Richard O. Whitten

Differentiating the pathologies of cerebral malaria by postmortem parasite counts

To study the pathogenesis of fatal cerebral malaria, we conducted autopsies in 31 children with this clinical diagnosis. We found that 23% of the children had actually died from other causes. The remaining patients had parasites sequestered in cerebral capillaries, and 75% of those had additional intra- and perivascular pathology. Retinopathy was the only clinical sign distinguishing malarial from nonmalarial coma. These data have implications for treating malaria patients, designing clinical trials and assessing malaria-specific disease associations.

The neuropathology of fatal cerebral malaria in malawian children.

We examined the brains of 50 Malawian children who satisfied the clinical definition of cerebral malaria (CM) during life; 37 children had sequestration of infected red blood cells (iRBCs) and no other cause of death, and 13 had a nonmalarial cause of death with no cerebral sequestration. For comparison, 18 patients with coma and no parasitemia were included. We subdivided the 37 CM cases into two groups based on the cerebral microvasculature pathology: iRBC sequestration only (CM1) or sequestration with intravascular and perivascular pathology (CM2). We characterized and quantified the axonal and myelin damage, blood-brain barrier (BBB) disruption, and cellular immune responses and correlated these changes with iRBC sequestration and microvascular pathology. Axonal and myelin damage was associated with ring hemorrhages and vascular thrombosis in the cerebral and cerebellar white matter and brainstem of the CM2 cases. Diffuse axonal and myelin damage were present in CM1 and CM2 cases in areas of prominent iRBC sequestration. Disruption of the BBB was associated with ring hemorrhages and vascular thrombosis in CM2 cases and with sequestration in both CM1 and CM2 groups. Monocytes with phagocytosed hemozoin accumulated within microvessels containing iRBCs in CM2 cases but were not present in the adjacent neuropil. These findings are consistent with a link between iRBC sequestration and intravascular and perivascular pathology in fatal pediatric CM, resulting in myelin damage, axonal injury, and breakdown of the BBB.

Tissue distribution of migration inhibitory factor and inducible nitric oxide synthase in falciparum malaria and sepsis in African children

BackgroundThe inflammatory nature of falciparum malaria has been acknowledged since increased circulating levels of tumour necrosis factor (TNF) were first measured, but precisely where the mediators downstream from this prototype inflammatory mediator are generated has not been investigated. Here we report on the cellular distribution, by immunohistochemistry, of migration inhibitory factor (MIF) and inducible nitric oxide synthase (iNOS) in this disease, and in sepsis.MethodsWe stained for MIF and iNOS in tissues collected during 44 paediatric autopsies in Blantyre, Malawi. These comprised 42 acutely ill comatose patients, 32 of whom were diagnosed clinically as cerebral malaria and the other 10 as non-malarial diseases. Another 2 were non-malarial, non-comatose deaths. Other control tissues were from Australian adults.ResultsOf the 32 clinically diagnosed cerebral malaria cases, 11 had negligible histological change in the brain, and no or scanty intravascular sequestration of parasitised erythrocytes, another 7 had no histological changes in the brain, but sequestered parasitised erythrocytes were present (usually dense), and the remaining 14 brains showed micro-haemorrhages and intravascular mononuclear cell accumulations, plus sequestered parasitised erythrocytes. The vascular walls of the latter group stained most strongly for iNOS. Vascular wall iNOS staining was usually of low intensity in the second group (7 brains) and was virtually absent from the cerebral vascular walls of 8 of the 10 comatose patients without malaria, and also from control brains. The chest wall was chosen as a typical non-cerebral site encompassing a range of tissues of interest. Here pronounced iNOS staining in vascular wall and skeletal muscle was present in some 50% of the children in all groups, including septic meningitis, irrespective of the degree of staining in cerebral vascular walls. Parasites or malarial pigment were rare to absent in all chest wall sections. While MIF was common in chest wall vessels, usually in association with iNOS, it was absent in brain vessels.ConclusionsThese results agree with the view that clinically diagnosed cerebral malaria in African children is a collection of overlapping syndromes acting through different organ systems, with several mechanisms, not necessarily associated with cerebral vascular inflammation and damage, combining to cause death.

Plasmodium falciparum-infected erythrocytes induce Tissue Factor expression in endothelial cells and support the assembly of multimolecular coagulation complexes

Summary.  Background: Plasmodium falciparum malaria infects 300–500 million people every year, causing 1–2 million deaths annually. Evidence of a coagulation disorder, activation of endothelial cells (EC) and increase in inflammatory cytokines are often present in malaria. Objectives: We have asked whether interaction of parasitized red blood cells (pRBC) with EC induces tissue factor (TF) expression in vitro and in vivo. The role of phosphatidylserine‐containing pRBC to support the assembly of blood coagulation complexes was also investigated. Results: We demonstrate that mature forms of pRBC induce functional expression of TF by EC in vitro with productive assembly of the extrinsic Xnase complex and initiation of the coagulation cascade. Late‐stage pRBC also support the prothrombinase and intrinsic Xnase complex formation in vitro, and may function as activated platelets in the amplification phase of the blood coagulation. Notably, post‐mortem brain sections obtained from P. falciparum‐infected children who died from cerebral malaria and other causes display a consistent staining for TF in the EC. Conclusions: These findings place TF expression by endothelium and the amplification of the coagulation cascade by pRBC and/or activated platelets as potentially critical steps in the pathogenesis of malaria. Furthermore, it may allow investigators to test other therapeutic alternatives targeting TF or modulators of EC function in the treatment of malaria and/or its complications.

The systemic pathology of cerebral malaria in African children

Pediatric cerebral malaria carries a high mortality rate in sub-Saharan Africa. We present our systematic analysis of the descriptive and quantitative histopathology of all organs sampled from a series of 103 autopsies performed between 1996 and 2010 in Blantyre, Malawi on pediatric cerebral malaria patients and control patients (without coma, or without malaria infection) who were clinically well characterized prior to death. We found brain swelling in all cerebral malaria patients and the majority of controls. The histopathology in patients with sequestration of parasites in the brain demonstrated two patterns: (a) the “classic” appearance (i.e., ring hemorrhages, dense sequestration, and extra-erythrocytic pigment) which was associated with evidence of systemic activation of coagulation and (b) the “sequestration only” appearance associated with shorter duration of illness and higher total burden of parasites in all organs including the spleen. Sequestration of parasites was most intense in the gastrointestinal tract in all parasitemic patients (those with cerebral malarial and those without).

Liver pathology in Malawian children with fatal encephalopathy.

A common clinical presentation of Plasmodium falciparum is parasitemia, complicated by an encephalopathy for which other explanations cannot be found, termed cerebral malaria-an important cause of death in young children in endemic areas. Our objective was to study hepatic histopathology in Malawian children with fatal encephalopathy, with and without P falciparum parasitemia, to assess the contributions of severe malaria. We report autopsy results from a series of 87 Malawian children who died between 1996 and 2008. Among 75 cases with P falciparum parasitemia, 51 had intracerebral sequestered parasites, whereas 24 without sequestered parasites had other causes of death revealed by autopsy including 4 patients with clinicopathologic findings which may represent Reye syndrome. Hepatic histology in parasitemic cases revealed very limited sequestration of parasites in hepatic sinusoids, even in cases with extensive sequestration elsewhere, but increased numbers of hemozoin-laden Kupffer cells were invariably present with a strong association with histologic evidence of cerebral malaria by quantitative analysis. Of 12 patients who were consistently aparasitemic during their fatal illness, 5 had clinicopathologic findings which may represent Reye syndrome. Hepatic sequestration of parasitized erythrocytes is not a feature of fatal malaria in Malawian children, and there is no structural damage in the liver. Reye syndrome may be an important cause of fatal encephalopathy in children in Malawi with and without peripheral parasitemia and warrants close scrutiny of aspirin use in malaria-endemic areas.

Salicylates, nitric oxide, malaria, and Reye's syndrome

Reyes syndrome virtually disappeared from much of the world after the use of salicylate in febrile children was successfully discouraged. This severe sepsis-like disease was thought to be caused by a hypersensitivity to salicylates in children with mild viral infections, although no mechanism consistent with this proposal was ever established. Salicylate toxicity in African children has been noted to have many clinical features in common with severe falciparum malaria, including acidosis, altered consciousness, convulsions, and hypoglycaemia. Salicylates are widely available in various formulations in many African countries, and are commonly used for initial treatment of the symptoms that malaria shares with other diseases. There is now experimental evidence that salicylate increases and prolongs the activity of key elements along the signalling pathway through which interferon gamma generates inducible nitric oxide synthase (iNOS), and we have shown that iNOS is strongly expressed in fatal malaria and other acute fevers in African children. We further propose that, in areas where salicyaltes are still used to treat the symptoms of febrile illnesses in children, this mechanism could exacerbate potentially serious infectious diseases, including falciparum malaria. In contrast, the absence of salicylate use in children in some Pacific islands could contribute to the milder outcome of falciparum malaria than is observed in Africa. Widespread expression of iNOS has also been seen in the tissues of a patient with fatal clinically defined Reyes syndrome. This finding suggests that Reyes syndrome can be mediated through salicylate enhancement of iNOS expression, the initial trigger in this instance usually being a viral infection.

Fatal Pediatric Cerebral Malaria Is Associated with Intravascular Monocytes and Platelets That Are Increased with HIV Coinfection

ABSTRACT  Cerebral malaria (CM) is a major contributor to malaria deaths, but its pathophysiology is not well understood. While sequestration of parasitized erythrocytes is thought to be critical, the roles of inflammation and coagulation are controversial. In a large series of Malawian children hospitalized with CM, HIV coinfection was more prevalent than in pediatric population estimates (15% versus 2%, P < 0.0001, chi-square test), with higher mortality than that seen in HIV-uninfected children (23% versus 17%, P = 0.0178, chi-square test). HIV-infected (HIV+) children with autopsy-confirmed CM were older than HIV-uninfected children (median age, 99 months versus 32 months, P = 0.0007, Mann-Whitney U test) and appeared to lack severe immunosuppression. Because HIV infection is associated with dysregulated inflammation and platelet activation, we performed immunohistochemistry analysis for monocytes, platelets, and neutrophils in brain tissue from HIV+ and HIV-uninfected children with fatal CM. Children with autopsy-confirmed CM had significantly (>9 times) more accumulations of intravascular monocytes and platelets, but not neutrophils, than did children with nonmalarial causes of coma. The monocyte and platelet accumulations were significantly (>2-fold) greater in HIV+ children than in HIV-uninfected children with autopsy-confirmed CM. Our findings indicate that HIV is a risk factor for CM and for death from CM, independent of traditional measures of HIV disease severity. Brain histopathology supports the hypotheses that inflammation and coagulation contribute to the pathogenesis of pediatric CM and that immune dysregulation in HIV+ children exacerbates the pathological features associated with CM. IMPORTANCE There are nearly 1 million malaria deaths yearly, primarily in sub-Saharan African children. Cerebral malaria (CM), marked by coma and sequestered malaria parasites in brain blood vessels, causes half of these deaths, although the mechanisms causing coma and death are uncertain. Sub-Saharan Africa has a high HIV prevalence, with 3 million HIV-infected (HIV+) children, but the effects of HIV on CM pathogenesis and mortality are unknown. In a study of pediatric CM in Malawi, HIV prevalence was high and CM-attributed mortality was higher in HIV+ than in HIV-uninfected children. Brain pathology in children with fatal CM was notable not only for sequestered malaria parasites but also for intravascular accumulations of monocytes and platelets that were more severe in HIV+ children. Our findings raise the possibility that HIV+ children at risk for malaria may benefit from targeted malaria prophylaxis and that adjunctive treatments targeting inflammation and/or coagulation may improve CM outcomes. There are nearly 1 million malaria deaths yearly, primarily in sub-Saharan African children. Cerebral malaria (CM), marked by coma and sequestered malaria parasites in brain blood vessels, causes half of these deaths, although the mechanisms causing coma and death are uncertain. Sub-Saharan Africa has a high HIV prevalence, with 3 million HIV-infected (HIV+) children, but the effects of HIV on CM pathogenesis and mortality are unknown. In a study of pediatric CM in Malawi, HIV prevalence was high and CM-attributed mortality was higher in HIV+ than in HIV-uninfected children. Brain pathology in children with fatal CM was notable not only for sequestered malaria parasites but also for intravascular accumulations of monocytes and platelets that were more severe in HIV+ children. Our findings raise the possibility that HIV+ children at risk for malaria may benefit from targeted malaria prophylaxis and that adjunctive treatments targeting inflammation and/or coagulation may improve CM outcomes.

A histological method for quantifying Plasmodium falciparum in the brain in fatal paediatric cerebral malaria

BackgroundThe sequestration of Plasmodium falciparum-infected erythrocytes in brain microvasculature through cytoadherence to endothelium, is the hallmark of the definitive diagnosis of cerebral malaria and plays a critical role in malaria pathogenesis. The complex pathophysiology, which leads each patient to the final outcome of cerebral malaria, is multifaceted and thus, metrics to delineate specific patterns within cerebral malaria are needed to further parse patients.MethodsA method was developed for quantification utilizing counts of capillary contents (early-stage parasites, late-stage parasites and fibrin) from histological preparations of brain tissue after death, and compared it to the standard approach, in which the percentage of parasitized vessels in cross-section is determined.ResultsWithin the initial cohort of 50 patients, two different observers agreed closely on the percentage of vessels parasitized, pigmented parasites and pigment globules (ICC = 0.795-0.970). Correlations between observers for correct diagnostic classification were high (Kendall’s tau-b = 0.8779, Kappa = 0.8413). When these methods were applied prospectively to a second set of 50 autopsy samples, they revealed a heterogeneous distribution of sequestered parasites in the brain with pigmented parasites and pigment globules present in the cerebellum > cortex > brainstem. There was no difference in the distribution of early stages of parasites or in the percentage of vessels parasitized across the same sites. The second cohort of cases was also used to test a previously published classification and regression tree (CART) analysis; the quantitative data alone were able to accurately classify and distinguish cerebral malaria from non-cerebral malaria. Classification errors occurred within a subclassification of cerebral malaria (CM1 vs CM2). A repeat CART analysis for the second cohort generated slightly different classification rules with more accurate subclassification, although misclassification still occurred.ConclusionsThe traditional measure of parasite sequestration in falciparum malaria, the percentage of vessels parasitized, is the most reliable and consistent for the general diagnosis of cerebral malaria. Methods that involve quantitative measures of different life cycle stages are useful for distinguishing patterns within the cerebral malaria population; these subclassifications may be important for studies of disease pathogenesis and ancillary treatment.