Clin. exp. Immunol (1991) 86, 19-21

Human cerebral malaria: characterization of malarial antibodies in cerebrospinal fluid S. MITRA, B. RAVINDRAN*, B. K. DAS, R. K. DAS, P. K. DAS & R. N. RATH Department of Medicine, S.C.B. Medical College, Cuttack, and *Department of Immunology, Regional Medical Research Centre, Indian Council of Medical Research, Bhubaneswar, India (Acceptedfor publication 26 April 1991)

SUMMARY Anti-malarial antibodies were quantified in cerebrospinal fluid (CSF) of 17 cases of cerebral malaria, 16 presumptive cases (no demonstrable parasitaemia in peripheral blood but responding to i.v. quinine therapy) of cerebral malaria, and 15 controls. A schizont-enriched Plasmodium knowlesi antigen was used in an ELISA. Anti-malarial antibodies of IgA and IgM isotypes were not detectable in most of the CSF samples analysed, although serum antibody titres were high. However, 88% of CSF from cerebral malaria and 56% of presumptive cerebral malaria cases had significant levels of IgG anti-malarial antibodies in comparison to control CSF. The antibody levels did not correlate with the severity of coma but correlated well with the duration of coma. The CSF malarial antibody titres were independent of degree of parasitaemia. The possible role of CSF anti-malarial antibodies in cerebral malaria in the light of recent demonstrations of intrathecal synthesis of immunoglobulins and deposition of immune complex in cerebral tissues is discussed.

Keywords cerebral malaria Plasmodiumfalciparum cerebrospinal fluid antibodies immunodiagnosis

study was undertaken in order to identify and quantify antimalarial antibodies, if any, in CSF of human cerebral malaria. While no significant anti-malarial activity was found in IgM and IgA isotypes, 88% of cerebral malaria cases had significant IgG anti-malarial antibodies and their presence in CSF correlated well with duration of coma. The results also indicate the possibility of developing an immunodiagnostic test for cerebral malaria. This, to our knowledge, is the first report demonstrating the presence of anti-malarial antibodies in CSF of human cerebral malaria.

INTRODUCTION Cerebral malaria is a severe clinical presentation of human Plasmodiumfalciparum infections accounting for nearly 80% of deaths due to malaria (World Health Organization Malaria Action Programme, 1986). The factors contributing to the pathogenesis of cerebral complications are not understood completely. Increased blood-brain barrier permeability (Maegraith & Fletcher, 1972) leading to cerebral oedema (Looaresuwan et al., 1983) and mechanical obstruction of microcirculation due to capillaries clogged with parasitized erythrocytes (Jerusalem et al., 1983; MacPherson et al., 1985) have been some of the implicating factors. However, recent findings have implicated immunological mechanisms such as immune complex vasculitis (Oo et al., 1987; Aikawa, 1988) and various cytokines in cerebral malaria (Grau et al., 1987, 1989; Kwiatkowski et al., 1990). In this context the report by Chapel et al. (1987) assumes importance wherein intrathecal synthesis of immunoglobulins was demonstrated in many cases of acute cerebral malaria indicating a local immune response in the central nervous system. However, the antigen specificity of IgG in cerebrospinal fluid (CSF) was not investigated. The present

SUBJECTS AND METHODS

Clinical cases Patients with clinical presentation of short history of fever associated with unarousable coma with microscopically detectable peripheral parasitaemia for P. falciparum were diagnosed as cerebral malaria cases and were admitted to the department of Internal Medicine of the S.C.B. Medical College, Cuttack. Other causes of encephalopathy such as hypoglycaemia, trauma or intoxication were excluded; only patients in whom coma had persisted for 24 h or more (at the time of reporting to the hospital) were included in the study. The severity of coma was graded by Glasgow coma scale: a grade of 1-5 was considered severe; 6-10 moderate; and 11-15 mild. The following other clinical complications were seen in these patients: acute renal

Correspondence: Dr B. Ravindran, Assistant Director, Department of Immunology, Regional Medical Research Centre, I.C.M.R., Bhubaneswar 751 016, India.

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failure (four patients); pulmonary oedema (two patients); anaemia (eight patients); hyperbilirubinaemia (six patients); haemoglobinuria (three patients); aspiration pneumonia (three patients); and convulsions (two patients). Multiple complications were found in 12 of the 17 cases, of whom 15 were males. The mean age was 36 6 years (range 17-60) for this group of cerebral malaria cases. The second group of 16 patients (14 men, two women) presented with clinical symptoms of cerebral malaria but were free of microscopically detectable parasites in peripheral blood on three successive examinations. However, all of them responded within 48 h after initiation of i.v. therapy with quinine dihydrochloride. This group of patients were taken as presumptive cerebral malaria cases; the mean age of this group was 30-1 years (range 14-62). CSF and blood for sera were collected within 24 h of admission after informed consent from the accompanying relatives. Fifteen control CSF samples were collected from patients undergoing surgery by spinal anaesthesia for reasons unrelated to malaria. Utmost care was taken to avoid any trauma at the time of collection of CSF samples; only those with microscopically undetectable erythrocytes were included in the study.

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P. knowlesi antigen Freed parasites prepared by saponin lysis of P. knowlesi (WI strain) infected schizonts were ultrasonicated in a Soniprep ultrasonicator (three 5-min cycles at 50% efficiency with an output of 20 W), centrifuged at 8000 g and the supernatant was stored at 20'C until further use.

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ELISA Essentially the procedure outlined elsewhere for P. knowlesi antigen was used (Srivastava et al., 1983). Polystyrene plates (Costar) were coated with 4 gg/well of P. knowlesi antigen and five-fold-diluted CSF or 200-fold-diluted sera were incubated in the coated plates for 90 min at 370C. The plates were washed with phosphate-buffered saline (PBS), pH 7 2, containing 0 1% Tween 20 and 500-fold-diluted affinity-purified anti-human IgG or IgM or IgA conjugated with alkaline phosphatase (Sigma) were incubated at 37°C for 90 min. The plates were then washed and the bound enzyme activity was quantified by the addition of p-nitrophenyl phosphate (1 mg/ml in 0-15 M NaHCO3 containing 1 nM MgCI2) and the OD was measured at 405 nm in an ELISA reader (BioRad). Mean + 3 s.d. of the OD values of the control group was taken as a cut-off level for the CSF samples. The results were analysed using Students' t-test and Pearson's correlation co-efficiency. RESULTS AND DISCUSSION ELISA with P. knowlesi schizont antigen indicated the presence of IgG malarial antibodies in CSF of cerebral malarial cases as well as in presumptive cases of cerebral malaria in comparison to normal controls (Fig. 1). When an OD of 0 21 was taken as a cut-off value (mean+3 s.d. of controls) 88 3% of cerebral malaria and 56-3% of presumptive cerebral malaria cases had significant levels of malarial antibodies in CSF; however, the levels of IgM malarial antibodies were scanty (Fig. 1). When the CSF samples were tested for antibodies of IgA isotype there was no demonstrable malarial antibody activity (data not shown). However, serum malarial antibodies of IgM and IgA were high

Fig. 1. ELISA. Malarial antibodies in CSF of cerebral malaria (A), presumptive cases of cerebral malaria (B) and normal controls (C). The dotted line indicates the cut-off value corresponding to the mean + 3 s.d. of control CSF for IgG and IgM.

in these cerebral malaria cases: the OD values were 1 66 + 0-29 and 1-07 + 0 39, respectively. The presence of IgG malarial antibodies in CSF correlated well with the duration of coma (Fig. 2). All cases of cerebral malaria and 64-2% of presumptive cerebral malaria cases had significant levels of CSF malarial antibodies if the duration of coma was 48 h or more. However, the levels of IgG malarial antibodies in CSF did not correlate well with either the degree of parasitaemia (P > 0 5) or with the severity of coma as measured by the Glasgow coma scale (P> 0 5). Pathogenesis of human cerebral malaria appears to involve immunologically mediated mechanisms, and experimental findings in recent years do not substantiate the earlier held theories on increased permeability or mechanical damage due to vessels clogged with parasitized erythrocytes (Lambert & Gru, 1989). The possibility of a malarial antigen or mitogen-induced B lymphocyte proliferation in the brain was proposed by Chappel et al. (1987), who demonstrated intrathecal synthesis of immunoglobulins in human cerebral malaria. Such a possibility has been strengthened by a recent report on the presence of two low molecular weight malarial antigens in CSF of patients with cerebral malaria (Jayshree et al., 1989). The findings reported in this communication offer direct evidence for the presence of CSF malarial antibodies of IgG isotype in human cerebral malaria-their titres being more consistent if the duration of coma was 48 h or more. The results of the present study also indicate the possibility of developing an immunodiagnostic test

CSF malarial antibodies in human cerebral malaria 1-6

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ACKNOWLEDGMENTS i2s

We gratefully acknowledge Dr Q. Z. Hussain, National Institute of Communicable Diseases, Delhi, for providing P. knowlesi-infected erythrocytes from an infected Rhesus monkey. The technical assistance of Mr Praksh K. Sahoo and secretarial assistance of Mr L. Satyanarayana Rao are gratefully acknowledged. The Regional Medical Research Centre, Bhubaneswar is supported by grants from the Indian Council of Medical Research, New Delhi.

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for cerebral malaria, the necessity of which is acutely felt in endemic areas. Due to widespread availability of anti-malarials or inadequate microscopy it is not uncommon to encounter clinical cases resembling cerebral malaria with no demonstrable circulating parasites but readily responding to i.v. quinine therapy. An immunological test for confirming the clinical diagnosis in these patients will be beneficial in P. falciparumendemic areas. The possible role played by CSF malarial antibodies in the pathogenesis ofcerebral malaria can only be conjunctural at this stage. Although the presence of malarial antibodies in CSF may not be in itself a contributory factor for development ofcerebral complications in P. falciparum malaria, it is reasonable to attribute a role for these antibodies in the immunopathology associated with cerebral malaria; immune-complex-mediated vasculitis in cerebral malaria (Aikawa, 1988) could have its origin in intrathecally synthesized malarial antibodies. Although the present investigation has shown significant levels of CSF malarial antibodies in cases of cerebral malaria in comparison to normal controls, it is not known whether such antibodies are absent in CSF of uncomplicated P. falciparum malaria. Further studies in CSF of cerebral malaria cases on antibody forming cells, characterization of isotypes of malarial antibodies, parasite-derived antigens and/or mitogens if any

AIKAWA, M. (1988) Human cerebral malaria. Am. J. trop. Med. Hyg. 39, 3. CHAPEL, H.M., WARRELL, D.A., LOOAREESUWAN, S., WHITE, N.J., PHILLIPS, R.E., WARRELL, M.J., SUPAEANTA, V. & THARAVANIJI, S. (1987) Intrathecal immunoglobulin synthesis in cerebral malaria. Clin. exp. Immunol. 67, 524. GRAU, G.E., FAJARDO, L.F., PIQUET, P.F., ALLET, B., LAMBERT, P.H. & VASSALLI, P. (1987) Tumour necrosis factor/cachectin as an essential mediator in murine cerebral malaria. Science, 237, 1210. GRAU, G.E., TAYLOR, T.E., MOLYNEUX, M.E., WIRIMA, J.J., VASALLI, P., HOMMEL, M. & LAMBERT, P.H. (1989) Tumour necrosis factor and disease severity in children with falciparum malaria. N. Engl. J. Med. 320, 1586. JAYSHREE, R.S., DUBEY, M.L., GANGULY, N.K. & MAHAJAN, R.C. (1989) Detection of malaria antigen in cerebrospinal fluid by counter-current immunoelectrophoresis. Lancet,, ii, 871. JERUSALEM, E., POLDER, T., WIJERS-Rouw, P., HEINEN, U., ELING, W., OSUNKOYA, B.O. & TRINH, P. (1983) Comparative clinical and experimental study on the pathogenesis of cerebral malaria. Contr. Microbiol. Immunol. 7, 130. KWIATKOWSKI, D., HILL, A.V.S., SAMBOU, I., TWUMASI, P., CASTRACANE, J., MANOGUE, K.R., CERAMI, A., BREWSTER, D.R. & GREENWOOD, B.M. (1990) TNF concentration in fatal cerebral, non-fatal cerebral and uncomplicated Plasmodiumfalciparum malaria. Lancet, 336, 1201. LAMBERT, P.H. & GRAU, G.E. (1989) Cerebral malaria. In New Strategies in Parasitology (ed. by K. P. W. J. McAdam) p. 51. Churchill Livingstone, Edinburgh. LOOARESUWAN, S., WARRELL, D.A., WHITE, N.J., SUTHARASAMAI, P., CHANTHARANICH, D., SUNDARARAJ, K., JUEL-JENSEN, B.E., BUNNAG, D. & HARINASUTA, T. (1983) Do patients with cerebral malaria have cerebral oedema? Lancet, i. 434. MACPHERSON, G.G., WARRELL, M.J., WHITE, N.J., LOOARESUWAN, S. & WARRELL, D.A. (1985) Human cerebral malaria. A quantitative ultrastructural analysis of parasitized erythrocytes sequestration. Am. J. trop. Med. Hyg. 119, 385. MAEGRAITH, B.G. & FLETCHER, A. (1972) The pathogenesis of mammalian malaria. Adv. Parasitol. 10, 49. Oo, M.M., Aikawa, M., Than, T., Aye, T.M., Myint, P.T., Igarashi, I. & Schoene, W.C. (1987) Human cerebral malaria: a pathological study. J. Neuropathol. exp. Neurol. 46, 223. SRIVASTAVA, I.K., SHARMA, P., NATH, A., AGARWAL, S.S. & DUTTA, G.P. (1983) Enzyme linked immunosorbent assay with Plasmodium knoulesi antigen in diagnosis of malaria. Ind. J. med. Res. 77, 43 1. WORLD HEALTH ORGANIZATION MALARIA ACTION PROGRAMME (1986) Severe and complicated malaria. Trans. R. Soc. trop. Med. Hyg. 80, 61.

Human cerebral malaria: characterization of malarial antibodies in cerebrospinal fluid.

Anti-malarial antibodies were quantified in cerebrospinal fluid (CSF) of 17 cases of cerebral malaria, 16 presumptive cases (no demonstrable parasitae...
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