Severity of retinopathy parallels the degree of parasite sequestration in the eyes and brains of malawian children with fatal cerebral malaria.

BACKGROUND: Malarial retinopathy (MR) has diagnostic and prognostic value in children with Plasmodium falciparum cerebral malaria (CM). A clinicopathological correlation between observed retinal changes during life and the degree of sequestration of parasitized red blood cells was investigated in ocular and cerebral vessels at autopsy. METHODS: In 18 Malawian children who died from clinically defined CM, we studied the intensity of sequestration and the maturity of sequestered parasites in the retina, in nonretinal ocular tissues, and in the brain. RESULTS: Five children with clinically defined CM during life had other causes of death identified at autopsy, no MR, and scanty intracerebral sequestration. Thirteen children had MR and died from CM. MR severity correlated with percentage of microvessels parasitized in the retina, brain, and nonretinal tissues with some neuroectodermal components (all P < .01). In moderate/severe MR cases (n = 8), vascular congestion was more intense (ρ = 0.841; P < .001), sequestered parasites were more mature, and the quantity of extraerythrocytic hemozoin was higher, compared with mild MR cases (n = 5). CONCLUSIONS: These data provide a histopathological basis for the known correlation between degrees of retinopathy and cerebral dysfunction in CM. In addition to being a valuable tool for clinical diagnosis, retinal observations give important information about neurovascular pathophysiology in pediatric CM.

Ocular Pathology of Cerebral Malaria.

Investigation of post-mortem eyes from children with malarial retinopathy has helped to explain the retinal pathology of cerebral malaria, and also demonstrated histological associations between evolving retinal pathogenesis-visible clinically-and similar cerebral features which can only be examined at autopsy. The pathology of malarial retinopathy has been well-described and correlates with brain pathology. Some clinical and pathological features are associated with outcome. This chapter describes the materials and methods needed to study the pathological features of malarial retinopathy. Some are common to histopathology in general, but accurate spatial correlation between retinal features observed in life and their associated pathology in post-mortem specimens requires special techniques.

Investigating Interactions Between Endothelial Cells and Parasitized Red Blood Cells in Skin and Subcutaneous Tissue.

In this chapter we present the methods for using biopsies of skin or subcutaneous tissue to examine the interactions between parasitized red blood cells and endothelial cells in patients with malaria infection. Punch biopsy can be used to obtain all skin layers and needle biopsy to obtain subcutaneous tissue. Smears are useful for spreading vessels on a slide for immunofluorescence staining. Specimens can be fixed and embedded for sectioning and traditional histological or immunostaining techniques or confocal microscopy with three-dimensional reconstruction. Finally, endothelium can be dissociated, allowing individual cells to be isolated for culture and ex vivo assays or used for immunophenotyping.

Histological Identification of Sequestered Parasitized Red Cells.

The performance of complete post-mortem examinations of children with severe malaria has helped to explain the cause of death in cerebral malaria as well as show the global phenomenon of sequestration in tissues throughout the body, beyond the brain and eye. The pathology of the brain and other organs has been well described and shows a systemic disease with the most catastrophic features found in the brain (i.e., fatal cerebral edema).This chapter describes the materials and methods needed to study the pathological features of tissues outside of the eye, including the brain and other organs. The bulk of these techniques are standard to pathology including gross examination, histology, special stains, and immunohistochemistry.

Bridging and Clumping: Investigating Platelet Interactions with P. falciparum-Infected Red Blood Cells and Endothelial Cells in Cerebral Malaria.

The methods presented in this chapter describe how to perform ex vivo clumping and in vitro bridging assays in the context of cerebral malaria. Both the protocols are detailed, and emphasis is made on how to prepare platelet suspensions suitable to each technique, including description of specific buffers and reagents to minimize the risk of aggregation while maintaining the platelet properties.

Measuring the Impact of Malaria on the Living Human Retina.

Retinal examination and imaging are relatively simple methods for studying the dynamic impact of cerebral malaria on the microcirculation of the central nervous system. Retina and brain are affected similarly by Plasmodium falciparum. Unlike the brain, the human retina can be directly observed using commercially available clinical instruments in the setting of a critical care unit, and this can be done repeatedly and non-invasively. Additional information about blood-tissue barriers can be gained from fluorescein angiography. Non-ophthalmologist clinician scientists are usually unfamiliar with ophthalmoscopy and retinal imaging, and some readers may feel that these techniques are beyond them. This chapter aims to quell these fears by providing a step-by-step description of how to examine and photograph the human retina in children with cerebral malaria.

A proposed theoretical framework for retinal biomarkers.

Objective: Propose a theoretical framework for retinal biomarkers of Alzheimer's disease (AD). Background: The retina and brain share important biological features that are relevant to AD. Developing retinal biomarkers of AD is a strategic priority but as yet none have been validated for clinical use. Part of the reason may be that fundamental inferential assumptions have been overlooked. Failing to recognize these assumptions will disadvantage biomarker discovery and validation, but incorporating them into analyses could facilitate translation. New theory: The biological assumption that a disease causes analogous effects in the brain and retina can be expressed within a Bayesian network. This allows inferences about abstract theory and individual events, and provides an opportunity to falsify the foundational hypothesis of retina-brain analogy. Graphical representation of the relationships between variables simplifies comparison between studies and facilitates judgements about whether key assumptions are valid given the current state of knowledge. Major challenges: The framework provides a visual approach to retinal biomarkers and may help to rationalize analysis of future studies. It suggests possible reasons for inconsistent results in existing literature on AD biomarkers. Linkage to other theories: The framework can be modified to describe alternative theories of retinal biomarker biology, such as retrograde degeneration resulting from brain disease, and can incorporate confounding factors such as co-existent glaucoma or macular degeneration. Parallels with analogue confirmation theory and surrogate marker validation suggest strengths and weaknesses of the framework that can be anticipated when developing analysis plans. Highlights: Retinal biomarkers hold great promise for Alzheimer's disease (AD), but none are currently used clinically.Assumptions about the cause of retinal and brain changes are often overlooked, and this may disadvantage biomarker discovery and validation.We present a new approach to retinal biomarkers that describes cause and effect graphically in a Bayesian network.We show how this allows a more complete assessment of how well a biomarker might reflect the brain, and how data from right and left eyes can be used to rule out poor biomarker candidates.

Neurovascular sequestration in paediatric P. falciparum malaria is visible clinically in the retina.

Retinal vessel changes and retinal whitening, distinctive features of malarial retinopathy, can be directly observed during routine eye examination in children with P. falciparum cerebral malaria. We investigated their clinical significance and underlying mechanisms through linked clinical, clinicopathological and image analysis studies. Orange vessels and severe foveal whitening (clinical examination, n = 817, OR, 95% CI: 2.90, 1.96-4.30; 3.4, 1.8-6.3, both p<0.001), and arteriolar involvement by intravascular filling defects (angiographic image analysis, n = 260, 2.81, 1.17-6.72, p<0.02) were strongly associated with death. Orange vessels had dense sequestration of late stage parasitised red cells (histopathology, n = 29; sensitivity 0.97, specificity 0.89) involving 360° of the lumen circumference, with altered protein expression in blood-retinal barrier cells and marked loss/disruption of pericytes. Retinal whitening was topographically associated with tissue response to hypoxia. Severe neurovascular sequestration is visible at the bedside, and is a marker of severe disease useful for diagnosis and management.