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The Brigham and Women's Hospital, Boston, Massachusetts
Blantyre Malaria Project, University of Malawi College of Medicine
Malawi/Liverpool/Wellcome Trust Clinical Research Programme, Blantyre, Malawi
College of Osteopathic Medicine, Michigan State University, East Lansing
The clinical diagnosis of cerebral malaria in Plasmodium falciparumendemic regions is strengthened by demonstration of cerebral sequestration at autopsy. Parasitized comatose patients dying of other causes are less likely to have cerebral sequestration but can be difficult to distinguish, on clinical grounds, from patients dying of cerebral malaria. Sequestered parasites in a cytological preparation of a supraorbital brain sample, obtained after death, can be studied by use of standard thin blood-film staining. We show that, when confirmation by autopsy is not possible, this procedure is a reliable surrogate for histological study of tissue and that it can accurately identify patients with or without sequestered parasites in cerebral capillaries.
The standard clinical case definition of cerebral malaria encompasses a spectrum of diseases that may include syndromes stemming from different pathologic processes [1, 2]. To satisfy the definition, patients must have Plasmodium falciparum parasitemia, coma (Blantyre coma score, 2), and no other obvious cause of coma (e.g., meningitis, hypoglycemia, or postictal state) [3].
P. falciparum is distinguished from the other 3 Plasmodium species infecting humans by its capacity to sequester in the capillaries of many tissues, including brain, bowel, muscle, and skin. This sequestration results from the receptor-mediated cytoadherance of infected erythrocytes to microvascular endothelial cells and may serve to protect parasitized red cells from being culled by the spleen. Although sequestration occurs in all patients infected with P. falciparum, the incidence of cerebral malariathe devastating complication thought to result from extensive sequestration in the brainis relatively rare [4].
In endemic areas, it is common for children who are not sick to have P. falciparuminfected erythrocytes in peripheral blood (a condition termed "asymptomatic parasitemia"). Some overdiagnosis of cerebral malaria is therefore inevitable when only the clinical definition is used. Unsuspected causes of death have been identified at autopsy in 23% of parasitized comatose children who satisfied the clinical case definition of cerebral malaria; these causes include unreported head trauma, ruptured vascular malformations, Reye syndrome, HIV encephalitis, pneumonia, sepsis, and hepatic necrosis [5]. Exclusive use of the clinical case definition probably exaggerates estimates of overall malaria mortality rates and makes the study of associations more difficult. Clinicopathological correlates are few, but recent autopsy-based data suggest that the percentage of cerebral capillaries containing sequestered parasitized erythrocytes can be reliably used to distinguish between parasitized patients with cerebral malaria and those with incidental parasitemia [5].
Obtaining samples of brain tissue during autopsy can be complicated by families' reluctance to consent to the procedure and by a lack of institutional capacity to conduct autopsies and to process histological samples. For these reasons, a rapid, easy, and convenient method to evaluate cerebral sequestration in fatal cases of suspected cerebral malaria would be useful.
As part of our ongoing clinicopathological study of fatal cerebral malaria in children in Malawi, a core-tissue sample from the frontal lobe is obtained by introducing a biopsy needle through the supraorbital plate. The puncture is not externally visible. Samples can be processed for routine histological examination (in a procedure consisting of fixation in formalin, embedding in wax, and staining with hematoxylin-eosin [H-E]), immunohistochemical evaluation, cryopreservation, and/or rapid diagnosis of sequestration. Unlike the 2-dimensional view provided by histological sections, which allows for precise quantification of parasites within blood vessels of a particular caliberand, thereby, for case-to-case comparison of numbers of parasitesthe 3-dimensional view provided by cytological smears produces much higher resolution of the contents of blood vessels. Accurate quantification is not possible with smears, but, when fixed and stained in the same manner as is used for a thin blood film, they can reveal intact blood-vessel segments ranging from 50 to many 100s of microns in length. Because the brain smears can be processed and stained in any lab capable of testing thin smears for the presence of malaria parasites, we chose to compare the utility, with regard to the assessment of cerebral sequestration, of core-tissue smears versus standard histological preparations.
After obtaining permission from the families of the deceased, autopsies were performed. A brain sample from the frontal lobe was collected via a trochar passed through the supraorbital plate (figure 1). Smears were prepared by placing a small (maximum dimension, 2 mm) piece of brain tissue on a glass slide, near the frosted end. A second slide was placed, in the opposite orientation, over the tissue; then gentle pressure was applied, and the tissue was smeared away from the frosted end of the slide. The slides were air-dried and fixed in methanol for 30 s. Staining was performed by reverse Field's stain (figure 2A and 2B) and Giemsa stain (2.5% for 1 h). The contents (unpigmented and pigmented parasites) of 3 blood-vessel segments per slide were counted and categorized as follows: rare parasites (<5/blood vessel), few parasites (510/blood vessel), and many parasites (>10/blood vessel); smears with <3 visible blood vessels were considered to be inadequate and were not included in the study. Smear counts were performed by 2 individuals (a clinician and a pathologist) blinded with regard to the source, and discrepancies were discussed and resolved. During formal open autopsy, tissue samples were collected from 13 additional brain sites (frontal lobe, parietal lobe, occipital lobe, temporal lobe, hippocampus, thalamus, caudate, cerebellum, cerebellar tonsils, midbrain, pons, medulla, and spinal cord); tissue sections were fixed in formalin, embedded in paraffin, and stained with H-E (figure 2C and 2D). As described elsewhere, the contents (unpigmented and pigmented parasites) of 100 capillaries in cross-section were counted and categorized as follows: rare parasites (0.5/blood vessel), few parasites (0.51.0/blood vessel), and many parasites (>1.0/blood vessel) [5]. The histological results were scored by 2 observers blinded to the source and were compared for interobserver variability. Between-site comparisons confirmed that sequestration was homogeneous in the cerebral cortex (data not shown); therefore, we compared the results of frontal-lobe histological examination with those for frontal-lobe brain smears. We employed Fisher's exact test (2-tailed) to compare the 2 variables: histological and cytological evidence of sequestration (table 1).
The high degree of agreement between the histological and cytological assessments of sequestration suggest that a single supraorbital brain smear can help to increase the specificity of the clinical case definition of cerebral malaria by distinguishing between those parasitized patients who die with significant cerebral sequestration and those in whom the parasitemia was probably unrelated to the cause of death. To preserve anatomic structure in advance of complete autopsy, only a single core-tissue sample was takenand only a single smear was examinedfor the purposes of comparison. Multiple core-tissue samples could easily have been taken either from the same entry point or from a second site in the opposite supraorbital plate, and many smears could have been made from the tissue obtained. Both of these procedures would improve the likelihood that an adequate smear would be generated. If we had included several smears from multiple core-tissue samples, we most likely would have had perfect correlations between the results for smears and the results of histological examination.
Semiquantitative measures of cerebral sequestration in parasitized erythrocytes obtained from brain smears compare favorably to those derived from histological preparations of brain tissue collected at autopsy. The probability that such sequestration can be identified in a cytological smear (which comprises a small number of blood vessels) is less than the probability that it can be identified in a histological preparation (which comprises 100s of blood vessels). In cases of cerebral malaria, sequestered parasites are often present in 75%100% of cross-sectioned capillaries, and there is a high probability that such sequestration will be identified in a cytological preparation [5]. Parasitized patients who die for reasons other than cerebral malaria have many fewer sequestered parasites in the cerebral microvasculature; therefore, there is less probability that random sampling will identify a cytologically positive area. To calculate the precise statistical probabilities and reasonable confidence intervals, additional cases will be required. However, the value of this technique for the health-care provider in the field is that it allows for confirmation of the clinical diagnosis of cerebral malaria when complete autopsy is not possible. Elsewhere, we have reported 18 cases of death in African children (7 cases of clinical cerebral malaria and 11 cases of clinical nonmalaria coma) demonstrating no sequestration on routine histology and with causes of death that were attributed to other etiologies [5]. Unfortunately, when sequestration cannot be demonstrated cytologically, the final diagnosis usually will not be discernible unless there is an autopsy to identify other causes of death.
Clinicians working in either remote locations or settings where autopsy is not possible can improve on the clinical diagnosis of fatal cerebral malaria by using this simple, relatively noninvasive technique to demonstrate the presence or absence of sequestration. Use of this technique would also enhance efforts targeted at the dissection of the disease process and of its associations.
Acknowledgments
This study would not have been possible without the support of the Malawian clinicians and nurses who obtained permission for the autopsies or without the willingness of many bereaved families to permit the autopsies. We also thank Karl Seydel, for an early critical reading of the manuscript; Richard Carr, for his patient instruction on reading and interpreting both the histological preparations and the brain smears; and Wales Namanya, for his faithful assistance with all of the autopsies.
References
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