1. Human Biology and Medicine
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Neurovascular sequestration in paediatric P. falciparum malaria is visible clinically in the retina

  1. Valentina Barrera  Is a corresponding author
  2. Ian James Callum MacCormick
  3. Gabriela Czanner
  4. Paul Stephenson Hiscott
  5. Valerie Ann White
  6. Alister Gordon Craig
  7. Nicholas Alexander Venton Beare
  8. Lucy Hazel Culshaw
  9. Yalin Zheng
  10. Simon Charles Biddolph
  11. Danny Arnold Milner
  12. Steve Kamiza
  13. Malcolm Edward Molyneux
  14. Terrie Ellen Taylor
  15. Simon Peter Harding
  1. University of Liverpool, United Kingdom
  2. College of Medicine, Malawi
  3. University of British Columbia and Vancouver General Hospital, Canada
  4. Liverpool School of Tropical Medicine, United Kingdom
  5. Royal Liverpool University Hospital, United Kingdom
  6. American Society for Clinical Pathology, United States
  7. University of Malawi, Malawi
  8. Michigan State University, United States
Research Article
Cite this article as: eLife 2018;7:e32208 doi: 10.7554/eLife.32208
13 figures, 6 tables and 2 additional files

Figures

Figure 1 with 1 supplement
Principal features of malarial retinopathy (MR).

(A) Montage image showing MR pathological features, including orange vessels (asterisks), white centred haemorrhages and whitening. (B) Corresponding fluorescein angiogram showing capillary nonperfusion (asterisks) mapping to retinal whitening. (C–D) Colour fundus image of retinopathy positive eyes (C, right; D, left eye; eyes were from different cases) showing orange intravascular material in large (arrowheads), small and postcapillary venules (asterisks), and capillaries; note retinal whitening also present.

https://doi.org/10.7554/eLife.32208.002
Figure 1—figure supplement 1
Flow chart describing clinical dataset.
https://doi.org/10.7554/eLife.32208.003
Vessel changes in malarial retinopathy.

(A–B) Vessel colour changes (panels A-B) and intravascular filling defects (panel B, arrowheads) were identified during gross pathology examination (representative images of superior calotte and PO block from histology cases n. 5 and 7, respectively) N = 12. Abnormal vessels were sampled during gross pathology examination and analysed separately (see marked quadrant in panel A). (C–D) H and E staining of parasitised venules from MR cases sampled by punch biopsies from a retinal quadrant with (panel C shows the same orange vessel as in panel A) and without (panel D, case n. 15) vessel discolouration. (C) The margin of the vessel lumen has a near-complete layer of pigment-containing pRBCs (that stain less intensely pink than the adjacent non-parasitised RBC) on the endothelium. (D) Mild sequestration of pRBCs which is marked by an arrowhead. Scale bars (50 μm, (C–D).

https://doi.org/10.7554/eLife.32208.007
Figure 3 with 1 supplement
Severe pRBC sequestration in large venules and arterioles in MR with visible vessel discolouration.

(A–B) Longitudinal section of large retinal venule from retinal area affected by intravascular filling defects on fluorescein angiography (histopathology case no. 9) analysed by H&E staining (A) and anti-fibrinogen IHC (B). Clusters of pRBC are seen within the vessel lumen and attached to the wall. (C) Cross section of a large retinal arteriole with moderate pRBC sequestration (case n. 5). Arteriole is surrounded by haemorrhage, probably of a venular origin as arteriolar vessel wall appeared intact (in multiple sections). Scale bars: 50 μm (all panels).

https://doi.org/10.7554/eLife.32208.008
Figure 3—figure supplement 1
Detection of thrombi in post-mortem retinal periphery using a combination of MSB staining (panels A,B; arrows: intravascular thrombi are stained bright pink), and anti-CD61 platelet marker immunostaining (panel C, red stained).

Scale bars: 50 μm.

https://doi.org/10.7554/eLife.32208.009
Vascular changes in retinal vessels in malarial retinopathy.

(A–I) Expression of endothelial CD34 (panel A: case n. 3, inset: case n. 25 and D: box plot), pericytic SMA (panel B: case n. 12, inset: case n. 27 and E: box plot) and pericytic PDGFRβ (panel C: case n. 13, inset: case n. 26 and F: box plot) markers. Insets show normal annular staining in absence of pRBC sequestration, whereas this annular pattern is lost in the sequestrated vessels seen in A-C. SMA was only reported for venules as it does not produce an annular staining pattern in normal capillaries: panel E. N = 17 for CD34; N = 29 for SMA and PDGFRβ immunostaining. ANOVA was used to compare means. **p<0.005. Scale bars: 20 μm (A–C), 5 μm (insets).

https://doi.org/10.7554/eLife.32208.011
Figure 4—source data 1

Vascular changes in retinal vessels inmalarial retinopathy.

https://doi.org/10.7554/eLife.32208.012
Activation of retinal glial cells in malarial retinopathy (MR).

(A–B) Anti-ICAM-1 staining of MR-positive cases with (case n. 16, panel A) and without (case n. 13, panel B) vessel discolouration. Haematoxylin (blue) counterstain was used. (C–D) Anti-GFAP staining of orange-discoloured vessels in punch biopsy from MR-positive case n. 5, and in MR-negative case n. 25. Haematoxylin counterstaining was omitted here. In A and C, peri-vascular activated astrocytes and Műller cells are marked with arrowheads, and asterisks label Műller cell bodies. Scale bars: 50 μm (all panels).

https://doi.org/10.7554/eLife.32208.013
Clinicopathological association between retinal whitening in the macula and increased VEGFR1 expression in malarial retinopathy.

(A–B) Immunostaining pattern in macula affected by whitening (case no 9) (low (A) and high (B) magnification; VEGFR1 +ve ganglion cell bodies indicated by arrowheads). (C) Cluster column chart showing densitometrically assessed intensity of immunoreactivity (‘value’) of VEGFR1 expression plotted by retinal layer against whitening severity, compared with MR –ve cases. Ganglion cell layer = GCL (blue); inner plexiform layer = IPL (green); inner nuclear layer = INL (light brown); outer plexiform layer = OPL (purple). (D) VEGFR1 levels in the GCL plotted against MR severity classification groups (grade 0 = none, 1 = mild, two moderate/severe). Means ± SD are reported in both charts; ANOVA was used to compare means (N = 26). *p≤0.05 and **p≤0.001. Scale bars: 50 μm (panel A); 20 μm (panel B).

https://doi.org/10.7554/eLife.32208.014
Figure 6—source data 1

Clinicopathological association between retinal whitening in the macula and increased VEGFR1 expression inmalarial retinopathy.

https://doi.org/10.7554/eLife.32208.015
Figure 7 with 1 supplement
Clinicopathological association between retinal whitening in the macula and increased AQP4 expression in malarial retinopathy.

(A–C) Immunostaining pattern in the macula with (A-B, case no 13) and without whitening (C, case no 21). Parasitised vessels are marked by arrows. The vertical linear pattern indicates Műller cell immunoreactivity for AQP4. (D) Cluster column chart showing densitometrically assessed intensity of immunoreactivity (‘value’) of AQP4 levels measured by IHC in the macula by retinal layers: nerve fibre layer = NFL (red), ganglion cell layer = GCL (blue), inner plexiform layer = IPL (green), outer plexiform layer = OPL (purple). (E): AQP4 levels in the nerve fibre layer plotted against MR severity classification groups (grade 0 = none, 1 = mild, two moderate/severe). Means ± SD are reported in all graphs; ANOVA was used to compare means (N = 26). *p<0.05 and **p<0.001. Scale bars: 50 μm (panels C, E, F and G); 10 μm (panel D).

https://doi.org/10.7554/eLife.32208.016
Figure 7—source data 1

Clinicopathological association between retinal whitening in the macula and increased AQP4 expression inmalarial retinopathy.

https://doi.org/10.7554/eLife.32208.018
Figure 7—figure supplement 1
Clinicopathological association between retinal whitening in the peripheral retina and increased AQP4 expression in malarial retinopathy.

(A–B) Immunostaining pattern is shown in MR-positive case with whitening (A, case n. 13) and MR-negative case (B, case n. 23). Parasitised vessels are marked by arrows. The vertical linear pattern indicates Műller cell immunoreactivity for AQP4. (C) Cluster column chart showing densitometrically assessed intensity of immunoreactivity (‘value’) of AQP4 levels measured by IHC in the peripheral retina by retinal layers: nerve fibre layer = NFL (red); ganglion cell layer = GCL (blue); inner plexiform layer = IPL (green); outer plexiform layer = OPL (purple). (D) AQP4 levels in the nerve fibre layer plotted against MR severity classification groups (grade 0 = none, 1 = mild, two moderate/severe). For details on grading zones see Appendix 2. Means ± SD are reported in all graphs; ANOVA was used to compare means (N = 26). *p≤0.05 and **p≤0.001. Scale bars: 50 μm (panels A-B).

https://doi.org/10.7554/eLife.32208.017
Figure 7—figure supplement 1—source data 1

Clinicopathological association between retinal whitening in the peripheral retina and increased AQP4 expression inmalarial retinopathy.

https://doi.org/10.7554/eLife.32208.019
Flow chart describing fluorescein angiography dataset.
https://doi.org/10.7554/eLife.32208.020
Visible sequestration in the retinal neurovasculature.

(A–D): Orange intravascular material is seen in the retinal venule (A, C) which co-localises to the intravascular filling defects on fluorescein angiography (D) (see arrows). Chart (B) shows the frequency of visible sequestration in six microvessel types in 259 subjects with retinopathy +ve CM and the odds ratios of death within the admission.

https://doi.org/10.7554/eLife.32208.021
Figure 9—source data 1

Visible sequestration in the retinal neurovasculature.

https://doi.org/10.7554/eLife.32208.022
Semiautomated quantitative analysis of sequestration by length of affected vessel.

(A) Example image of semiautomated system to show vessels affected by sequestration (red). (B) Chart showing distribution of proportion of detected vessel affected by sequestration related to survival in 251 eyes (one eye per case).

https://doi.org/10.7554/eLife.32208.024
Figure 10—source data 1

Semiautomated quantitative analysis of sequestration by length of vessel involved.

https://doi.org/10.7554/eLife.32208.025
Appendix 1—figure 1
Orientation and topographical association in whole eye histology blocks (A) and in eye sections (B), used to perform correlation studies between fundal images and histology.
https://doi.org/10.7554/eLife.32208.030
Appendix 1—figure 2
Panel A: retinal structure on light microscopy (H&E staining).

Panel B shows the specific feature of >1 cell thickness in the ganglion cell layer, used to identify the macula.

https://doi.org/10.7554/eLife.32208.031
Appendix 2—figure 1
Retinal zones used for clinical grading.
https://doi.org/10.7554/eLife.32208.033

Tables

Table 1
Associations with death in 817 subjects with admission retinal exam and retinopathy-positive paediatric cerebral malaria, 137 of whom died and 680 survived.

Retinal features are presented for the worse eye. Estimates are from unadjusted logistic regression. p≤0.01 is bold.

https://doi.org/10.7554/eLife.32208.004

Variable name
UnitsDiedSurvivedAssociation with death
Numerical characteristicsNumerical characteristicsOR95% CIp
Demographics
Age
(median, IQR)
months3523–591363927–58.756800.990.00–1.000.43
Weight
(median, IQR)
kg119–151371210–156800.970.93–1.020.22
Height
(median, IQR)
cm8979–1031359283–1036710.990.98–1.000.15
Sex
(%)
boy48.96650.296801.060.73–1.530.77
girl51.16949.71
Clinical
Coma score
(%)
023.3329.85673.572.13–5.88<0.001
141.65737.72562.131.28–3.570.003
235.04852.5357reference
Respiratory distress
(%)
Present48.96739.02651.51.04–2.170.03
Absent51.17061.0415
Convulsions at admission (%)Present12.41714.9980.830.45–1.440.51
Absent87.612085.4574
Temperature
(median, IQR)
degrees C38.737.8–39.513738.938–39.76800.890.77–1.030.12
Systolic BP
(median, IQR)
mmHg10090–11012710090–1106520.990.99–1.010.63
Pulse
(median, IQR)
beats/min156136.5–170.5137152136.75–1696781.00.99–1.010.98
Duration of coma
(median, IQR)
Hours74–1811074–175580.990.98–1.010.29
Duration of fever
(median, IQR)
Hours4833.25–721306043.25–726520.990.99–1.000.09
Hypoglycaemia on ward (%)Present14.6207.81532.021.16–3.50.012
Absent85.411792.1626
Laboratory
Parasitaemia
(median, IQR)
#cells7905216695–3570001346807611700–2980006491.00.99–1.000.27
White cell count
(median, IQR)
#cells113006925–1822512092006600–137256301.01.00–1.000.004
Haematocrit
(median, IQR)
%19.515–24.751362015.8–256730.990.97–1.020.69
Lactate
(median, IQR)
mmol/L8.755.38–12.78925.33.2–9.95191.111.06–1.16<0.001
HRP2
(median, IQR)
ng/ml8838.54435.5–15102.312057652471.5–100316091.01.00–1.000.004
HIV
(%)
Positive22.52914.9881.661.03–2.660.036
Negative77.510085.1503
Ophthalmoscopy
Retinal haemorrhage
(%)
>5016.0224.7323.41.78–6.5<0.001
21 to 5011.0156.50441.690.85–3.340.14
6 to 2013.11819.01290.690.38–1.270.23
1 to 532.94542.92910.760.48–1.230.27
None27.03727.0183reference
Macular whitening
(%)
>123.93214.81002.311.16–4.590.017
1/3 to 128.43825.11701.610.83–3.120.16
<1/337.35045.23061.180.63–2.220.61
None10.51414.9101reference
Foveal whitening
(% of foveal zone)
>2/323.33111.5783.391.83–6.26<0.001
1/3 to 2/318.12415.21031.991.05–3.740.03
<1/342.85746.83161.540.90–2.620.11
none15.82126.5179reference
Temporal whitening
(%)
310.01312.9870.830.41–1.660.60
224.63218.41241.430.83–2.470.20
141.55443.12901.030.64–1.670.89
none23.93125.6172reference
Orange vessels, temporal quadrant (%)present44.65821.71452.91.96–4.3<0.001
absent55.47278.3523
White vessels, temporal quadrant (%)present25.43324.31621.060.69–1.640.78
absent74.69775.8506
White capillaries
(%)
present26.93533.12210.750.49–1.130.17
absent73.19566.9447
Papilloedema
(%)
present39.05321.81482.291.55–3.38<0.001
absent61.08378.2530
Disc hyperaemia
(%)
present48.75435.32121.731.15–2.610.008
absent51.45764.7388
Table 2
Summary of clinicopathology dataset.
https://doi.org/10.7554/eLife.32208.005
Clinicopathological investigation (per MR feature)Number of cases analysedNumber of retinal layers analysedNumber of vessels counted
Vessel changes (H and E; GFAP; FGN; ICAM-1)
PO block analysis27--100
Calotte analysis6--100
Punch biopsies4--50
Retinal whitening (VEGFR1; AQP4)
Macular analysis204--
Peripheral retinal analysis214--
Table 3
Retinal pathological features and scores for 29 study subjects in the clinicopathology dataset
https://doi.org/10.7554/eLife.32208.006

Case n.
MR* GradeEyeVessel changes
(Q)Vessels§Localization#HaemMacular whitening¥Central retinal whitening (overall score)**Peripheral whitening (score)Whitening: retinal quadrantsPapill- oedemah†† (score)
12RE4 QVen + CapAll quadrants>501/3–1 DA434 Q2
22RE4 QVen + CapAll quadrants1–5≥1 DA634 Q2
32LE4 QVenAll quadrants1–5≥1 DA61.754 Q2
42RE4 QVenAll quadrants>501/3–1 DA51.5T + N0
52RE3 QVen + CapT + N + S1–5≥1 DA62.7T + N + S0
62RE2 QVenT + S>50<1/3 DA20.75T + S2
72LENoneNone06–20≥1 DA60.25T2
82RENoneNone00≥1 DA624 Q2
92LE4 QVen + CapAll quadrants0≥1 DA614 Q0
102LENoneNone021–501/3–1 DA41.54 Q0
112LE3 QNANA0≥1 DA424 Q0
122LENoneNone06–201/3–1 DA4002
132LENoneNone01–5≥1 DA614 Q0
142RENANANA1–51/3–1 DA4NANA2
152LE3 QVen + CapT + N + S1–5<1/3 DA20.7T + N + S0
162LE3 QVenT + N + S1–5<1/3 DA20.5I + N0
171RE1 QNone00<1/3 DA21T + S2
181RE1 QCapT0<1/3 DA214Q0
191RENoneNone01–5<1/3 DA21T + N0
201LENoneNone01–5<1/3 DA20NA0
211LENoneNone0NoneNone00.2500
220RENoneNone0NoneNone0000
230LENoneNone0NoneNone0000
240RENoneNone0NoneNone0000
250LENoneNone0NoneNone0000
260LENoneNone0NoneNone0000
270RENoneNone0NoneNone0000
280LENoneNone0NoneNone0000
290RENoneNone0>50None0000
  1. *MR = malarial retinopathy. Grade was defined based on percentage of retinal vessels with sequestration (Beare et al., 2004) as explained in Methods. Last peripheral parasitaemia (expressed as asexual pRBCs/μl blood), geometric means reported) was: 42,200 (Grade 0), 43,212 (Grade 1) and 9357 (Grade 2).

    Eye: RE = right eye; LE = left eye vessel changes:

  2. (Q)=number of retinal quadrants affected.

    §Vessels: Ven = venules; Cap = capillaries.

  3. #Localisation of vessel changes: I = inferior; N = Nasal; S = superior; T = temporal.

    Haem = no. of retinal haemorrhages.

  4. ¥Extent of whitening is shown for macula in disc areas (DA).

    **Central whitening (overall score)=sum of macular and foveal whitening scores assigned as: 1 =<1/3 DA or FA, 2 = 1/3–1 DA or 1/3-2/3FA, 3 =>1 DA or >2/3FA.

  5. ††Papilloedema is the swelling of optic disc caused by increased intracranial pressure. The significance of papilloedema in cerebral malaria is not clear; however, it is the strongest risk factor for poor outcome among comatose children with clinical cerebral malaria.

Table 4
Relationship between severe sequestration (pigmented/late parasitised RBCs sequestered around 360° of the lumen circumference) and orange discoloration visible clinically and on gross pathology in 412 venules (diameter 10–50 μm) from nine cases
https://doi.org/10.7554/eLife.32208.010
Orange discolouration
+-
Severe sequestration+1885
-24195
Table 5
Frequency of intravascular filling defects (worse eye) on fluorescein angiography manual grading by involvement of retinal vessel in 259 children with MR-positive disease and FA within 24 hr of admission and unadjusted association with death (n = 35) and coma recovery of consciousness (BCS ≥3; n = 225)
https://doi.org/10.7554/eLife.32208.023

Retinal vessel
SequestrationDied*Survived*Association with death
N%TotalN%TotalOR95% CIp
large venulespresent2686.73017279.32171.700.56–5.120.35
absent413.34520.7
small venulespresent2996.73021198.12150.880.71–1.090.23
absent13.3341.86
post-capillary venulespresent2596.22620198.52040.370.04–3.700.4
absent13.8531.47
pre-capillary arteriolespresent1976.02510956.21942.470.94–6.450.065
absent624.08543.8
small arteriolespresent1551.7299342.92171.430.66–3.110.37
absent1448.312457.1
large arteriolespresent930.0302913.22192.811.17–6.720.02
absent2170.019086.8
Key resources table
Antibodies used for immunohistochemistry analysis of the clinicopathology dataset
https://doi.org/10.7554/eLife.32208.026
AntigenSpecificityMR featureManufacturer
(clone); RRID*
Host (class)Ag retrievalDilution§Chromogen#Staining
quantification
Ref
VEGFR1Retinal cellRetinal whitening
Tissue effects
Abcam (Y103);
AB_778798
Rb mAb (IgG)Heat (High pH)1:2,000, 30 min RTDABAutomated(Kaur et al., 2008a)
Aquaporin 4 (AQP4)NeurogliaRetinal whitening
Tissue effects
Intracellular oedema
Abcam (EPR7040);
AB_11143780
Rb mAb (IgG)Heat (Low pH)1:500, 60 min RTAECAutomated(Medana et al., 2011)
Glial fibrillaryacidicprotein (GFAP)Neuroglia
(late activation)
Vessel discolourationDako;
AB_10013482
Rb pAbProteinase K1:2,000, o.n. 4°CAECManual(Hiscott et al., 1984)
ICAM-1Endothelium
Neuroglia(early
activation)
Vessel discolourationAbcam (EP1442Y);
AB_870702
Rb mAb (IgG)Heat (High pH)1:100, 30 min RTDABManual(Lee et al., 2000)
CD61Platelets and
precursors
Retinal whitening
Vessel discolouration
Thermo Scientific;
AB_929194
Ms mAb (IgG1)Heat (High pH)1:100, 32 min RTDAB or AECManual(White et al., 2009)
CD34 (II)EndotheliumVessel discolourationDako (QBEnd-10);
AB_2074478
Ms mAb (IgG1k)Heat (High pH)1:100, 30 min RTDABManual(Kaur et al., 2008a)
Smooth muscle
actin (SMA)
Pericyte
(venules only)
Vessel discolourationDako (1A4);
AB_2223500
Ms mAb (IgG2ak)Heat (Low pH)1:2,000, o.n. 4°CAECManual(Kaur et al., 2008b)
Platelet derived
growth factor
receptor β
(PDGFRβ)
Pericyte
(signalling)
Vessel discolourationAbcam (Y92);
AB_777165
Rb mAb (IgG)Heat (Low pH)1:100, 30 min RTDABManual(Armulik et al., 2005)
  1. *RRID: Research Resource Identifiers.

    Host: Rb = rabbit; Ms = mouse; mAb = monoclonal antibody; pAb = polyclonal antibody.

  2. Ag retrieval: heat-mediated antigen retrieval was performed in high pH solution (10 mM Tris/1 mM EDTA, pH 9.0) or low pH solution (trisodium citrate 10 mM, pH 6.0). Proteinase K was from Dako (ready-to-use solution).

    §Dilution and incubation time: RT = room temperature; o.n. = over night.

  3. #Chromogen: AEC: 3-amino-9-ethylcarbazole; DAB = 3,3'-diaminobenzidine. Reported references are from main manuscript.

Additional files

Supplementary file 1

Comparison of children without and with admission retinal exam data.

https://doi.org/10.7554/eLife.32208.027
Transparent reporting form
https://doi.org/10.7554/eLife.32208.028

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