Conceived and designed the experiments: VAW SL NAVB MEM TET. Performed the experiments: VAW SL NAVB MEM TET. Analyzed the data: VAW SL TET. Contributed reagents/materials/analysis tools: VAW SL TET. Wrote the paper: VAW. Obtained funding: MEM TET.
The authors have declared that no competing interests exist.
The causes of coma and death in cerebral malaria remain unknown. Malarial retinopathy has been identified as an important clinical sign in the diagnosis and prognosis of cerebral malaria. As part of a larger autopsy study to determine causes of death in children with coma presenting to hospital in Blantyre, Malawi, who were fully evaluated clinically prior to death, we examined the histopathology of eyes of patients who died and underwent autopsy.
Children with coma were admitted to the pediatric research ward, classified according to clinical definitions as having cerebral malaria or another cause of coma, evaluated and treated. The eyes were examined by direct and indirect ophthalmoscopy. If a child died and permission was given, a standardized autopsy was carried out. The patient was then assigned an actual cause of death according to the autopsy findings. The eyes were examined pathologically for hemorrhages, cystoid macular edema, parasite sequestration and thrombi. They were stained immunohistochemically for fibrin and CD61 to identify the components of thrombi, β-amyloid precursor protein to detect axonal damage, for fibrinogen to identify vascular leakage and for glial fibrillary acidic protein to detect gliosis. Sixty-four eyes from 64 patients were examined: 35 with cerebral malaria and 29 with comas of other causes. Cerebral malaria was distinguished by sequestration of parasitized erythrocytes, the presence and severity of retinal hemorrhages, the presence of cystoid macular edema, the occurrence and number of fibrin-platelet thrombi, the presence and amount of axonal damage and vascular leakage.
We found significant differences in retinal histopathology between patients who died of cerebral malaria and those with other diagnoses. These histopathological findings offer insights into the etiology of malarial retinopathy and provide a pathological basis for recently described retinal capillary non-perfusion in children with malarial retinopathy. Because of the similarities between the retina and the brain it also suggests mechanisms that may contribute to coma and death in cerebral malaria.
In endemic countries, asymptomatic malaria parasitemia is common. Individuals with coma of a non-malarial cause may happen to be parasitemic and their illness may be wrongly identified as cerebral malaria (CM). In an autopsy study in Malawi all children with
Malarial retinopathy consists of one or more of the following ocular fundus findings: hemorrhages, whitening of the retina, orange to white discoloration of the retinal vessels and papilledema (
There has not been a previous description of ophthalmic pathology from a single large series of children dying of CM. The specific aims of this manuscript are to describe the retinal pathology in patients dying of CM, to correlate pathological features with clinical findings; and to compare these with observations in children dying from fatal comas of other causes (COC).
Comatose children were admitted to the Research Ward of the Queen Elizabeth Central Hospital, Blantyre, Malawi. The clinical definition of cerebral malaria was
Children's pupils were dilated and the fundi examined with direct and indirect ophthalmoscopy upon admission. The findings of an ophthalmologist or experienced clinician were recorded on standardized forms.
If the patient died, informed written consent was requested from the child's guardian in the local language, for autopsy and the use of human tissues. If this was obtained, a standardized autopsy was carried out. Representative sections were taken from multiple areas of the brain and all other organs. The eyes were removed and replaced by artificial glass eyes. The consensus classification of cause of death was made by collaborating pathologists other than the authors of this paper, and only after the complete microscopic examination of all blocks of tissue and review of microbiological cultures obtained prior to death as previously described.
After fixation in formalin, the eyes were examined externally, opened horizontally in the pupil-optic nerve plane and the superior calotte removed. They were then examined under a dissecting microscope for hemorrhages and these were quantified according to the method used in the clinical examination (Grade 1:1–5 hemorrhages, Grade 2: 5–20, Grade 3: 20–50, Grade 4: >50).
Hemorrhages were semi-quantitatively evaluated from 1–4 by the number of quadrants of the retina that were involved in one cross-section through the pupil-optic nerve section of the globe. The presence of cystoid macular edema (CME) was recorded when adequate sections through the macular area were obtained. The presence of fibrin thrombi was assessed as 0: absent, 1: occasional and 2: many, on both H&E and PAS stains.
All slides were stained by a routine avidin-biotin immunoperoxidase method with primary antibodies directed against β-amyloid precursor protein (APP) to assess axonal damage
For APP, foci of positive staining associated and not associated with hemorrhage were counted in the retina and optic nerve. The sizes of the foci were also measured with a standard micrometer and the total area expressed in square mm. Only focal, dense staining that mirrored the structure of the underlying axon was counted.
For fibrinogen, the areas of focal staining of the retina, associated and not associated, with hemorrhage were counted. The presence of fibrinogen staining of the edema spaces of CME in cases in which CME was visible were documented as positive or negative.
For GFAP, the number of cells with positively staining cytoplasm was counted in the entire area of optic nerve present on a slide and standardized to 10 square mm. In the retina GFAP antibody staining of Muller cells was recorded as 0: absent, 1: peripheral staining on one side, 2: halfway to the equator on one side, 3: to the equator on one side, and 4: present on both sides to the equator.
The study protocol was reviewed and approved by the Institutional Review Boards of the University of Malawi, University of British Columbia and Michigan State University.
All statistics were calculated using S-PLUS (Insightful Corporation, Seattle), version 6.2.1. Categorical values were assessed for difference with the Chi-square test or Fisher's exact test. P<0.05 was taken as the level of a significant difference between groups.
Sixty-four eyes from 64 patients were examined pathologically. This included 13 cases that were diagnosed clinically as CM, but were found on autopsy to have other causes of death. For technical reasons, not all eyes were examined for all features described.
Thirty-five patients had the systemic gross and microscopic pathology typical of cerebral malaria and no other possible cause of death identified after full autopsy.
There were significantly more hemorrhages in CM patients than in patients with non-malarial disease (
A. Representative gross photo of malarial retinopathy showing multiple white-centered hemorrhages. B. Low power photomicrograph of hemorrhages involving all layers of the retina, including beneath the internal limiting membrane as well as subretinal hemorrhage with shallow detachment.
CM | % | COC |
% | p-value | |
Clinical | 17/26 | 65.38 | 4/19 | 21.05 | 0.003 |
Gross | 25/32 | 78.13 | 2/29 | 6.90 | <0.001 |
Microscopic | 25/35 | 71.43 | 3/29 | 10.34 | <0.001 |
No. cases positive | 17/32 | 53.13 | 1/27 | 3.70 | <0.001 |
No. cases positive | 26/35 | 74.29 | 2/29 | 6.90 | <0.001 |
No. cases positive | 20/35 | 57.14 | 6/29 | 20.69 | 0.003 |
Mean no. of foci (SD) | 1.43 (1.67) | 0.34 (0.72) | 0.003 | ||
Mean area (sq mm) (SD) | 1.31 (1.66) | 1.14 (3.57) | 0.017 | ||
No. cases positive | 11of 35 | 31.43 | 2/29 | 6.90 | 0.004 |
No. of foci (SD) | 1.2 (2.61) | 0.21 (1.11) | <0.001 | ||
Retina, grade of severity (SD) | 2.15 (1.12) | 1.79 (1.01) | 0.22 | ||
Optic nerve (#cells/10 sq mm) (SD) | 117.22 (81.62) | 126.5 (105.07) | 0.86 |
CM = cerebral malaria.
COC = comas of other causes.
includes 13 cases of clinically, but not pathologically, defined CM.
SD = standard deviation.
The retinal hemorrhages were usually located in the inner layers, but when numerous and large, involved all layers and were associated with subretinal hemorrhage and a shallow retinal detachment (
0 | 1 | 2 | 3 | 4 | NA | |
Clinical | 9 | 10 | 2 | 5 | 0 | 9 |
Gross | 7 | 9 | 2 | 11 | 3 | 3 |
Micro | 10 | 11 | 2 | 2 | 10 | 0 |
0 = none.
1 = 1–5.
2 = 5–20.
3 = 20–50.
4 = 50+.
CM = cerebral malaria.
NA = data not available.
CME was significantly more common in the CM group than the non-CM group (
Thrombi were frequently seen in the retinal vessels in cerebral malaria (
A. Photomicrograph of retinal hemorrhage with a central vessel containing a thrombus sectioned obliquely (arrow). B. Thrombus in a larger vessel without surrounding hemorrhage. Note that some of the surrounding capillary-sized vessels contain thrombi (arrows) while others do not (arrowhead). C&D. Immunohistochemical staining for fibrin (C), and CD61 to identify platelets (D). (C: Anti-fibrin, hematoxylin counterstain; D: Anti-CD61, hematoxylin counterstain.)
The number of cases positive for any APP staining in the CM group was significantly higher than the number in the COC group (
(Anti-β-APP, hematoxylin counterstain.)
In CM staining for fibrinogen was present around all vessels with hemorrhage, but was also present around blood vessels that were not associated with hemorrhage (
(Anti-fibrinogen, hematoxylin counterstain.)
(Anti-GFAP, hematoxylin counterstain.)
In this study we have documented the gross and microscopic pathology of the retina in the eyes of a large series of Malawian children dying from illnesses causing coma, including cerebral malaria and a variety of other conditions. We have attempted to relate the pathological findings to ophthalmological features observed during the illness. The extent of all pathological features, except gliosis, differed significantly between the malaria and non-malaria cases.
Retinal hemorrhages are frequent in cerebral malaria, being reported in 40–60% of cases.
Retinal hemorrhages are similar histopathologically to ring hemorrhages in the brain in CM. They usually occur in the inner and middle layers of the retina, but when more frequent and larger, involve all retinal layers and can be associated with subretinal hemorrhage and shallow retinal detachment.
The presence of thrombi has been described previously in cases of cerebral malaria.
Retinal thrombi are present in diseases that produce a fundoscopic retinal appearance called Purtscher retinopathy, similar in some respects to the retinal whitening in malarial retinopathy.
The concept of cerebral edema in CM is not new, but evidence has been lacking. At gross examination, the brain in fatal CM is commonly swollen, but uncal herniation is rare and there is limited histopathological evidence of cerebral edema.
In support of the breakdown of the BRB is our frequent histologic finding of CME and of staining of the fluid within the intraretinal cystic spaces for fibrinogen in CM patients. In CME, fluid accumulates between the Muller cell and photoreceptor cell processes in the outer plexiform layer of the macular area of the retina producing an appearance of multiple small cystic intraretinal spaces on ophthalmoscopic exam. It can occur after intraocular surgery, in diabetics, retinal vein occlusion and ocular inflammatory diseases, all conditions where vascular integrity is impaired.
Axonal damage as demonstrated by immunohistochemical staining for APP is seen in numerous CNS diseases.
In this study, gliosis was the only finding that was not different in CM and non-CM cases in both the retina and optic nerve. We postulate that this is because the causes of death in the non-malarial cases would also lead to gliosis, although not necessarily by the same mechanism as in CM.
We have described the histopathological correlates of malarial retinopathy. We found significant differences in retinal pathology between patients who died of CM and those with other diagnoses. CM was distinguished by the presence and severity of retinal hemorrhages, the presence of cystoid macular edema, the occurrence and number of fibrin-platelet thrombi, and the presence and amount of axonal damage and vascular leakage. The retinal pathology we have described offers insights into the etiology of malarial retinopathy and, because of the similarities between the retina and the brain, suggests mechanisms that may contribute to coma and death in cerebral malaria.
We acknowledge the guardians of the patients who gave permission for the autopsies, the nurses of the Pediatric Department research ward at the Queen Elizabeth Central Hospital, Blantyre, Malawi and the laboratory technologists of Vancouver General Hospital.
This work was presented in part at:
Symposium on Malarial Retinopathy: Nov 14, 2006. American Society of Tropical Medicine and Hygiene 55th Annual Meeting, Atlanta GA, Nov 12–16, 2006.
United States and Canadian Academy of Pathology 93rd Annual Meeting, Vancouver BC, Mar 6–12, 2004.
American Society of Tropical Medicine and Hygiene 52nd annual meeting, Philadelphia PA, Dec 3–7, 2003.
Association for Research in Vision and Ophthalmology Annual Meeting, Fort Lauderdale, Florida, April 29-May 5, 2000