P) adjacent to an endothelial cell (EC) (bar = 2 μm). C, A cerebral microvessel from a case with cerebral malaria tightly packed with PRBCs, many of which contain mature schizonts (arrows) and a PRBC ghost (arrowhead) (bar = 2 μm). D, An empty cerebral capillary from a case with cerebral malaria in which the intact endothelial cells show extensive pseudopodia formation into the lumen (arrows) (bar =1 μm). E, Detail through the periphery of cerebral vessel from a case with cerebral malaria showing the interaction between the PRBC and the endothelial cell at the knobs (arrows). The PRBC contains a parasite at the segmentor stage showing the rhoptries (R) of the developing merozoites and the food vacuole (F) containing pigment (bar = 1 μm). F, Enlargement of the periphery of cerebral venule from a case with cerebral malaria showing the numerous invaginations of the PRBC (PR) plasmalemma and the penetration of the endothelial cell (EC) pseudopodia into the invaginations (arrows), thus increasing the surface area of interaction (bar = 1 μm).

  • View in gallery

    A larger cerebral vessel from a case with cerebral malaria containing a number of loosely packed red blood cells (RBCs) (R), marginated parasitized red blood cells (PRBCs) (PR), and PRBC ghosts (arrows). Note that two PRBCs appear to be surrounded by uninfected RBCs (arrowhead), which may represent rosette formation (bar = 5 μm). Insert. Detail from a case with cerebral malaria showing two RBCs around a PRBC, which may represent rosette formation (bar = 1 μm).

  • View in gallery

    A, Cerebral vessel from a case with cerebral malaria containing parasitized red blood cells (PRBCs) (PR) and a number of inflammatory cells consisting of monocytes (M) plus a few neutrophils (N) and lymphocytes (L). Some of the monocytes contain phagocytosed malarial pigment (arrow) (bar = 5 μm). B, Microvessel from a case with cerebral malaria containing a number of PRBCs (PR), a PRBC ghost (arrow) and an activated monocyte (M). Note the interaction of cytoplasmic processes from the monocyte and the PRBCs (arrowheads) (bar = 2 μm). C, A cerebral vessel from a case with cerebral malaria packed with degenerate material in which numerous packets of malarial pigment were present (arrows), but in which no intact parasites were present (bar = 2 μm). D, A single monocyte from a case with non-cerebral malaria with a phagolysosome containing malarial pigment (arrow) that is located within a small vessel (bar = 2 μm). E, A cerebral microvessel from a case with cerebral malaria appears blocked by a mass of fibrillar material (arrowheads) that is surrounded by red blood cells (R) and PRBCs (PR). The parasites within the PRBCs show evidence of structural degeneration (bar = 1 μm).

  • View in gallery

    A, A microvessel from a case with cerebral malaria tightly packed with parasitized red blood cells (PRBCs) (PR) showing perivascular vacuolation suggestive of edema (arrows). Note that the parasites are at different stages of development (bar = 2 μm). B, Detail of the periphery of a microvessel from a case with cerebral malaria showing a gap (arrow) between the endothelial cells (EC) (bar = 1 μm). C, A small microvessel from a case with cerebral malaria with the lumen occluded by a PRBC ghost (PR). The endothelial cell (EC) and a rounded pericyte (P) containing large lipofuscin granules (L) show evidence of degeneration (bar = 2 μm). D, Section through a ruptured vessel from a case with cerebral malaria showing both PRBCs (PR) and RBCs (R) escaping from the gap in the vessel wall (arrows) (bar = 2 μm).

  • View in gallery

    A, Part of a ring hemorrhage from a case with cerebral malaria showing numerous extravascular red blood cells (RBCs) (R) within the brain parenchyma. Note the intact microvessel containing a monocyte (M) within the lesion (bar = 5 μm). B, Part of a lesion from a case with non-cerebral malaria showing empty (arrow) or partly filled microvessels with adjacent extravascular parasitized red blood cells (PRBCs) (PR), RBCs (R), and a lymphocyte (Ly). Note the large lipofuscin granules within a pericyte (L) (bar = 5 μm). C, Part of a ring hemorrhage from a case with cerebral malaria showing numerous PRBCs (PR) and RBCs (R) within the brain parenchyma consisting of myelinate nerves, astrocytes (A), oligodendrocytes (O), and microgial cells (M), but with no obvious interactions (bar = 5 μm). D, Low-power view from a case with cerebral malaria showing distended foot processes (arrowheads) from an astrocyte (A) partially surrounding a microvessel occluded by PRBCs (PR) (bar = 5 μm).

  • View in gallery

    Histogram showing the parasite load (number of parasitized red blood cells [PRBC] per grid square) of the cerebral (CM) and noncerebral (NCM) malaria patients for each of the three areas of the brain examined (cerebellum, cerebrum, and medulla). Note the significant difference between CM and NCM for each of the areas. *P = 0.010; **P = 0.012; ***P = 0.029.

  • View in gallery

    Histogram showing the sequestration index (SI) of the cerebral (CM) and noncerebral (NCM) malaria patients. The SI is significantly higher in CM group compared with the NCM group. *P =0.042. and ruptures, the remaining ghost RBC continues to adhere to the endothelial cell, in addition to residual malaria pigment. Pigment and ghosts were seen being phagocytosed by macrophages in some cases in our study. This may provide continued stimulation to vascular endothelium after rupture of sequestered schizonts.

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AN ULTRASTRUCTURAL STUDY OF THE BRAIN IN FATAL PLASMODIUM FALCIPARUM MALARIA

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  • 1 Department of Tropical Pathology, and Wellcome Trust Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Nuffield Department of Clinical Laboratory Sciences, and Center for Tropical Diseases, Nuffield Department of Medicine, The John Radcliffe Hospital, Oxford, United Kingdom; Oxford University Clinical Research Unit, and Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam

Cerebral malaria (CM) is a major cause of death in severe Plasmodium falciparum malaria. We present quantitative electron microscopic findings of the neuropathologic features in a prospective clinicopathologic study of 65 patients who died of severe malaria in Thailand and Vietnam. Sequestration of parasitized red blood cells (PRBCs) in cerebral microvessels was significantly higher in the brains of patients with CM compared with those with non-cerebral malaria (NCM) in all parts of the brain (cerebrum, cerebellum, and medulla oblongata). There was a hierarchy of sequestration with more in the cerebrum and cerebellum than the brain stem. When cerebral sequestration was compared with the peripheral parasitemia pre mortem, there were 26.6 times more PRBCs in the brain microvasculature than in the peripheral blood. The sequestration index was significantly higher in CM patients (median = 50.7) than in NCM patients (median = 6.9) (P = 0.042). The degree of sequestration of P. falciparum-infected erythrocytes in cerebral microvessels is quantitatively associated with pre-mortem coma.

INTRODUCTION

The pathophysiology of severe malaria is complex and poorly understood.1–3 Nearly all severe and fatal malaria in humans is caused by Plasmodium falciparum, and manifests clinically as a spectrum of disease ranging from asymptomatic infection through mild, severe, and fatal disease. Death can result from a variety of syndromes including renal failure, severe anemia, cardiovascular shock, multi-system organ failure, and cerebral malaria (CM). Cerebral malaria is the most common clinical presentation and accounts for the majority of deaths in severe malaria4. The pattern of vital organ dysfunction is different in adults and children, although CM is common at all age groups.5–7

Previous studies of the pathology of malaria have identified several specific features of the disease.2 As the Plasmodium parasite matures within host erythrocytes, it disappears from the peripheral circulation and localizes preferentially within the vascular beds of the vital organs such as the brain, lung, and kidney. This process is termed sequestration. There has been some debate as to the role of sequestration in causing disease within a particular organ such as the brain in CM. Some investigators believe that malaria infection causes generalized vital organ dysfunction as a result of the release of systemic cytokines such as tumor necrosis factor alpha, or local release of mediators such as nitric oxide.2,7 It has thus been important to try and relate the presence of sequestration within a particular organ to clinical disease within that organ before death.

The importance of a quantitative approach in answering this question has been addressed in several major studies8–10 aided by the use of electron microscopy.11 These studies confirmed that when patients dying of CM were compared with those dying of non-cerebral malaria (NCM), there was significantly more cerebral vascular sequestration and denser packing of red blood cells (RBCs) in the CM group compared with deaths due to NCM, but no difference in other pathologic features such as endothelial cell changes.

Whereas all patients dying of CM have histologic evidence of cerebral sequestration, some case of NCM also have appreciable levels of sequestration.12 Coma requires sequestration, but is not an inevitable consequence of parasitized red blood cells (PRBCs) being present within the brain.2 As well as estimating sequestration by counting affected vessels histologically, it may be relevant to try and combine measures of vessel involvement with an estimate of vessel packing by PRBCs. This can be done using electron microscopy to analyze brain samples, which allows more accurate ultrastructural quantitation of the degree of parasite packing within vessels. This in turn will allow calculation of a measure of the total parasite load, which combines measurement of the variability of vessel numbers, parasite sequestration, and packing of PRBCs, to see if this correlates with the clinical incidence of coma. Because peripheral parasitemia is a poor predictor of overall parasite burden and tissue levels of sequestration, we examined a series of brain tissues from patients who died of CM and NCM to calculate parasite load in the brain.

We have completed a major clinicopathologic autopsy-based study of the pathology of severe malaria in Vietnam12–15 (Turner GDH and others, unpublished data). As part of this study, which included other cases from Thailand, we have examined brain samples from 65 adult Southeast Asian patients who died of malaria. We have performed a qualitative survey of the ultrastructural features of fatal malaria and compared these features between patients dying of CM and those dying of NCM. Using careful quantitation of parasite sequestration, and comparing this with peripheral blood films and staging from the same patient before death, we have also examined the relationship between cerebral microvascular sequestration, the sequestration index (SI), and coma.

MATERIALS AND METHODS

Clinical diagnosis.

Specimens were collected from patients dying of severe falciparum malaria in Thailand and Vietnam. On admission, the presence of falciparum malaria parasites in the peripheral blood was detected by routine parasitologic examination in all patients. The cases detailed included deaths in a large double-blind trial of the effects of artemether versus quinine in Vietnam,16 and fatal cases from Thai adults treated in Kanchanaburi and Bangkok, Thailand.

These patients were divided into two groups: subjects with CM and those with NCM. Cerebral malaria was defined initially as unrousable coma (a non localizing response to a painful stimulus), and subsequently as a Glasgow Coma score ≤11.4,17 Other causes of unconsciousness in these patients, e.g., hypoglycemia, meningitis, or other encephalopathy, were excluded by clinical examination and testing of cerebrospinal fluid. Full clinical information and treatment histories were obtained for each patient and kept blinded until the end of the statistical analysis.

Autopsy protocols and specimen collection.

Protocols for autopsies, tissue samples, and data collection were reviewed and approved by the Central Scientific and Ethics Committee of the Center for Tropical Diseases and Mahidol University, for patients in Vietnam and Thailand, respectively. Autopsy was performed after obtaining informed consent from the patient’s family. Autopsies were conducted as soon as possible after death (median = 7 hours, interquartile range [IQR] =3–12 hours). Small 1-mm3 tissue samples were taken from multiple areas of the brain for analysis by electron microscopy, including the temporal cortex, cerebellum, and medulla oblongata. In some patients where a full autopsy was refused, permission for a blind needle biopsy was obtained. Post mortem needle biopsies were performed immediately after death.

Preparation of tissues for transmission electron microscopy.

Tissues were fixed in 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, post-fixed with 1% osmium tetroxide in 0.1 M phosphate buffer, pH 7.4, dehydrated in a graded ethanol series, infiltrated with propylene oxide, and embedded in Spurr’s epoxy resin (TABB Laboratories, Reading, United Kingdom). Thin sections were cut with glass knives and/or diamond knives on an ultramicrotome. Copper grids (200-mesh squares) (Agar Scientific, Stansted, United Kingdom) were used to collect the thin sections, which were stained with uranyl acetate and lead citrate prior to examination by transmission electron microscopy using either a Jeol (Tokyo, Japan) 1200 EX II or a Hitachi (Tokyo, Japan) H-7000 electron microscope.

Qualitative ultrastructural analysis.

A qualitative examination of pathologic features from these samples was performed. Within vessels, the nature and severity of the following changes was recorded: presence or absence of sequestered PRBCs, host leukocytes, fibrin thrombi, and platelets. Endothelial cells were assessed for pseudopodia formation, hyperplasia or cell swelling, vacuolation, changes in organelle structure, and intercellular bridges. In the perivascular space the presence of perivascular edema was sought, along with extravasation of PRBCs, uninfected RBCs, or leukocytes into the surrounding parenchyma as hemorrhages. These features were then quantified in three different areas of the brain from the same patient, namely the cerebrum, cerebellum, and medulla oblongata. Electron micrographs were taken of relevant areas.

Quantitation.

Quantitation of sequestration within the brain was examined systematically using the squares of the copper grids as fixed reference areas. Blood vessels were identified morphologically and counted to give a vascular count per grid square. All intravascular RBCs were counted within the vessels seen in each grid square, and assigned as either normal (RBCs) or parasitized (PRBCs). Multiple grid squares were examined for each case (at least 10 grid squares for each sample or more if available, depending on the size of the sample). Sequestration of PRBCs was quantified both in terms of PRBCs per vessel and PRBCs per grid square. In addition, the vascular density in different areas of the brain was compared, using the counts of vessels per grid square.

Parasite stage was quantified by counting PRBCs showing knob protein expression (knob-positive) on their surface compared with knob-negative infected cells. Since drug treatment may arrest the development of the parasite at the trophozoite stage, the only other morphologic division of staging used was the recognition of schizont forms. These were defined as the parasites having at least three segmental nuclei present in a single RBC.

Quantitation of the degree of pathologic changes was performed by counting all vessels within a section of the same specimen. We quantified the following features for each samples: number of grid squares counted, number of vessels per grid square, number of uninfected RBCs, number of PRBCs expressing knob proteins on their surface (knob-positive PRBCs) and those without knobs (knob-negative PRBCs), number of schizonts (defined as multisegmented parasites with more than three nuclei per PRBC), number of intravascular white blood cells (WBCs), presence or absence of platelets and fibrin, and presence of hemorrhages.

We compared several ways of measuring parasite load (degree of sequestration) in the brain and compared this to the peripheral blood. These included the total number of PRBCs/grid square = parasite load, the percentage of PBRCs as calculated from the total number of RBCs and PRBCs from all grid squares, and the sequestration index (SI).

Comparison of the SI.

The SI has been described previously12 and is defined as the percentage parasite count (by electron microscopy) in the organ (ie. brain) divided by the last percentage parasite count in peripheral blood.

Sequestration Index (SI)=(%) Parasite count (EM) in the brain(%) Last parasite count in peripheral blood

It represents one way to estimate the difference between PRBC accumulation in a tissue vascular bed compared with the peripheral blood parasitemia. Thus the ratio of (PRBCs/100 RBCs in the brain)/(PRBCs/100 RBCs in peripheral blood) just before death was calculated. The SI for some cases had been calculated previously using light microscopy; it compared the final peripheral parasitemia before death with the sequestered parasite load as estimated from brain smears taken in the same three brain areas. The same procedure was used for the SI calculated from electron microscopic examination of the brain.

Data analysis.

To summarize the number of PRBCs, RBCs, knob-positive and knob-negative PRBCs, schizonts, WBCs, fibrin, and platelets observed intravascularly and extravascularly, the total number per brain section per subject was calculated and divided by the total number of grid squares examined. This gave the number of PRBCs, RBCs, etc. observed per grid square per subject per brain section. These variables are given as the median (range) and compared between CM and NCM patients for each brain section using the Mann-Whitney U test. For some of the variables, e.g., WBCs, there were no WBCs observed microscopically in many of the patients. Therefore, these variables were categorized as WBCs present or absent, and comparisons between CM and NCM patients for each brain section were evaluated by the chi-square test with Yates’ correction or Fisher’s exact test.

Counting parameters per grid square were then compared between different parts of the brains (cerebrum, cerebellum, and medulla oblongata), using matched samples. Only patients with sample tissue from all three parts of the brain available were included in the analysis (n = 35). Continuous parameters were compared using non-parametric analysis of variance, and categorized variables were analyzed by conditional logistic regression.

The SI was compared between different treatment groups (artemether versus quinine) and clinical groups (CM versus NCM) using the Mann-Whitney U test. Association with time to death, which corresponds to the duration of treatment, was measured by the Spearman correlation. In addition, the SI was logarithmically transformed to a normal distribution and regression modeling was used to explore the relationship between SI, clinical diagnosis, and type and duration of drug treatment. Results given as the 90% value are the range of values from the 5th percentile to the 95th percentile.

RESULTS

Summary of clinical data.

The clinical summaries of the cases are shown in Table 1. There were 65 patients in this study, of which 56% (37 cases) had CM and 44% (28 cases) had NCM. The two groups of patients were similar with respect to time from admission to death (CM: median = 36 hours, NCM: median = 45 hours; P = 0.415), numbers of patients treated with artemether (CM = 47%, NCM = 37%; P = 0.388), and age (CM: median = 30 years, NCM: median = 31 years; P = 0.592).

Pathologic and ultrastructural features of P. falciparum infection of the brain.

Despite the short post mortem intervals in many of the cases, tissue ultrastructure was highly susceptible to degenerative post mortem artifacts. This was seen in a number of cases, and emphasized the need to guard against over interpretation of the pathologic changes. Changes commonly caused by tissue degeneration, fixation, and processing included vessel retraction (giving an appearance of perivascular space expansion and edema), loss of nuclear morphology, and secondary endothelial cell vacuolation.

Despite these caveats, there were a number of obvious pathologic processes observed in the brains of the patients with malaria. Examples of these are shown in Figures 1–5. The foremost among these was the extensive sequestration of parasitized erythrocytes. Adhesion of infected erythrocytes to the endothelial cells of blood vessels of varying sizes was observed, irrespective of the parasitemia and packing of the vessels (Figures 1A, 1B, 1C, 2, and 4A). However, the degree of sequestration varied between cases and between different vessels from the same area of the brain in individual cases. In addition, PRBC ghosts, characterized by the presence of knobs, were observed to remain attached to the endothelial cells (Figures 1A, 1C, and 4C). In capillaries, the lumen could be occluded by PRBCs (Figure 5D) or PBRC ghosts (Figure 4C).

In many of the small congested vessels, the vast majority of the RBCs were infected (Figures 1C and 3A). In the larger congested venules, there was a distinct margination of the PRBCs with a central core of uninfected RBCs (Figure 1B). In some cases, the parasites within the sequestered PRBCs exhibited developmental synchronization, in which parasites in a given vessel tended to be at a similar stage of development; for example mid trophozoites (Figure 1B) or mature schizonts (Figure 1C). However, examples showing mixed stages were also observed (Figure 4A). In addition, in several cases the parasites were damaged, probably as a result of drug treatment, and appeared as small pyknotic (Figure 1A) or vacuolated (Figure 2) structures. Infected cells could still be identified by the presence of knobs on the red cell membrane even if no parasite was included in the plane of section. In the larger, loosely packed, vessels, a detailed search was undertaken to identify rosette formation, the process whereby uninfected RBCs surround and adhere to PRBCs. Examples of possible rosette formation were occasionally observed (Figure 2, insert), but these were extremely rare. In addition, there was no evidence of uninfected RBCs adhering to PRBCs sequestered to the endothelium (Figures 1A and 2). In these cases, PRBC adhesion and margination appeared to occur as a monolayer phenomenon with no bridging.

Detailed examination of the interaction of the endothelial cells and PRBCs showed that attachment was mediated by contacts between the PRBC surface, particularly at the site of electron-dense knobs and the endothelial plasmalemma (Figure 1E). In the examination of many thousands of adherent PRBCs in larger vessels in 65 different patients, no clear example of cytoadherence of a knob-negative PRBC was observed. The brain endothelium of malaria-infected patients, irrespective of the presence of PRBCs, displayed numerous pseudopodia-like protuberances (Figure 1D). In certain instances, there were numerous invaginations of the PRBC plasmalemma, specifically on the aspect facing the endothelial cell, and endothelial cell processes were observed penetrating into these invaginations (Figure 1F). This interdigitation of cell processes increased the area of contact between the PRBCs and endothelial cell and may assist adhesion. However this was not a universal finding, and other vessels were packed with more rounded less deformed PRBCs (Figure 1B).

In some cases, phagocytes were seen intermixed with the PRBCs, many of which contained malaria pigment (Figure 3A and D). In some scattered vessels, numerous monocytes or lymphocytes were seen, but neutrophils, eosinophils, and plasma cells were observed rarely (Figure 3A and B). These accumulations of host leukocytes were focal and were not a consistent finding. Certain monocytes could be seen making pseudopodial contacts with PRBCs (Figure 3B) and cerebral endothelial cells (Figure 3D). There was little evidence of phagocytosis of intact PRBCs by monocytes, but there were numerous examples of phagocytosis of PRBC ghosts and malarial pigment (Figures 3A, 3C, 3D, and 5A).

Fibrin deposition was also a rare finding within vessels (Figure 3E). When present, it did not co-localize with sites of hemorrhage. Occasional clumps of RBCs were seen bound together by fibrin and fibrillar material, but no evidence for fibrin platelet thrombi was seen in any of the hundreds of sections examined. In some cases, the cerebral microvessels showed no intact RBCs or malaria parasites, but residual malaria pigment was clearly visible either within residual adherent membranes or deposited within a mass of degenerating material (Figure 3C).

Gross parenchymal edema was not seen, although there was a widespread appearance of perivascular clearing. Some of these areas appeared to show artifactual change with retraction of the vessel. However, there did appear to be a degree of perivascular edema in some cases (Figure 4A). Other perivascular changes included rounding up of pericytes, with occasional vacuolation of their cytoplasm, and formation of large lipofuscin granules (Figures 4C and 5B). There was no perivascular demyelination observed in this study and little evidence of an inflammatory response within the brain parenchyma (Figure 5A, B, and D). However, distension of astrocytic foot process around vessels was noted (Figure 5D).

Gaps between endothelial cells were observed (Figure 4B), leading to perivascular diapedesis and hemorrhages with release of RBCs around vessels (Figure 4D). The hemorrhages contained infected and uninfected RBCs and occasional leukocytes (Figure 5A–C). When the incidence of hemorrhages and the numbers and types of cells involved were compared, no significant differences were noted between the CM and NCM groups. All stages of parasite development were found in the brain parenchyma, including schizonts and PRBC ghosts (Figure 4D). These extravasated cells intermixed with neurons, astrocytes, oligodendrocytes, and microglial cells, but active interaction between these cells was not observed (Figure 5A–C).

It should be noted that the features described were observed in the brains of both CM and NCM patients. There was no distinct feature specific for CM, but, as described later in this report, there were a number of significant quantitative differences.

Quantitation of pathologic features between clinical groups.

The quantitative data, which show a comparison between various quantitative measures of sequestration and other factors compared in the three different areas of the brain for both the CM and NCM groups, is shown in Table 2.

Quantitation of differential vascularity in different areas of the brain.

Statistical analysis showed significant differences in vascularity between patients with CM and those with NCM in all areas of the brain examined (cerebrum: P = 0.035, cerebellum: P = 0.001, and medulla: P = 0.008) (Table 2). This may reflect a greater degree of sequestered parasites and congestion of vessels with uninfected RBCs in CM cases, thus leading to an increase in the number of vessels seen per unit area. Significantly more uninfected RBCs were seen in the cerebrum and cerebellum of CM cases (Table 2), implying some degree of vascular congestion compared with the NCM cases.

Quantitation of the total parasite load.

The parasite load, the total number of sequestered parasites per grid square for each of the areas of the brain, was significantly increased in CM cases compared with NCM cases (Table 2 and Figure 6). When these data were used to estimate the overall parasite load (total PRBCs per grid square), it was significantly greater in 36 CM patients (median = 1.93, 90% range = 0–10.23) compared with 28 NCM patients (median = 0.44, 90% range = 0–3) (P < 0.001, by Mann-Whitney U test) (Table 3). This difference in parasite load was independent of the type of treatment (either artemether or quinine). In both CM and NCM cases, there was a hierarchy of sequestration with the cerebrum and cerebellum showing higher sequestration than in the brain stem (Figure 6). However, PRBC sequestration was significantly higher for each of the areas of the brain of patients with CM compared with those with NCM (cerebrum: P = 0.010, cerebellum: P = 0.012, medulla: P = 0.029) (Table 2 and Figure 6). Although there was no ultra-structural evidence of K PRBC cytoadhesion, there were significantly more of these cells in the brains of patients with CM than NCM (Table 2).

Quantitation of other pathologic features.

Quantitation of leukocytes showed no difference in the numbers of WBCs between the CM and NCM groups. Quantitative examination of the incidence of other possible pathologic features such as fibrin deposition, platelet aggregation, or hemorrhages showed no significant differences between the CM and NCM cases (Table 2). In addition, no statistically significant differences were seen between the incidence of specific endothelial cell changes, including pseudopodia formation, cytoplasmic vacuolation, and cell swelling or gaps between cells in patients with CM or NCM.

Comparative quantitation of pathologic features between different areas of the brain.

In the subgroup of 35 cases (18 CM and 17 NCM) where three matched samples of the brain were available for quantitation, a direction comparison of the incidence of the pathologic features in the different areas of the brain was undertaken. All the parameters examined were similar between the different brain areas, with the exception of the numbers of sequestered PRBCs, numbers of knob-positive PRBCs, and number of cases with leukocytes (Table 4).

There were significantly more PRBCs and knob-positive PRBCs in the cerebrum compared with both the cerebellum and medulla and cerebellum compared with the medulla (Table 4). In addition, while there were no differences in the number of leukocytes between the cerebrum and cerebellum, there were significantly more leukocytes in both the cerebrum and cerebellum compared with the medulla (Table 4).

These parameters were examined further using the non-parametric sign test to compare each possible pair of the brain sections. The P values for all paired comparisons for both the CM and NCM groups plus the combined data are shown in Table 4. The highest counts of PRBCs and knob-positive PRBCs were found in the cerebrum and the lowest were found in the medulla. For WBCs, similar numbers were found in the cerebrum and cerebellum, but the levels in the medulla were significantly lower.

Quantitation of the SI.

We then compared the parasite burden in the brain with the peripheral parasite count immediately before death. Among the 65 patients studied, one did not have parasites in the brain or in the peripheral blood immediately before death (case AQ424). Of the 53 patients with parasites in the brain, five had no parasites in the blood (4 of 34 CM patients and 1 of 19 NCM patients; P =0.64). Of the 57 patients with peripheral blood parasites, nine showed no parasites in the brain (1 of 31 CM patients and 8 of 26 NCM patients; P = 0.028). Clinical details for these patients are shown in Table 1. These cases tended to be patients who had survived longest and were therefore treated for a prolonged period after admission. Patients who had no observable parasites in their brain by electron microscopy had a significantly longer time to death (median = 104.4 hours, IQR = ± 90.5 hours) than the rest of the group (median = 27.3 hours, IQR = ± 45.7 hours) (P < 0.02, by unpaired t-test). For the remaining 48 patients (combining CM and NCM cases) in which cerebral and peripheral parasitemia could be compared, the parasite counts in the brains (median = 58.3%, 90% range = 6.7–91.5%) were significantly higher than in the peripheral blood (median = 2.5%, 90% range = 0.0007–34.9%) (P < 0.001). Peripheral parasitemia was not significantly different between the CM and NCM groups (P = 0.621, by Mann-Whitney U test). This emphasizes that peripheral parasitemia is a poor guide to the tissue-sequestered parasite burden.

When all 63 patients with peripheral blood parasitemia (including with those with no brain parasites) were included in the analysis (Table 5), the median sequestration index showed 26.6 times (range = 0–3.2 × 106) more PRBCs in the brain microvasculature than in the peripheral blood. In comparing K and K+ PRBCs, the SI for K PRBCs, although much lower (median = 3.2, range 1–4.9 × 104) than for K+ PRBCs (median = 23.4, range 0–3.2 × 106), was still higher than expected from a free mixing model. The SI was significantly higher in CM patients (median = 50.7, range = 0–3.6 ×106) compared with NCM patients (median = 6.9, range = 0–29,520) (P = 0.042) (Figure 7). Sequestration patterns were not affected by drug treatment with either quinine or artemether (Table 5).

DISCUSSION

Cerebral involvement in severe malaria is confined to infection with P. falciparum, but there is still debate as to the pathogenesis of coma in CM. There have been many detailed pathologic descriptions of malaria infection in humans in the more than 100 years since the seminal observations of Marchiafava and Bignami.9,18–22 The Italian pathologists were struck by the extent of sequestration of parasitized erythrocytes in the brains of fatal cases and surmised that this was a direct cause of coma and death.18 Since then, many different theories have been advanced. The striking pattern of apparent vascular occlusion resulting from PRBC sequestration suggested to Marchiafava and Bignama that microvascular obstruction would lead to hypoxia and ischemia. Pathophysiologic studies in humans do indicate significant hypoxia/ischemia and consequent anaerobic glycolysis. However, this ultrastructural study, in conjunction with neuropathologic and immunohistochemical examination,15 does not show evidence of widespread hypoxic-ischemic patterns of brain damage, and a predominantly ischemic etiology is at odds with the complete recovery of the majority of survivors.

The role of cerebral edema in the pathogenesis of coma has been debated for decades. Increased intracranial pressure is relatively common in African children7 and to a lesser extent in adults with CM. It results largely from the increased intra-cerebral blood volume consequent upon sequestration.23 However, increased intracranial pressure per se does not cause coma unless cerebral blood perfusion is compromised. Cerebral edema is not common in radiologic examinations of southeast Asian adults with CM,24 a finding consistent with the morphologic observation of a limited degree of edema in both the CM and NCM cases in this series.

The rapidly reversible nature of CM has also led to the proposal that soluble, neuroactive mediators such as nitric oxide may be released locally within the brain parenchyma following PRBC sequestration.25,26 Reversible disturbances of blood brain barrier function27 or biochemical disruption resulting from metabolic competition by sequestered PRBCs may be contributing factors. The key to dissecting these different possibilities lies in prospective clinicopathologic studies using a variety of techniques including histology, electron microscopy, and immunohistochemistry.

Ultrastructural studies of the neuropathology of CM have been conducted previously.8,11,28 We have used electron microscopy to investigate the neuropathologic features of fatal malaria in a large series of southeast Asian adults. To date, this is the largest autopsy series of malaria cases ever examined using electron microscopy. This extensive series also provides detailed clinical information on the patients pre mortem, which could be correlated with pathologic features. Thus, we have been able to compare ultrastructural features in patients with fatal CM and those who died without cerebral involvement to try and delineate differences and similarities in the pathology related to coma.

Common to all pathologic descriptions of CM is the presence of large numbers of PRBCs in the microvasculature (sequestration) and to a lesser extent the margination of infected RBCs in the medium and large vessels. Quantitative studies of sequestration in different organs from fatal cases con firmed that the sequestration of PRBCs in the cerebral microvasculature was significantly associated with clinical CM (i.e., coma).9–11 Sequestration in the brains of CM patients was higher than in the other organs and also higher than in NCM patients.9 There was more sequestration in CM in terms of the overall total parasite load (total number of PRBCs) and the SI (proportion sequestered in the brain). In three different areas of the brain (cerebrum, cerebellum, and medulla oblongata), there is a significant increase in the sequestered parasite load in CM compared with NCM, which is independent of the potential confounding factors of varying time to death, type of treatment, or vascularity.

Taken together with our findings from light microscopic examination of histologic sections and brain smears from these patients12 (Turner GDH and others, unpublished data), we have shown that all patients who developed coma before death had cerebral sequestration. Measurement of the SI showed that compared with peripheral blood, brain microvessels contain 26 times more PRBCs than would be expected from a free-mixing model. Most of those PRBCs contained mature (K+) parasites, but in 61% of cases increased numbers of PRBCs containing immature (K) parasites were also found. We found no evidence to support the earlier conjectures that coma could develop in the absence of PRBC sequestration. Clearly, some patients who do not develop CM by the time they die did have significant degrees of cerebral sequestration, despite being clinically labeled as having NCM. Sequestration in the brain to some degree probably occurs in all cases of falciparum malaria. This may explain the increased frequency of seizures in falciparum malaria compared with other malarias. Thus, while the presence of the PRBCs may initiate the processes that cause coma in CM, sequestration does not necessarily do so in all patients. Therefore, sequestration is necessary, but not sufficient alone, to cause coma. Other factors must be involved, several of which are being studied further in this group of patients.

In a subset of these patients (n = 23), we have also compared peripheral parasitemia and sequestration in the brain with that in the kidney, and showed that sequestration in the brain greatly exceeds that in the kidney in these cases (Pongponratn E and others, unpublished data). Ultrastructural details of renal microvascular sequestration showed much less PRBC sequestration and higher rates of WBC sequestration compared with the brain of the same case. Thus, high levels of cerebral sequestration are specific to the brain in cases of CM, and not just a reflection of increased parasite burden and sequestration in all tissues.

Examination of multiple areas of the brain showed a hierarchy of sequestration with significantly more parasitized vessels in the cortex and cerebellum than in the brain stem. Nagatake and others29 found preferential sequestration in white matter versus gray matter, and Sein and others30,31 observed differential sequestration in the cerebellum compare with cerebral cortex in both a simian model and in humans. The use of electron microscopy is open to sampling error implicit in examining a small fragment of tissue from a large organ. However the measurement of total parasite load, the use of counts in multiple vessels, and comparison between different areas was an attempt to address this potential problem. Reassuringly, the findings of this study of differential vascularity, differential sequestration, and an increased SI in the brain were similar to our findings using light microscopy or brain smears on these cases12 (Turner GDH and others, unpublished data).

In previous post mortem studies, animal models, and in vitro culture systems, PRBCs have been shown to elicit morphologic changes in endothelial cells. These included formation of pseudopodia, phagocytosis of PRBCs, fusion with platelets, and vacuolar changes.11,32–35 Immunohistochemical studies have confirmed the presence of an activated endothelial phenotype with increased expression of intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-selectin.13 The endothelial cell junctional protein distribution is also disrupted and these changes co-localize with parasite sequestration.14 Once the parasite undergoes schizogony

In this study, we did not identify phagocytosis of pigment by endothelial cells. However, there were numerous ruptured RBC ghosts still adherent to endothelial cells via knob proteins left in the ghost membranes. Should signaling events in cerebral endothelial cells be initiated by knob protein contacts, then the persistence of membrane ghosts after clearance of living parasites (either by drug treatment or due to schizont rupture) may explain the persistence of symptoms. Monocyte phagocytosis appeared to be predominantly of these ghosted erythrocytes rather than whole, infected but intact PRBCs. This may reflect a local resistance to phagocytosis by PRBCs, possibly by contact inhibition. Monocyte pseudopodia could be seen contacting PRBC surfaces via electron-dense knobs, but this did not seem to be accompanied by active phagocytosis. Contact with PRBCs has been shown to affect the functions of other cells during adhesive events, including endothelial14,34 and dendritic cells.36 This emphasizes that PRBC adhesion is not a passive process, but can elicit functional and ultrastructural changes in host cells. In macrophages and circulating monocytes, this was shown by activation and the formation of numerous secondary vacuoles.

The major changes seen in endothelial cells were some vacuolar degeneration and marked pseudopodia formation, which were similar in degree and extent in both CM and NCM patients. MacPherson and others11 reported that cerebral endothelial cells showed numerous pseudopodia in both CM and NCM patients, some of which link to RBC membrane knobs via thin electron-dense strands. Similar pseudopodia were not seen in control brain sections or in blood vessels from other organs in CM and NCM patients. The cause of these changes to cerebral endothelial cells in patients with CM is not known, although in in vitro and animal model systems they have been associated with free oxygen radical production37 and endotoxic shock.38

Fibrin deposition as fibrillary protein within the vessel lumen was not a major feature of our series, nor was the presence of fibrin platelet thrombus formation. No association between hemorrhages and thrombus formation was seen in contrast to a previous report suggesting the involvement of platelet extravasation.39 In many patients, focal ring hemorrhages were seen centered on small subcortical vessels.40,41 It has been suggested that platelet adhesion to and fusion with cerebral endothelial cells may mediate damage and predispose to hemorrhages,35 but in this series we saw little evidence for fibrin or platelet deposition in association with hemorrhages.

Parenchymal responses were variable and rather limited in this study. We saw no evidence for gross demyelination or perivascular leukocyte collections. Our ultrastructural and histologic studies in this series of patients have indicated that individual vessels may show marked leukocyte sequestration, but that this is a very focal finding, even within one brain area in an individual case12,13 (Turner GDH and others, unpublished data). Extravasation of host monocytes was present, but intravascular host leukocyte sequestration was not marked. This is in marked distinction to the pathology observed in rodent models of CM. One histopathologic study of human falciparum malaria in India42 reported that vascular clogging by PRBCs was associated with margination of mononuclear cells. The investigators suggested that sequestration of PRBCs leads to blockage of vessels, inducing local hypoxia, but the pathology is aggravated and abetted by the marginating mononuclear cells, resulting in vascular endothelial injury and cerebral edema. However MacPherson and others11 and the present large study found little evidence of edema and local inflammatory responses to the sequestered PRBCs in the vessels.

Differences between published studies may in part be due to sampling error because histologic and immunohistochemical studies on the patients indicate variable and in some cases quite appreciable degrees of edema and parenchymal stress responses15 (Turner GDH and others, unpublished data). Clinical and pathologic features also vary between different populations, and features such as intravascular leukocyte sequestration, fibrin deposition, and platelets are more common in pediatric African patients. Whether this difference has pathologic significance remains to be determined.

The first impact of electron microscopy on malaria research was to reveal the electron-dense knob regions on the surface of some strains of infected erythrocytes,43 indicating an anatomic basis for the phenomenon of cytoadherence. Cultured malaria parasites in the laboratory show cytoadherence to a variety of cells such as endothelial cells, leukocytes, platelets, and uninfected RBCs. A common feature of all the interactions with host cells seen in this study was that the contact points between PRBCs and other cells were clearly at electron-dense areas. Several factors may underlie this, including the location of specific parasite adhesins such as P. falciparum erythrocyte membrane protein 1 and rifins44 at knob proteins, and the apparent overall negative charge at knobs demonstrated by atomic force microscopy.45,46 This would allow both specific molecular adhesion to endothelial adhesion ligands, or nonspecific attraction to the positive charge on the endothelial cell surface.47

Non-parasitized erythrocytes will agglutinate around RBCs containing the mature form of the parasite in vitro,48 a phenomenon termed rosetting. Rosettes could cause greater resistance to microvascular flow, and the erythrocytes aggregate readily in the larger venules.49 In this study, we found only one rosette-like structure. However, we cannot definitively prove that this structure represents true rosette formation since it was found intermixed with areas of RBC aggregation. We are thus unable to provide evidence for rosette formation playing a major role in microvascular obstruction in this series. Agglutination of infected erythrocytes is more difficult to confirm or refute within the packed sequestered microvasculature.

This study has been the first to investigate possible effects of different types and duration of treatment on the pathologic features observed in CM. Although treatment had caused a degree of morphologic damage, the presence of parasites in brain sections was consistent and recognizable. No differences in parasite clearance from the cerebral vessels, parasite load, or SI were found between patient groups treated with artemether or quinine. This differs from the picture in the bloodstream in which rates of peripheral parasite clearance were significantly more rapid following artemether.16 Parasitized red blood cells, once bound to an endothelial cell, do not appear to recirculate and die in-situ, a conclusion that is supported by quantitative analysis on brain smears.12 The PRBCs containing dead parasites that had developed to the knob-positive stage before death and ghosted PRBC membranes left by schizogony and their associated pigment granules remained cytoadherent to the endothelium, and this may contribute to continued pathologic consequences even after effective drug treatment.

In conclusion, we have performed the largest study to date of electron microscopic findings in an autopsy study of patients with fatal malaria. This provides, in conjunction with histopathologic and immunohistochemical studies on these patients, conclusive evidence for the key role of sequestration in initiating the coma that characterizes CM. The range of ultrastructural data described confirms previous reports and shows how PRBCs interact within the cerebral vasculature with endothelial cells and host leukocytes, and after schizogony leaves ghosted erythrocyte membranes and pigment behind. These interactions may give some indication of how sequestration initiates other pathologic processes that contribute to and maintain coma in patients with CM.

Table 1

Clinical details of the malaria cases (n = 65)*

Case numberAge (years)SexDiagnosisDrugTime to death (hours)Admission parasitecount/μLMaximum parasitecount/μLLast parasite count/μL (%)EM brain parasite count (%)Other complications of disease
* EM = electron microscopy; CM = cerebal malaria; A = artemether; ARF = acute renal failure; J = jaundice; Hypogly = hypoglycemia; NCM = non-cerebral malaria; Q = quinine; S = shock; An = anemia; Pulm ed = pulmonary edema; C = coma; Hyperp = hyperparasitemia; TB = tuberculosis; ARDS = acute respiratory distress syndrome; NA = not available; DIC = disseminated intravascular coagulation; AT = arteether; ND = not done; AS = artesunate;
** Single small biopsy containing few RBCs.
AQ1224MCMA3.867,82467,82467,824 (1.2)79.36ARF
AQ1928MCMA7099,475585,8086,782 (0.3)0J, ARF, Hypogly
AQ2050FNCMQ21.58,29017,0826,531 (0.2)6.90S
AQ3663MCMA2384,40384,403560 (0.015)78.66S, J
AQ5951FCMQ3.269,20698,34598,345 (3.1)54.30An, ARF, Hypogly
AQ6126MNCMA453,14021,101023.08Hypogly
AQ6327MNCMQ1851,044,8661,085,81271,215 (2.1)0An, Hypogly, Pulm ed, S
AQ9843MCMA36105,5041,254,7441,125,878 (24.9)56.74C, Pulm ed, S, Hyperp
AQ10534MNCMQ4805,912805,912805,912 (22.3)87.31C
AQ12840FNCMQ1324,5224,55220 (0.0016)0An, Hypogly, Pulm ed
AQ13333MNCMQ441,450,6801,450,68050,240 (2.5)74.57An, Hyperp
AQ13438MNCMA394692,810790,14900An, S
AQ14144FCMA2415,07215,0721,706 (0.05)80.90C, S
AQ17628MCMA4546,360958,830958,830 (34.9)55.73S, Hyperp
AQ17933MNCMA23.5121,330121,32922,105 (0.8)35.22S
AQ19428MNCMQ44619,208619,208128,865 (5.4)72.75Ane, S, C
AQ21947MNCMQ2.51,161,2981,161,2971,161,297 (20.1)56.19Pulm ed, S, Hyperp
AQ22228MCMQ95.845,34245,34103.66An, Hypogly
AQ23134MCMQ6.6150,720150,72053,882 (3.3)93.86S, J, An, ARF
AQ23422FNCMQ27.3802,584802,58456,520 (1.8)79.42Pulm ed, Hyperp, Hypogly, S
AQ24324MNCMQ6.31,026,1521,026,152813,762 (25)34.94Hyperp, Pulm ed, S
AQ25154MNCMQ353,51735,1682809 (0.05)0An, S
AQ25356FNCMA4.721,101177,096117,096 (4.7)43.29None
AQ25728MNCMA18.5629,256629,256148,836 (7.9)35.90An, Hypogly, Pulm ed
AQ26536MCMQ20.7193,424636,540395,640 (15)50.95C, S, J, ARF
AQ26822MCMA6.5112,538112,53778,500 (2.5)61.63C, S, J, ARF
AQ28222MNCMQ11347,72847,72820 (0.0008)0An, ARF
AQ31230MCMQ36412,847412,84735,922 (2.6)91.47S, Pulm ed, ARF, An
AQ32244MCMQ6.31,112,3141,112,313919,392 (36.6)73.87S, J, ARF, Pulm ed, Hyperp
AQ32450FNCMA26476,36576,36420 (0.0009)5.00An, S
AQ32736FCMQ5230034020 (0.0016)23.08S, J, An, ARF
AQ32822MNCMQ6768,421768,420102,490 (4.4)85.34An, S, Hyperp
AQ35025MNCMA15148048020 (0.0019)6.67An, Hypogly, ARF
AQ35743FNCMQ94.5567,712824,81560 (0.0025)0An, S
AQ37569MCMA33692,442112,53820 (0.0008)1.79S, J, Hapogly
AQ39263MCMQ16120,576120,5765,778 (0.2)24.49C, S, Pulm ed, Hypogly
AQ39922MCMA38.5297,421596,851596,851 (14.4)61.87C, S, J, Hypogly, Hyperp
AQ40563MNCMQ50136,653136,653420 (0.02)27.83An, Pulm ed, ARF, S
AQ42426MCMA171115,552115,55200S, ARF, J, Pulm ed
AQ42832MNCMQ1811,30419,46860 (0.002)0ARF, S
AQ42915MNCMA52144,440144,440460 (0.02)0AN, ARF, S
AQ43541MNCMA51271,296271,29640 (0.0005)14.76Pulm ed, S, TB
AQ44274FCMQ191281,344281,34420 (0.0006)7.7Septic shock
AQ44745FCMA6989,553297,42040 (0.0014)59.80S, J, Pulm ed
AQ45954MCMQ59406,944406,94421,478 (0.4)64.71S, An
AQ48030MNCMQ254406020 (0.0007)18.19An
AQ48633MCMA49.536,424129,242220 (0.05)44.45Pulm ed
AQ49021MCMA285.3109,021109,02103.23S, ARDS, ARF
AQ50129MCMQ9118,692118,69220,724 (0.3)66.14C, J, ARF, Pulm ed
AQ50220FNCMA113676,984676,984140,421 (8.6)0An
DN10013MCMQ40529,530529,53018.1 (0.001)66.67**ARF, An, Hyperp, Hypogly, Pulm ed
HD19NA ~25MCM4324,048324,048324,048 (10.38)0
MK345MCMQ53.5160,454160,454042.86S, J, ARF
MK3126FCMQ8444,624444,624444,624 (17.5)69.23S, J, ARF
MK3219MCMQ1672,84872,84862,848 (2.87)92.25J, An, ARF, Acidosis, Sepsis
MK4657MCMQ57179,503179,50316,192 (0.34)80.56J, S
MK5826MCMQ3166,882735,890564,572 (18.73)77.34S, J, ARF, Hyperp
MK7721MCMQ65842,806842,8064,174 (0.1)26.87Hyperp, ARF, Pulm ed
KAN1940MCMQ3858,696455,400455,400 (13.95)65.90None
KAN2719FNCMQ53215,176215,17629,601 (1.2)70.00J, An, DIC, Sepsis, Miscarriage
KAN5520MNCMQ31,360,1281,360,1281,360,128 (44.8)71.43J, An, Hyperp
A3800125MCMAT13.5967,120967,120815,509 (29.51)65.66ARDS, ARF, S, Acidosis, Hypogly
A3900116MCMAS1.5NDND564,740 (18.7)11.56Pulm ed, ARF
A3900361MCMAS296.53,692,6403,692,6401,195,335 (30.7)29.16Pulm ed, ARF, Hyperp, Asplenic
A4000122MCMAS17189,028189,0288,540 (0.2)43.06ARDS
Table 2

Comparative quantitation of pathologic features between different areas of the brains and different clinical groups*

CerebrumCerebellumMedulla
ParametersCM (n = 35)NCM (n = 26)PCM (n = 24)NCM (n = 20)PCM (n = 18)NCM (n = 19)P
* Values are the median (range) or number (%). CM = cerebral malaria; NCM = non-cerebral malaria; RBCs = red blood cells; PRBCs = parasitized red blood cells; K = knobs; WBCs = white blood cells. Significant P values (by Mann-Whitney U test) are shown in bold.
Grid squares counted17 (4–28)17.00 (6–33)0.90114.5 (17–27)20 (10–33)0.07516.5 (10–37)16 (10–52)0.867
Blood vessels per grid square1.18 (0.5–5)0.91 (0.23–1.87)0.0351.32 (0.72–3.14)0.79 (0.19–1.55)0.0010.70 (0–3)0.28 (0–0.81)0.008
RBCs per grid square0.86 (0.23–10.92)0.46 (0.03–2.81)0.0061.75 (0.03–10.61)0.82 (0–2.37)0.0250.94 (0–4)0.38 (0–5.67)0.086
Number (%) of cases with RBCs35/35 (100%)25/26 (96%)0.42623/24 (96%)17/20 (85%)0.31617/18 (94%)16/19 (84%)0.604
PRBCs per grid square % with PRBCs1.81 (0–15)0.47 (0–6.87)0.0102.60 (0–8.88)0.37 (0–5.00)0.0120.82 (0–12.07)0.07 (0–2.94)0.029
32/35 (91%)18/26 (69%)0.04219/24 (79%)12/20 (60%)0.31116/18 (89%)11/19 (58%)0.062
K+ PRBCs per grid square % with K+ PRBCs1.32 (0–14.7)0.34 (0–6.64)0.0811.49 (0–8.07)0.12 (0–4.79)0.0940.64 (0–11.4)0.07 (0–2.94)0.058
28/35 (80%)16/26 (62%)0.11215/24 (63%)12/20 (60%)0.86513/18 (72%)10/19 (53%)0.219
K PRBCs per grid square % with K PRBCs1.04 (0–2.7)0.02 (0–0.55)0.0400.18 (0–2.95)0.08 (0–0.40)0.0770.06 (0–1.36)0 (0–1.17)0.056
25/35 (71%)13/26 (50%)0.05717/24 (71%)11/20 (55%)0.27712/18 (67%)6/19 (32%)0.033
Schizonts per grid square0 (0–0.88)0 (0–0)0.0580 (0–0.7)0 (0–0.19)0.2480 (0–0.88)0 (0–0.17)0.346
% with schizonts11/35 (31%)1/26 (4%)0.0096/24 (25%)1/20 (5%)0.0715/18 (28%)2/19 (11%)0.232
% with WBCs21/35 (60%)17/26 (65%)0.66816/24 (67%)10/20 (50%)0.2637/18 (39%)3/19 (16%)0.151
% with fibrin6/35 (17%)3/26 (12%)0.7205/24 (21%)1/20 (5%)0.1984/18 (22%)00.046
% with platelets2/35 (6%)1/26 (4%)1.0000 (0%)1/20 (5%)0.455001.000
% with hemorrhages22 (63%)13 (50%)0.31511 (45%)11 (55%)0.54510 (56%)10 (53%)0.858
Table 3.

Total parasite load per grid square averaged for whole brain*

Clinical categoryNo.Median (90% range)
* P < 0.001 by Mann-Whitney U test.
Cerebral malaria361.93 (0–10.23)
Non-cebral malaria280.44 (0–3)
Table 4.

Statistical analysis of the differences between pathologic features in three different brain areas*

Cerebrum versus cerebellumCerebrum versus medullaCerebellum versus medulla
* Shown is a comparison between major pathologic changes quantitated in three different areas of the brain, where matched samples from each patient were available (n = 35). Only features that showed significant differences between brain areas are shown (see Results). The upper values show the difference between the two brain areas compared (median and 90% range) (e.g., [PRBCs per grid square (cerebrum)–PRBCs per grid square (cerebellum)]). The statistical significance was calculated using the nonparametric sign test. Statistically significant results are shown in bold with P values in italics. For example, there is a significant difference between the number of white blood cells quantitated in the cerebrum of patients compared with the medulla, but no difference between those in the cerebrum compared with the cerebellum.
Parasitized red blood cells (PRBCs)0.107 (−1.702–9.28)0.723 (−2.062–5.446)0.273 (−4.693–3.434)
0.006<0.0010.035
Percentage of PRBCs [PRBCs/(PRBCs + RBCs)]0.104 (−0.220–0.448)0.128 (−0.063–0.617)0 (−0.198–0.451)
<0.001<0.0010.232
Number of knob+ (K+) PRBCs0.234 (−1.163–9.152)0.856 (−1.559–6.049)0.04 (−3.836–2.933)
0.002<0.0010.013
Percentage of K+ PRBCs [K+ PRBCs/(K+ PRBCs + RBCs)]0.076 (−0.221–0.571)0.088 (−0.049–0.681)0 (−0.230–0.309)
<0.001<0.0010.305
White blood cells0 (−0.231–0.334)0.029 (−0.047–0.211)0.011 (−0.182–0.255)
0.564<0.0010.012
Table 5.

Comparison of sequestration index between clinical and treatment groups*

No.Median sequestration index90% rangeP
*CM = cerebral malaria; NCM = non-cerebral malaria. Significant P value is shown in bold.
Overall6426.550–3,230,000
Treatment
    Artemether2724.650–3,230,000
    Quinine3728.440–3,660,0000.865
CM3650.660–3,660,000
NCM286.880–29,5200.042
Figure 1.
Figure 1.

A, A cerebral microvessel from a case with non-cerebral malaria showing loosely packed parasitized red blood cells (PRBCs) (PR) and a PRBC ghost containing parasite debris (arrow) adhering to an endothelial cells (EC) (bar = 2 μm). B, A congested cerebral venule from a case with cerebral malaria showing margination of the PRBCs (PR) with uninfected red blood cells (R) in the center of the lumen. Note that many of the parasites are at a similar stage of development. There is vacuolation of the pericyte (P) adjacent to an endothelial cell (EC) (bar = 2 μm). C, A cerebral microvessel from a case with cerebral malaria tightly packed with PRBCs, many of which contain mature schizonts (arrows) and a PRBC ghost (arrowhead) (bar = 2 μm). D, An empty cerebral capillary from a case with cerebral malaria in which the intact endothelial cells show extensive pseudopodia formation into the lumen (arrows) (bar =1 μm). E, Detail through the periphery of cerebral vessel from a case with cerebral malaria showing the interaction between the PRBC and the endothelial cell at the knobs (arrows). The PRBC contains a parasite at the segmentor stage showing the rhoptries (R) of the developing merozoites and the food vacuole (F) containing pigment (bar = 1 μm). F, Enlargement of the periphery of cerebral venule from a case with cerebral malaria showing the numerous invaginations of the PRBC (PR) plasmalemma and the penetration of the endothelial cell (EC) pseudopodia into the invaginations (arrows), thus increasing the surface area of interaction (bar = 1 μm).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 4; 10.4269/ajtmh.2003.69.345

Figure 2.
Figure 2.

A larger cerebral vessel from a case with cerebral malaria containing a number of loosely packed red blood cells (RBCs) (R), marginated parasitized red blood cells (PRBCs) (PR), and PRBC ghosts (arrows). Note that two PRBCs appear to be surrounded by uninfected RBCs (arrowhead), which may represent rosette formation (bar = 5 μm). Insert. Detail from a case with cerebral malaria showing two RBCs around a PRBC, which may represent rosette formation (bar = 1 μm).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 4; 10.4269/ajtmh.2003.69.345

Figure 3.
Figure 3.

A, Cerebral vessel from a case with cerebral malaria containing parasitized red blood cells (PRBCs) (PR) and a number of inflammatory cells consisting of monocytes (M) plus a few neutrophils (N) and lymphocytes (L). Some of the monocytes contain phagocytosed malarial pigment (arrow) (bar = 5 μm). B, Microvessel from a case with cerebral malaria containing a number of PRBCs (PR), a PRBC ghost (arrow) and an activated monocyte (M). Note the interaction of cytoplasmic processes from the monocyte and the PRBCs (arrowheads) (bar = 2 μm). C, A cerebral vessel from a case with cerebral malaria packed with degenerate material in which numerous packets of malarial pigment were present (arrows), but in which no intact parasites were present (bar = 2 μm). D, A single monocyte from a case with non-cerebral malaria with a phagolysosome containing malarial pigment (arrow) that is located within a small vessel (bar = 2 μm). E, A cerebral microvessel from a case with cerebral malaria appears blocked by a mass of fibrillar material (arrowheads) that is surrounded by red blood cells (R) and PRBCs (PR). The parasites within the PRBCs show evidence of structural degeneration (bar = 1 μm).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 4; 10.4269/ajtmh.2003.69.345

Figure 4.
Figure 4.

A, A microvessel from a case with cerebral malaria tightly packed with parasitized red blood cells (PRBCs) (PR) showing perivascular vacuolation suggestive of edema (arrows). Note that the parasites are at different stages of development (bar = 2 μm). B, Detail of the periphery of a microvessel from a case with cerebral malaria showing a gap (arrow) between the endothelial cells (EC) (bar = 1 μm). C, A small microvessel from a case with cerebral malaria with the lumen occluded by a PRBC ghost (PR). The endothelial cell (EC) and a rounded pericyte (P) containing large lipofuscin granules (L) show evidence of degeneration (bar = 2 μm). D, Section through a ruptured vessel from a case with cerebral malaria showing both PRBCs (PR) and RBCs (R) escaping from the gap in the vessel wall (arrows) (bar = 2 μm).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 4; 10.4269/ajtmh.2003.69.345

Figure 5.
Figure 5.

A, Part of a ring hemorrhage from a case with cerebral malaria showing numerous extravascular red blood cells (RBCs) (R) within the brain parenchyma. Note the intact microvessel containing a monocyte (M) within the lesion (bar = 5 μm). B, Part of a lesion from a case with non-cerebral malaria showing empty (arrow) or partly filled microvessels with adjacent extravascular parasitized red blood cells (PRBCs) (PR), RBCs (R), and a lymphocyte (Ly). Note the large lipofuscin granules within a pericyte (L) (bar = 5 μm). C, Part of a ring hemorrhage from a case with cerebral malaria showing numerous PRBCs (PR) and RBCs (R) within the brain parenchyma consisting of myelinate nerves, astrocytes (A), oligodendrocytes (O), and microgial cells (M), but with no obvious interactions (bar = 5 μm). D, Low-power view from a case with cerebral malaria showing distended foot processes (arrowheads) from an astrocyte (A) partially surrounding a microvessel occluded by PRBCs (PR) (bar = 5 μm).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 4; 10.4269/ajtmh.2003.69.345

Figure 6.
Figure 6.

Histogram showing the parasite load (number of parasitized red blood cells [PRBC] per grid square) of the cerebral (CM) and noncerebral (NCM) malaria patients for each of the three areas of the brain examined (cerebellum, cerebrum, and medulla). Note the significant difference between CM and NCM for each of the areas. *P = 0.010; **P = 0.012; ***P = 0.029.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 4; 10.4269/ajtmh.2003.69.345

Figure 7.
Figure 7.

Histogram showing the sequestration index (SI) of the cerebral (CM) and noncerebral (NCM) malaria patients. The SI is significantly higher in CM group compared with the NCM group. *P =0.042. and ruptures, the remaining ghost RBC continues to adhere to the endothelial cell, in addition to residual malaria pigment. Pigment and ghosts were seen being phagocytosed by macrophages in some cases in our study. This may provide continued stimulation to vascular endothelium after rupture of sequestered schizonts.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 4; 10.4269/ajtmh.2003.69.345

Authors’ addresses: Emsri Pongponratn, Parnpen Viriyavejakul, and Sornchai Looareesuwan, Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. Gareth D. H. Turner and David J. P. Ferguson, Nuffield Department of Clinical Laboratory Sciences, Level 1 Pathology, Electron Microscopy Unit, The John Radcliffe Hospital, Oxford OX3 9 DU, United Kingdom, Telephone: 44-1865-220-514, Fax: 44-1865-220-524, E-mail: david.ferguson@ndcls.ox.ac.uk. Nicholas P. J. Day, Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Ho Chi Minh City, Viet Nam and Center for Tropical Diseases, Nuffield Department of Medicine, The John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom. Nguyen Hoan Phu, Nguyen Thi Hoan Mai, Tran Tinh Hien, Hospital for Tropical Diseases, Ho Chi Minh City, Viet Nam. Julie A. Simpson and Kasia Stepniewska, Wellcome Trust Clinical Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. Nicholas J. White, Center for Tropical Diseases, Nuffield Department of Medicine, The John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom and Wellcome Trust Clinical Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.

Acknowledgments: We thank the staff in Vietnam and Thailand for their assistance in clinical care and specimen collection. We also thank Marie Rychetinic and Andrew Skinner (Electron Microscopy Unit in Oxford) for help with processing the electron microscopic samples.

Financial support: This work was supported by grants from the Faculty of Tropical Medicine of Mahidol University and The Wellcome Trust of Great Britain directly to the research unit in Vietnam and as part of the Wellcome Trust-Mahidol University, Oxford Tropical Medicine Research Programme. Emsri Pongponratn received a Wellcome Trust traveling fellowship as part of this project.

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