Paediatric cerebral malaria (CM) is a frequently fatal complication of Plasmodium falciparum malaria that disproportionately afflicts children in sub-Saharan Africa; the WHO Malaria Report estimated that malaria killed 429,000 people worldwide in 2016, about 70% of whom were African children under 5 years of age (World Malaria Report 2016). CM is clinically defined as peripheral parasitaemia with coma not directly attributable to convulsions, hypoglycaemia, meningitis or any other identifiable cause (Newton et al., 1998). This definition is broad and is likely to over-diagnose a significant proportion of cases. The presence of a retinopathy known as malarial retinopathy (MR), and described by us with other colleagues (Lewallen et al., 1993; Hero et al., 1997; Beare et al., 2004; Harding et al., 2006), increases specificity when included in the diagnostic criteria (Taylor et al., 2004; Beare et al., 2011; Barrera et al., 2015).

Sequestration of parasitised red blood cells (pRBC) in the cerebral neurovasculature is the key underlying pathophysiological feature in P. falciparum CM (Turner, 1997). Unlike in the brain, the degree and location of neurovascular abnormalities can be observed clinically in the retina using routinely available ophthalmological techniques (Harding et al., 2006). Features comprise orange or white retinal vessels, patchy or confluent retinal whitening and white centred retinal haemorrhages (Figure 1). Severity of MR predicts the risk of death and duration of coma (Beare et al., 2004; Lewallen et al., 2008; Beare et al., 2006).

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The management of P. falciparum malaria is changing. The incidence has fallen but is notoriously difficult to enumerate. Clearly, malaria is still causing significant numbers of deaths each year despite widespread use of artesunate-based combination therapies and moves to improve the early diagnosis of CM in district general hospitals (World Malaria Report 2016). New diagnostic and therapeutic interventions are being developed and tested. Our group has developed an automated algorithm platform for detection of MR from colour photographs (Joshi et al., 2017).

We with other colleagues have previously reported descriptive pathological investigations of the features of MR (Lewallen et al., 2000), including clinical associations (White et al., 2009) and suggesting mechanisms. We have previously hypothesised that the orange vessels in the retina (Lewallen et al., 2000) and the intravascular material seen on fluorescein angiography may indicate sequestration (Beare et al., 2009), but definitive evidence is required. We have previously identified that retinal whitening is caused by capillary nonperfusion, but the relationship of this nonperfusion to sequestration is unclear.

We studied orange and white vessels and retinal whitening to understand sequestration and its effects on the retina, and to inform clinical management of CM. We addressed these complex questions in a large series of children with CM recruited over 15 years all of whom had retinal examinations (clinical dataset), and in two subgroups, one comprising children who died and from whom eyes were available for histopathology (clinicopathology dataset) and a second of children who underwent retinal angiography (image analysis dataset). Findings from other cohorts and subcohorts from our programme have been reported previously by our group, addressing other research questions. The further analysis of our clinical dataset is an extension of our previous association study, while all other analyses presented in this manuscript are new.

The clinicopathological findings from our unique cohort provide strong evidence that the orange appearance of retinal vessels in comatose children with a clinical diagnosis of CM is caused by sequestered late-stage pRBCs. Our dataset of clinical outcomes, the largest to date, and our independently graded angiographic data show that this visible sequestration is strongly associated with death, with an increased risk when arterioles are involved. The tissue effects of sequestration are widespread within the neurovascular unit, including novel findings of severe loss/disruption of pericytes. Retinal whitening, also strongly associated with death, is associated with features of cytotoxic oedema, consistent with sequestration causing ischaemia.

We used three datasets to investigate if the features seen clinically in the retina represent sequestration, which is the principal underlying pathophysiological event in P. falciparum malaria. Our data from 817 children point definitively to the importance of sequestration seen clinically as visible orange vessels, associated with a 2.71-fold increased odds of death. Our data add to previous work by us (Beare et al., 2004) but with greater confidence and with specific reference to orange vessels rather than all retinal vessel abnormalities.

The orange colour of the sequestered intravascular material appears to be a result of a mix of sequestered late-stage pRBCs (containing haemozoin) adherent to the endothelium, surrounding a central narrowed blood column consisting of uninfected RBCs. Our numbers of cases and controls are typical for this type of pathological study and the numbers of vessels sampled were high. Our findings add to those reported by some of us previously (Lewallen et al., 2000), which described dehaemoglobinised RBCs in sequestration, by adding new topographical clinicopathology data. We found that sequestration involving 360° of the circumference of the vessel lumen was strongly associated with the presence of orange discolouration clinically. We think orange vessels can be considered as an indication of severe sequestration and, as such, are clinically extremely valuable. This severe sequestration is easily visible with indirect ophthalmoscopy after pupil dilation. Less severe sequestration may be detectable with the newly available technology of hand-held optical coherence tomography (OCT) of the retina.

Retinal capillary involvement, in contrast to orange vessels, appears to be a phenomenon in CM not associated with death. Clinically, this is visible as white vessels and histologically predominantly as ruptured RBCs and extra-erythrocytic haemozoin, with no intact or non-parasitised RBCs. This feature was associated with non-perfusion on FA.

Our large fluorescein angiography (FA) study, which extended over eight seasons, shows that the retinal intravascular material was seen in nearly all MR-positive cases, especially the post-capillary and small venules (98.3% and 87.9% of gradeable vessels, respectively). These findings are novel, whereas our and others’ earlier data have been descriptive. The limitations of imaging in comatose young children mean that our grading method was unable to reliably identify capillaries, and so we were unable to robustly investigate the capillaries angiographically. We believe that capillary involvement is typical of pRBC sequestration in the neurovasculature. The presence of intravascular material in the arterioles was much less likely (pre-capillary 58.4%, small 43.9%, large 15.3%). However, arteriolar intravascular material was associated with longer recovery times (p<0.01- < 0.02) and greater risk of death, with involvement of the large arterioles conferring a 2.81-fold increased risk of death. It appears that the involvement of the arteriolar side can be taken as a clinical marker of severity, indicating a greater extent or load of sequestration. We have previously described the features of intraretinal material coining the term ‘intravascular filling defects’. This FA term can now be replaced by ‘retinal sequestration’.

We identified an association between sequestration and profound changes in the cells of the retinal neurovascular unit. These cells are critical to the preservation of BRB function (Kaur et al., 2008b) and the changes have important parallels in the brain, especially for swelling (Seydel et al., 2015). Reduced expression of CD34 in endothelial cells and of SMA and PDGRFβ in pericytes indicates significant dysfunction of both cell types. Our pericyte data are novel; pericytes have not been extensively studied in malaria before, with only one study reporting pericyte vacuolation in adult fatal CM (Pongponratn et al., 2003). Reduction of SMA immunoreactivity may be related to two pathological mechanisms: vessel dilatation with altered pericyte function, or pericyte loss. PDGF-signalling is critical for the survival of endothelium in physiological conditions (Armulik et al., 2005). Pericytes are highly sensitive to hypoxia (Kamouchi et al., 2012), especially in brain and retina where they are most abundant, and when vessels lose or develop abnormal pericytes they become hyperdilated, show signs of vessel dysfunction, and haemorrhage may occur (Bergers and Song, 2005). Within the MR cases, we found more retinal vessels with abnormal pericyte staining in those cases presenting with retinal haemorrhages than those cases without. The retina and brain present similar pathological features in CM, including haemorrhages (Greiner et al., 2015). We found the same significant loss of pericytic SMA and PDGFRβ in a further small analysis comparing brain microvessels in the presence of pRBC sequestration (median %, min-max% of vessels with SMA intact: 15%, 9–20%; PDGFRβ: 13%, 6–24%) with non-parasitaemic vessels (SMA intact: 92%, 79–100%; PDGFRβ: 96%, 91–100%) (p<0.001 for all) (n = 5; Barrera V et al, unpublished). These data suggest that retina and brain may have similar dysfunction/loss of pericytes in fatal paediatric CM.

We also identified effects on astrocytes and Müller cells indicating wider effects on neural retinal cells than previously identified. Late reactive (Hiscott et al., 1984) GFAP was upregulated, but the greater effect was seen for the early-responsive (Ortinski et al., 2010) perivascular ICAM-1 perhaps reflecting the short survival time of children with fatal CM. Our group has also previously identified upregulation of β-amyloid precursor protein as evidence of axonal damage (White et al., 2009). These neuroglial effects of retinal sequestration are likely to be widespread and include disturbance of tight junction regulation causing BRB/BBB breakdown with vasogenic oedema, an implicated pathway for brain swelling and death (Taylor et al., 2004; Mohanty et al., 2017).

Retinal whitening is a key feature of MR. Our finding of whitening at the fovea conferring a 3.4-fold increased risk of death strengthens our previous findings (Beare et al., 2004). We have previously shown that retinal whitening is topographically associated with capillary non-perfusion and is found in watershed zones of the retina, sites of high metabolic demand (Beare et al., 2009), suggesting that tissue hypoxia is a principal pathogenic pathway (White et al., 2009).

Our immunohistochemistry data provide further evidence that the inner retina is affected by tissue hypoxia and intracellular oedema. Ganglion cells showed increased expression of VEGFR1, which, in combination with VEGF, is neuroprotective during ischaemia (Saint-Geniez et al., 2008). Glia in retinal zones where whitening is mainly localised were found to express AQP4, a water channel protein linked to hypoxic oncotic swelling. This observation is supported by our previous electrophysiologcal study, which showed abnormal B wave implicit time indicating inner retinal dysfuction in retinal whitening (Lochhead et al., 2003). This all supports inner retinal neuronal ischaemia as opposed to dysfunction of the outer retinal photoreceptors and choroidal circulation. Further studies with the OCT may shed new light on the retinal whitening.

We have some conflicting evidence on the importance of capillary non-perfusion (CNP). There are undoubted tissue effects of sequestration-induced hypoxia in the vessel and extending into the neuroretina causing tissue swelling and opacification. However, the whitening seen in capillaries was not associated with death, and sequestration seen in the post-capillary venules on FA, a frequent association with CNP, showed a trend for survival. Sequestration in post-capillary venules is more common than arterioles, and this suggests these children as a group were not as critically ill as those with sequestration extending additionally into arterioles.

So how can our findings affect the clinic management and future research directions in CM? The detection of orange vessels on clinical examination has a high sensitivity and specificity for a severe degree of sequestration, which is associated with death. Sequestration detectable on FA in the arterioles, and especially the large arterioles, is also predictive of death and probably indicates a high parasite load. Orange vessels can be seen clinically with the indirect and direct ophthalmoscope through a dilated pupil by a trained physician (Taylor et al., 2004), but these skills are mainly available in research or tertiary centres in malaria endemic areas (Swamy et al., 2018). We have with others recently developed MR detection algorithms offering a potential automated diagnostic tool for severe malaria in district hospitals (Joshi et al., 2017). Our new clinical markers of severe disease and poor outcome (visible orange vessels and arteriolar involvement indicating severe sequestration, and severe foveal whitening) should be a focus for diagnosis and management. It should be recognised that including children without MR in clinical trials is likely to reduce their power to detect an effect of an intervention on CM outcomes.

There is good evidence that the clinicopathological features of CM in the retina parallel those seen in the brain (Barrera et al., 2015; MacCormick et al., 2014): ring-shaped haemorrhages (White et al., 2009; Dorovini-Zis et al., 2011), pathology of sequestration, associations between retinal features and that key neurological pathways seem to be non-functioning ('pathways to neural cell death'). Mendis K and others (Mendis et al., 2009) have argued that the long-term goal of eliminating malaria remains dependent on continuing research and the development of new drugs and therapeutic strategies to sustain control programmes. Better identification and treatment of severe malaria will also be needed. Our findings from manual and semiautomated image analysis provide an indication that quantification of the load of retinal sequestration is promising as a useful metric in clinical trials and merits further development to identify a severity cut-off.

The results we have presented in this paper from our long-term programme of research strongly support the concept that sequestration can be identified clinically in the retina at the bedside, and offer important new insights into the widespread effects of sequestration on the neural microvasculature and cells of the neurovascular unit. This sequestration can be seen in clinical practice at a critical time in the management of the comatose child in malaria endemic areas offering opportunities to study the effects of new therapies, as well as an early concrete diagnosis and a marker of severe disease.

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Author details

1. Valentina Barrera

   Department of Eye and Vision Science, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing

   Contributed equally with

   Ian James Callum MacCormick

   For correspondence

   v.barrera@liverpool.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0515-5901

2. Ian James Callum MacCormick

   1. Department of Eye and Vision Science, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom
   2. Malawi-Liverpool-Wellcome Trust Clinical Research Programme, College of Medicine, Blantyre, Malawi

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing

   Contributed equally with

   Valentina Barrera

   Competing interests

   No competing interests declared

3. Gabriela Czanner

   1. Department of Eye and Vision Science, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom
   2. Department of Biostatistics, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Validation, Methodology, Writing—review and editing, oversaw data quality and statistical analyses

   Competing interests

   No competing interests declared

4. Paul Stephenson Hiscott

   Department of Eye and Vision Science, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom

   Contribution

   Conceptualization, Data curation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

5. Valerie Ann White

   1. Department of Pathology and Laboratory Medicine, University of British Columbia and Vancouver General Hospital, Vancouver, Canada
   2. Department of Ophthalmology and Visual Science, University of British Columbia and Vancouver General Hospital, Vancouver, Canada

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

6. Alister Gordon Craig

   Liverpool School of Tropical Medicine, Liverpool, United Kingdom

   Contribution

   Conceptualization, Resources, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

7. Nicholas Alexander Venton Beare

   1. Department of Eye and Vision Science, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom
   2. St Paul’s Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom

   Contribution

   Conceptualization, Data curation, Validation, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

8. Lucy Hazel Culshaw

   Department of Eye and Vision Science, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom

   Contribution

   Formal analysis, Writing—review and editing

   Competing interests

   No competing interests declared

9. Yalin Zheng

   Department of Eye and Vision Science, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom

   Contribution

   Conceptualization, Data curation, Formal analysis, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

10. Simon Charles Biddolph

    National Specialist Ophthalmic Pathology Service, Royal Liverpool University Hospital, Liverpool, United Kingdom

    Contribution

    Data curation, Investigation, Writing—review and editing

    Competing interests

    No competing interests declared

11. Danny Arnold Milner

    Center for Global Health, American Society for Clinical Pathology, Chicago, United States

    Contribution

    Conceptualization, Resources, Validation, Investigation, Methodology, Writing—review and editing

    Competing interests

    No competing interests declared

12. Steve Kamiza

    Department of Histopathology, College of Medicine, University of Malawi, Blantyre, Malawi

    Contribution

    Conceptualization, Resources, Methodology, Writing—review and editing

    Competing interests

    No competing interests declared

13. Malcolm Edward Molyneux

    1. Malawi-Liverpool-Wellcome Trust Clinical Research Programme, College of Medicine, Blantyre, Malawi
    2. Liverpool School of Tropical Medicine, Liverpool, United Kingdom

    Contribution

    Conceptualization, Resources, Data curation, Funding acquisition, Methodology, Project administration, Writing—review and editing

    Competing interests

    No competing interests declared

14. Terrie Ellen Taylor

    1. Blantyre Malaria Project, College of Medicine, University of Malawi, Blantyre, Malawi
    2. Department of Osteopathic Medical Specialties, College of Osteopathic Medicine, Michigan State University, East Lansing, United States

    Contribution

    Conceptualization, Resources, Data curation, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing—review and editing

    Competing interests

    No competing interests declared

15. Simon Peter Harding

    1. Department of Eye and Vision Science, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom
    2. St Paul’s Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom

    Contribution

    Conceptualization, Resources, Data curation, Formal analysis, Funding acquisition, Validation, Investigation, Writing—original draft, Project administration, Writing—review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-4676-1158

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