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Seizures in children with acute falciparum malaria : risk factors, mechanisms of neuronal damage and neuro-protection

Idro, R.I.

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Download date:23 Sep 2021 SEIZURES IN CHILDREN WITH ACUTE FALCIPARUM MALARIA Risk factors, mechanisms of neuronal damage and neuro-protection

Proefschrift.indb 1 14-1-2008 13:09:04 Copyright © Richard Iwa Idro, Amsterdam, 2007 No part of this thesis may be reproduced, stored or transmitted, in any form or by any means, without prior permission of the author

Lay-out: Chris Bor, Medical Photography and Illustration, Academic Medical Center, Amsterdam Printed by: Buijten en Schipperheijn, Amsterdam

Proefschrift.indb 2 14-1-2008 13:09:04 SEIZURES IN CHILDREN WITH ACUTE FALCIPARUM MALARIA Risk factors, mechanisms of neuronal damage and neuro-protection

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus prof. dr. D.C. van den Boom

ten overstaan van een door het college voor promoties ingestelde commissie, in het openbaar te verdedigen in de Agnietenkapel der Universiteit

op dinsdag 19 februari 2008, te 14.00 uur

door Richard Iwa Idro geboren te Moyo, Oeganda.

Proefschrift.indb 3 14-1-2008 13:09:04 PROMOTIECOMMISSIE

Promotores: Prof. dr. P.A. Kager Prof. dr. C.R.J.C. Newton Co-promotor: Prof. dr. B.G.R. Neville

Overige leden: Prof. dr. B.J. Brabin Prof. dr. J. Stam Prof. dr. B.T. van der Poll Dr. J.C.F.M. Wetsteyn Dr. M. Boele van Hensbroek

Faculteit der Geneeskunde

Proefschrift.indb 4 14-1-2008 13:09:04 CONTENTS

I. General introduction 7 II. The incidence, etiology and outcome of acute seizures in children admitted to a 17 rural Kenyan district hospital Idro R, Gwer S, Kahindi M, Gatakaa H, Kazungu T, Ndiritu M, Maitland K, Neville BGR, Kager PA, Newton CJRC Submitted. III. Haptoglobin HP2-2 genotype, α-thalassaemia and acute seizures in children 35 living in a malaria endemic area of Kenya Idro R, Williams TN, Gwer S, Uyoga S, Macharia A, Opi H, Atkinson S, Maitland K, Kager PA, Kwiatkowski D, Neville BGR, Newton CRJC Submitted. IV. Burden, features and outcome of neurological involvement in acute falciparum 45 malaria in Kenyan children Idro R, Ndiritu M, Ogutu B, Mithwani S, Maitland K, Berkley JA, Crawley J, Fegan G, Bauni E, Peshu N, Marsh K, Neville BGR, Newton CJRC JAMA, 2007; 297 (20): 2232 – 40 V. Decorticate, decerebrate and opisthotonic posturing and seizures in Kenyan 63 children with cerebral malaria Idro R, Otieno G, White S, Kahindi A, Fegan G, Ogutu B, Mithwani S, Maitland K, Neville BGR, Newton CJRC Malaria Journal, 2005 Dec 7; 4: 57 VI. Continuous electroencephalographic monitoring and seizures in children with 81 cerebral malaria Gwer S, Idro R, Otieno G, Chengo E, Boga M, Aketch S, Maitland K, Neville BGR, White S, Newton CJRC VII. Pathogenesis, clinical features and neurological outcome of cerebral malaria 101 Idro R, Jenkins NE, Newton CJRC Lancet Neurology, 2005; 4(12): 827 – 40 VIII. Risk factors for persisting neurological and cognitive impairments following 133 cerebral malaria Idro R, Carter JA, Fegan G, Neville BGR, Newton CRJC Arch Dis of Childhood, 2006; 91 (2): 142-48 IX. Axonal and astrocyte injury markers in the cerebrospinal fluid of Kenyan 149 children with severe malaria Medana I, Idro R, Newton CJRC Journal of the Neurological Sciences, 2007; 258 (1-2): 93-8

Proefschrift.indb 5 14-1-2008 13:09:04 X. High plasma levels of erythropoietin are associated with protection against 163 neurological sequelae in African children with cerebral malaria. Casals-Pascual C, Idro R, Gicheru N, Gwer S, Kitsao B, Gitau E, Mwakesi R, Roberts DJ, Newton CJRC Submitted. Summary 179 Samenvatting 189 Acknowledgments 199 Curriculum vitae 201

Proefschrift.indb 6 14-1-2008 13:09:04 Chapter 1

General introduction

Proefschrift.indb 7 14-1-2008 13:09:04 Proefschrift.indb 8 14-1-2008 13:09:04 GENERAL INTRODUCTION Epidemiology Malaria is a major public health problem in many tropical countries. Of the four species that cause malaria in man, Plasmodium falciparum, which is transmitted by female anopheles mosquitoes, causes the most severe illness. Recent estimates indicate that worldwide, over 2 billion people are at risk of contracting malaria annually and in 2002, there were 515 million clinical episodes of acute falciparum malaria. The majority (70%) of these clinical events occurred in sub-Saharan Africa. South East Asia contributed 25% of the cases[1].

Although Africa accounts for the majority of cases of falciparum malaria, the distribution of malaria in this region is not uniform. The pattern is greatly influenced by the annual entomological inoculation rate (the number of infected mosquito bites per person per year)[2] which in turn is influenced by climatic factors[3] and land use[4]. About 75% of the population in sub-Saharan Africa lives in areas with stable malaria transmission where children under the age of 5 years are most at risk and 18% live in areas where transmission is seasonal and unstable such that all age groups are vulnerable to infection and disease. Such areas are prone to malaria epidemics.

Severe malaria The majority of clinical episodes of falciparum malaria are uncomplicated. In African children under the age of 5 years, 1 out of every 100 clinical episodes may progress to severe and complicated disease often, within 48 hours of fever onset[5] such that between 25–40% of paediatric admissions to hospitals in malaria endemic areas are due to malaria. Children with severe malaria may present with life threatening complications such as impaired consciousness, severe anaemia or severe metabolic acidosis. Other features include repeated seizures, prostration, hypoglycaemia and shock (table I).[6] Children with these severe features have a mortality >5%. It is estimated that at least one million deaths occurring in children living in sub-Saharan Africa are a result of the direct effects of falciparum malaria. Deaths are particularly common among children who develop impaired consciousness, respiratory distress, shock or hypoglycaemia[6].

Among survivors, gross neurological deficits have been observed in those admitted with recurrent and prolonged seizures, repeated episodes of hypoglycaemia or prolonged and deep coma[8, 9]. More recently, severe malaria in children has been associated with deficits in cognition (memory, attention, executive functions, speech and language), behavioural disorders[10-13] and subsequent epilepsy[14, 15].

Proefschrift.indb 9 14-1-2008 13:09:04 Table I Manifestations of severe malaria in African Children Clinical manifestations and laboratory findings Frequency of feature Prognostic value Prostration +++ + Impaired consciousness +++ +++ Respiratory distress +++ +++ Multiple convulsions +++ + Circulatory collapse (shock) + +++ Pulmonary oedema +/- +++ Abnormal bleeding +/- +++ Jaundice +/- ++ Haemoglobinuria +/- + Severe anaemia +++ + (Adapted from WHO 2000)[7]

The manifestations of severe malaria in children in endemic areas is dependent on age and transmission intensity[16]. In areas of intense transmission, infection and clinical disease are rare until about 6 months. Symptoms are mild as a result of the passive immunity offered by maternal antibodies. The highest disease burden is borne by children in their first two years of life and by four years, they experience few clinical episodes. In areas with less intense transmission, the peak incidence of severe disease falls at a later age[17]. Repeated infections over several years provide protection against disease. Immunity is effective but partial and declines in the absence of continuous exposure.

Neurological involvement in falciparum malaria Falciparum malaria appears to have a particular propensity to involve the brain in children. Many children are brought to hospital with seizures, confusion, prostration, impaired consciousness and/or coma. Seizures are the most frequent manifestation of central nervous system (CNS) involvement[18]. In those with cerebral malaria, seizures may manifest as convulsions, have subtle features like noisy breathing or only be detected by electroencephalographic monitoring[19]. Brainstem signs and abnormalities of posture, tone and reflexes are also observed[20, 21]. However, apart from cerebral malaria, the features of CNS involvement in acute falciparum malaria have not been systematically described. It is also not known why some children develop CNS disease and others do not.

Risk factors and precipitants of seizures in falciparum malaria Up to 20% of children admitted to some district hospitals in sub-Saharan Africa may have a history of seizures. Infection with falciparum malaria is an important cause

Proefschrift.indb 10 14-1-2008 13:09:05 of admission to hospitals and malaria is thought to be the primary diagnosis in the majority of children admitted with seizures[22-24]. Although most children who present to hospital with seizures have P. falciparum parasitaemia, in an endemic area where asymptomatic parasitaemia is common, it is unclear if malaria causes these seizures or there are other factors that precipitate them. A greater proportion of seizures are complex (focal, prolonged or multiple) when compared to simple febrile seizures despite occurring in the context of a febrile illness[24]. Parasite and host factors may be important underlying risk factors. Specific alleles, mutations and polymorphisms may be risk factors. It is possible that seizures are precipitated by co-infection with viral infections[25] and the seizure-threshold and frequency modulated by specific micronutrient deficiencies[26].

Outcome of seizures, mechanisms of neuronal damage and neuroprotective therapy Earlier studies suggested that recovery from cerebral malaria is almost complete[27] but recent data suggest otherwise. Multiple and prolonged seizures are associated with increased mortality and neurological and cognitive deficits. Neurological assessments performed after discharge have indicated that most deficits thought to arise from mild degrees of ischaemia like blindness, ataxia and paresis may be transient and resolve over a 6-month period[8] but long-term neurological and cognitive impairments are common. These include spasticity, epilepsy, hearing, language, memory, and learning impairments. It is estimated that, over 20% of children who survive cerebral malaria may have persistent impairments. These deficits are not only limited to cerebral malaria but also follow other features of CNS involvement such as malaria with multiple seizures[12]. The risk factors for such persistent impairments are unknown yet identifying them may allow early initiation of interventions for patients at risk. This is particularly critical in centres across Africa where assessments to detect neuro-cognitive impairments are either poorly developed or non-existent.

The mechanisms by which neuronal damage develops are also poorly understood. Studies that have examined the role of neuro-protection[28] and adjuvant therapies[29, 30] aimed at improving outcome have been disappointing. There is need for a clearer understanding of the pathogenesis of neuronal damage and for a study of other adjuvant and neuro-protective therapies.

Proefschrift.indb 11 14-1-2008 13:09:05 Hypotheses In this thesis, I have examined the following hypotheses: 1. Seizures are the most common feature of central nervous system involvement in children with acute falciparum malaria. 2. Seizures in children with acute falciparum malaria are associated with specific haptoglobin genotypes and deletions in the α-globin gene (α-thalassaemia). 3. Seizures in cerebral malaria may manifest as convulsions, tonic posturing or only be detected by continuous electroencephalographic monitoring. 4. Seizures in children with falciparum malaria cause neuronal injury. 5. Erythropoietin may be associated with an improved neurological outcome of children with cerebral malaria.

Studies conducted To answer these questions, I undertook a series of studies at the Kenya Medical Research Institute/Wellcome Trust - Centre for Geographic Medicine Research – Coast in Kilifi, Kenya. The patients described in the thesis attended Kilifi District Hospital and came from the hospital’s catchment area – a well mapped area that undergoes regular census and has a population of about 230,000[31], (figure 1).

Figure 1 Kilifi Demographic Surveillance study (DSS) area

A map of the study area (marked DSS) in Kilifi District, Kenya

Proefschrift.indb 12 14-1-2008 13:09:05 The results of these studies are presented in chapters 2 to 10. Chapter 2 provides the incidence and a description of the aetiological risk factors for acute seizures in the Kilifi demographic surveillance area. In chapter 3, I examined the relationship between common host polymorphisms in malaria endemic areas and acute seizures. Chapter 4 is an estimate of the burden and a description of the features of CNS involvement in children with acute falciparum malaria. Chapter 5 examines the relationship between abnormal motor posturing and seizures in children with cerebral malaria while chapter 6 describes electroencephalographic features of cerebral malaria on continuous monitoring. In chapter 7, we review the published literature of the clinical presentation, pathogenesis and neurological outcome of cerebral malaria. In chapter 8, I examined the risk factors for long-term deficits following cerebral malaria and in chapter 9, some of the mechanisms involved. Chapter 10 describes the potential of erythropoietin as a neuro-protective therapy for cerebral malaria. Lastly, I summarised the findings and suggested further studies, some of which my colleagues and I have started.

REFERENCES 1. Snow RW, Guerra CA, Noor AM, Myint HY, Hay SI: The global distribution of clinical episodes of Plasmodium falciparum malaria. Nature 2005, 434:214-217. 2. Hay SI, Rogers DJ, Toomer JF, Snow RW: Annual Plasmodium falciparum entomological inoculation rates (EIR) across Africa: literature survey, Internet access and review. Trans R Soc Trop Med Hyg 2000, 94:113-127. 3. Snow RW, Craig MH, Deichmann U, le Sueur D: A preliminary continental risk map for malaria mortality among African children. Parasitol Today 1999, 15:99-104. 4. Lindblade KA, Walker ED, Onapa AW, Katungu J, Wilson ML: Land use change alters malaria transmission parameters by modifying temperature in a highland area of Uganda. Trop Med Int Health 2000, 5:263-274. 5. Greenwood BM: The epidemiology of malaria. Ann Trop Med Parasitol 1997, 91:763-769. 6. Marsh K, Forster D, Waruiru C, Mwangi I, Winstanley M, Marsh V, Newton C, Winstanley P, Warn P, Peshu N, et al.: Indicators of life-threatening malaria in African children. N Engl J Med 1995, 332:1399-1404. 7. WHO: Severe falciparum malaria. Trans R Soc Trop Med Hyg 2000, 94 Suppl 1:S1-90. 8. van Hensbroek MB, Palmer A, Jaffar S, Schneider G, Kwiatkowski D: Residual neurologic sequelae after childhood cerebral malaria. J Pediatr 1997, 131:125-129. 9. Bondi FS: The incidence and outcome of neurological abnormalities in childhood cerebral malaria: a long-term follow-up of 62 survivors. Trans R Soc Trop Med Hyg 1992, 86:17-19. 10. Boivin MJ, Bangirana P, Byarugaba J, Opoka RO, Idro R, Jurek AM, John CC: Cognitive impairment after cerebral malaria in children: a prospective study. Pediatrics 2007, 119:e360-366. 11. Carter JA, Lees JA, Gona JK, Murira G, Rimba K, Neville BG, Newton CR: Severe falciparum malaria and acquired childhood language disorder. Dev Med Child Neurol 2006, 48:51-57.

Proefschrift.indb 13 14-1-2008 13:09:05 12. Carter JA, Mung’ala-Odera V, Neville BG, Murira G, Mturi N, Musumba C, Newton CR: Persistent neurocognitive impairments associated with severe falciparum malaria in Kenyan children. J Neurol Neurosurg Psychiatry 2005, 76:476-481. 13. Holding PA, Stevenson J, Peshu N, Marsh K: Cognitive sequelae of severe malaria with impaired consciousness. Trans R Soc Trop Med Hyg 1999, 93:529-534. 14. Ngoungou EB, Dulac O, Poudiougou B, Druet-Cabanac M, Dicko A, Mamadou Traore A, Coulibaly D, Farnarier G, Tuillas M, Keita MM, et al: Epilepsy as a consequence of cerebral malaria in area in which malaria is endemic in Mali, West Africa. Epilepsia 2006, 47:873-879. 15. Carter JA, Neville BG, White S, Ross AJ, Otieno G, Mturi N, Musumba C, Newton CR: Increased prevalence of epilepsy associated with severe falciparum malaria in children. Epilepsia 2004, 45:978-981. 16. Idro R, Aloyo J, Mayende L, Bitarakwate E, John CC, Kivumbi GW: Severe malaria in children in areas with low, moderate and high transmission intensity in Uganda. Trop Med Int Health 2006, 11:115-124. 17. Reyburn H, Mbatia R, Drakeley C, Bruce J, Carneiro I, Olomi R, Cox J, Nkya WM, Lemnge M, Greenwood BM, Riley EM: Association of transmission intensity and age with clinical manifestations and case fatality of severe Plasmodium falciparum malaria. Jama 2005, 293:1461- 1470. 18. Newton CR, Warrell DA: Neurological manifestations of falciparum malaria. Ann Neurol 1998, 43:695-702. 19. Crawley J, Smith S, Kirkham F, Muthinji P, Waruiru C, Marsh K: Seizures and status epilepticus in childhood cerebral malaria. Q J Med 1996, 89:591-597. 20. Molyneux ME, Taylor TE, Wirima JJ, Borgstein A: Clinical features and prognostic indicators in paediatric cerebral malaria: a study of 131 comatose Malawian children. Q J Med 1989, 71:441- 459. 21. Newton CR, Crawley J, Sowumni A, Waruiru C, Mwangi I, English M, Murphy S, Winstanley PA, Marsh K, Kirkham FJ: Intracranial hypertension in Africans with cerebral malaria. Arch Dis Child 1997, 76:219-226. 22. Angyo IA, Pam SD, Szlachetka R: Clinical pattern and outcome in children with acute severe falciparum malaria at Jos University Teaching Hospital, Nigeria. East Afr Med J 1996, 73:823-826. 23. Birbeck GL: Seizures in rural Zambia. Epilepsia 2000, 41:277-281. 24. Waruiru CM, Newton CR, Forster D, New L, Winstanley P, Mwangi I, Marsh V, Winstanley M, Snow RW, Marsh K: Epileptic seizures and malaria in Kenyan children. Trans R Soc Trop Med Hyg 1996, 90:152-155. 25. Schubart CD, Mturi N, Beld MG, Wertheim PM, Newton CR: Role of viruses in Kenyan children presenting with acute encephalopathy in a malaria-endemic area. Am J Trop Med Hyg 2006, 75:1148-1150. 26. Kobrinsky NL, Yager JY, Cheang MS, Yatscoff RW, Tenenbein M: Does iron deficiency raise the seizure threshold? J Child Neurol 1995, 10:105-109. 27. Muntendam AH, Jaffar S, Bleichrodt N, van Hensbroek MB: Absence of neuropsychological sequelae following cerebral malaria in Gambian children. Trans R Soc Trop Med Hyg 1996, 90:391- 394. 28. Crawley J, Waruiru C, Mithwani S, Mwangi I, Watkins W, Ouma D, Winstanley P, Peto T, Marsh K: Effect of phenobarbital on seizure frequency and mortality in childhood cerebral malaria: a randomised, controlled intervention study. Lancet 2000, 355:701-706. 29. Smith HJ, Meremikwu M: Iron chelating agents for treating malaria. Cochrane Database Syst Rev 2003:CD001474.

Proefschrift.indb 14 14-1-2008 13:09:05 30. Warrell DA, Looareesuwan S, Warrell MJ, Kasemsarn P, Intaraprasert R, Bunnag D, Harinasuta T: Dexamethasone proves deleterious in cerebral malaria. A double-blind trial in 100 comatose patients. N Engl J Med 1982, 306:313-319. 31. Cowgill KD, Ndiritu M, Nyiro J, Slack MP, Chiphatsi S, Ismail A, Kamau T, Mwangi I, English M, Newton CR, et al: Effectiveness of Haemophilus influenzae type b Conjugate vaccine introduction into routine childhood immunization in Kenya. Jama 2006, 296:671-678.

Proefschrift.indb 15 14-1-2008 13:09:05 Proefschrift.indb 16 14-1-2008 13:09:05 Chapter 2

The incidence, etiology and outcome of acute seizures in children admitted to a rural Kenyan district hospital

Richard Idro, Samson Gwer, Michael Kahindi, Helen Gatakaa, Tony Kazungu, Moses Ndiritu, Kathryn Maitland, Brian GR

Centre for Geographic Medicine Research – Coast, Kenya Medical Research Institute, Kilifi, Kenya; Department of Paediatrics and Child Health, Mulago Hospital/Makerere University, Kampala, Uganda; Department of Paediatrics, Faculty of Medicine and The Wellcome Trust Centre for Tropical Medicine, Imperial College, London – UK; Neurosciences Unit, The Wolfson Centre, University College London, Institute of Child Health, UK; Department of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Centre, Amsterdam, The Netherlands

Submitted

Proefschrift.indb 17 14-1-2008 13:09:06 ABSTRACT Background Acute seizures are a common cause of paediatric admissions to hospitals in resource poor countries and a risk factor for neuro-cognitive impairment and epilepsy. We determined the incidence, aetiological factors and the immediate outcome of seizures in a rural malaria endemic area in coastal Kenya.

Methods We recruited all children with and without seizures, aged 0-13 years and admitted to Kilifi District hospital over 2 years from st1 December 2004 to 30th November 2006. Only incident admissions from a defined area were included. Patients with epilepsy were excluded. The population denominator, the number of children in the community on 30th November 2005 (study midpoint), was modelled from the census data.

Results Seizures were reported in 900/4,921(18.3%) incident admissions and at least 98 had status epilepticus. The incidence of acute seizures in children 0-13 years was 425/100,000/ year and was 879/100,000/year in children <5 years. Over 80% of the seizures were associated with infections. Neonatal infections (28/43[65.1%]) and falciparum malaria (476/821[58.0%]) were the main diseases associated with seizures in neonates and in children 6 months or older respectively. Falciparum malaria was also the main illness (56/98[57.1%]) associated with status epilepticus. Other illnesses associated with seizures included pyogenic meningitis, respiratory tract infections and gastroenteritis. Twenty- eight children (3.1%) with seizures died and 11 surviving children (1.3%) had gross neurological deficits on discharge. Status epilepticus, focal seizures, coma, metabolic acidosis, bacteraemia, and pyogenic meningitis were independently associated with mortality; while status epilepticus, hypoxic ischaemic encephalopathy and pyogenic meningitis were independently associated with neurological deficits on discharge.

Conclusions There is a high incidence of acute seizures in children living in this malaria endemic area of Kenya. The most important causes are diseases that are preventable with available public health programs.

Proefschrift.indb 18 14-1-2008 13:09:06 BACKGROUND Acute seizures are a common neurological symptom in sick children. In patients with fever, they include febrile seizures[1, 2], acute symptomatic seizures (e.g. in a child with pyogenic meningitis)[3] or initial seizures in a child with epilepsy or epilepsy syndrome[2].

Worldwide, febrile seizures are the most common type of acute seizures in children[4]. Most are associated with infections and have a good outcome[5]. In tropical countries, febrile seizures are common but the prevalence of acute symptomatic seizures (which have a poorer outcome) may be higher than Western countries[6-8]. The incidence of both acute seizures and febrile status epilepticus is higher[2, 9] and the outcome is worse since the aetiology is different from that in Western countries[6, 8, 10, 11]. Acute seizures are therefore a major risk factor for neurological and cognitive impairment[12-14] and for the development of epilepsy[15-17] in children living in these regions. However few studies have examined the burden and risk factors for acute seizures in sub-Saharan Africa. Most of the available reports are of rates in hospital in the 1990’s and are limited to the diagnoses associated with the seizure-events [18-20].

In this study, we recruited all children with acute seizures from a defined area in coastal Kenya admitted to a district hospital to determine the incidence, the aetiology and outcome of the seizures.

METHODS Study participants Participants were children who were admitted with a history of acute convulsions, to Kilifi District Hospital over a period of 2 years from st1 December 2004 to 30th November 2006 and who were resident in a defined area in coastal Kenya. Children from the same study area but admitted without a history of seizures formed the comparison group. This hospital admits about 5000 children <14 years annually and is the only hospital in the area that admits very sick children. The study area undergoes a regular census three times per year and is defined by an area where over 60% of patients attending the hospital live. Each person in the study area has a unique identifier and data from hospital admissions is individually linked to the census data. The annual entomological inoculation rate (the number of times an individual is bitten by mosquitoes infected with Plasmodium falciparum in one year) in the catchment area ranges from <1 to 120 infectious bites per year[21].

Proefschrift.indb 19 14-1-2008 13:09:06 Admission procedures Ethical approval for the study was granted by the Kenya Medical Research Institute. Study participants were recruited consecutively as they presented to hospital. At admission, emergency care and resuscitation procedures such as correction of hypoglycemia, hypovolemia, oxygen and anticonvulsant therapy were administered according to Kenyan guidelines. Parents were then invited to consent to participate in the study. Children with epilepsy (two or more lifetime episodes of unprovoked seizures) were excluded. The history included the number and a parental description of the seizures. Level of consciousness was determined using the Blantyre Coma Scale[22]. Status epilepticus was defined as a seizure lasting 30 minutes or longer or as three or more seizures from which the patient did not regain consciousness after one hour[23].

Venous blood was obtained for a full blood count, blood glucose, malaria parasites, microbiological culture, and plasma from children with life threatening features (acidotic breathing, shock, prostration or impaired consciousness) was obtained for acid base status, electrolytes and creatinine. A growth of micrococci, coagulase negative staphylococci or gram-positive Bacillus species was considered a contaminant. Severe anemia was defined as hemoglobin <50g/L and metabolic acidosis as a base deficit >8. Hypokalemia was defined as plasma + K <3.0mmol/L, hyperkalemia as K+>5.0mmol/ L, hyponatremia (moderate–severe) as Na+<125mmol/L and impaired renal function as plasma creatinine >80µmol/L. Lumbar punctures were performed according to a standard protocol to exclude pyogenic meningitis[24]. Malaria was treated with parenteral quinine and for bacterial infections a combination of gentamicin and ampicillin (for children ≤2 months) or crystalline penicillin and chloramphenicol (for older children) was used. There were no facilities to identify viral causes of meningo- encephalitis and acyclovir was unavailable.

DATA MANAGEMENT AND ANALYSIS Individual patient clinical data was directly entered in a FileMaker 5.5 database at admission. Data was analysed using Stata version 9 (Stata Corporation, Texas). Using the unique patient identification numbers, the first admission during the two-year period was defined as the incident admission. The denominator, the population of children 0- 13 years was 105,992 and was obtained by modelling the census data to the date of 30 November 2005 (the midpoint of the study period). This age and sex specific estimate of the population denominator was obtained by fitting a linear regression line through the census counts (on a log scale) of October 2005 and February 2006. The incidence rates of acute seizures and status epilepticus are expressed as events per 100,000/year. A

Proefschrift.indb 20 14-1-2008 13:09:06 map was constructed by plotting the incidence by area of patient origin. In addition, we plotted the monthly admissions with acute seizures and compared this with the number of admissions with malaria. We then compared the clinical and laboratory features and the diagnoses in patients with and without seizures to describe the clinical risk factors for seizures and examined the risk factors for poor outcome. Normally distributed continuous data were compared using the unpaired Student’s t-test while skewed data were compared using Mann Whitney-Wilcoxon’s rank-sum test. Pearson’s chi square test (or Fischer’s exact test as appropriate) was used to compare proportions. Results are presented with crude odds ratios and a p value <0.05 is considered significant. To determine risk factors independently associated with mortality or neurological deficits, clinical and laboratory parameters with a p value <0.2 at univariate analysis were entered in a logistic regression model using a stepwise entry system.

RESULTS General description A total of 9,960 children aged 0-13 years were admitted to KDH during the 2-year period. We excluded the 3,745 children from outside the study area from the analysis. In addition, 190 children with epilepsy, 36 whose residence status in the DSS could not be ascertained and 126 children not assessed for a history of seizures were excluded (figure 1).

Figure 1 – Admissions to KDH, December 2004 to November 2006

The incidence of acute seizures and status epilepticus in Kilifi DSS Among the remaining 5,863 admissions from the study area, there were 942(16.1%) children defined as re-admissions. These non-incident admissions were excluded from further analysis. Nine hundred (18.3%) out of the 4,921 incident admissions had seizures.

Proefschrift.indb 21 14-1-2008 13:09:06 The incidence of acute seizures in children 0-13 years was 425 per 100,000/year and was 879 per 100,000/year in children <5 years. The incidence was highest (1,403 per 100,000/year) in neonates, lowest in infants aged 2-5 months with a second peak of 1,226 per 100,000/year in children 13-24 months (figure 2). At least 98(10.9%) children with seizures had status epilepticus. The overall incidence rate of status epilepticus was 46 per 100,000/year and was 95 per 100,000/year in children <5 years.

Figure 2 The incidence of acute seizures and status epilepticus by age group in children in Kilifi study area

In general, zones nearest to the hospital had a higher incidence rate of acute seizures (figure 3). Children with seizures were admitted throughout the year, with increases in May– August and December–January, the months that followed the long and short rains respectively (figure 4).

Risk factors for acute seizures and aetiological diagnoses associated with seizures

Clinical and laboratory features in children with acute seizures Of the 900 children with seizures, 427(47.5%) had single seizures and the remaining 473(52.3%) had 2 or more seizures. The median number of seizures in an individual child

Proefschrift.indb 22 14-1-2008 13:09:06 Figure 3 Incidence of acute seizures in Kilifi study area

Figure legend Incidence for acute seizures in Kilifi demographic surveillance study area by sub-location. In general, the incidence is highest in areas nearest to the district hospital and decreases with distance away from the hospital.

was 2 (IQR 1-3). The median age of children admitted with seizures was higher than that of children without seizures and fewer children with seizures were severely wasted. There were no differences in gender (table 1).

A history of fever was more common in patients with seizures. The mean axillary temperature at admission was higher (38.2 [SD 1.3]oC) in children with seizures compared to patients without seizures (37.6 [SD 1.2]oC), p<0.001. However, the median duration of fever was shorter (2 days) in patients with seizures compared to those without seizures (3 days), p<0.001 (table 1).

The median white blood cell counts and the proportions with severe anemia were similar in both groups of patients. A total of 4,546 children had blood glucose levels determined at admission. Overall, hypoglycemia was observed in 163 (3.6%) patients. The proportions of patients with hypoglycemia were similar in both groups (table 1). Of the 2,066 patients with life threatening-features who had acid base status measured, 793(38.4%) had metabolic acidosis. The proportions of patients with metabolic acidosis admitted with

Proefschrift.indb 23 14-1-2008 13:09:07 Figure 4 - Monthly admissions with malaria, seizures and rainfall in Kilifi

Figure legend Monthly total admissions, admissions with malaria and seizures and rainfall: The number of patients admitted with seizures has a seasonal pattern with peaks in December-January (after the short rains) and May-August (after the long rains). These peaks coincide with that for patients with presenting with malaria.

and without seizures were also similar. However, impaired renal function, hypokalemia, and moderate-severe hyponatremia were more common in children admitted without seizures (table 1).

Etiology of acute seizures Over 80% of the seizures were associated with an infectious illness (table 2). Malaria was the commonest aetiological factor associated with seizures and over 50% of the children admitted with acute seizures had malaria as the primary diagnosis. The number of children with seizures and a positive malaria slide was even higher: 489/885 (55.3%) of the children with seizures had positive malaria slides compared to 647/3,924 (16.5%) children without seizures, p<0.001. The risk of seizures increased with the parasite density. Children who experienced two or more seizures had a higher geometric mean parasite density (34,167 [95%CI 25,910-45,054]/µL) compared to those with single or no seizures (23,962 [95%CI 20,135-28,517]/ µL), p<0.001.

Proefschrift.indb 24 14-1-2008 13:09:07 Table 1 - Clinical and laboratory features on admission Patient characteristics Patients with Patients without Crude OR, P value seizures seizures (95% CI) Median (IQR) age, months 25.1 (12.5, 41.4) 13.1 (3.7, 36.3) - <0.001 Males, (%) 488/900 (54.2) 2,311/4,020 (57.5) 0.88 (0.76, 1.01) 0.074 Median (IQR) duration of illness, days 2 (1 – 3) 3 (2 – 4) - <0.001 Fever, (%) 829/900 (92.1) 2,649/4,021 (65.9) 6.05 (4.69, 7.89) <0.001 Severe wasting, (%) 12/900 (1.3) 257/4,019 (6.4) 0.20 (0.10, 0.35) <0.001 Severe anemia, (%) 56/900 (6.2) 291/4,021 (7.2) 0.85 (0.62, 1.15) 0.282 Mean (SD) WBC, x103/ml 13.8 (9.9) 14.0 (9.5) - 0.612 Hypoglycemia, (%) 27/864 (3.1) 136/3,682 (3.7) 0.84 (0.53, 1.29) 0.418 Acidosis, (%) 141/401 (35.2) 652/1,665 (39.2) 0.84 (0.67, 1.06) 0.140 Moderate – severe hyponatremia, (%) 14/438 (3.2) 41/2,064 (2.0) 1.63 (0.81, 3.08) 0.117 Hypokalemia, (%) 16/438 (3.7) 243/2,061 (11.8) 0.28 (0.16, 0.48) <0.001 Hyperkalemia, (%) 37/438 (8.5) 239/2,061 (11.6) 0.70 (0.48, 1.02) 0.056 Impaired renal function, (%) 60/407 (14.7) 408/1,864 (21.9) 0.62 (0.45, 0.83) 0.001 Bacteremia, (%) 23/882 (2.6) 225/3,720 (6.1) 0.42 (0.26, 0.64) <0.001

Table 2 Etiological diagnoses associated with acute seizures and outcome Diagnosis Patients with Patients without Crude OR, P value seizures, (%, n=900) seizures, (%, n=4,021) (95% CI) Diagnosis Malaria 479 (53.2) 482 (12.0) 8.35 (7.08, 9.85) <0.001 Respiratory tract infections 138 (15.3) 956 (23.8) 0.58 (0.47, 0.71) <0.001 Pyogenic meningitis 24 (2.7) 20 (0.5) 5.48 (2.89, 10.5) <0.001 Encephalopathy of unknown 15 (1.7) 12 (0.3) 5.66 (2.46, 13.3) <0.001 cause Gastroenteritis 39 (4.3) 564 (14.0) 0.28 (0.19, 0.39) <0.001 Severe malnutrition 13 (1.4) 230 (5.7) 0.24 (0.13, 0.42) <0.001 Trauma 4 (0.4) 96 (2.4) 0.18 (0.05, 0.48) <0.001 Poisoning 1 (0.1) 42 (1.0) 0.11 (0.003, 0.62) 0.007 Prematurity 1 (0.1) 82 (2.0) 0.05 (0.001, 0.31) <0.001 Hypoxic ischemic 5 (0.6) 98 (2.4) 0.23 (0.07, 0.55) <0.001 encephalopathy Neonatal sepsis 33 (3.7) 406 (10.1) 0.34 (0.23, 0.49) <0.001 Others 149 (16.6) 1,076 (26.8) 0.54 (0.45, 0.66) <0.001 Outcome Died 28 (3.1) 226 (5.6) 0.54 (0.35, 0.81) 0.002 Neurological deficits 13 (1.4) 0 (0) - <0.001

Apart from being the commonest illness associated with seizures, malaria was also associated with the highest seizure frequency and status epilepticus. The median number of seizures in children with malaria was 2[IQR 1-4] compared to 1[IQR 1-3], (p=0.004) in those with other illnesses. Out of the 98 children with status epilepticus,

Proefschrift.indb 25 14-1-2008 13:09:07 malaria was the primary diagnoses in 56 (57.1%). In addition, seizures in children with malaria had the longest duration.

Table 3 – Seizures manifestation, main etiological factors and outcome by age group of children with seizures Description of seizures Age group of patients 0 – 1 month, 2 – 5 months, 6 – 36 months, 37 – 72 >72 months, N=43, (%) N=36, (%) N=535, (%) months, N=49, (%) N=237, (%) Proportion with seizures 43/736 (5.8) 36/514 (7.0) 580/2,374 (22.5) 258/742 (31.9) 55/555 (8.8) Parental description of seizure event Focal 13 (30.2) 7 (19.4) 72 (13.5) 21 (8.9) 8 (16.3) Focal, secondarily 6 (14.0) 0 (0) 25 (4.3) 7 (3.0) 0 (0) generalised Generalised 21 (48.8) 25 (69.4) 423 (79.2) 199 (84.0) 39 (79.6) Not described 3 (7.0) 4 (11.1) 16 (3.0) 10 (4.2) 2 (4.1) Number of seizures, 3 (2, 5) 1 (1, 3) 2 (1, 3) 2 (1, 3) 1 (1, 2) median (IQR)

Level of consciousness at admission Normal consciousness 31 (72.1) 26 (72.2) 401 (74.9) 171 (72.2) 33 (67.4) Prostration or mild 8 (18.6) 6 (16.7) 73 (13.6) 39 (16.5) 5 (10.2) impairment of consciousness Agitation 3 (7.0) 3 (8.3) 8 (1.5) 3 (1.3) 3 (6.1) Coma 1 (2.3) 1 (2.7) 61 (10.5) 29 (11.2) 8 (14.6)

Leading diagnoses associated with seizures by age group 1 Neonatal sepsis Respiratory Malaria, Malaria, Malaria, 28 (65.1%) tract infections, 303 (56.6%) 155 (65.4%) 18 (36.7%) 10 (27.8%) 2 Pyogenic Acute Respiratory tract Respiratory Respiratory meningitis, gastroenteritis, infections, tract tract 7 (16.3%) 6 (16.7%) 85 (15.9%) infections, infections, 6 37(15.6%) (12.2%) 3 Hypoxic Pyogenic Acute Acute Others ischaemic meningitis, gastroenteritis, 21 (42.9) encephalopathy, 6 (16.2%) 27 (5.1%) 5 (11.6%) Outcome Death 7 (16.3) 0 (0) 15 (2.8) 3 (1.3) 3 (6.1) Neurological sequelae 3 (7.0) 1 (2.8) 5 (0.9) 3 (1.3) 1 (2.0)

Proefschrift.indb 26 14-1-2008 13:09:07 Two hundred and forty eight children out of 4,602 (5.4%) had positive blood cultures and bacteremia was more common among patients without seizures (table 1). Streptococcus pneumoniae was the commonest cause of bacteremia in both groups of children but was more common among patients with seizures, (8/23[34.8%] vs 31/225[13.8%], p<0.001). Other bacteria in children with seizures included Haemophilus influenzae, Escherichia coli, Salmonella species and Streptococcus viridans.

Among patients without seizures the causative organisms included Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, Acinetobacter, Streptococcus viridans, Salmonella species and several other gram-negative rods. The aetiology of seizures varied with age. Sepsis was the most important cause of neonatal seizures. Pyogenic meningitis, gastroenteritis and respiratory tract infections were the most common diagnoses in children 2-5 months while malaria was the most common illness in children 6 months or older (table 3). Staphylococcus aureus, E. coli and other gram-negative rods,

Table 4 Risk factors for death among children with seizures Risk factors for death Deaths, Survivors, Crude Odds ratio P- Adjusted odds P- (%) (%) (95% CI) value ratio (95% CI) value Median (IQR) duration 3 (1-4) 2 (1-3) - 0.170 - - of illness, days Median age (IQR), 17.2(3.4-32.9) 25.4(12.7-41.5) - 0.048 - - months Focal seizures 8/25 (32.0) 149/839 (17.8) 2.18 (0.80, 5.45) 0.069 - - Status epilepticus 12/28 (42.9) 86/872 (9.9) 6.85 (2.85, 16.0) <0.001 - - Coma 14/28 (50.0) 73/872 (8.4) 11.0 (4.62, 25.7) <0.001 10.3 (3.21, 33.1) <0.001 Severe wasting 4/28 (14.3) 8/872 (0.9) 18.0 (3.67, 72.3) <0.001 - - Severe anemia 4/28 (14.3) 52/872 (6.0) 2.63 (0.64, 8.05) 0.073 - - Acidosis 16/20 (80.0) 125/381 (32.8) 8.19 (2.56, 34.2) <0.001 7.19 (1.94, 26.7) 0.003 Hypoglycemia 6/27 (22.2) 21/837 (2.5) 11.1 (3.30, 33.3) <0.001 7.06 (1.73, 28.7) 0.006 Hyperkalemia 5/24 (20.8) 32/414 (7.7) 3.14 (.86, 9.45) 0.025 - - Hypokalemia 2/24 (8.3) 14/414 (3.4) 2.60 (0.27, 12.4) 0.209 - - Hyponatremia 2/24 (8.3) 12/414 (2.9) 3.05 (0.31, 15.0) 0.141 - - Impaired renal function 14/24 (58.3) 46/383 (12.0) 10.3 (3.94, 27.2) <0.001 - - Bacteremia 4/27 (14.8) 19/855 (2.2) 7.65 (1.75, 25.6) <0.001 16.9 (3.39, 33.1) 0.001 Malaria 11/28 (39.3) 468/872 (53.7) 0.56 (0.23, 1.28) 0.133 - - Respiratory tract 0/28 (0) 138/872 (16.4) - 0.022 - - infections Gastroenteritis 1/28 (3.6) 38/872 (4.4) 0.81 (0.02, 5.42) 0.841 - - Pyogenic meningitis 4/28 (14.3) 20/872 (2.3) 7.1 (1.63, 23.5) <0.001 - - Neonatal sepsis 2/28 (7.1) 31/872 (3.6) 2.08 (0.23, 8.98) 0.320 - - Hypoxic ischemic 2/28 (7.1) 3/872 (0.3) 22.28 (1.77, 200) <0.001 - - encephalopathy

Proefschrift.indb 27 14-1-2008 13:09:08 Streptococcus viridans, Acinetobacter, and Streptococcus pneumoniae were the organisms most commonly isolated in neonatal sepsis.

Immediate outcome of acute seizures Overall, mortality was lower among patients with seizures compared to patients without seizures; 28/900(3.1%) children with seizures died compared to 225/4,021(5.4%) children without seizures. The children with seizures who died presented to hospital after a longer median duration of illness (3 IQR 1, 4 days) compared to survivors (2 IQR 1, 3 days, p<0.001). Mortality was highest in neonates, in patients admitted with coma, focal seizures or status epilepticus and among those with complications such as acidosis, hypoglycemia, hyperkalemia and impaired renal function, bacteremia and pyogenic meningitis. The risk factors independently associated with death were coma, bacteremia, hypoglycaemia and metabolic acidosis (table 4).

At discharge, gross neurological deficits were observed in 11/872 (1.3%) surviving children. These included motor deficits (spasticity and central hypotonia) in 7 children, speech and hearing difficulties each in 2 children and dystonia/athetosis in one child. Neurological sequela was independently associated with coma, status epilepticus, severe wasting and hypoxic ischaemic encephalopathy (table 5).

Table 5 Risk facors for neurological deficits at discharge Risk factors Deficits on No deficits at Crude Odds P- Adjusted odds P- discharge, (%) discharge, (%) ratio (95% CI) value ratio (95% CI) value Median (IQR) age, 12.9 (0.2, 47.7) 25.5(12.9, 41.3) - 0.644 - - months Focal seizures 1/11 (9.1) 148/828 (17.9) 0.46 (0.01, 3.28) 0.449 - - Status epilepticus 5/11 (45.5) 81/861 (9.4) 8.02 (1.88, 32.2) <0.001 6.56 (1.05, 44.9) 0.045 Coma 4/11 (36.4) 69/861 (8.0) 6.56 (1.37, 26.4) 0.001 18.4 (3.03, 112) 0.002 Severe wasting 1/11 (9.1) 7/861 (0.8) 12.2 (0.24, 110) 0.097 23.9 (2.15, 266) 0.010 Hypoglycemia 1/11 (9.1) 20/826 (2.4) 4.03 (0.09, 30.8) 0.160 - - Malaria 5/11 (45.5) 463/861 (53.8) 0.72 (0.17, 2.84) 0.582 - - Bacteremia 1/11 (9.1) 18/844 (2.1) 4.59 (0.10, 35.4) 0.120 - - Pyogenic meningitis 1/11 (9.1) 19/861 (2.2) 4.43 (0.10, 34.0) 0.130 - - Neonatal sepsis or 1/11 (9.1) 30/861 (3.5) 2.77 (0.06, 20.6) 0.318 - - tetanus Hypoxic ischamic 2/11 (18.2) 1/861 (0.1) 191 (8.61, 11278) <0.001 158 (8.78, 2827) 0.001 encephalopathy

Proefschrift.indb 28 14-1-2008 13:09:08 DISCUSSION Children living in this rural area of Kenya experience a very high burden of acute seizure disorders. The incidence of acute seizures in children 0-13 years was 425 per 100,000 / year and the incidence of non-epilepsy associated status epilepticus was 46 per 100,000 per year. Younger children were more at risk: the incidence of acute seizures in children younger than 5 years was 879/100,000/year and of that of non-epilepsy associated status epilepticus was 95 per 100,000/year. Malaria and neonatal sepsis were the major aetiological factors for acute seizures and malaria for status epilepticus. Mortality was 3.1% while 1.3% of the surviving children had gross neurological deficits at the time of discharge from hospital.

The incidence of acute seizures The incidence rate is age dependent and is highest in neonates in whom infections are the predominant cause. However, the peaks are seasonal and follow seasons of malaria transmission, which in turn is dictated by the rainfall pattern in the area. The numbers we describe excludes seizures in children with epilepsy and this incidence data is comparable to rates described in West Africa[9] except that the proportion of malaria related seizures is higher[19]. At least 11% of the children with acute seizures had status epilepticus.

This incidence data is an absolute minimum. It does not account for children from the study area that did not attend Kilifi district hospital, from previous studies only about 20% of children who have seizures in the community are admitted to hospital (A Ngugi, personal communication) and that up to two thirds of deaths in children <5 years occur outside the hospital[25]. The map of incidence suggests that distance from the hospital could have affected hospital attendance.

In a recent epidemiological study in the UK, the incidence of convulsive status epilepticus in children under the age of 16 years was estimated to be 18-20 per 100,000 per year[26], a rate similar to that in other developed countries[27]. Despite the fact that we excluded status epilepticus associated with the epilepsies, the incidence of status epilepticus in this setting was at least twice that described in the UK. The data clearly shows that malaria accounts for the majority of the excess cases.

Etiological factors associated with acute seizures in children Infections were the predominant precipitants of acute seizures - over 80% of the seizures were associated with an infection. Malaria was not only the predominant infection

Proefschrift.indb 29 14-1-2008 13:09:08 in children 6 months and older but was also the main cause of status epilepticus. The median number of seizures in children infected with malaria was higher than that in children with other causes of seizures. The risk of seizures also increased with parasite density supporting the hypothesis that the parasite, Plasmodium falciparum, may be epileptogenic[28]. This is probably caused by the sequestration of the parasite in deep capillary beds in the cerebral microvasculature leading local ischemia/hypoxia (reviewed in[29]).

The main causes of acute seizures in children living in this setting (malaria and neonatal infections) and other important causes (pyogenic meningitis and hypoxic ischemic encephalopathy) are illnesses that are preventable with available public health measures. Impregnated mosquito nets and other treated mosquito materials or residual in-door spraying are proven methods for malaria control[30]. Scaling up these measures can greatly reduce the number of children exposed to malaria related seizures. Immunization against Haemophilus influenzae type b (Hib) has virtually stopped this organism from causing severe childhood illnesses in Western countries and significant progress has already been achieved with Streptococcus pneumoniae. Improved antenatal, perinatal (delivery by competent personnel and neonatal resuscitation programs) and postnatal care (cord care) can decrease the number of neonates exposed to seizures. Already, there is evidence to suggest that some of these programs are working in sub- Saharan Africa; in Kenya, there has been a drastic decline in the number of children with Haemophilus influenzae type b infections after introduction of the pentavalent (DTP-HBV-Hib) vaccine[31] and in Eritrea, the incidence rate of malaria declined by over 80% after the introduction of multiple vector control methods, case management and surveillance[32].

Outcome of acute seizures Overall, mortality in children with seizures was low except in patients admitted with bacteremia and neonatal infections, or those with complications such as hypoglycemia, severe acidosis and coma. Unlike patients with malaria, pyogenic meningitis, neonatal infections or hypoxic ischemic encephalopathy, almost all those with respiratory tract infections had single seizures and none died suggesting that these were probably febrile seizures. Put together, these findings suggest that the poor outcome of seizures in tropical settings[12] is related either to the severity of the illness associated with the seizures or the presence of complications such as coma, hypoglycaemia, acidosis and status epilepticus.

In this community, annually, at least 12 children <5 years per 100,000 develop gross neurological impairments following exposure to acute seizures. Status epilepticus is a

Proefschrift.indb 30 14-1-2008 13:09:08 prominent risk factor and malaria is the main cause of status epilepticus. The association between infection with falciparum malaria and repeated or prolonged seizures may therefore be a major risk factor for neurological impairments in children in malaria endemic Africa. Other risk factors include a background of severe malnutrition and hypoxic ischaemic encephalopathy.

CONCLUSION In conclusion, children living in this rural malaria endemic area of Kenya have a high incidence of acute seizure disorders. The main causes are diseases that are preventable with available public health programs. Poor outcomes are associated with severe infection, concurrent biochemical complications, coma, status epilepticus and malnutrition.

ACKNOWLEDGEMENTS We are grateful to the clinical and nursing staff of Kilifi District Hospital and the census staff. We specifically thank Ramos and Josephine Kazungu who stored, helped retrieve and organised the records. The Wellcome Trust sponsored this study, through funding Charles Newton as a Wellcome Trust Senior Fellowship in Clinical Tropical Medicine (No. 070114).

REFERENCES 1. Nelson KB, Ellenberg J.H: Febrile Seizures. New York: Raven Press; 1981. 2. Sadleir LG, Scheffer IE: Febrile seizures. Bmj 2007, 334:307-311. 3. Huang CC, Chang YC, Wang ST: Acute symptomatic seizure disorders in young children--a population study in southern Taiwan. Epilepsia 1998, 39:960-964. 4. Hauser WA: The prevalence and incidence of convulsive disorders in children. Epilepsia 1994, 35 Suppl 2:S1-6. 5. Verity CM, Greenwood R, Golding J: Long-term intellectual and behavioral outcomes of children with febrile convulsions. N Engl J Med 1998, 338:1723-1728. 6. Akpede GO, Abiodun PO, Sykes RM: Pattern of infections in children under-six years old presenting with convulsions associated with fever of acute onset in a children’s emergency room in Benin City, Nigeria. J Trop Pediatr 1993, 39:11-15. 7. Birbeck GL: Seizures in rural Zambia. Epilepsia 2000, 41:277-281. 8. Waruiru CM, Newton CR, Forster D, New L, Winstanley P, Mwangi I, Marsh V, Winstanley M, Snow RW, Marsh K: Epileptic seizures and malaria in Kenyan children. Trans R Soc Trop Med Hyg 1996, 90:152-155. 9. Iloeje SO: Febrile convulsions in a rural and an urban population. East Afr Med J 1991, 68:43-51.

Proefschrift.indb 31 14-1-2008 13:09:08 10. Owusu-Ofori A, Agbenyega T, Ansong D, Scheld WM: Routine lumbar puncture in children with febrile seizures in Ghana: should it continue? Int J Infect Dis 2004, 8:353-361. 11. Iloeje SO: Paediatric neurologic emergencies at the University of Nigeria Teaching Hospital, Enugu. West Afr J Med 1997, 16:80-84. 12. Carter JA, Mung’ala-Odera V, Neville BG, Murira G, Mturi N, Musumba C, Newton CR: Persistent neurocognitive impairments associated with severe falciparum malaria in Kenyan children. J Neurol Neurosurg Psychiatry 2005, 76:476-481. 13. Boivin MJ, Bangirana P, Byarugaba J, Opoka RO, Idro R, Jurek AM, John CC: Cognitive impairment after cerebral malaria in children: a prospective study. Pediatrics 2007, 119:e360-366. 14. Annegers JF, Hauser WA, Elveback LR, Kurland LT: The risk of epilepsy following febrile convulsions. Neurology 1979, 29:297-303. 15. Carter JA, Neville BG, White S, Ross AJ, Otieno G, Mturi N, Musumba C, Newton CR: Increased prevalence of epilepsy associated with severe falciparum malaria in children. Epilepsia 2004, 45:978-981. 16. Ngoungou EB, Dulac O, Poudiougou B, Druet-Cabanac M, Dicko A, Mamadou Traore A, Coulibaly D, Farnarier G, Tuillas M, Keita MM, et al: Epilepsy as a consequence of cerebral malaria in area in which malaria is endemic in Mali, West Africa. Epilepsia 2006, 47:873-879. 17. Ngoungou EB, Koko J, Druet-Cabanac M, Assengone-Zeh-Nguema Y, Launay MN, Engohang E, Moubeka-Mounguengui M, Kouna-Ndouongo P, Loembe PM, Preux PM, Kombila M: Cerebral malaria and sequelar epilepsy: first matched case-control study in Gabon. Epilepsia 2006, 47:2147- 2153. 18. Akpede GO, Sykes RM: Convulsions with fever of acute onset in school-age children in Benin City, Nigeria. J Trop Pediatr 1993, 39:309-311. 19. Obi JO, Ejeheri NA, Alakija W: Childhood febrile seizures (Benin City experience). Ann Trop Paediatr 1994, 14:211-214. 20. Axton JH, Siebert SL: Aetiology of convulsions in Zimbabwe children three months to eight years old. Cent Afr J Med 1982, 28:246-249. 21. Mbogo CM, Mwangangi JM, Nzovu J, Gu W, Yan G, Gunter JT, Swalm C, Keating J, Regens JL, Shililu JI, et al: Spatial and temporal heterogeneity of Anopheles mosquitoes and Plasmodium falciparum transmission along the Kenyan coast. Am J Trop Med Hyg 2003, 68:734-742. 22. Molyneux ME: The clinical features of cerebral malaria in children. Med Trop (Mars) 1990, 50:65- 68. 23. Guidelines for epidemiologic studies on epilepsy. Commission on Epidemiology and Prognosis, International League Against Epilepsy. Epilepsia 1993, 34:592-596. 24. Berkley JA, Mwangi I, Ngetsa CJ, Mwarumba S, Lowe BS, Marsh K, Newton CR: Diagnosis of acute bacterial meningitis in children at a district hospital in sub-Saharan Africa. Lancet 2001, 357:1753-1757. 25. Snow RW, Mung’ala VO, Foster D, Marsh K: The role of the district hospital in child survival at the Kenyan Coast. Afr J Health Sci 1994, 1:71-75. 26. Chin RF, Neville BG, Peckham C, Bedford H, Wade A, Scott RC: Incidence, cause, and short-term outcome of convulsive status epilepticus in childhood: prospective population-based study. Lancet 2006, 368:222-229. 27. Chin RF, Neville BG, Scott RC: A systematic review of the epidemiology of status epilepticus. Eur J Neurol 2004, 11:800-810. 28. Idro R, Ndiritu M, Ogutu B, Mithwani S, Maitland K, Berkley J, Crawley J, Fegan G, Bauni E, Peshu N, et al: Burden, features, and outcome of neurological involvement in acute falciparum malaria in Kenyan children. Jama 2007, 297:2232-2240.

Proefschrift.indb 32 14-1-2008 13:09:08 29. Idro R, Jenkins NE, Newton CR: Pathogenesis, clinical features, and neurological outcome of cerebral malaria. Lancet Neurol 2005, 4:827-840. 30. Lengeler C: Insecticide-treated bed nets and curtains for preventing malaria. Cochrane Database Syst Rev 2004:CD000363. 31. Cowgill KD, Ndiritu M, Nyiro J, Slack MP, Chiphatsi S, Ismail A, Kamau T, Mwangi I, English M, Newton CR, et al: Effectiveness of Haemophilus influenzae type b Conjugate vaccine introduction into routine childhood immunization in Kenya. Jama 2006, 296:671-678. 32. Mufunda J, Nyarango P, Usman A, Gebremeskel T, Mebrahtu G, Ogbamariam A, Kosia A, Ghebrat Y, Gebresillosie S, Goitom S, et al: Roll back malaria--an African success story in Eritrea. S Afr Med J 2007, 97:46-50.

Proefschrift.indb 33 14-1-2008 13:09:08 Proefschrift.indb 34 14-1-2008 13:09:08 Chapter 3

Haptoglobin HP2-2 Genotype, α-ThalasSaemia and acute seizures in children living in a malaria endemic area of Kenya

Richard Idro, Thomas N Williams, Samson Gwer, Sophie Uyoga, Alex Macharia, Herbert Opi, Sarah Atkinson, Kathryn Maitland, Piet A Kager, Dominic Kwiatkowski, Brian GR Neville, Charles RJC Newton

Centre for Geographic Medicine Research – Coast, Kenya Medical Research Institute, Kilifi, Kenya; Department of Paediatrics and Child Health, Mulago Hospital/Makerere University, Kampala, Uganda; Department of Paediatrics, John Radcliffe Hospital, Oxford, UK; Centre for Clinical Vaccinology and Tropical Medicine, University of Oxford, UK; Department of Paediatrics, Faculty of Medicine and The Wellcome Trust Centre for Tropical Medicine, Imperial College, London – UK, Department of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Centre, Amsterdam, The Netherlands, The Wellcome Trust Centre for Human Genetics, University of Oxford, UK, Neurosciences Unit, University College London, Institute of Child Health, UK

Submitted

Proefschrift.indb 35 14-1-2008 13:09:08 ABSTRACT Background Polymorphisms of the haptoglobin (HP) gene and deletions in the α-globin gene (α- thalassaemia) are both found at high frequencies in many parts of malaria-endemic Africa. The same regions also have very high incidence rates for childhood acute seizures. We investigated the hypothesis that the HP2-2 genotype and the common African α- globin gene deletions are associated with an increased risk of seizures in children.

Methods Two hundred and eighty eight children aged 3–156 months, consecutively admitted to Kilifi District Hospital in coastal Kenya with acute seizures were matched for ethnicity to an equal number of community controls. We compared the proportions of cases and controls with the HP2-2 genotype and the common African 3.7kb α-globin gene deletions.

Results The proportion of cases (72/288 [25.0%]) and controls (80/288 [27.8%]) with the HP2-2 genotype was similar, p=0.499. The allele frequency of the HP2 gene in cases (49.3%) and controls (48.6%) was also similar, p=0.814. Similarly, we found no significant difference between the proportion of cases (177/267 [66.3%]) and controls (186/267 [69.7%]) with deletions in the α-globin gene (p=0.403). Among cases, the HP2-2 polymorphism and deletions in the α-globin gene were neither associated with a change in the type (focal or generalized), number or duration of the seizures nor did they affect the outcome of treatment.

Conclusions Polymorphisms in the haptoglobin gene and deletions in the α-globin gene are not a risk factor for acute seizures in children. Future studies should examine other susceptibility genes.

Proefschrift.indb 36 14-1-2008 13:09:08 INTRODUCTION Seizures are the commonest neurological symptom in sick children admitted to hospital. In parts of malaria-endemic Africa, over 15% of paediatric admissions have a history of seizures[1-3] and acute seizures are a major risk factor for epilepsy later in life[4, 5]. Most seizures occur in febrile children and are precipitated by infections[2, 3, 6] but in some children, genetic factors may be important[2, 7].

Polymorphisms of the haptoglobin (HP) gene and deletions in the α-globin gene (α- thalassaemias) are very common in malaria endemic Africa[8, 9] and both are associated with protection against malaria [8-11]. The same region also has high incidence rates for childhood acute seizure disorders [1, 12]. It is unclear whether these polymorphisms or deletions and the high incidence of seizures are associated.

Haptoglobin is a protein that binds free haemoglobin in circulation. The gene for this protein has 3 main polymorphisms: HP1-1, HP1-2 and HP2-2. Studies have associated the HP2-2 genotype with idiopathic generalized epilepsy[13, 14] and the mean plasma levels of haptoglobin in patients with idiopathic epilepsy is lower than that in normal population controls[15]. It has been suggested that in persons with the HP2-2 genotype, a defective free haemoglobin clearing system after central nervous system micro- hemorrhage events is involved in the development of chronic seizure disorders[14, 15].

The relationship between theα -thalassaemias and acute seizures has not been reported. It is postulated that, children with thalassaemia have an increased oxidative stress on neurons and an altered iron metabolism. The change in iron metabolism may interfere with activities of gamma amino butyric acid and the glutamate system[16] and alter the risk for seizures. Only one retrospective study has examined the incidence of seizures in children with β-thalassaemia and the incidence was lower than that in the general population[16].

We hypothesized that compared to children with HP1-1 and HP1-2 genotypes or those without deletions in the α-globin gene, children with HP2-2 genotype or the common African α-globin gene deletion have an increased risk of developing acute seizures. We recruited children hospitalized with acute seizures and compared their HP genotypes and α-thalassaemia types to that of a matched cohort of community controls.

Proefschrift.indb 37 14-1-2008 13:09:08 MATERIALS AND METHODS Study participants The study was conducted in Kilifi District on the coast of Kenya where most residents are collectively known as the Mijikenda, a Bantu grouping of nine tribes, with the Giriama (45%), Chonyi (33%), and Kauma (11%) forming the major groups. A system of continuous demographic surveillance (DSS) is maintained in a population of roughly 230,000 living in the immediate catchment area of Kilifi District hospital[17]. Cases were children from within the DSS aged 3–156 months, consecutively admitted to the paediatric wards of the hospital with acute seizures. Children with epilepsy (2 lifetime episodes of unprovoked seizures) were excluded. Controls were children born in the hospital who had cord blood samples collected and archived, and were frequency matched to cases on the basis of ethnicity. The main exposure factor of interest was haptoglobin HP2-2 genotype. We estimated that a sample size of 248 cases and 248 controls would give 90% power to detect an OR of 2.0 at 5% level of significance given a prevalence of HP2-2 in the community of 25%[8]. Ethical approval for the study was granted by the Kenya Medical Research Institute.

Procedures The cases received emergency care and resuscitation based on standard guidelines[18]. Parents were then invited to participate in the study and consent obtained. The history included the number, duration, and a description of the seizures and a history of previous seizures in the child. Level of consciousness was assessed using the Blantyre Coma Scale[19]. Specific anti-microbial therapy was given for malaria and for bacterial infections. At discharge, all patients were assessed for the presence of neurological deficits.

Venous blood was drawn and plasma from both cases and controls was immediately separated by centrifugation and the cell pellet frozen and stored at -80oC. DNA was extracted using commercially available kits according to the manufacturers instructions (PUREGENE DNA extraction kit, GENTRA SYSTEMS, Boston, MA) and haptoglobin and thalassaemia genotyping was performed by PCR as previously described[9, 10, 20].

STATISTICAL ANALYSIS We compared the prevalence of HP2-2 genotypes, α-thalassaemia deletion types and allele frequency in cases and controls using Pearson’s chi square test to investigate the association with acute seizures in children. In addition, we compared the seizure types

Proefschrift.indb 38 14-1-2008 13:09:08 (generalized, focal or secondarily generalized) and the median number of seizures in children with the different genotypes. We also performed a sub analysis comparing the genotypes of cases with Plasmodium falciparum infection.

RESULTS Three hundred and twenty five children, consecutively admitted with acute seizures to Kilifi District hospital, between December 2004 and August 2005 were recruited. Haptoglobin genotyping was successful in 294 (90.5%) of whom, 288 were frequency matched for ethnicity to an equal number of controls from the birth cohort (six cases did not have matching ethnic groups in the birth cohort). In 267 out 288 (92.7%) children, both the haptoglobin genotype and α-thalassaemia type was available and the cases and controls could be matched for ethnicity. The majority of cases, 167 (58.0%) were from the Giriama ethnic group, 67 (23.3%) were Chonyi, 32 (11.1%) Kauma while the remaining 22 cases belonged to the Digo, Duruma, Jibana, Kambe, Rabai or the migrant Luo ethnic groups.

The median (IQR) age of the cases was 25.6 (14.0 - 41.1) months and 147 (51.0%) were males. The majority, 276 (95.8%), presented with fever and 104 (36.1%) reported previous episodes of provoked seizures. Malaria was the leading primary diagnosis associated with acute seizures and was seen 186 (64.6%) cases. Other primary diagnoses included respiratory tract infections, otitis media, acute diarrhoea or childhood viral fevers in 78 (27.1%), acute bacterial meningitis/septicaemia in 7 (2.4%) and suspected viral meningo- encephalitis in 7 (2.4%). Ten children (3.5%) had other encephalopathies.

Table I Haptoglobin genotypes in children with and without acute seizures Subject group Haptoglobin genotypes Total, Allele frequency Total, HP1-1, HP1-2, HP2-2, N HP1, HP2, N n (%) N (%) n (%) n (%) n (%) Cases 76 (26.4) 140 (48.6) 72 (25.0) 288 292 (50.7) 284 (49.3) 576 Controls 88 (30.5) 120 (41.7) 80 (27.8) 288 296 (51.4) 280 (48.6) 576

Table II a-thalassaemia genotypes in children with and without acute seizures Subject group a-3.7 kd globin deletion types Total, Allele frequency Total No Heterozygous, Homozygous, N No Any N deletion, N (%) n (%) deletion, deletion, N (%) n (%) n (%) Cases 90 (33.7) 128 (47.9) 49 (18.4) 267 308 (57.7) 226 (42.3) 534 Controls 81 (30.3) 150 (56.2) 36 (13.5) 267 314 (58.4) 222 (41.6) 534

Proefschrift.indb 39 14-1-2008 13:09:08 Table III Haptoglobin genotypes, a-thalassaemia and acute seizures Seizure characteristics Haptoglobin genotype Unadjusted odds P value a-3.7-globin deletions Unadjusted odds P value HP2-2, HP1-2 or HP1-1, ratio (95%CI) Any deletion, No deletion, ratio (95%CI) N=72, (%) n=216, (%) n=177, (%) n=90, (%) Past seizures 21 (29.2) 83 (38.4) 0.66 (0.35-1.21) 0.157 65 (36.7) 33 (36.7) 1.00 (0.57-1.76) 0.993 Seizure type Generalized 58 (80.6) 176 (81.5) 0.94 (0.46-2.01) 0.862 146 (82.5) 73 (81.1) 1.10 (0.53-2.20) 0.782 Focal 9 (12.5) 28 (13.0) 0.96 (0.38-2.23) 0.919 21 (11.9) 12 (13.3) 0.88 (0.39-2.06) 0.730 Secondarily generalized 3 (4.2) 4 (1.9) 2.30 (0.33-13.94) 0.269 4 (2.3) 3 (3.3) 0.67 (0.11-4.69) 0.604 Not described 2 (2.8) 8 (3.7) 0.74 (0.08-3.85) 0.710 6 (3.4) 2 (2.2) 1.54 (0.27-15.92) 0.597 Median (IQR) number of seizures 1 (1-2) 1 (1-3) - 0.734 1 (1-3) 1 (1-3) - 0.851 Level of consciousness at admission Coma (BCS 0-2) 6 (8.3) 24 (11.1) 0.73 (0.23-1.93) 0.504 17 (9.6) 12 (13.3) 0.69(0.29-1.67) 0.355 Impaired consciousness, (BCS 3& 4) 11 (15.3) 24 (11.1) 1.44 (0.60-3.27) 0.349 21 (11.9) 14 (15.6) 0.73 (0.33-1.65) 0.398 Full consciousness, (BCS 5) 55 (76.4) 168 (77.8) 0.92 (0.46-1.86) 0.807 139 (78.5) 64 (71.1) 1.49 (0.79-2.75) 0.179 Malaria 49 (68.1) 137 (63.4) 1.23 (0.67-2.28) 0.477 116 (65.5) 56 (62.2) 1.15 (0.66-2.02) 0.593 Deaths 0 (0) 4 (1.9) 0 (0-2.88) 0.245 1 (0.6) 3 (3.3) 0.16 (0.003-2.10) 0.078

Haptoglobin genotype, α-thalassaemia and acute seizures No significant association was found between seizures and haptoglobin HP2-2 genotype (table I). The allele frequency of the HP2-2 genotype in cases and controls was similar (χ2=0.57, p=0.499). Similarly, we found no association between acute seizures and α- thalassaemia (table II). The proportion of cases and controls with any deletion in the globin gene was similar, (χ2=0.70, p=0.403).

A parental description of the seizure event was available in 278/288 cases; the seizures did not occur in the presence of the consenting parent in 10 cases. Two hundred and thirty four children (84.2%) had generalized seizures, 37 (13.3%) had focal seizures while 7 (2.5%) had secondarily generalized seizures. HP2-2 genotype and deletions in the α- globin gene were not associated with any particular seizure manifestation, number of seizures, presentation with status epilepticus or with impairment of consciousness. In addition, there was no association between the HP2-2 polymorphism or deletions in the α-globin gene with malaria related seizures or outcome (table III).

DISCUSSION Increasingly, genetic factors are being recognised as important risk factors for both acute (febrile seizures, reviewed in [21]) and chronic (epilepsies, reviewed in [22]) seizure disorders. Studies have demonstrated a strong association between the haptoglobin HP2-2 polymorphism and idiopathic generalized epilepsies[13, 14]. In this study we

Proefschrift.indb 40 14-1-2008 13:09:09 Table III Haptoglobin genotypes, a-thalassaemia and acute seizures Seizure characteristics Haptoglobin genotype Unadjusted odds P value a-3.7-globin deletions Unadjusted odds P value HP2-2, HP1-2 or HP1-1, ratio (95%CI) Any deletion, No deletion, ratio (95%CI) N=72, (%) n=216, (%) n=177, (%) n=90, (%) Past seizures 21 (29.2) 83 (38.4) 0.66 (0.35-1.21) 0.157 65 (36.7) 33 (36.7) 1.00 (0.57-1.76) 0.993 Seizure type Generalized 58 (80.6) 176 (81.5) 0.94 (0.46-2.01) 0.862 146 (82.5) 73 (81.1) 1.10 (0.53-2.20) 0.782 Focal 9 (12.5) 28 (13.0) 0.96 (0.38-2.23) 0.919 21 (11.9) 12 (13.3) 0.88 (0.39-2.06) 0.730 Secondarily generalized 3 (4.2) 4 (1.9) 2.30 (0.33-13.94) 0.269 4 (2.3) 3 (3.3) 0.67 (0.11-4.69) 0.604 Not described 2 (2.8) 8 (3.7) 0.74 (0.08-3.85) 0.710 6 (3.4) 2 (2.2) 1.54 (0.27-15.92) 0.597 Median (IQR) number of seizures 1 (1-2) 1 (1-3) - 0.734 1 (1-3) 1 (1-3) - 0.851 Level of consciousness at admission Coma (BCS 0-2) 6 (8.3) 24 (11.1) 0.73 (0.23-1.93) 0.504 17 (9.6) 12 (13.3) 0.69(0.29-1.67) 0.355 Impaired consciousness, (BCS 3& 4) 11 (15.3) 24 (11.1) 1.44 (0.60-3.27) 0.349 21 (11.9) 14 (15.6) 0.73 (0.33-1.65) 0.398 Full consciousness, (BCS 5) 55 (76.4) 168 (77.8) 0.92 (0.46-1.86) 0.807 139 (78.5) 64 (71.1) 1.49 (0.79-2.75) 0.179 Malaria 49 (68.1) 137 (63.4) 1.23 (0.67-2.28) 0.477 116 (65.5) 56 (62.2) 1.15 (0.66-2.02) 0.593 Deaths 0 (0) 4 (1.9) 0 (0-2.88) 0.245 1 (0.6) 3 (3.3) 0.16 (0.003-2.10) 0.078

investigated whether this risk extends to acute seizures and whether another common genetic trait in Africa - a 3.7kd deletion in the α-globin gene - is associated with the high incidence of acute seizures in this region. The findings do not support our hypothesis: the prevalence of the HP2-2 genotype and α-thalassaemia genotypes were similar in cases and controls; the median number of seizures during the current illness and the frequency of past seizures in children with these polymorphisms/deletions were similar to that in cases without the polymorphisms/deletions.

Accumulation of iron and iron containing products such as haemoglobin in the interstitial tissues of the brain can initiate and enhance the generation of reactive oxygen species and cause neuronal damage by peroxidation of cell membrane lipids[15, 23]. Haptoglobin is an acute phase protein, which binds free haemoglobin released into circulation following haemolysis. Experimental evidence suggests that hypo-haptoglobinaemia is associated with poor clearance of free haemoglobin from the central nervous system and may lead to seizure disorders [15]. It was postulated that the inefficient clearance of free haemoglobin in patients with HP2-2 genotype increases unbound free haemoglobin in the brain (and other tissues) and may be associated with increased haemoglobin mediated oxidant stress on cell membranes, oxidative neuronal damage and an increased risk of seizures. In addition, α-thalassaemia deletions may affect a child’s susceptibility to acute seizures due to possible changes in neurotransmitter metabolism. Our findings suggest that, unlike the idiopathic generalized epilepsies in which the HP2-2 genotype may be one of many contributory genes involved in the polygenic inheritance of the seizure disorder[13, 14], this polymorphism is not a risk factor for acute seizure disorders.

Proefschrift.indb 41 14-1-2008 13:09:09 Although we did not measure plasma haptoglobin levels to directly correlate genotype with phenotype, the evidence for the lack of association between HP2-2 genotype and acute seizures is compelling. It is possible that there is a time lag between the onset of free haemoglobin induced “oxidative neuronal damage” and manifestation of the seizure disorder i.e. epilepsy. Alternatively, repeated episodes of “neuronal oxidative damage” are necessary before the seizure disorder can develop.

Despite this finding, family studies in children from this community do provide strong evidence to support a genetic risk factor for acute seizures [7]. It is likely that there are other susceptibility genes. Mutations and single nucleotide polymorphisms in voltage and ligand gated channels such as those described in children with febrile seizures or generalized epilepsy with febrile seizures plus may be possible candidates [24-26]. Screening for these genetic disorders may be a useful step.

CONCLUSION In conclusion, the haptoglobin HP2-2 genotype and deletions in the α-globin gene are not risk factors for acute seizures in Kenyan children with and without falciparum malaria infection. Future studies should examine other susceptibility genes.

ACKNOWLEDGEMENTS The Wellcome Trust and the European Union network 6 BioMalpar consortium funded this study. We thank Michael Kahindi and Tony Kazungu for setting up the system for identifying eligible patients, Robert Mwakesi and Barnes Kitsao for managing the blood sample storage system and, Emmanuel Ouma and Frances Ibison for helping out with DNA extraction and gel electrophoresis. CN is supported by the Wellcome Trust and the Wellcome Trust and the European Network 6 BioMalpar consortium support TW and DK.

REFERENCES 1. Berkley JA, Ross A, Mwangi I, Osier FH, Mohammed M, Shebbe M, Lowe BS, Marsh K, Newton CR: Prognostic indicators of early and late death in children admitted to district hospital in Kenya: cohort study. Bmj 2003, 326:361. 2. Obi JO, Ejeheri NA, Alakija W: Childhood febrile seizures (Benin City experience). Ann Trop Paediatr 1994, 14:211-214.

Proefschrift.indb 42 14-1-2008 13:09:09 3. Waruiru CM, Newton CR, Forster D, New L, Winstanley P, Mwangi I, Marsh V, Winstanley M, Snow RW, Marsh K: Epileptic seizures and malaria in Kenyan children. Trans R Soc Trop Med Hyg 1996, 90:152-155. 4. Carter JA, Neville BG, White S, Ross AJ, Otieno G, Mturi N, Musumba C, Newton CR: Increased prevalence of epilepsy associated with severe falciparum malaria in children. Epilepsia 2004, 45:978-981. 5. Ngoungou EB, Dulac O, Poudiougou B, Druet-Cabanac M, Dicko A, Mamadou Traore A, Coulibaly D, Farnarier G, Tuillas M, Keita MM, et al: Epilepsy as a consequence of cerebral malaria in area in which malaria is endemic in Mali, West Africa. Epilepsia 2006, 47:873-879. 6. Akpede GO, Abiodun PO, Sykes RM: Pattern of infections in children under-six years old presenting with convulsions associated with fever of acute onset in a children’s emergency room in Benin City, Nigeria. J Trop Pediatr 1993, 39:11-15. 7. Versteeg AC, Carter JA, Dzombo J, Neville BG, Newton CR: Seizure disorders among relatives of Kenyan children with severe falciparum malaria. Trop Med Int Health 2003, 8:12-16. 8. Atkinson SH, Mwangi TW, Uyoga SM, Ogada E, Macharia AW, Marsh K, Prentice AM, Williams TN: The haptoglobin 2-2 genotype is associated with a reduced incidence of Plasmodium falciparum malaria in children on the coast of Kenya. Clin Infect Dis 2007, 44:802-809. 9. Williams TN, Wambua S, Uyoga S, Macharia A, Mwacharo JK, Newton CR, Maitland K: Both heterozygous and homozygous alpha+ thalassemias protect against severe and fatal Plasmodium falciparum malaria on the coast of Kenya. Blood 2005, 106:368-371. 10. Atkinson SH, Rockett K, Sirugo G, Bejon PA, Fulford A, O’Connell MA, Bailey R, Kwiatkowski DP, Prentice AM: Seasonal childhood anaemia in West Africa is associated with the haptoglobin 2-2 genotype. PLoS Med 2006, 3:e172. 11. Wambua S, Mwangi TW, Kortok M, Uyoga SM, Macharia AW, Mwacharo JK, Weatherall DJ, Snow RW, Marsh K, Williams TN: The effect of alpha+-thalassaemia on the incidence of malaria and other diseases in children living on the coast of Kenya. PLoS Med 2006, 3:e158. 12. Idro R, Ndiritu M, Ogutu B, Mithwani S, Maitland K, Berkley J, Crawley J, Fegan G, Bauni E, Peshu N, et al: Burden, features, and outcome of neurological involvement in acute falciparum malaria in Kenyan children. Jama 2007, 297:2232-2240. 13. Saccucci P, Verdecchia M, Piciullo A, Bottini N, Rizzo R, Gloria-Bottini F, Lucarelli P, Curatolo P: Convulsive disorder and genetic polymorphism. Association of idiopathic generalized epilepsy with haptoglobin polymorphism. Neurogenetics 2004, 5:245-248. 14. Sadrzadeh SM, Saffari Y, Bozorgmehr J: Haptoglobin phenotypes in epilepsy. Clin Chem 2004, 50:1095-1097. 15. Panter SS, Sadrzadeh SM, Hallaway PE, Haines JL, Anderson VE, Eaton JW: Hypohaptoglobinemia associated with familial epilepsy. J Exp Med 1985, 161:748-754. 16. Auvichayapat P, Auvichayapat N, Jedsrisuparp A, Thinkhamrop B, Sriroj S, Piyakulmala T, Paholpak S, Wattanatorn J: Incidence of febrile seizures in thalassemic patients. J Med Assoc Thai 2004, 87:970-973. 17. Berkley JA, Lowe BS, Mwangi I, Williams T, Bauni E, Mwarumba S, Ngetsa C, Slack MP, Njenga S, Hart CA, et al: Bacteremia among children admitted to a rural hospital in Kenya. N Engl J Med 2005, 352:39-47. 18. WHO: Severe falciparum malaria. Trans R Soc Trop Med Hyg 2000, 94 Suppl 1:S1-90. 19. Molyneux ME, Taylor TE, Wirima JJ, Borgstein A: Clinical features and prognostic indicators in paediatric cerebral malaria: a study of 131 comatose Malawian children. Q J Med 1989, 71:441- 459.

Proefschrift.indb 43 14-1-2008 13:09:09 20. Chong SS, Boehm CD, Higgs DR, Cutting GR: Single-tube multiplex-PCR screen for common deletional determinants of alpha-thalassemia. Blood 2000, 95:360-362. 21. Audenaert D, Van Broeckhoven C, De Jonghe P: Genes and loci involved in febrile seizures and related epilepsy syndromes. Hum Mutat 2006, 27:391-401. 22. Berkovic SF, Mulley JC, Scheffer IE, Petrou S: Human epilepsies: interaction of genetic and acquired factors. Trends Neurosci 2006, 29:391-397. 23. Suzer T, Coskun E, Demir S, Tahta K: Lipid peroxidation and glutathione levels after cortical injection of ferric chloride in rats: effect of trimetazidine and deferoxamine. Res Exp Med (Berl) 2000, 199:223-229. 24. Baulac S, Huberfeld G, Gourfinkel-An I, Mitropoulou G, Beranger A, Prud’homme JF, Baulac M, Brice A, Bruzzone R, LeGuern E: First genetic evidence of GABA(A) receptor dysfunction in epilepsy: a mutation in the gamma2-subunit gene. Nat Genet 2001, 28:46-48. 25. Ito M, Nagafuji H, Okazawa H, Yamakawa K, Sugawara T, Mazaki-Miyazaki E, Hirose S, Fukuma G, Mitsudome A, Wada K, Kaneko S: Autosomal dominant epilepsy with febrile seizures plus with missense mutations of the (Na+)-channel alpha 1 subunit gene, SCN1A. Epilepsy Res 2002, 48:15- 23. 26. Wallace RH, Scheffer IE, Parasivam G, Barnett S, Wallace GB, Sutherland GR, Berkovic SF, Mulley JC: Generalized epilepsy with febrile seizures plus: mutation of the sodium channel subunit SCN1B. Neurology 2002, 58:1426-1429.

Proefschrift.indb 44 14-1-2008 13:09:09 Chapter 4

Burden, features and outcome of neurological involvement in acute falciparum malaria in Kenyan children

Richard Idro, Moses Ndiritu, Bernardhs Ogutu, Sadik Mithwani, Kathryn Maitland, James A Berkley, Jane Crawley, Gregory Fegan, Evasius Bauni, Norbert Peshu, Kevin Marsh, Brian Neville, Charles C.R.J. Newton

Centre for Geographic Medicine Research – Coast, Kenya Medical Research Institute, Kilifi, Kenya; Department of Paediatrics and Child Health, Mulago Hospital/Makerere University, Kampala, Uganda; Walter Reed Project - Centre for Clinical Research, Kenya Medical Research Institute, Kisumu, Kenya; Department of Paediatrics, Faculty of Medicine and The Wellcome Trust Centre for Tropical Medicine, Imperial College, London, UK; Centre for Clinical Vaccinology and Tropical Medicine, University of Oxford, UK; Infectious Diseases Epidemiology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London – UK; Neurosciences Unit, University College London, Institute of Child Health, UK

JAMA, 2007; 297 (20): 2232 – 40

Proefschrift.indb 45 14-1-2008 13:09:09 ABSTRACT

Context Plasmodium falciparum appears to have a particular propensity to involve the brain, but the burden, risk factors and full extent of neurological involvement have not been systematically described.

Objective Determine the incidence and describe the clinical phenotypes and outcome of neurological involvement in African children with acute falciparum malaria.

Design, setting and patients A review of records of all children younger than14 years admitted to a Kenyan District Hospital with malaria, from January 1992 through December 2004. Neurological involvement was defined as convulsive seizures, agitation, prostration, impaired consciousness or coma.

Outcome measures The incidence, pattern and outcome of neurological involvement.

Results Of 58,239 children admitted, 19,560(33.6%) had malaria as the primary clinical diagnosis. Neurological involvement was observed in 9,313(47.6%) children and manifested as seizures (6,563/17,517, 37.5%), agitation (316/11,193, 2.8%), prostration (3,223/15,643, 20.6%), impaired consciousness or coma (2,129/16,080, 13.2%). In children <5 years, the mean annual incidence of admissions with malaria was 2,694/100,000 and the incidence of malaria with neurological involvement was 1,156/100,000. However, re-admissions may have led to a 10% overestimate in the incidence. Children with neurological involvement were older (median [inter quartile range] age 26[15-41] vs 21[10-40] months, p<0.001), had a shorter duration of illness (2[1-3] vs 3[2-3] days, p<0.001) and a higher geometric mean parasite density (42.0 [95%CI 40.0-44.1] vs 30.4 [95%CI 29.0-31.8]x103/µl, p<0.001). Factors independently associated with neurological involvement included past history of seizures (adjusted OR 3.50 95%CI 2.78-4.42), fever ≤2 days (adjusted OR 2.02 95%CI 1.64-2.49), delayed capillary refill time (adjusted OR 3.66 95% CI 2.40-5.56), acidosis (adjusted OR 1.55 95% CI 1.29-1.87) and hypoglycemia (adjusted OR 2.11 95% CI 1.32-3.37). Mortality was higher in patients with neurological involvement (4.4% [95% CI, 4.2%-5.1%] vs 1.3% [95% CI, 1.1%-1.5%], p<0.001), and at discharge, 159 out of 7281 (2.2%) had neurological deficits.

Conclusions Neurological involvement is common in children with acute falciparum malaria. It is associated with metabolic derangements, impaired perfusion, parasitemia and has increased mortality and neurological sequelae. The study suggests that malaria exposes many African children to brain insults.

Proefschrift.indb 46 14-1-2008 13:09:09 INTRODUCTION Malaria is a leading cause of ill health in tropical countries. In 2002, over 2 billion people were exposed to malaria and there were an estimated 515 million clinical episodes of acute Plasmodium falciparum infection. Over 70% of these episodes occurred in sub- Saharan Africa and mainly affected children <5 years1.

Plasmodium falciparum is the most common cause of severe malaria and in children this typically manifests as severe anemia, prostration, repeated seizures, impaired consciousness, hypoglycemia and metabolic acidosis2. Uniquely, Plasmodium falciparum infected erythrocytes sequester within deep vascular beds, particularly in the brain3. Neurological involvement (NI) may manifest as seizures, impaired consciousness or coma4. Recent studies demonstrate that nearly a quarter of the children who survive cerebral malaria or malaria with complicated seizures are at risk of persistent neurological and cognitive impairments and/or epilepsy5-7. Apart from cerebral malaria8, the burden, manifestations, risk factors and consequences of NI in malaria have not been fully described. This study determined the incidence, clinical phenotypes, associated factors and outcome of NI in acute falciparum malaria in Kenyan children.

METHODS Study design Records of all children admitted to Kilifi District Hospital (KDH) with a parasitological diagnosis of malaria over a 13-year period from January 1992 through December 2004 were reviewed. We compared clinical and laboratory features of patients with and without NI on admission.

Study area KDH is located on coastal Kenya and admits approximately 5,000 children annually. It serves a predominantly rural population and is the only hospital that admits very sick children from the surrounding communities. The annual entomological inoculation rate (the number of times an individual is bitten by mosquitoes infected with Plasmodium falciparum in one year) in the catchment area ranges from <1 to 120 infectious bites per year9. The area has been described in detail elsewhere10.

Proefschrift.indb 47 14-1-2008 13:09:09 Study population Eligible children had asexual forms of P. falciparum parasites detected on blood films with malaria as the only or main diagnosis. Neurological involvement4 was defined as: i. history of convulsive seizures during the presenting illness reported by the parent or observed on admission to hospital. ii. the presence of agitation (abnormally high level of activity or irritability), iii. prostration (inability to sit upright or breast feed)2 , iv. impaired consciousness (Blantyre coma score less than 5)11, 12 v. coma (unable to localise a painful stimulus i.e cerebral malaria)11, 13 . We included prostration as a neurological feature since it is difficult to differentiate neurological and severe systemic causes of prostration in children.

Admission procedures and inpatient care Admission data was collected on standardized proformas that had a conserved basic format and otherwise varied according to ongoing clinical studies. The proformas detailed the medical history and the physical examination14. As part of the research activities of the center, parents of children admitted to the hospital were invited to consent for their clinical data to be used for disease surveillance.

Emergency resuscitation and inpatient care was provided according to standard protocols13, 15. Severe malaria was treated with parenteral quinine or arthemeter until patients could tolerate oral medication, when antimalarial therapy was completed with a full course of first line drugs according to the national guidelines in Kenya. Those with life threatening features were closely monitored on the high dependency unit. Nursing staff performed clinical assessments of vital signs, seizures and level of consciousness every 4-6 hours. Physicians’ reassessments were at 4 and 24 hours after admission and then, daily. Mechanical ventilation was not available. Patients without life threatening features (impaired consciousness, repeated seizures or respiratory distress)2 had daily assessments on the general ward.

Laboratory procedures At admission, blood was drawn for a full blood count, blood glucose, thick and thin films (stained for malaria parasites with 10% Giemsa) and from 1998, for blood culture16. Among those with life threatening features, a venous sample was drawn for acid base status and plasma electrolytes. Metabolic acidosis was defined as a base deficit >8. Hypokalemia was defined as plasma +K <3.0mmol/L, hyperkalemia as K+>5.0mmol/L and hyponatremia as Na+<135mmol/L. Lumbar punctures were performed according to a standard protocol to exclude pyogenic meningitis17. A cerebrospinal fluid (CSF) leukocyte

Proefschrift.indb 48 14-1-2008 13:09:09 count of up to 10/µl was considered normal18. All tests including the parasitological diagnosis of malaria are performed in a certified research laboratory with a system for regular internal and external audits.

DATA MANAGEMENT AND STATISTICAL ANALYSIS For this analysis, we classified admissions as with or without NI. We determined the mean incidence of admissions with malaria and of malaria with NI in a defined study area using population denominators projected from the 1989 (for the years 1992–1998) and the 1999 (for years 2000-2004) Government of Kenya census. The yearly population estimate was calculated from the intercensal population growth rate of 2.9% between the censuses19, 20. Admission clinical characteristics of the two groups of patients were compared to describe features associated with NI and a p-value<0.05 was considered statistically significant. Comparisons were performed only for patients for whom data was available. Categorical variables were compared using Pearson’s Chi square test. For approximately normally distributed data, means were compared with the student’s t- test and the Wilcoxon Rank-sum test used for skewed data. We performed multivariable logistic regression analysis with NI as the dependent variable to identify features independently associated with NI. In building the multiple regression model, we included clinical and laboratory features with p values<0.1 on univariate analysis and identified the independent variables by backward elimination. Similarly, we compared the characteristics of patients with prostration to describe the associated features. Using the conservative Bonferroni approach to multiple comparisons, we adjusted the level of significance α for the additional comparisons (prostration α<0.025) and death or neurological sequelae (α<0.017). Data analysis was performed using Stata Version 9 (Stata Corporation Inc. Texas).

RESULTS Burden of malaria with neurological involvement A total 58,239 children were admitted to KDH during the study period. Of these, 22,441 had malaria parasites detected on blood smears and 19,560 (33.6%) had malaria as the primary clinical diagnosis (figure 1).

Of the 19,560 admissions with malaria, neurological features were present in 9,313 (47.6%) at admission, compared to 7,794 of the 35,798 (21.8%) admissions without malaria (p<0.001). The mean annual incidence of admissions with malaria among children<5 years over the study period was 2,694 (range 1,506 – 3,744) per 100,000 and of malaria

Proefschrift.indb 49 14-1-2008 13:09:09 Figure 1 Children admitted with malaria to KDH from 1992 – 2004

with NI was 1,156 (range 474 – 2075) per 100,000 (figure 2). The peak incidences (3,794 and 1,181 per 100,000 respectively) were in the first year of life. A marked decline in incidence was observed in children older than 5 years: the incidence of admissions with malaria or malaria with NI were 344 per 100,000 and 120 per 100,000 in children 5–9 years and, 39 and 9 per 100,000 in children 10-14 years respectively.

Figure 2 The incidence of admissions with malaria and malaria with neurological involvement in children <13 years in Kilifi District Hospital, 1992–2004

Proefschrift.indb 50 14-1-2008 13:09:10 We were unable to exclude re-admissions due to malaria over the whole study period, a fact that could have led to an over-estimate of the incidence. In order to examine this, we estimated the proportion of children who could have had re-admissions for malaria using data of children admitted from mid April 2002 to December 2004, when each child obtained a unique identification number that was used during all subsequent admissions. Using this identifier, 348/3421 (10.2%) children were found to have had more than one admission. Therefore, about 10% of the incidence data provided here may be an over estimate contributed by re-admissions with malaria.

Features of neurological involvement on admission Seizures Seizures were the commonest neurological feature of acute falciparum malaria and were reported or observed (at admission) in 37.5% (table 1).

Table 1 Neurological involvement in children with falciparum malaria on admission to hospital and treatment outcome, 1992-2004 Neurological involvement on admission to No. of cases/Total No. of No. (%) who died, hospital patients assessed, (%) N=542* No neurological involvement at admission 10,247/19560 (52.4) 130 (1.3) Types of neurological involvement at admission Seizures 6,563/17,517 (37.5) 251 (3.8) Agitation 316/11,193 (2.8) 15 (4.7) Prostration 3,223/15643 (20.6) 215 (6.7) Impaired consciousness or coma 2,129/16,080 (13.2) 190 (8.9) *Categories are not mutually exclusive (i.e. some children had both seizures and agitation or other combinations)

Seizures were uncommon before the age of 6 months. After 12 months, the age specific prevalence increased rapidly reaching a peak prevalence of 48.6% among patients 27–33 months. Patients with seizures had a shorter duration of illness and higher parasitemia (table 2). Multiple seizures were common with 56% of those with a history of seizures reporting two or more episodes during the illness. In 22% of children, seizures lasted longer than 30 minutes, fulfilling the definition of status epilepticus.

During the course of the inpatient stay, seizures were observed in 13.3% of children with NI. These were not associated with hypoglycemia or hyponatremia. Temperature appeared to influence seizure manifestation; 63% of generalized seizures were reported in febrile children compared to 54% secondarily generalized seizures and 44% focal seizures (χ2 for linear trend=7.235, p=0.007). The recurrence of seizures in the ward was associated with increased mortality (12.8 vs 1.2 %, p<0.001).

Proefschrift.indb 51 14-1-2008 13:09:10 Among 6,212 children asked about seizures during previous illnesses, a history of seizures was more common (41.5%) among those admitted with seizures compared to those without (15.1%) p<0.001. The majority of these past seizures were associated with febrile illnesses including respiratory tract infections and malaria.

Prostration Sixty percent of patients with prostration reported a seizure. We examined the clinical risk factors for prostration among 2,967 consecutive children assessed for both seizures and prostration. Children without seizures were more likely to have severe anemia, acidosis or hypoglycemia. Prostration with seizures was associated with higher parasitemia. Seizures (adjusted OR 2.44 95%CI 2.04-2.93, p<0.001), delayed capillary refill time (adjusted OR 5.24 95%CI 3.72-7.38, p<0.001), hypoglycemia (adjusted OR 3.85 95%CI 2.73-5.43, p<0.001), metabolic acidosis (adjusted OR 1.92 95%CI 1.61-2.29, p<0.001) and high parasite density (adjusted OR 1.01 95%CI 1.00-1.10, p=0.001) were independently associated with prostration.

Impaired consciousness Impaired consciousness was associated with seizures, older age, higher parasite density, hypoglycemia and acidosis but not hyponatremia (table 2). Patients with seizures and normal consciousness on admission had less metabolic derangements compared to those with impaired consciousness. Comatose patients were ill for longer prior to admission compared to either those with agitation or prostration.

Table 2 Clinical, hematological and biochemical factors associated with changes in consciousness Patient characteristics Normal consciousness Agitation Prostration Impaired consciousness No Seizures Seizures or coma Age, median (IQR), mo 22 (10-41) 25 (14-38)*** 24 (12-42)* 27 (15-43) 28 (16-44)*** Seizures 0 (0) 3,951/3951 (100)*** 146/291 (50.2)*** 1742/2967 (58.7)*** 1512/2129 (71.0)*** Duration of illness, median (IQR), d 3 (1-3) 2 (1-3)*** 1(0-3)*** 2 (1-3)*** 3 (0-3)** Admission temperature, mean (SD), o C 38.6 (1.2) 38.7 (1.2)*** 38.2 (1.3)* 38.0 (1.4)*** 38.1 (1.3)*** Delayed capillary refill time 212/4680 (4.5) 98/2724 (3.6)* 56/291 (19.2)*** 336/2151 (15.6)*** 264/1574 (16.8)*** Parasite density, geometric mean (95% CI), x103/ml, 36.5 (33.4-39.8) 42.2 (39.2-45.4)*** 38.8 (29.8-42.8) 42.6 (38.6-46.9)*** 45.0 (36.8-55.0)*** Hypoglycemia 70/1457 (4.8) 56/1442 (3.9) 16/150 (10.7) *** 208/1466 (14.2) *** 126/785 (16.0)*** Hemoglobin mean (SD), g/L 77 (28) 95 (31) *** 86 (36) *** 100 (33) *** 85 (44)*** Metabolic acidosis 735/2007 (36.6) 466/1366 (34.1) 101/182 (55.5) *** 955/1665 (57.4) *** 595/1017 (58.5)*** Hyperkalemia 50/1917 (2.6) 82/1236 (6.6)* 22/147 (14.0) *** 303/1720 (17.7)*** 239/1229 (19.5)*** Hyponatremia 1026/1922 (53.4) 696/1236 (56.3) 81/157 (51.6) 941/1724 (54.6) 641/1232 (52.0) Abbreviations: CI, confidence interval; IQR, interquartile range. †Values are expressed as number (percentage) unless otherwise indicated *P<0.05 (All values compared to normal consciousness with no seizures). **P<0.01 ***P<0.001

Proefschrift.indb 52 14-1-2008 13:09:10 Overlap between the features of NI was common, particularly seizures and prostration or seizures and impaired consciousness (table 2). All patients with agitation had at least one other feature of neurological involvement i.e. seizures, prostration or impaired consciousness.

Deterioration in level of consciousness after admission Deterioration in consciousness during admission was observed in 219/1,533(14.3%) children with NI admitted to the high dependency unit. It was associated with recurrence of seizures and abnormal motor posturing but not duration of illness, admission temperature, parasite density, hypoglycemia, metabolic acidosis, hyponatremia or severe anemia. However, secondary deterioration in consciousness was associated with higher mortality than established impaired consciousness (38.6 vs 12.8%, p<0.001). Some children developed agitation during the course of the admission. This was associated with worsening level of consciousness and increased mortality.

Factors associated with NI in falciparum malaria Children with NI were older and had a shorter history of illness (table 3). The median duration of illness was 2 days in patients with NI compared to 3 days in those without NI and the majority of children admitted with <2 days of fever had NI (65.4 vs 34.6%, p<0.001). Apart from seizures, the previous medical history in the two groups was similar. Features of shock or impaired perfusion and metabolic acidosis were associated with NI. Vomiting, diarrhea and cough were less common in patients with NI. Additional

Proefschrift.indb 53 14-1-2008 13:09:10 Table 3 Admission characteristics Patient Characteristics Neurological involvement, P value No. (%) of patients* Present Absent Male, sex 4884/9310 (52.5) 5361/10246 (52.3) 0.85 Age, median (IQR) in months 26 (15 – 41) 21 (10 – 40) <0.001 Head circumference <-2 SD for age+ 371/3305 (11.2) 226/1935 (11.7) 0.62 Weight for height Z score <-2+, (wasting) 1241/5732 (21.7) 810/4408 (18.4) <0.001 Past seizures 1338/3841 (34.8) 349/2371 (14.7) <0.001 Fever 6019/6231 (96.6) 4718/4894 (96.4) 0.57 Vomiting 1336/5339 (25.0) 1638/4361 (37.6) <0.001 Cough 1996/6230 (32.0) 2144/4893 (43.8) <0.001 Diarrhoea 530/5585 (9.5) 646/4883 (13.2) <0.001 Duration of illness, median (IQR), days 2 (1 – 3) 3 (2 – 3) <0.001 Axillary temperature, mean (SD), oC 38.4 + 1.4 38.6 +1.5 >0.99 Jaundice 102/6042 (1.7) 83/4529 (1.8) 0.58 Delayed capillary refill 646/6392 (10.1) 212/4680 (4.5) <0.001 Temperature gradient† 809/3840 (21.1) 351/2371 (14.8) <0.001 Splenomegaly 4271/9313 (45.9) 6404/10247 (62.5) <0.001 Hepatomegaly 662/6391 (10.4) 286/4679 (6.1) <0.001 Respiratory distress‡ 1209/6521 (18.5) 906/5331 (17.0) 0.03 Sustained nasal flaring 528/4884 (10.8) 249/3022 (8.2) <0.001 Deep breathing 760/6520 (11.7) 221/5329 (4.1) <0.001 Subcostal retractions 658/6227 (10.6) 579/4890 (11.8) 0.03 Severe anemia 870/9307 (9.3) 2039/9744 (20.9) <0.001 Metabolic acidosis 1910/3904 (48.9) 735/2007 (36.6) <0.001 White blood count, mean (SD), x 103/ml 13.7 (8.9) 14.7 (10.3) <0.001 Parasite density, geometric mean (95% CI), x103/ml 42.0 (40.0-44.1) 30.4 (29.0-31.8) <0.001 Hypoglycemia 322/3441 (9.4) 70/1457 (4.8) <0.001 Thrombocytopenia 1295/3652 (35.5) 929/2859 (32.5) 0.01 Hyponatremia 2249/4094 (54.9) 1026/1922 (53.4) 0.26 Potassium level Hyperkalemia 70/4088 (1.7) 50/1917 (2.6) 0.02 Hyperkalemia 595/4088 (14.6) 188/1917 (9.8) <0.001 Impaired renal function (creatinine >80 mmol/L]) 665/3659 (18.2) 311/1619 (19.2) 0.37 Bacteremia 126/5627 (2.2) 122/4151 (2.9) 0.03 Comorbidity§ 924/9313 (9.9) 1901/10247 (18.6) <0.001 Abbreviations: CI, confidence interval; IQR, interquartile range * Unless otherwise indicated + Using the 1978 WHO standards † Subjective temperature difference between the trunk and the peripheries ‡ Presence of sustained nasal flaring, deep acidotic breathing or subcostal retractions § Presence of an additional diagnosis when the primary diagnosis is malaria

Proefschrift.indb 54 14-1-2008 13:09:10 diagnoses, particularly respiratory tract infections, were less common among children with NI.

Overall, 15.3% of patients had severe anemia (Hb<50 g/L). There was no clear association between the hemoglobin concentration and level of consciousness (table 2). Although NI was observed at low levels of parasitemia, the proportion with NI increased with rising parasitemia: NI was present in 40% of patients with parasite densities <100,000/µl, 50% of patients with 100,000-500,000/µl and in over 60% of those with densities >1,000,000/µl (χ2 for trend =120, p<0.001).

Among patients with life threatening features, the commonest biochemical derangements were hyponatremia, acidosis, hyperkalemia, hypoglycemia and elevated plasma creatinine (table 3). Only metabolic acidosis, hypoglycemia and hyperkalemia were significantly associated with NI.

Factors independently associated with neurological involvement Clinical and laboratory features associated with NI on univariate analysis with a p value <0.1 (table 3) were entered in a logistic regression model to identify those features independently associated with NI. These included past history of seizures, hypoglycemia, acidosis, delayed capillary refill and duration of fever shorter than 2 days. An additional diagnosis (co-morbidity) and severe anemia were independently associated with absence of NI (table 4).

Table 4 Variables independently associated with neurological involvement Variables Unadjusted OR Adjusted OR P value (95%CI) (95% CI) Past history of seizures 3.10 (2.71 – 3.54) 3.50 (2.78 – 4.42) <0.001 Fever £ 2 days 1.58 (1.45 – 1.72) 2.02 (1.64 – 2.49) <0.001 Delayed capillary refill 2.37- (2.01 – 2.79) 3.66 (2.40 – 5.56) <0.001 Metabolic acidosis 1.66 (1.48 – 1.85) 1.55 (1.29 – 1.87) <0.001 Hypoglycemia 2.05 (1.56 – 2.71) 2.11 (1.31 – 3.37) 0.002 Co-morbidity 0.48 (0.44 – 0.52) 0.38 (0.30 – 0.47) <0.001 Severe anemia 0.39 (0.36 – 0.42) 0.78 (0.62 – 0.98) 0.04

Outcome The median duration of hospital stay including deaths was 3 (IQR 2 – 4) days and was similar in patients with and without NI overall. However, compared to those without any alteration of consciousness, patients with impaired consciousness stayed longer in the wards, (3 [IQR 2-4] vs 2 [1-4] days, p<0.001).

Proefschrift.indb 55 14-1-2008 13:09:10 Mortality Overall, 542 children died. Neurological involvement was associated with increased mortality: 4.4% (95% CI 4.2-5.1) died compared to 1.3% (95% CI 1.1-1.5) of those without NI. Mortality increased with lower levels of consciousness at admission (table 1). Children with cerebral malaria had the highest mortality, especially those admitted without a history of seizures (22.2 vs 13.6%, p=0.006). Respiratory arrest (often associated with brainstem signs) occurred more commonly in children with NI (37.2 vs 18.2% of all deaths, p=0.031) while cardio-respiratory arrests (associated with severe metabolic acidosis or anemia) were more common in children without NI. At univariate analysis, several clinical and laboratory features (coma, respiratory distress, a temperature gradient between the periphery and the trunk, delayed capillary refill time, splenomegaly, hepatomegaly, severe anemia, high parasitemia, hypoglycemia, bacteremia, hyperkalemia, thrombocytopenia, and leucocytosis) were associated with increased mortality. Factors independently associated with death were impaired consciousness/coma, hypoglycemia, severe anemia, bacteremia, hyperkalemia and respiratory distress (table 5). The same risk factors were associated with death in the subset of patients with NI.

Table 5 Variables independently associated with mortality Variables Unadjusted OR Adjusted OR P value (95%CI) (95%CI) Impaired consciousness or coma 15.86 (12.11-20.80) 4.06 (2.33 – 7.07) <0.001 Respiratory distress 8.25 (6.40 – 10.64) 4.47 (2.66 – 7.54) <0.001 Severe anemia 1.84 (1.49 – 2.25) 3.08 (1.75 – 5.42) <0.001 Hypoglycemia 10.95 (8.04– 14.88) 2.46 (1.40 – 4.35) 0.002 Hyperkalemia 10.86 (8.49– 13.90) 3.83 (2.27 – 6.45) <0.001 Bacteremia 4.86 (3.04 -7.50) 5.01 (1.93 – 13.04) 0.001 Seizures on the ward 10.25 (7.20 – 14.38) 3.56 (2.03 – 6.20) <0.001

Neurological deficits on discharge One hundred fifty nine children out of 7,281 (2.2%) with NI had neurological deficits at discharge. Neurological deficits were observed in 2.1% of survivors with seizures, 4.4% with agitation, 3.6% with agitation and 6.4% of those with impaired consciousness or coma. Deficits included motor disorders (spasticity and central hypotonia), ataxia, movement disorders (choreo-athetoid, tremors and dystonic posturing), visual, hearing and speech impairments, and continuing epileptic seizures. Behavioral problems were reported in 11% of those with neurological deficits and included hyperactivity, violent and impulsive behavior, hallucinations, excessive eating and fear/anxiety. Presentation with impaired consciousness (adjusted OR 6.9 95%CI 4.6-10.2, p<0.001) and recurrence

Proefschrift.indb 56 14-1-2008 13:09:10 of seizures in hospital (adjusted OR 2.7 95%CI 1.9-4.1, p<0.001) were independently predictive of neurological deficits at discharge.

DISCUSSION Neurological involvement occurred in almost half of all children admitted with acute falciparum malaria and commonly manifested as seizures, prostration, impaired consciousness or coma. It was associated with increased mortality and neurological sequelae. Although prostration may be a general feature of severe systemic illness, the occurrence of seizures in 60% suggests frequent involvement of the CNS.

Burden of NI in children with acute falciparum malaria In children aged <5years, the mean incidence of admission with malaria in this area with <1-120 infectious mosquito bites a year was 2,694/100,000 and at least 1,156/100,000 were exposed to malaria related brain insults annually between 1992-2004. The risk was lower in children older than 5 years.

In 1998, the mid-term year for this study, there were an estimated 94.3 million children <5 years and 77.8 million children 5-9 years living in areas with stable malaria transmission in sub-Saharan Africa21. Applying our estimates to the population denominator, at a minimum 2,540,442 children <5 years were admitted to hospitals with malaria of who 1,090,108 had exposure to potential brain injury every year from 1992 to 2004. Similar annual figures for children 5-9 years are 267,632 and 93,360 respectively. These numbers are an absolute minimum as they do not account for children from the study area who did not attend our hospital. Previous estimates from the study area indicate that two thirds of deaths in children <5 years occur outside the hospital22. In addition, the annual EIR in the study area (<1–121)9 is lower than the overall figures for the continent (0–884)23. This estimates also do not include areas with unstable (epidemic prone) transmission in Africa. However, 10% of the incidence may be an over estimate due to the inclusion of re- admissions in the numerator.

Traditionally, NI in childhood falciparum malaria has been defined as cerebral malaria, the extreme form of NI, and has been characterised by seizures and coma15. The study shows that emphasis on cerebral malaria alone may be missing other syndromes with the potential to damage the brain. We found that <20% of those with neurological illness attributable to malaria fulfilled the definition of cerebral malaria and propose the term “malaria with neurological involvement” to include all. The public health importance of such NI is enormous: recent studies described a combination of long-term neurological and cognitive impairments in 24% of children exposed to cerebral malaria or malaria

Proefschrift.indb 57 14-1-2008 13:09:11 with multiple seizures5 and an increased risk for epilepsy6, 24. In addition, the longer duration of inpatient stay and more severe illness may translate into higher healthcare costs. It is estimated that the cost of treating malaria with cerebral features in a child in district hospitals in sub-Saharan Africa is US$ 44-105 compared to US$ 33-57 for that without cerebral features25.

Risk factors for neurological involvement Seizures were reported in almost 40% of children admitted with malaria compared to 12% of children with other acute medical illnesses. The proportion of patients with NI and in particular seizures, increased until 1997. Possible reasons for this include an increasing awareness of the problem, patient selection or a growing confidence of the community in the ability of the hospital to manage seizures26. Seizures were associated with high parasitemia, but not fever or hyponatremia, supporting the hypothesis that falciparum malaria might be epileptogenic per se27-30. The presence of additional neurological features supports the hypothesis of direct cerebral involvement. However, it is also likely that some seizures in malaria are simple febrile seizures similar to those seen in non-malaria endemic areas. It is also notable that not all children with cerebral malaria reported seizures and those without seizures had a worse outcome suggesting there are other mechanisms by which coma might develop.

Several factors may contribute to the pathogenesis of NI in childhood malaria; biochemical perturbations (hypoglycemia, acidosis), impaired perfusion, high parasitemia and, some children may have a predisposition to seizures (higher frequency of past history of seizures, figure 3). Although peripheral parasitemia correlates poorly with vascular sequestration3, 31, high parasite density does predict poor outcome in cerebral malaria11. This may be due to systemic derangements in immunological, metabolic and cardio- respiratory functions. These changes may be associated with altered consciousness32 and may precipitate or lower the threshold for seizures.

We propose that NI in falciparum malaria may arise from: i) A direct effect of sequestered parasites possibly through parasite induced toxins or immune responses to sequestered parasites on neuronal/blood-brain barrier function or mechanical vascular blockage. ii) An indirect effect from parasite induced local and systemic metabolic derangements or impaired perfusion (impaired delivery of substrates).

Genetic susceptibility to seizures33 including febrile seizures may be important. Acute and long-term imaging studies, especially magnetic resonance imaging, will greatly assist in determining pathogenesis.

Proefschrift.indb 58 14-1-2008 13:09:11 Outcome of malaria with neurological involvement Overall, involvement of the CNS was associated with increased mortality and neurological sequelae in survivors. The risk of death and neurological damage increased with lower levels of consciousness at admission (table 2). The proportion of children admitted with metabolic acidosis or hypoglycemia also increased with worsening level of consciousness and rising mortality. Children with single seizures without additional neurological features had low mortality (minimal risk) but the presence of prostration, impaired consciousness or secondary deterioration in consciousness was associated with increased mortality (high risk). Identification and supportive treatment may improve outcome in these patients34.

Two percent of the patients with NI and in particular those with impaired consciousness or repeated seizures, had gross neurological deficits at discharge similar to that previously described32. At least one tenth of those with deficits had behavioral problems. Future studies should characterise these behavior disorders and suggest intervention measures. Apart from the difficulties in ascertaining re-admissions, the study suffers from the limitations of relying on retrospective data from one hospital, based only on inpatients, possible incomplete parental reporting and the possibility that some children in the study area could have received care from other health units. These have the consequence of under reporting the extent of the burden of malaria and its sequelae. Our data also does not allow analysis of any contribution of HIV infection to NI. This is clearly an important area that needs further study.

CONCLUSION Neurological involvement is common in children admitted with acute falciparum malaria and goes beyond what has been traditionally regarded as “cerebral malaria”. It is associated with a history of seizures in previous illnesses, impaired perfusion, perturbations in biochemical functions, high parasitemia, mortality and neurological deficits. The high frequency of NI suggests that annually, many children in sub-Saharan Africa are exposed to malaria related brain insults.

ACKNOWLEDGEMENTS The data for this paper accumulated from clinical studies supported by the Wellcome Trust and Kenya Medical Research Institute in Kilifi. CRJC Newton (070114), J Berkley (053439), K Maitland (045194) and K Marsh are supported by the Wellcome Trust. Were

Proefschrift.indb 59 14-1-2008 13:09:11 are grateful to the clinical and nursing staff of KDH who over the years, diligently kept patients’ records, the records and data management staff who stored, helped retrieve and organised the records, and the Medical Superintendent KDH. This paper is published with the permission of the Director of KEMRI.

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Proefschrift.indb 60 14-1-2008 13:09:11 18. Berkley JA, Mwangi I, Mellington F, Mwarumba S, Marsh K. Cerebral malaria versus bacterial meningitis in children with impaired consciousness. Q J Med. Mar 1999;92(3):151-157. 19. (CBS) CBoS. Kenya Population Census, 1989. Nairobi: CBS, Ministry of Planning and National Development, Kenya; 1994. Vol. 1. 20. (CBS) CBoS. Population distribution by administrative areas and urban centres, Kenya 1999 Population and Housing Census. Nairobi: CBS, Ministry of Planning and National Development, Kenya; 2001. Vol 1. 21. Murphy SC, Breman JG. Gaps in the childhood malaria burden in Africa: cerebral malaria, neurological sequelae, anemia, respiratory distress, hypoglycemia, and complications of pregnancy. Am J Trop Med Hyg. Jan-Feb 2001;64(1-2 Suppl):57-67. 22. Snow RW, Mung’ala VO, Foster D, Marsh K. The role of the district hospital in child survival at the Kenyan Coast. Afr J Health Sci. May 1994;1(2):71-75. 23. Hay SI, Rogers DJ, Toomer JF, Snow RW. Annual Plasmodium falciparum entomological inoculation rates (EIR) across Africa: literature survey, Internet access and review. Trans R Soc Trop Med Hyg. Mar-Apr 2000;94(2):113-127. 24. Ngoungou EB, Koko J, Druet-Cabanac M, et al. Cerebral malaria and sequelar epilepsy: first matched case-control study in Gabon. Epilepsia. Dec 2006;47(12):2147-2153. 25. Kirigia JM, Snow RW, Fox-Rushby J, Mills A. The cost of treating paediatric malaria admissions and the potential impact of insecticide-treated mosquito nets on hospital expenditure. Trop Med Int Health. Feb 1998;3(2):145-150. 26. Mwenesi HA, Harpham T, Marsh K, Snow RW. Perceptions of symptoms of severe childhood malaria among Mijikenda and Luo residents of coastal Kenya. J Biosoc Sci. Apr 1995;27(2):235- 244. 27. Akpede GO, Sykes RM, Abiodun PO. Convulsions with malaria: febrile or indicative of cerebral involvement? J Trop Pediatr. Dec 1993;39(6):350-355. 28. Idro R, Aloyo J, Mayende L, Bitarakwate E, John CC, Kivumbi GW. Severe malaria in children in areas with low, moderate and high transmission intensity in Uganda. Trop Med Int Health. Jan 2006;11(1):115-124. 29. Wattanagoon Y, Srivilairit S, Looareesuwan S, White NJ. Convulsions in childhood malaria. Trans R Soc Trop Med Hyg. Jul-Aug 1994;88(4):426-428. 30. Waruiru CM, Newton CR, Forster D, et al. Epileptic seizures and malaria in Kenyan children. Trans R Soc Trop Med Hyg. Mar-Apr 1996;90(2):152-155. 31. Gravenor MB, van Hensbroek MB, Kwiatkowski D. Estimating sequestered parasite population dynamics in cerebral malaria. Proc Natl Acad Sci U S A. Jun 23 1998;95(13):7620-7624. 32. Newton CR, Krishna S. Severe falciparum malaria in children: current understanding of pathophysiology and supportive treatment. Pharmacol Ther. Jul 1998;79(1):1-53. 33. Versteeg AC, Carter JA, Dzombo J, Neville BG, Newton CR. Seizure disorders among relatives of Kenyan children with severe falciparum malaria. Trop Med Int Health. Jan 2003;8(1):12-16. 34. Idro R, Aketch S, Gwer S, Newton CR, Maitland K. Research priorities in the management of severe Plasmodium falciparum malaria in children. Ann Trop Med Parasitol. Mar 2006;100(2):95- 108.

Proefschrift.indb 61 14-1-2008 13:09:11 Proefschrift.indb 62 14-1-2008 13:09:11 Chapter 5

Decorticate, decerebrate and opisthotonic posturing and seizures in kenyan children with cerebral malaria

Richard Idro, Godfrey Otieno, Steven White, Anderson Kahindi, Greg Fegan, Bernhards Ogutu, Sadik Mithwani, Kathryn Maitland, Brian GR Neville, Charles RJC Newton

Centre for Geographic Medicine Research–Coast, Kenya Medical Research Institute/ Wellcome Trust Research Labs, Kilifi, Kenya; Department of Paediatrics and Child Health, Mulago Hospital/Makerere University, Kampala, Uganda; Department of Clinical Neurophysiology, Great Ormond Street Hospital for Children London, UK; Infectious Disease Epidemiology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK; Department of Paediatrics, Faculty of Medicine and the Wellcome Trust Centre for Tropical Medicine, Imperial College, London, UK; Neurosciences Unit, University College London, Institute of Child Health, UK

Malaria Journal, 2005 Dec 7; 4: 57

Proefschrift.indb 63 14-1-2008 13:09:11 ABSTRACT Background Abnormal motor posturing is often observed in children with cerebral malaria, but the aetiology and pathogenesis is poorly understood. This study examined the risk factors and outcome of posturing in Kenyan children with cerebral malaria.

Methods Records of children admitted to Kilifi district hospital with cerebral malaria from January 1999 through December 2001 were reviewed for posturing occurring on or after admission. The clinical characteristics, features of raised intracranial pressure, number of seizures and biochemical changes in patients that developed posturing was compared to patients who did not.

Results Of the 417 children with complete records, 163 (39.1%) had posturing: 85 on admission and 78 after admission to hospital. Decorticate posturing occurred in 80, decerebrate in 61 and opisthotonic posturing in 22 patients. Posturing was associated with age ≥3 years (48.1 vs 35.8%, p=0.01) and features of raised intracranial pressure on funduscopy (adjusted OR 2.1 95%CI 1.2–3.7, p=0.009) but not other markers of severity of disease. Unlike decorticate posturing, decerebrate (adjusted OR 1.9 95%CI 1.0–3.5) and opisthotonic postures (adjusted OR 2.9 95%CI 1.0–8.1) were, in addition, independently associated with recurrence of seizures after admission. Opisthotonus was also associated with severe metabolic acidosis (OR 4.2 95%CI 3.2–5.6, p<0.001). Thirty-one patients with posturing died. Of these, 19 (61.3%) had features suggestive of transtentorial herniation. Mortality and neurological deficits on discharge were greatest in those developing posturing after admission.

Conclusion Abnormal motor posturing is a common feature of cerebral malaria in children. It is associated with features of raised intracranial pressure and recurrence of seizures, although intracranial hypertension may be the primary cause.

Proefschrift.indb 64 14-1-2008 13:09:11 BACKGROUND Cerebral malaria is one of the most common non-traumatic encephalopathies affecting children worldwide and the most severe neurological complication of falciparum malaria [1, 2]. Patients present with a one to three day history of fever, seizures, unarouseable coma and brainstem signs [1, 3, 4]. Abnormal motor posturing (AMP), manifesting as decorticate, decerebrate or opisthotonus, is common, but the aetiology and pathogenesis of these signs in cerebral malaria and the prognosis of each type of posturing is poorly understood [3-9].

Raised intracranial pressure (ICP), cerebral ischaemia, hypoxia, hypoglycaemia, and hyponatraemia are recognized causes of AMP [10, 11] and raised ICP is a feature of cerebral malaria in African children [6, 7, 12]. In a study of cerebral malaria in which ICP was monitored, all children in whom AMP was documented had raised ICP, but in some patients, the timing of the AMP was not directly associated with episodes of severe intracranial hypertension [7]. Based on current guidelines, children with similar features presenting to emergency services in areas where malaria is absent will receive management for raised ICP, including paralysis and ventilation. On the other hand, seizures have been described in over 60% of patients with cerebral malaria and other acute encephalopathies associated with AMP [10, 13]. The hypothesis that AMP may be caused by seizures has led to the use of anticonvulsants in their management but there is little evidence to support this management. Other treatment practices have included non-interventional observation or therapy with mannitol.

The frequent occurrence of AMP in children with cerebral malaria, lack of ventilation facilities in most centres that manage such patients and the apparent resolution in most patients without active intervention warrants further description of the associated risk factors. In this study, records of children admitted to a Kenyan district hospital with cerebral malaria were examined to determine the risk factors associated with AMP, in particular to determine any association between AMP and features of raised ICP or seizures. The relationship between AMP and outcome was also examined.

METHODS Study design Clinical records of all children admitted to Kilifi District Hospital with cerebral malaria from January 1999 through December 2001 were examined for AMP. This hospital is situated in a malaria-endemic area and the setting was described previously[14, 15].

Proefschrift.indb 65 14-1-2008 13:09:11 Definition of cerebral malaria Cerebral malaria was defined as unarouseable coma (unable to localize a painful stimulus, Blantyre Coma Score ≤2) [3], at least one hour after termination of a seizure, administration of diazepam or correction of hypoglycaemia, with asexual forms of falciparum malaria parasites on a Giemsa stained blood smear and cerebrospinal fluid (CSF) examination not suggestive of bacterial meningitis [2, 16]. Children with epilepsy, cerebral palsy or sickle cell disease were excluded.

Definition of decorticate, decerebrate and opisthotonic posturing Abnormal motor posturing, classified as decorticate, decerebrate or opisthotonic posturing is characterised by generalised extension of the trunk and lower limbs with increased muscular tone [11]. Decorticate posturing was defined as semi-flexion, adduction and internal rotation at the shoulders and semi-flexion or flexion at the elbows. Decerebrate posturing was defined as extension of upper limbs, adduction and internal rotation of the shoulders, with pronation of the forearms. Opisthotonic posturing was defined as decerebrate posturing where the neck and back are arched posteriorly [10]. For the purposes of the study, if a patient exhibited more than one type of AMP, the more severe type ordered as: decorticate, decerebrate and opisthotonus was assigned.

Admission procedures Ethical permission for the study was obtained from the Kenya Medical Research Institute Scientific and Ethical Review Committees. At admission, standard proforma were completed detailing the medical history and physical examination. Emergency care and resuscitation were performed according to standard protocols [2, 17]. The number, duration and type of seizures prior to admission were documented. Level of coma was assessed using the Blantyre coma scale (BCS) [3]. Features of AMP were documented and assigned to decorticate, decerebrate or opisthotonic categories.

Laboratory procedures Patients had venous blood drawn for a full blood count and blood glucose, parasite density, plasma electrolytes and microbiological culture. A heparinized venous sample was drawn to determine acid base status. Lumbar punctures were performed when the level of consciousness improved (BCS>2) and the child did not have brainstem signs[5].

Proefschrift.indb 66 14-1-2008 13:09:11 Inpatient course While in the ward, patients were closely monitored during the first hour. Thereafter, clinical assessments, level of consciousness and blood glucose measurements were repeated every four hours by the nursing staff. Physicians assessed the children at four and 24 hours and on a daily basis thereafter. Patients were re-assessed if the nursing team observed seizures, AMP, deterioration in consciousness or worsening vital signs. Seizures on admission and those that developed after admission and lasted longer than five 5 minutes were treated with intravenous diazepam 0.3 mg/kg. Paraldehyde (0.4 ml/kg intramuscularly) was the second line antiepileptic drug, and phenytoin (18 mg/kg intravenously) or phenobarbital (18 mg/kg) were the third line antiepileptic drugs. Clinical signs within one hour of a seizure or anticonvulsant were not reported. Observed seizures were documented, timed and classified as partial, partial with secondary generalization or generalized tonic-clonic and recorded on a proforma sheet. Direct ophthalmoscopy for retinal signs was performed by the attending physicians on admission, at 4 hours and then, daily until full consciousness was regained. In the first year, some of these examinations were performed by an ophthalmologist who trained the other physicians in the recognition of abnormal retinal features, but there was no formal validation of the quality of funduscopy performed by the attending physicians against that of the ophthalmologist. AMP and other brainstem signs were documented. Treatment and nursing care were according to standard protocols [2, 17]. Patients were deemed to have regained full consciousness when they scored a BCS of 5 (or BCS 4 in children <9 months) [18].

Cerebral function analyser monitoring Fifty-eight of the study patients participated in pharmacokinetic studies of anti-epileptic drugs [19, 20] and had continuous monitoring with a 4-channel cerebral function analyser (CFAM3c RDM, East Sussex) for the duration of coma. The CFAM recordings were examined for evidence of seizure activity and changes in amplitude during episodes of AMP.

DATA MANAGEMENT AND STATISTICAL ANALYSIS Patients’ records were entered directly into a database using FoxPro software and analysed using SPSS 11.5 (SPSS Chicago Inc, 2003). Patients were divided into three groups: those with AMP at admission, those with AMP after admission and those

Proefschrift.indb 67 14-1-2008 13:09:11 without. Children who had AMP at admission and additional episodes after admission were regarded as having AMP at admission and the most severe form of AMP was assigned to the patient. Univariate analysis was performed to identify features associated with AMP. Patients who had AMP on admission were compared with those without AMP. A similar comparison was also made between patients posturing after admission and those without AMP. Categorical variables were compared using Pearson’s Chi square or Fisher’s exact test as appropriate. Continuous variables were compared with the student’s t-test. The mean parasite densities were compared after logarithmic transformation and the median and inter quartile range (IQR) used for other skewed data. Logistic regression analysis was performed to identify risk factors independently associated with the development of AMP.

RESULTS General description A total of 15,506 children were admitted to KDH during the three-year period. Of these, 5,767 had malaria as a primary diagnosis. Four hundred and twenty-six patients fulfilled the criteria for cerebral malaria, of whom nine had incomplete records and were excluded. Of the remaining four hundred and seventeen, 215 (51.6%) were males. The median age was 27 (IQR 15–41) months and median duration of fever prior to admission was two (IQR one – three) days. Overall, 163 children (39.1%) had AMP: 85 (20.4%) presented with AMP and an additional 78 (18.7%) developed AMP after admission. Of the 85 patients who had AMP on admission, 63 (74%) had further episodes after admission. Most AMP occurred spontaneously and manifested as decorticate posturing in 80 (49.1%), decerebrate posturing in 61 (37.4%) and opisthotonus in 22 (13.5%).

Presentation and risk factors for abnormal motor posturing at admission Admission clinical characteristics of patients with AMP at admission, during admission or without AMP are summarized in Table 1. Overall, patients with AMP were older: 48.1% of those 3 years or older had AMP compared to only 35.8% of children <3 years, p=0.01. No association was observed between AMP and duration of illness, history of seizures or parasite density. No relationship was also observed between AMP and other markers of severity of illness at admission such as a history of multiple convulsions, profound coma, deep breathing, hypotension, hypoglycaemia, severe anaemia or hyperparasitaemia. No significant differences were found in the CSF white blood cell count, protein, sugar or CSF: blood sugar ratio.

Proefschrift.indb 68 14-1-2008 13:09:11 Using multiple logistic regression analysis, age three years or older was independently associated with a child presenting with AMP (adjusted OR 2.0 95% CI 1.7 – 2.4, p<0.001). Unlike decorticate or decerebrate posturing, opisthotonic posturing was also independently associated with severe acidosis, (adjusted OR 4.2 95%CI 3.2–5.6, p<0.001).

Abnormal motor posturing after admission Of the 78 children who developed posturing after admission, 38 (49%) had decorticate, 30 (38%) decerebrate and 10 (13%) opisthotonic posturing. This distribution was similar to the overall distribution of AMP. Apart from being younger, no differences in admission characteristics and in particular, the duration of illness or coma, history of seizures, level of coma, parasite density, blood glucose, electrolytes, acid base status or haemodynamic state were observed when compared to those who presented with AMP (Table 1). The median time from admission to the time of AMP was 4.0 (IQR 1.9 - 8.8) hours. Fifty- two patients developed posturing within six hours and the remaining 26 patients had posturing after six hours (Figure 1).

Figure 1 Time from admission to onset of posturing

Two children developed opisthotonus 32 and 55 hours after admission. No significant age, sex or clinical differences were observed between those who developed posturing before or after six hours. However, most of those who developed opisthotonic posturing after admission did so after more than six hours of admission: of the 26 patients who developed AMP after 6 hours, 6 (23.3%) had opisthotonic posturing compared to only four out of 52 (7.7%) with AMP within six hours of admission, (OR 2.0 95% CI 1.1 – 3.8). Features of RICP on funduscopy (congested retinal veins, blurred disc margins or overt

Proefschrift.indb 69 14-1-2008 13:09:12 Table 1 Characteristics of patients with posturing Clinical and laboratory characteristics, and No AMP AMP at P AMP after P changes during admission (n=254) admission value admission value (n=85) (n=78) Clinical characteristics on admission Sex, male (%) 130 (51.2) 43 (50.6) 0.93 42 (53.8) 0.68 Mean (SD) age in months 28.4(20.1) 38.0(25.7) 0.01 35.0 (21.0) 0.01 Median (IQR) duration of illness in days 2.0 (1 – 3) 2.0 (1 – 3) - 2.0 (0.3 – 3) - History of seizures, (%) 217 (85.4) 72 (85.7) 0.95 66 (84.6) 0.86 Median (IQR) duration of coma before admission in hrs 3 (2 – 6) 3 (2 – 6) - 4 (2 – 6) - Mean (SD) axillary temperature oC 38.0 (4.3) 38.7 (7.0) 0.25 38.8 (7.2) 0.22 Deep (acidotic) breathing, (%) 69 (27.2) 26 (30.6) 0.54 24 (30.8) 0.54 Profound coma (BCS 0, (%)) 58 (22.8) 15 (17.6) 0.31 19 (24.4) 0.78 Retinal haemorrhages, (%) 18 (7.1) 15 (17.6) 0.01 11 (14.1) 0.07 Laboratory investigations on admission Severe anaemia, [haemoglobin <5.0 g/dl, (%)] 54 (22.3) 17 (20.5) 0.73 14 (18.4) 0.47 Hypoglycaemia, [Glucose < 2.2 mmol/L, (%)] 51 (21.3) 14 (17.7) 0.50 17 (23.3) 0.71 Severe acidosis, [Base excess < - 15, (%)] 56 (24.6) 19 (25.3) 0.89 23 (32.9) 0.17 Hyponatraemia, [Sodium <135 mmol/L, (%)] 114 (47.7) 39 (51.3) 0.58 32 (43.8) 0.56 Mean log10 (SD) of parasite density 4.8 (1.2) 4.8 (1.2) 0.77 4.7 (1.3) 0.39 Hyperparasitaemia, [>20% parasitaemia, (%)] 77 (31.8) 22 (26.5) 0.36 24 (31.6) 0.97 Brainstem features of raised ICP observed during admission Deterioration in level of consciousness, (%) 64 (25.2) 39 (45.9) <0.01 43 (55.1) <0.001 Sluggish papillary reaction, (%) 82 (32.4) 37 (43.5) 0.06 30 (38.5) 0.32 Non-reactive pupils, (%) 12 (4.7) 3 (3.5) 0.77* 4 (5.1) 1.00* Pupillary sizes, (%) Constricted 42 (16.5) 6 (7.1) 0.03 8 (10.3) 0.18 Dilated 41 (16.2) 24 (28.6) 0.01 19 (24.4) 0.10 Unequal 3 (1.2) 3 (3.5) 0.17* 3 (3.8) 0.14* Dysconjugate gaze, (%) 42 (17.1) 28 (33.3) <0.01 20 (26.0) 0.08 Congested retinal veins, blurred disc margins or overt 16 (6.3) 14 (16.5) <0.01 18 (23.1) <0.001 papilloedema, (%) Horizontal oculocephalic eye deviation, (%) No deviation 17 (6.7) 5 (5.9) 0.79 6 (7.7) 0.76 Minimal deviation 44 (17.3) 16 (18.8) 0.75 11 (14.1) 0.50 Abnormalities in respiration, (%) Irregular 49 (19.3) 18 (21.2) 0.71 20 (25.6) 0.23 Shallow 42 (16.5) 6 (7.1) 0.03 14 (17.9) 0.77 Respiratory arrest 12 (4.7) 9 (10.6) 0.05 10 (12.8) 0.01 Seizures Clinical seizures witnessed in the ward, (%) 129 (50.80 49 (57.6) 0.27 58 (74.4) <0.001 Type of witnessed seizures, (%) Partial 74 (29.1) 28 (32.9) 0.51 38 (48.7) <0.01 Partial with secondary generalisation 29 (11.4) 11 (12.9) 0.71 16 (20.5) 0.04 Generalised 81 (31.9) 28 (32.9) 0.86 39 (50.0) <0.01 Outcome Mortality, (%) 32 (12.6) 15 (17.6) 0.24 16 (20.5) 0.08 Neurological deficits, (%) 17 (6.7) 7 (8.2) 0.63 15 (19.5) <0.01 *Fisher’s exact test (two tailed)

Proefschrift.indb 70 14-1-2008 13:09:12 papilloedema) were the only clinical feature predictive for the development of posturing after admission, (adjusted OR 1.9 95% CI 1.7 - 2.2, p<0.001).

Clinical events after admission Deterioration in consciousness observed during the four hourly assessments, recurrence of seizures and brainstem features of raised ICP were the most significant clinical events after admission. The level of consciousness deteriorated in over 50% of patients with AMP, within 24 hours of admission and often, just before further posturing episodes (Table 1).

Abnormal motor posturing and features of intracranial hypertension Several brainstem signs consistent with different stages of raised ICP were observed. Abnormalities of pupillary size and reaction, eye movements, disorders of conjugate gaze, and changes on funduscopy were the most common features (Table 1). Motor abnormalities of tone and reflexes were also seen. Raised ICP was associated with all three types of posturing (Table 2), although patients with decorticate posturing had earlier funduscopic features such as congested retinal veins, unlike those with decerebrate posturing who had papilloedema more frequently. Thirty-two patients thought to have severe intracranial hypertension usually with papilloedema or clinical signs compatible with progressive herniation (23 of whom had AMP), were given mannitol (0.5g/kg infused over 20 minutes up to a maximum of three doses). No further episodes of AMP were observed in 12 (52.2%) after the administration of mannitol. Fifteen of the 32 patients (46.9%) died and five (15.6%) showed severe neurological deficits. Fourteen of the 15 deaths occurred in patients with AMP.

Herniation syndromes Patients were assessed for features of transtentorial herniation based upon the Plum and Posner criteria adapted for children with cerebral malaria [5, 11]. Forty-eight patients (11.5%) fulfilled a criterion for herniation (table 3). Thirty-three (68.8%) of these died compared to only 30 (8.1%) of those without features of herniation, (OR 12.4 95% CI 7.1 – 21.4, p<0.001). Most patients with AMP who died had features of herniation (19/31), with most deaths following a respiratory arrest.

Seizures after admission Clinical seizures were observed in 107/163 (65.6%) patients with AMP compared to 129/254 (50.8%) of those without AMP (Table 1). Most patients with AMP had multiple short seizures, often lasting one to three minutes and 20% had five or more seizures witnessed over the duration of admission. Seizures were particularly common among

Proefschrift.indb 71 14-1-2008 13:09:12 Table 2 Brainstem features of raised ICP, seizures after admission and type of posturing Brainstem signs No Decorticate P Decerebrate P Opisthotonus P and seizures after Posturing Posturing, value posturing, (%) value Posturing, value admission (%) (%) (%) Number 254 80 61 22 Deterioration in level of 64 (25.2) 43 (53.8) < 0.001 25 (41.0) 0.01 14 (63.6) <0.001 consciousness Sluggish pupillary 82 (32.4) 32 (40.0) 0.21 25 (41.0) 0.20 10 (45.5) 0.21 reaction Fixed pupils 12 (4.7) 1 (1.3) 0.32* 4 (6.6) 0.52* 2 (9.1) 0.31* Pupil sizes Constricted 42 (16.5) 6 (7.5) 0.05 5 (8.2) 0.10 3 (13.6) 1.00* Dilated 41 (16.2) 20 (25.0) 0.08 17 (28.3) 0.03 6 (27.3) 0.19 Unequal 3 (1.2) 2 (2.5) 0.60* 3 (4.9) 0.09* 1 (4.5) 0.28* Dysconjugate vision 42 (17.1) 27 (34.2) < 0.01 13 (21.3) 0.44 8 (38.1) 0.02 Ocular fundi Congested retinal veins 11 (4.3) 11 (13.8) < 0.01 2 (3.3) 1.00* 4 (18.2) 0.02* Blurred disc margins 6 (2.4) 6 (7.5) 0.03 6 (9.8) <0.01 2 (9.1) 0.13* Papilloedema 4 (2.4) 3 (3.8) 0.37* 7 (11.5) <0.001 1 (4.5) 0.34* Retinal haemorrhages 18 (7.1) 12 (15) 0.03 11 (18.0) <0.01 3 (13.6) 0.23* Any fundoscopic 16 (6.3) 15 (18.8) < 0.01 12 (19.7) 0.001 5 (22.3) <0.01 evidence of Raised ICP Horizontal oculocephalic deviation No deviation 17 (6.7) 6 (7.5) 0.80 3 (4.9) 0.78* 2 (9.1) 0.66* Minimal deviation 44 (17.3) 12 (15) 0.63 10 (16.4) 0.86 5 (22.7) 0.53 Respiration Irregular 49 (19.3) 13 (16.3) 0.54 20 (32.8) 0.02 5 (22.7) 0.70 Shallow 42 (16.5) 5 (6.3) 0.02 12 (19.7) 0.56 3 (13.6) 1.00* Respiratory arrest with 12 (4.7) 10 (12.5) 0.01 6 (9.8) 0.12 3 (13.6) 0.11* good cardiac output Clinical seizures 129 (50.8) 48 (60.0) 0.15 42 (68.9) 0.01 17 (77.3) 0.02 observed in the ward 5 or more seizures in the 29 (11.4) 13 (16.3) 0.26 13 (21.3) 0.04 6 (27.3) 0.03 ward Type of clinical seizure Partial 74 (29.1) 31 (38.8) 0.11 22 (36.1) 0.29 13 (59.1) <0.01 Partial with secondary 29 (11.4) 13 (16.3) 0.26 9 (14.8) 0.47 5 (22.7) 0.12 generalisation Generalised 81 (31.9) 29 (36.3) 0.47 30 (49.2) 0.01 8 (36.4) 0.67 * Fisher’s exact test

those who developed AMP after admission, often involving the mouth, face and limbs and were twice as common as that in patients without AMP. An association was observed between the type of AMP and recurrence of seizures in the ward; no posturing 50.8%, decorticate posturing, 60.0%, decerebrate, 68.9% and opisthotonus, 77.3%

Proefschrift.indb 72 14-1-2008 13:09:12 Table 3 Herniation syndromes and posturing Herniation syndromes Posturing No posturing P (n=163) (n=254) Value Uncal[1] 2 (1.2) 2 (0.8) 0.64 Diencephalic[2] 8 (4.9) 2 (0.8) 0.02 Midbrain/upper pontine[3] 0 (0.0) 2 (0.8) 0.52 Lower pontine[4] 2 (1.2) 1 (0.4) 0.56 Medullary[5] 19 (11.7) 12 (4.7) 0.01 [1]Unilateral mydriasis and unilateral fixed pupil, unilateral ptosis, minimal or no oculocephalic eye deviation and hemiparesis [2]Cheynes-Stokes respiration, small or midpoint pupils reactive to light, full deviation of oculocephalic response, flexor response to pain and/or decorticate posturing, hypertonia and or hypereflexia with extensor planters [3]Hyperventilation, midpoint pupils fixed to light, minimal oculocephalic deviation, extensor response to pain or decerebrate posturing [4]Shallow or ataxic respiration, midpoint pupils fixed to light, no oculocephalic response, flexion of legs only, no response to pain or decorticate posturing, flaccid with extensor planters [5]Slow irregular or gasping respirations, dilated pupils fixed to light, respiratory arrest with adequate cardiac output

(χ2=11.4, p=0.01 for trend). The median number of witnessed seizures among patients with opisthotonus was three compared to one in patients with either decerebrate or decorticates posturing. Thirty-five of the 85 patients with AMP at admission received intravenous phenytoin. AMP recurred in 22/35 (62.9%) of those who received phenytoin compared to 41/50 (82.0%) of those who did not receive phenytoin, but this difference was not statistically significant.

Abnormalities on cerebral function analyser monitoring Twenty-one of the 58 patients (36%) monitored with the CFAM had AMP, a proportion similar to that seen in whole group. Recordings were examined for evidence of seizure activity and changes in background electrical activity during AMP. Patients with AMP had elevated peaks in the amplitude of the background electrical activity, which were not seen in those without AMP. During posturing, transient increases in the amplitude of the electroencephalographic (EEG) background activity were observed (Figure 2). These were most marked with opisthotonic posturing and more pronounced with decerebrate than with decorticate posturing. The mean peak-to-peak amplitude in patients with decorticate posturing increased to just below 100µV (CFAM voltage readings are in a logarithmic scale starting at 1, 10 and 100µV), while in decerebrate and opisthotonic posturing it exceeded 100µV. There was no evidence of seizure activity during AMP in the areas monitored by EEG (left and right fronto-central: F3-C3, F4-C4; left and right parieto-occipital: P3-O1, P4-O2).

Proefschrift.indb 73 14-1-2008 13:09:12 Figure 2 Cerebral function analyser monitoring tracings before and during opisthotonic posturing in a child with cerebral malaria

Seizures, posturing and resolution of coma Both seizures and AMP were associated with prolonged coma. The median time to full consciousness in patients with AMP was 32 (IQR 12-63) compared to 13 (IQR 4-26) hours in those without AMP. In patients who had seizures after admission, the median time to full consciousness was 26 (IQR 10.3–51.8) hours compared to nine (IQR 4–21.5) among those without seizures. On multiple linear regression analysis, time to full consciousness was significantly longer in both patients with AMP, β( = -21.4, t= -5.453, p<0.001) and those with seizures after admission, (β = -20.9, t= -5.485, p<0.001). Of the 374 patients alive 24 hours after admission, 163/232 (70.3%) without AMP had regained full consciousness compared to only 57/142 (40.1%) with AMP, p<0.001. Prolonged periods of unconsciousness were particularly common in patients who developed AMP after admission when compared with those posturing at admission, 53.8 (SD 55.1) vs 30.8 (SD 33.8) hours, p=0.002.

Independent risk factors for abnormal motor posturing Logistic regression analysis was performed to determine the main risk factor for AMP in particular, features associated with raised ICP or seizures. Only features of raised ICP on funduscopy (adjusted OR 2.1 95% 1.2 – 3.7, p=0.009) were independently associated with AMP. No independent association was observed with seizures. When the different types of AMP were considered separately, decorticate posturing was associated with raised ICP (adjusted OR 3.2 95% CI 1.5–6.9, p=0.003) but not seizures (adjusted OR 1.3 95% CI 0.7–2.1, p=0.378). However, decerebrate posturing was significantly associated with both raised ICP (adjusted OR 3.2 95% CI 1.4–7.1, p=0.006) and seizures (adjusted OR 1.9 95% CI 1.0–3.5, p=0.036). Similarly, opisthotonic posturing was also associated with both raised ICP (adjusted OR 3.4 95% CI 1.1–10.7, p=0.035) and seizures (adjusted OR 2.9 95% CI 1.0–8.1, p=0.049).

Proefschrift.indb 74 14-1-2008 13:09:12 Outcome Out of the 417 children with cerebral malaria, 63 (15.1%) died and 39 (11.0%) of the 354 survivors had neurological deficits at discharge. The mortality in the 163 patients with AMP was 19.0% compared to 12.6% of 254 patients without AMP, p=0.07. Primary respiratory arrest was the commonest cause of death among patients with AMP (19/31 [61.3%] compared to 12/32 [37.5%] in those without posturing, p=0.06) and the median time from admission to death was four hours longer (15 hours IQR 3-48 vs 11.5 hours IQR 3-106) among patients with AMP. Mortality increased with the type of AMP: mortality was 12.6% among children without AMP, 16.3% in those with decorticate posturing, 19.7% with decerebrate posturing and 27.3% in those with opisthotonic posturing. Patients who developed AMP after admission had the worst outcome; 40% either died or had gross neurological deficits at discharge (Table 1).

At discharge, of the 39 children with neurological deficits, motor impairments in 28 (18 with central hypotonia, six with paresis and four with ataxia), aphasia in 13, blindness in 12 and deafness in 6 were the most common impairments. These impairments were particularly common among patients with AMP: 22 (16.7%) compared to 17 (7.7%) of those without AMP, p=0.009). Other neurological deficits observed in patients with AMP included labile emotions with frequent mood changes, persistent visual hallucinations, lip smacking, choreoathetosis and repeated episodes of confusion. Recurrence of seizures in the ward was also associated with both increased neurological deficits (16.6 vs 4.3%, OR 4.4 95% CI 1.9–10.2, p<0.001) and mortality (18.2 vs 11.0%, OR 1.8 95% CI 1.0–3.2, p=0.04).

DISCUSSION This study has shown that abnormal motor posturing is a common feature of cerebral malaria in Kenyan children, occurring in 40% of patients. It is associated with features of raised ICP, seizures after admission, prolonged coma, increased mortality and neurological deficits. The evidence suggests that raised ICP may be the most important risk factor.

Risk factors for posturing With the exception of age, patients with or without AMP had similar demographic and clinical characteristics. The reduced risk of posturing in younger children may be explained by an open fontanel or uncalcified sutures that can dissipate increases in intracranial pressure. There was no association with well recognised metabolic causes such as hypoglycaemia or hyponatraemia [10, 11]. Evidence of raised ICP on funduscopy

Proefschrift.indb 75 14-1-2008 13:09:12 was the only risk factor that predicted AMP after admission. Decorticate posturing was associated with features of raised ICP only while decerebrate and opisthotonic posturing were associated with features of raised ICP and seizures after admission. Opisthotonic posturing had a third independent risk factor, severe metabolic acidosis. It would appear that although features of raised ICP are strongly associated with AMP, the type of AMP may be influenced by the degree of raised ICP and the presence of seizures or acidosis. Decorticate posturing developed in patients with earlier features of raised ICP such as congested retinal veins while decerebrate posturing was associated with a more overt feature of raised ICP, papilloedema, and with recurrent seizures. Opisthotonic posturing was independently associated with features of raised ICP, recurrent seizures and severe metabolic acidosis.

The clinical significance of the risk factors associated with the different types of AMP is not clear. Sherrington originally described decorticate and decerebrate AMP in the context of the trans-section of the brain of a cat at different levels[21]. Since then, AMP has been incorporated in the clinical assessment of coma. Raised ICP and coning are presumed to cause compression of the brainstem resulting in an ischaemic trans-section of the cerebrum, “separating” it from the brainstem [11, 22, 23]. This possibly may be the predominant mechanism in the causation of AMP in children with cerebral malaria, given the considerable clinical evidence for raised ICP observed in these patients.

Abnormal motor posturing, seizures and raised ICP AMP was associated with recurrence of seizures after admission, mostly as short multiple seizures, but not seizures before admission. CFAM recordings showed increases in the mean peak-to-peak amplitude of the EEG background activity, most marked during opisthotonic posturing. These amplitude increases may possibly reflect acute elevations of ICP. There was no indication of any epileptiform activity during AMP recorded from the four leads of the CFAM. It is however possible that these leads were not able to detect localized discharges.

The association between the more severe forms of AMP – decerebrate and opisthotonic

posturing – and seizures may arise because seizures increase ICP, PCO2 and cerebral blood flow, which then leads to AMP. Increased metabolic demands and cerebral blood flow during seizures may aggravate the critical perfusion state. It is less plausible that AMP itself represents an ictal phenomenon, since there is no evidence of a temporal association between seizure activity on CFAM and the occurrence of posturing. Continuous ICP and EEG monitoring would allow further investigation of the relationship between electrical seizures and AMP.

Proefschrift.indb 76 14-1-2008 13:09:13 Posturing and outcome In many encephalopathies, AMP is associated with a poor prognosis [3, 10]. A similar trend was observed in this study. In addition to being a sign of lower brainstem compression, the particularly high mortality in patients with opisthotonic posturing may be a result of the combined effects of a more severe encephalopathy with raised ICP, repeated seizures and severe metabolic acidosis. Severe metabolic acidosis in African children with malaria has been associated with shock/hypovolaemia, anaemia, lactic acidosis and other ketones [24, 25]. Those with cerebral malaria and concurrent severe metabolic acidosis have the highest severe malaria mortality [26]. The association of opisthotonic posturing with severe acidosis suggests that this type of abnormal posturing develops in very sick patients with the highest risk of death.

The median time to death in patients with AMP was four hours longer than that in patients without AMP and mortality was highest in those who developed AMP after six hours of admission. The majority of deaths had a respiratory arrest with initially an adequate cardiac output, a phenomenon associated with medullary herniation syndrome [5, 7, 11]. Herniation syndromes could be recognized in two-thirds of deaths in patients with posturing and over 50% of all deaths [13]. Effective treatment of intracranial hypertension may, therefore, be able to reduce the mortality of cerebral malaria in African children especially in patients who develop posturing after admission. The additional four hours in the median time to death provide an opportunity for the use of measures aimed at lowering ICP. Patients that develop AMP after admission may be the best group to target.

Posturing and therapy for raised ICP No guidelines exist for the use of mannitol in cerebral malaria [2], but it was administered to 32 patients in this series. Despite mannitol administration, 15 patients died and another five developed severe neurological deficits. Uncontrolled studies of mannitol in cerebral malaria show that it reduces ICP but may not prevent subsequent development of severe intracranial hypertension [5]. The use of mannitol or other osmotic diuretics requires further investigation. Most district hospitals across sub-Saharan Africa that also treat the majority of patients with cerebral malaria are unable to offer ventilation to patients with raised ICP [27]. Since most patients develop AMP several hours before death, posturing may be used as an early marker for clinicians to start initial treatment for raised ICP: patient positioning, adequate oxygenation and perfusion, temperature and seizure control, and possibly offer mannitol therapy. This can be done with the hope that at this stage, patients still have mild or moderately elevated intracranial pressures

Proefschrift.indb 77 14-1-2008 13:09:13 that may respond to mannitol [7]. However, the use of osmotic diuretics may have risks that can ideally be minimised by more intensive monitoring including that of ICP.

CONCLUSIONS In conclusion, AMP is a common feature of cerebral malaria especially in older children. Such patients are at an increased risk of multiple seizures after admission, prolonged coma, increased mortality and neurological deficits. Although raised ICP appears to be the most important risk factor for AMP, the association between the more severe forms of AMP and seizures needs to be further investigated. Continuous multi-lead EEG and ICP monitoring, a randomized trial of seizure prophylaxis and/or treatment for raised ICP may be useful.

ACKNOWLEDGEMENTS We thank the clinical, nursing and records staff of KDH who diligently kept and helped retrieve patients’ records, the Scientific Director Prof K Marsh, Centre Director Dr N Peshu, the Medical Superintendent and Scientists in Kilifi for their support. This paper is published with the permission of the Director of KEMRI. Professor CRJC Newton is supported by the Wellcome Trust, UK (070114). The Wellcome Trust has had no role in the collection, analysis, and interpretation of data, in writing this manuscript or in the decision to submit the manuscript for publication.

REFERENCES 1. Newton CR, Hien TT, White N: Cerebral malaria. J Neurol Neurosurg Psychiatry 2000, 69:433- 441. 2. WHO: Severe falciparum malaria. Trans R Soc Trop Med Hyg 2000, 94 Suppl 1:S1-90. 3. Molyneux ME, Taylor TE, Wirima JJ, Borgstein A: Clinical features and prognostic indicators in paediatric cerebral malaria: a study of 131 comatose Malawian children. Q J Med 1989, 71:441- 459. 4. Idro R, Karamagi C, Tumwine J: Immediate outcome and prognostic factors for cerebral malaria among children admitted to Mulago Hospital, Uganda. Ann Trop Paediatr 2004, 24:17-24. 5. Newton CR, Kirkham FJ, Winstanley PA, Pasvol G, Peshu N, Warrell DA, Marsh K: Intracranial pressure in African children with cerebral malaria. Lancet 1991, 337:573-576. 6. Waller D, Crawley J, Nosten F, Chapman D, Krishna S, Craddock C, Brewster D, White NJ: Intracranial pressure in childhood cerebral malaria. Trans R Soc Trop Med Hyg 1991, 85:362-364. 7. Newton CR, Crawley J, Sowumni A, Waruiru C, Mwangi I, English M, Murphy S, Winstanley PA, Marsh K, Kirkham FJ: Intracranial hypertension in Africans with cerebral malaria. Arch Dis Child 1997, 76:219-226.

Proefschrift.indb 78 14-1-2008 13:09:13 8. Newton CR, Krishna S: Severe falciparum malaria in children: current understanding of pathophysiology and supportive treatment. Pharmacol Ther 1998, 79:1-53. 9. Schmutzhard E, Gerstenbrand F: Cerebral malaria in Tanzania. Its epidemiology, clinical symptoms and neurological long term sequelae in the light of 66 cases. Trans R Soc Trop Med Hyg 1984, 78:351-353. 10. Brown JK, Ingram TT, Seshia SS: Patterns of decerebration in infants and children: defects in homeostasis and sequelae. J Neurol Neurosurg Psychiatry 1973, 36:431-444. 11. Plum F, Posner JB: The diagnosis of stupor and coma. Contemp Neurology Series 1972, 10:1-286. 12. Newton CR, Peshu N, Kendall B, Kirkham FJ, Sowunmi A, Waruiru C, Mwangi I, Murphy SA, Marsh K: Brain swelling and ischaemia in Kenyans with cerebral malaria. Arch Dis Child 1994, 70:281-287. 13. Solomon T, Dung NM, Kneen R, Thao le TT, Gainsborough M, Nisalak A, Day NP, Kirkham FJ, Vaughn DW, Smith S, White NJ: Seizures and raised intracranial pressure in Vietnamese patients with Japanese encephalitis. Brain 2002, 125:1084-1093. 14. Schellenberg JA, Newell JN, Snow RW, Mung’ala V, Marsh K, Smith PG, Hayes RJ: An analysis of the geographical distribution of severe malaria in children in Kilifi District, Kenya. Int J Epidemiol 1998, 27:323-329. 15. Mbogo CN, Snow RW, Khamala CP, Kabiru EW, Ouma JH, Githure JI, Marsh K, Beier JC: Relationships between Plasmodium falciparum transmission by vector populations and the incidence of severe disease at nine sites on the Kenyan coast. Am J Trop Med Hyg 1995, 52:201- 206. 16. Berkley JA, Mwangi I, Mellington F, Mwarumba S, Marsh K: Cerebral malaria versus bacterial meningitis in children with impaired consciousness. Q J Med 1999, 92:151-157. 17. Severe and complicated malaria. World Health Organization, Division of Control of Tropical Diseases. Trans R Soc Trop Med Hyg 1990, 84:1-65. 18. Newton CR, Chokwe T, Schellenberg JA, Winstanley PA, Forster D, Peshu N, Kirkham FJ, Marsh K: Coma scales for children with severe falciparum malaria. Trans R Soc Trop Med Hyg 1997, 91:161- 165. 19. Ogutu BR, Newton CR, Muchohi SN, Otieno GO, Edwards G, Watkins WM, Kokwaro GO: Pharmacokinetics and clinical effects of phenytoin and fosphenytoin in children with severe malaria and status epilepticus. Br J Clin Pharmacol 2003, 56:112-119. 20. Ogutu BR, Newton CR, Crawley J, Muchohi SN, Otieno GO, Edwards G, Marsh K, Kokwaro GO: Pharmacokinetics and anticonvulsant effects of diazepam in children with severe falciparum malaria and convulsions. Br J Clin Pharmacol 2002, 53:49-57. 21. Sherrington CS: Decerebrate rigidity and reflex coordination of movements. Journal of Physiology 1898, 22:319 - 332. 22. Walshe FMR: The decerebrate rigidity of Sherrington in man. Archives of Neurology and Psychiatry 1923, 10:1-28. 23. Wilson SAK: On decebrate rigidity in man and the occurrence of tonic fits. Brain 1920, 43:220- 268. 24. Maitland K, Newton CR: Acidosis of severe falciparum malaria: heading for a shock? Trends Parasitol 2005, 21:11-16. 25. Sasi P, English M, Berkley J, Lowe B, Shebe M, Mwakesi R, Kokwaro G: Characterisation of metabolic acidosis in Kenyan children admitted to hospital for acute non-surgical conditions. Trans R Soc Trop Med Hyg 2005.

Proefschrift.indb 79 14-1-2008 13:09:13 26. Marsh K, Forster D, Waruiru C, Mwangi I, Winstanley M, Marsh V, Newton C, Winstanley P, Warn P, Peshu N, et al.: Indicators of life-threatening malaria in African children. N Engl J Med 1995, 332:1399-1404. 27. Tomlinson RJ, Morrice J: Does intravenous mannitol improve outcome in cerebral malaria? Arch Dis Child 2003, 88:640-641.

Proefschrift.indb 80 14-1-2008 13:09:13 Chapter 6

CONTINUOUS ELECTROENCEPHALOGRAPHIC Decorticate, decerebrate and opisthotonic MONITORING AND SEIZURES IN CHILDREN WITH posturing and seizures in kenyan children with CEREBRAL MALARIA cerebral malaria

Samson Gwer, Richard Idro, Godfrey Otieno, Edwin Chengo, Mwanamvua Boga, Samuel Akech, Kathryn Maitland, Piet A Richard Idro, Godfrey Otieno, Steven White, Anderson Kahindi, Greg Fegan, Bernhards Kager, Brian GR Neville, Steve White, Charles RJC Newton Ogutu, Sadik Mithwani, Kathryn Maitland, Brian GR Neville, Charles RJC Newton

Centre for Geographic Medicine Research–Coast, Kenya Medical Research Institute/ Centre for Geographic Medicine Research – Coast, Kenya Medical Wellcome Trust Research Labs, Kilifi, Kenya; Department of Paediatrics and Child Research Institute, Kilifi, Kenya; Department of Paediatrics and Health, Mulago Hospital/Makerere University, Kampala, Uganda; Department Child Health, Mulago Hospital/Makerere University, Kampala, of Clinical Neurophysiology, Great Ormond Street Hospital for Children London, UK; Infectious Disease Epidemiology Unit, Department of Infectious and Tropical Uganda; Department of Paediatrics, Imperial College, London, Diseases, London School of Hygiene and Tropical Medicine, London, UK; Department UK; Department of Infectious Diseases, Tropical Medicine and of Paediatrics, Faculty of Medicine and the Wellcome Trust Centre for Tropical AIDS, Academic Medical Centre, University of Amsterdam, Medicine, Imperial College, London, UK; Neurosciences Unit, University College The Netherlands; Neurosciences Unit, University College London, Institute of Child Health, UK

Malaria Journal, 2005 Dec 7; 4: 57

Proefschrift.indb 81 14-1-2008 13:09:13 ABSTRACT Background Seizures are a common feature of cerebral malaria in children and multiple seizures are a risk factor for poor outcome. Continuous electroencephalogram (EEG) recordings in comatose patients in intensive care units have demonstrated a high incidence of non-convulsive seizures that may damage neurons. The aim of this study was to describe electrographic characteristics of children with cerebral malaria detected with continuous EEG monitoring, compare the detection of seizures by clinical staff with that using continuous EEG, and relate specific EEG features to outcome.

Methods Fifty-two children with cerebral malaria of whom, 38 were part of a randomized trial of fosphenytoin for prevention of seizures in acute non-traumatic coma, were prospectively enrolled on admission to hospital to undergo continuous EEG monitoring. Clinical seizures were recorded on a standard proforma that included the duration, type and manifestation of the seizures and interventions performed. The EEG recordings were analyzed by at least 2 clinicians and non-congruent results re-examined. At discharge, neurological assessment was performed to detect neurological deficits.

Results The background EEG was characterized by very slow generalized high amplitude waves. A total of 372 seizures (149[40%] electroclinical and 223[60%] electrographic) were detected in 20/52 (38.5%) children. Most seizures were observed in 7 children admitted with status epilepticus. The majority had a focal origin but seizures involving a whole hemisphere were common among seven children with status epilepticus. A higher frequency of the background EEG was associated with a shorter duration of coma while an asymmetrical background EEG, rhythmic runs and electrographic status epilepticus were associated with death or neurological deficits.

Conclusion Children with cerebral malaria and convulsive status epilepticus experience a big burden of electrographic seizures and electrographic status epilepticus is associated with poor outcome. Continuous EEG monitoring is a useful tool for the detection of such seizures and the acute EEG may be of prognostic value.

Proefschrift.indb 82 14-1-2008 13:09:13 INTRODUCTION Cerebral malaria is the leading cause of acute non-traumatic coma in tropical countries and the most severe neurological complication of infection with Plasmodium falciparum. It is associated with a high mortality and among survivors, up to 24% may have long- term neurological and cognitive deficits[1-3].

Seizures are a common feature of childhood cerebral malaria. Over 80% of children have a history of convulsive seizures on admission and 60% continue to have seizures after admission[4-6]. Recurrent and prolonged seizures are a risk factor for poor outcome (reviewed in [7]).These seizures may manifest as single convulsions, convulsive status epilepticus, subtle seizures or electrographic seizures detectable by electroencephalographic (EEG) monitoring. In a prospective observational study of serial EEG recordings of children with cerebral malaria in our unit, subtle and electrographic seizures were described in 15 out of 65 patients[8].

Recent studies of continuous EEG recordings among comatose patients in intensive care units have demonstrated a higher incidence of seizure episodes and in particular, subtle and electrographic seizures than on clinical observation alone[9-12]. These nonconvulsive seizures may have damaging effect on neurons similar to the clinically observed convulsive seizures. Continuous EEG monitoring may improve their detection, provide insight into the pathophysiology of disease, and guide prognosis[13]. Other than the Kenyan study, which relied on data from serial EEGs, the frequency and electrophysiological characteristics of seizures in children with cerebral malaria has not been described.

We carried out prospective observations on continuous EEG recordings to describe the types and the electrographic characteristics of seizures in children with cerebral malaria, estimate the frequency of seizures, compare seizure detection by clinical staff with detection using continuous EEG, and relate specific EEG features to the clinical course of the disease.

METHODS Setting The study was conducted in the high dependency ward of Kilifi district hospital, coastal Kenya. This hospital admits approximately 5000 children aged 0-13 years annually of

Proefschrift.indb 83 14-1-2008 13:09:13 whom, 15% are treated in the high dependency unit. Between 100 and 150 children are admitted with coma.

Study participants The study participants were children with cerebral malaria, aged between 9 months and 13 years. Cerebral malaria was defined as coma (inability to localize a painful stimulus [Blantyre coma score (BCS) ≤2])[5] at least 30 minutes after treatment of seizures or correction of hypoglycemia if present, with asexual forms of Plasmodium falciparum parasites on Giemsa stained peripheral blood smears and no other cause to explain the coma (in particular pyogenic meningitis). Patients with epilepsy, developmental delay, cerebral palsy, and sickle cell disease were excluded. Most of the study participants were part of a randomized placebo controlled trial of fosphenytoin for prevention of seizures and neuro-cognitive impairments in children with acute non-traumatic coma (ISRCTN11862726). Only half of the children who were eligible for this study between December 2004 and March 2006 underwent continuous EEG monitoring as per the trial protocol. Thereafter, continuous EEG monitoring was performed on all eligible and consented patients until March 2007. The Kenya Medical Research Institute Scientific and Ethical committees approved the study.

Emergency care At admission, patients received emergency care based on standard guidelines[14]. Anticonvulsants were administered for all seizures at admission and for convulsive seizures lasting longer than 5 minutes after admission. This entailed a sequential administration of Diazepam (0.3mg/kg intravenously) or Paraldehyde (0.4mls/kg intramuscularly) as first line therapy. First line drugs were repeated if seizures did not stop within 10 minutes. Phenobarbital (15mg/kg infused over 20 minutes) was administered for status epilepticus and sodium valproate (25 mg/kg infused over 20 minutes) was administered for refractory status epilepticus. Children with electrographic status epilepticus received similar treatment. Lumbar puncture was performed in the absence of features of raised intracranial pressure (ICP) or when patients regained a BCS greater than 2. All patients received intravenous Benzyl penicillin and chloramphenicol (for pyogenic meningitis) and quinine (for cerebral malaria) pending confirmation of diagnosis. Acyclovir is unavailable for routine use in this hospital. Antibiotics were discontinued when bacteremia and pyogenic meningitis were excluded.

Acquisition and analysis of EEG data After stabilization, consent was obtained and patients set up for continuous EEG monitoring. Two patients were allocated sequentially to a 16-lead Nervus EEG monitor

Proefschrift.indb 84 14-1-2008 13:09:13 (Taugagreining hf, Study room version 3.3.779, Iceland) for each child on a 21 lead video EEG machine (Grass-Telefactor Twin, Astro-Med [UK] version 3.4.57). Silver/silver chloride electrodes were applied to the child’s unshaved head with Elefix after cleaning with an abrasive gel and secured with colloidon and taped. The international 10-20 system was used for electrode placement[15]. EEG monitoring was performed till when the child regained full consciousness or for a maximum of 72 hours. Patients were closely monitored by the nursing staff and all seizure events recorded on a seizure monitoring chart stating the type and region of seizure onset and offset, the time, trans-cutaneous oxygen saturation, respiratory and heart rates at seizure onset and offset, blood glucose level and the interventions performed. These seizure events were classified as clinical seizures. The average patient to nurse ratio in the high dependency unit was 3:1.

Using pilot data from 16 patients, a standard proforma was developed and used for the analysis of EEG recordings. It included the background EEG activity at the start and end of recording, wave symmetry over the 2 hemispheres, region of seizure onset, the number and duration of each seizure episode and status epilepticus, transient attenuations and ictal discharges. An EEG seizure was described as a distinct episode of epileptiform activity observed over a minimum duration of 6 seconds and at least 9 seconds apart from another episode. Electrographic status epilepticus was defined as continuous epileptiform activity for 30 minutes. It was observed from the onset that almost all children with cerebral malaria had wave amplitudes higher than that seen in normal children. For the purposes of the study therefore, we used relative measurements and compared the wave amplitudes of the patients against each other and categorized them into three: low amplitude (<100mV), medium amplitude (100-300mV) and high amplitude (>300mV). Wave frequencies were grouped into 4: A - dominant delta activity with little or minimal theta activity (<1-2Hz), B - some delta and some theta activity (mixed frequency, 2-3 Hz), C - dominant theta activity with some delta activity and D - near normal. Each EEG recording was analyzed by two independent readers: all were analyzed by GO and some by SG and others by RI. The two reports were later checked for congruency and disparities in analysis were reviewed and discussed by the whole team. The interrater agreement between the investigators on seizure detection was excellent. Where consensus could not be reached, the recordings were sent to SW, a consultant Neurophysiologist, for clarification.

Neurological assessment at discharge All surviving patients underwent a neurological examination at discharge. Standard clinical classifications were used and children with gross deficits such as motor deficits (cranial nerve palsies, spasticity and central hypotonia), ataxia, movement disorders (tremors, dystonia and choreoathetoid movements), speech (speech difficulties or

Proefschrift.indb 85 14-1-2008 13:09:13 aphasia), visual (blindness) and hearing impairments, epileptic seizures, behavioral abnormalities (like new onset aggressive behavior and hyperactivity) were defined to have neurological deficits[16]. Trial participants had a similar neurological assessment 3 months after discharge from hospital. Children found to have neurological deficits are being followed up in a neurology clinic.

DATA ANALYSIS Data was analyzed with Stata version 9 (STATA corporation, Texas). We compared the characteristics of patients with recurrences of seizures to those without. A description of the background and ictal EEG recordings was made. The EEG features of patients who died, survived with or without neurological deficits were compared describe to features associated with poor outcome. Continuous data were compared using Mann Whitney-Wilcoxon’s rank-sum test. Pearson’s chi square test (or Fischer’s exact test as appropriate) was used to compare proportions.

RESULTS General description Between December 2004 and March 2007, fifty-four children with cerebral malaria were recruited for continuous EEG monitoring. The recordings of two children were unsatisfactory and were excluded (equipment failure occurred in one child while the second died before an appreciable recording was made). Thirty-six of the remaining 52 children were monitored with the Nervus EEG monitor while 16 were monitored on the Grass telefactor EEG machine. The median duration of recording was 20 (IQR 12, 42) hours. Table 1 is a summary of the clinical characteristics and outcome of these patients.

The background EEG In the majority of patients, the background EEG at admission was characterized by very slow wave activity (1-3Hz) with medium to high amplitudes (100-450µV), figure 1.

The background EEG of a 36-month old child with cerebral malaria showing generalised high amplitude slow wave activity. The vertical lines are at 1 sec intervals.

Waves with amplitudes <100µV were recorded in 7 children and dominant theta waves (4-7Hz) in only 3. No child had a normal background EEG. As the level of consciousness

Proefschrift.indb 86 14-1-2008 13:09:13 Table 1 Characteristics of 52 children with cerebral malaria monitored on continuous EEG Patient characteristics Seizures detected on EEG P value monitoring Yes, n=20 No, n=32 Median (IQR) duration of illness, days 2.5 (2, 3) 3 (2.5, 4) 0.196 Median (IQR) age, months 35 (27, 42) 32 (25, 43) 0.665 Seizures before admission, (%) 17 (85.0) 26 (81.3) 0.728 Median (IQR) number of seizures before admission 3 (2, 7) 2 (1, 3) 0.215 Mean (SD) admission temperature, o C 37.7 (1.2) 38.0 (1.2) 0.449 Features of shock (capillary refill>2 sec), (%) 6 (30.0) 6 (18.8) 0.349 Deep (acidotic) breathing, (%) 9 (45.0) 16 (50.0) 0.726 Depth of coma (BCS) 0 4 7 0.573* 1 4 8 2 12 17 Hypoglycaemia, (%) 4 (20.0) 2 (6.3) 0.131 Median (IQR) duration of EEG monitoring, hrs 39.0 (18.5, 60.5) 14.5 (10.5, 26.0) 0.002 Blood transfusion, (%) 2 (10.0) 10 (31.3) 0.077 Deaths, (%) 5 (25.0) 3 (9.4) 0.129 Neurological sequelae in survivors, (%)¶ 2 (13.3) 5 (17.2) 0.737 *Chi square for trend ¶ N=44, seizures recurred=15 and did not recur=29

Figure 1 The background EEG in a child with cerebral malaria

The background EEG of a 36-month old child with cerebral malaria showing generalised high amplitude slow wave activity. The vertical lines are at 1 sec intervals.

Proefschrift.indb 87 14-1-2008 13:09:14 improved, the dominant frequency increased above the admission frequency. Concurrently, there was a decrease in the amplitude. At the end of recording when patients had regained consciousness, all surviving patients had waves with amplitudes <300µV. Children admitted with higher frequency waves had a shorter duration of coma. In these patients, the frequency at admission was ≥4Hz and the amplitude 100-200 µV. Full consciousness was regained within 8 hours of admission.

Two children were admitted with profound coma (BCS 0) and an asymmetrical background EEG characterized by lower amplitude and frequency over one hemisphere (figure 2). One died. Two other children, who both had status epilepticus, developed asymmetrical background EEG waveforms during the course of admission. After a prolonged period of hospitalization, one was discharged with quadriparesis and impaired speech. The second child initially regained full consciousness but later developed further seizures and lapsed into coma (biphasic cerebral malaria). This second period of coma lasted 38 hours and neurological deficits were not observed on discharge from hospital.

Figure 2 Asymmetry in the background EEG in a child with cerebral malaria

Asymmetry of the background EEG of a 40-month old child with cerebral malaria on a Nervus monitor: waves over the right hemisphere have lower amplitudes in comparison with those over the left hemisphere.

Proefschrift.indb 88 14-1-2008 13:09:14 Transient attenuations in the background EEG were observed in 23/52 children. This was associated with status epilepticus and multiple anticonvulsants in 8 children or with single seizures (controlled with paraldehyde or diazepam before or during the recording) in 7 other cases. Of the 23 children with transient attenuations, 5 died and 4 developed neurological deficits. One child died after developing a burst suppression pattern.

The ictal EEG and seizures EEG recording started after patients had been resuscitated and stabilized. In almost all cases, this was within 2 hours of arrival to hospital. Children brought to the ward while convulsing received anticonvulsants and therefore such seizures were not captured on EEG. Only seizures after the resuscitation and stabilization period are documented.

Of the 52 children, 20(38.5%) had seizures detected on EEG. The EEG recording time in patients who had seizures detected on EEG was two times longer than that in patients who did not have any seizures detected (table1). A total of 372 seizure episodes were observed of which 149 (40%) were documented as electroclinical seizures. Seven children contributed most of the seizure episodes and in particular, one child who had 210 seizure episodes. Between them, the seven children had 343 seizures detected on EEG. Ictal activity was characterized by spikes and sharp waves and in a few episodes, spike and wave complexes. A generalized onset was observed in 155(41.7%) and a focal onset in 208(55.9%) seizure episodes. Focal seizures mostly originated over the parieto- occipital areas. Some seizures arose from the frontal, anterior and posterior temporal areas and rarely over the mid temporal region. In up to 55.3% of cases, the focal seizures especially those among the seven children with multiple seizures, involved the whole hemisphere of origin.

One child had prolonged periods of ictal activity. These were interspersed by short intervals when no ictal discharges were observed. Each seizure originated from different areas of the brain (temporal, frontal and parietal) and from either hemisphere although the left temporal area was most involved. The wave amplitude progressively decreased to extremely low values (4-30µV) but during this period, near normal bursts of brain electrical activity and seizure discharges would appear. The child eventually died.

Electroclinical and electrographic seizures on continuous EEG monitoring Of the 372 seizure episodes documented on EEG, only 149 had corresponding clinical manifestations (electoclinical seizures). The remaining 223 seizures were therefore

Proefschrift.indb 89 14-1-2008 13:09:14 Table 3 Clinical seizures and corresponding EEG seizure types and electrographic seizures EEG seizure types Clinical seizure types Electrographic Total Focal Secondarily Generalised Subtle seizures* generalised Focal 35 17 7 11 139 209 Secondarily generalised 2 0 0 0 6 8 Generalised 19 2 23 33 78 155 Total 56 19 30 44 223 372 *Seizures arising and localised over one hemisphere were considered focal

Table 4 Duration of seizures Duration of seizures, Electrographic seizures, Clinical seizures, All seizures, minutes N=223 (%) N=149 (%) N=372, (%) <1 44 (19.7) 17 (11.4) 61 (16.4) 1 – 4 107 (48.0) 69 (46.3) 176 (47.3) 5 – 14 47 (21.1) 41 (27.5) 88 (23.7) 15 – 29 12 (5.4) 11 (7.4) 23 (6.2) 30 – 59 8 (3.4) 8 (5.4) 16 (4.3) ³ 60 5 (2.2) 3 (2.0) 8 (2.2)

described as electrographic seizures. The manifestation of the electroclinical seizures (e.g. focal, generalized) corresponded with the expected EEG manifestations in only 39% of episodes: 34% of the seizures detected clinically as focal seizures appeared generalized on EEG and 75% of seizures with subtle manifestations were generalized on EEG. Subtle seizures made up 30% of all electoclinical seizures. These were often described as blank stares, twitching of oro-facial muscles and limb digits, nystagmoid eye movements, irregular respiration and frothing. Most electrographic seizures (139/223 [62.3%]) were focal seizures that originated over the frontal lobe, anterior and posterior temporal regions or the parietal and occipital lobes. Table 3 shows the types of clinical seizures compared with the corresponding seizure detected on EEG and the types of electrographic seizures.

Two thirds of both electroclinical and electrographic seizures lasted <5 minutes (table 4). On average, electroclinical seizures lasted one minute longer than electrographic seizures: the median duration of electrographic seizures was 3.2 (IQR 1.2, 8.0) minutes while that of electroclinical seizures was 4.3 (IQR 2.1, 9.9) minutes, p=0.004.

Status epilepticus was observed in 11 patients. Electrographic status epilepticus was observed in 6 children and was associated with poor outcome: three children died and the fourth was discharged with multiple neurological deficits. In all cases, electrographic status was observed in children who had electroclinical status epilepticus before or after admission to hospital and had been controlled with anticonvulsants.

Proefschrift.indb 90 14-1-2008 13:09:14 Abnormal EEG patterns and specific clinical events EEG during abnormal motor posturing Among patients with abnormal motor posturing, the background EEG was characterized by very high amplitude (300-500µV) and low frequency (1-3Hz) waves. During periods of decorticate posturing, the background EEG changed to a very slow (<1-2Hz) generalized high amplitude (450-600µV) waves. In one child, the waves reached a peak of 775µV. These changes were observed intermittently and each episode lasted 1-4 minutes. There were no epileptic discharges during any of these events. Similar changes in amplitude and frequency were observed in children with decerebrate posturing. However, in one child with decerebrate posturing, epileptic spike and sharp wave discharges were observed in addition.

Rhythmic runs We observed rhythmic runs in 3 children. These occurred both, in association with or without seizure discharges. One child, a 33-month old boy, had multiple and prolonged runs of such rhythmic activity over both hemispheres and not associated with any epileptiform discharges. This continued for several hours although there were short intervening periods of slow wave activity. On discharge from hospital, the child had a normal neurological examination. Another child, an 18-month old girl, was admitted with electrical status epilepticus refractory to multiple anticonvulsants. During the short interictal periods, she exhibited bilateral runs of rhythmic activity most marked over F3. She had decerebrate posturing associated with sharp waves and spikes during the periods of posturing and eventually died.

Non-seizure events Four children had short-lived clinical seizure-like events not accompanied by any epileptiform activity on the EEG. There were 10 such events in the four patients. Eight were described as “focal seizures” (manifested as clonic movements of the upper limb), one as a “generalized seizure” and another as a “subtle seizure”. Three of the four children had other seizure events at different times. All survived without deficits.

Outcome of cerebral malaria Eight children (15.3%) died and of the 44 surviving children, 7 (15.9%) had neurological deficits on discharge from hospital. The characteristics of the 15 patients with poor outcomes are presented in tables 4 and 5.

Proefschrift.indb 91 14-1-2008 13:09:15 Table 4 Characteristics of children with fatal cerebral malaria Patient 1 2 3 4 5 6 7 8 Age, months 40 19 90 22 20 33 17 40 Duration of illness, days 4 3 1 1 4 3 3 1 Duration of coma before admission, hours 2 26 6 4 6 6 1 1 Parasite density /ml 8,085 68,58 7,79 324 247,28 639,36 40,863 724 Status epilepticus before or on admission No Yes No Yes No No Yes Yes Status after admission to hospital No Yes No Yes No Yes Yes No Number of seizures after admission 0 32 - 25 0 29 2 0 Total duration of seizures after admission 0 638 - 52 0 149 34 0 to hospital, minutes Background EEG on admission Very slow <1-2Hz low 2-3Hz medium Very slow 1-2Hz 2-3Hz low amplitude Very slow 1-2Hz 2-3Hz high amplitude 2-3Hz medium Very slow <2Hz amplitude waves. amplitude waves. medium amplitude waves. Symmetrical high amplitude waves. Symmetrical amplitude waves. medium amplitude. Asymmetrical Symmetrical waves. background. waves. Symmetrical background. Symmetrical Symmetrical background background EEG Symmetrical background background. background. background Localisation of area of origin of ictal - Multiple areas: frontal, Generalised - Generalised Posterior temporal, - activity anterior and posterior generalised temporal, occipital, parietal, hemispheric and generalised Types of clinical seizures - Focal, generalised, Generalised - Secondarily Partial - secondarily Subtle generalised, Subtle generalised, subtle Electrographic seizures No Yes No Yes No Yes Yes No Other significant events Refractory status, Raised ICP. Non-seizure Refractory status. Recovered from Sudden given thiopentone Repeated posturing movements, Continuous earlier respiratory deterioration after Posturing midazolam arrest initial improvement Time from admission to death, hr 11 106 40 9 21 36 47 64 Type of arrest Respiratory Respiratory Cardio-respiratory Cardio-respiratory Cardio-respiratory Respiratory Cardio-respiratory Cardio-respiratory

Six out of eight deaths occurred within 48 hours of admission and in four patients, this was associated with status epilepticus. Respiratory arrests followed status epilepticus and multiple anticonvulsants. Among survivors, patients without additional seizures regained consciousness faster (median [IQR] duration 25 [10, 50] hours) than those with further seizures, (44 [22, 72] hours). Neurological deficits on discharge included hemiparesis, paraparesis, quadriparesis, ataxia, speech and visual impairments. Children who developed deficits also had a longer duration of coma, table 5. Neurological deficits were associated with status epilepticus (before or after admission to hospital) and features of raised ICP. Three other children had behavioral abnormalities (aggressive behavior and hyperactivity) documented 3 months after discharge.

Proefschrift.indb 92 14-1-2008 13:09:15 Table 4 Characteristics of children with fatal cerebral malaria Patient 1 2 3 4 5 6 7 8 Age, months 40 19 90 22 20 33 17 40 Duration of illness, days 4 3 1 1 4 3 3 1 Duration of coma before admission, hours 2 26 6 4 6 6 1 1 Parasite density /ml 8,085 68,58 7,79 324 247,28 639,36 40,863 724 Status epilepticus before or on admission No Yes No Yes No No Yes Yes Status after admission to hospital No Yes No Yes No Yes Yes No Number of seizures after admission 0 32 - 25 0 29 2 0 Total duration of seizures after admission 0 638 - 52 0 149 34 0 to hospital, minutes Background EEG on admission Very slow <1-2Hz low 2-3Hz medium Very slow 1-2Hz 2-3Hz low amplitude Very slow 1-2Hz 2-3Hz high amplitude 2-3Hz medium Very slow <2Hz amplitude waves. amplitude waves. medium amplitude waves. Symmetrical high amplitude waves. Symmetrical amplitude waves. medium amplitude. Asymmetrical Symmetrical waves. background. waves. Symmetrical background. Symmetrical Symmetrical background background EEG Symmetrical background background. background. background Localisation of area of origin of ictal - Multiple areas: frontal, Generalised - Generalised Posterior temporal, - activity anterior and posterior generalised temporal, occipital, parietal, hemispheric and generalised Types of clinical seizures - Focal, generalised, Generalised - Secondarily Partial - secondarily Subtle generalised, Subtle generalised, subtle Electrographic seizures No Yes No Yes No Yes Yes No Other significant events Refractory status, Raised ICP. Non-seizure Refractory status. Recovered from Sudden given thiopentone Repeated posturing movements, Continuous earlier respiratory deterioration after Posturing midazolam arrest initial improvement Time from admission to death, hr 11 106 40 9 21 36 47 64 Type of arrest Respiratory Respiratory Cardio-respiratory Cardio-respiratory Cardio-respiratory Respiratory Cardio-respiratory Cardio-respiratory

DISCUSSION We carried out prospective observations with continuous EEG in 52 children who fulfilled the WHO definition of cerebral malaria. Seizures were detected in 40% of patients after the initial period of resuscitation on admission. Majority of recurrent seizures were electrographic and occurred in a few individuals initially admitted with status epilepticus. There was little congruence between the electroclinical seizure manifestations and the EEG characteristics. Very slow or asymmetrical EEG background on or during the course of admission and electrographic status epilepticus were associated with poor outcome.

The characteristics of the background EEG in cerebral malaria and prognosis The background EEG in children with cerebral malaria is characterized by non-specific features – generalized slowing and high amplitude waves. The degree of slowing reflects the severity of the encephalopathy. Recovery is associated with an increase in the

Proefschrift.indb 93 14-1-2008 13:09:15 Table 5 Characteristics of children who survived with neurological sequelae Patient 1 2 3 4 5 6 7 Age, months 38 26 28 46 29 34 40 Duration of illness, days 2 3 6 7 3 4 2 Duration of coma before admission, hours 8 3 4 6 12 24 4 Parasite density, /ml 372,78 402,8 706,68 362,04 536 14,442 741,6 Status epilepticus before admission Yes No Yes, controlled Yes No Yes Controlled with No with diazepam and Phenobarbital paraldehyde Status epilepticus after admission No No Yes No No No Number of seizures after admission 1 0 0 210 0 0 0 Total duration of seizures after admission 10 0 0 1394 0 0 0 to hospital, minutes Background EEG on admission Slow 2-3Hz medium Very slow (1-2Hz) Very slow (1-2Hz) Very slow (1-2 Hz) low Very slow frequency Very slow (1-2 Hz) Very slow amplitude waves medium amplitude, medium amplitude amplitude (<100mV) (1-2Hz) medium medium amplitude (1-2Hz) and low slows with each symmetrical waves symmetrical waves, asymmetrical amplitude (100- (100-200mV) amplitude (<100mV) episode of posturing, background, several background background waves, symmetrical background episodes of transient developed during 300mV) waves, background waves, symmetrical symmetrical attenuations recording symmetrical background background Ictal discharges and localization Anterior frontal No further epileptic No further epileptic Multiple areas: No epileptic No further epileptic No epileptic of ictal activity discharges after initial discharges after frontal, anterior and discharges discharges after discharges status epilepticus initial status posterior temporal, initial status epilepticus parietal, occipital, epilepticus hemispheric and generalised Types of clinical seizures in ward Secondarily - - Focal, subtle, - - - generalised secondarily generalised, generalised refractory status Electrographic seizures No No No Yes Other significant events Decorticate posturing Opisthotonic Required multiple Decorticate Raised ICP HIV infected posturing anticonvulsants posturing Time to full consciousness, hours 40 61 64 >120 52 72 12 Neurological sequelae at discharge Impaired speech Hemiparesis Impaired vision Quadriplegia Paraparesis Impaired speech Ataxia Impaired speech

background frequency and a reduction in the amplitude. However, large decreases in amplitude are pre-terminal events. Specific features in the background EEG and in particular, asymmetry between the two hemispheres is associated with poor prognosis. The pathological changes resulting in asymmetrical waves are unclear. However, out of four children with asymmetry, one died, the second was discharged with quadriparesis and impaired speech and the third, developed biphasic cerebral malaria (relapsed into coma after an initial period of recovery of consciousness). Asymmetry of the background EEG was associated with profound coma and status epilepticus, both poor prognostic features in children with cerebral malaria[6]. Local events (e.g. ischemia) over the affected hemisphere may be important pathogenic factors. Imaging studies such as magnetic resonance imaging (which was unavailable to us) may be useful in establishing the underlying pathology. On the other hand, children with less severe encephalopathy had background EEG with wave frequencies ≥4Hz, a good outcome and rapidly regained

Proefschrift.indb 94 14-1-2008 13:09:15 Table 5 Characteristics of children who survived with neurological sequelae Patient 1 2 3 4 5 6 7 Age, months 38 26 28 46 29 34 40 Duration of illness, days 2 3 6 7 3 4 2 Duration of coma before admission, hours 8 3 4 6 12 24 4 Parasite density, /ml 372,78 402,8 706,68 362,04 536 14,442 741,6 Status epilepticus before admission Yes No Yes, controlled Yes No Yes Controlled with No with diazepam and Phenobarbital paraldehyde Status epilepticus after admission No No Yes No No No Number of seizures after admission 1 0 0 210 0 0 0 Total duration of seizures after admission 10 0 0 1394 0 0 0 to hospital, minutes Background EEG on admission Slow 2-3Hz medium Very slow (1-2Hz) Very slow (1-2Hz) Very slow (1-2 Hz) low Very slow frequency Very slow (1-2 Hz) Very slow amplitude waves medium amplitude, medium amplitude amplitude (<100mV) (1-2Hz) medium medium amplitude (1-2Hz) and low slows with each symmetrical waves symmetrical waves, asymmetrical amplitude (100- (100-200mV) amplitude (<100mV) episode of posturing, background, several background background waves, symmetrical background episodes of transient developed during 300mV) waves, background waves, symmetrical symmetrical attenuations recording symmetrical background background Ictal discharges and localization Anterior frontal No further epileptic No further epileptic Multiple areas: No epileptic No further epileptic No epileptic of ictal activity discharges after initial discharges after frontal, anterior and discharges discharges after discharges status epilepticus initial status posterior temporal, initial status epilepticus parietal, occipital, epilepticus hemispheric and generalised Types of clinical seizures in ward Secondarily - - Focal, subtle, - - - generalised secondarily generalised, generalised refractory status Electrographic seizures No No No Yes Other significant events Decorticate posturing Opisthotonic Required multiple Decorticate Raised ICP HIV infected posturing anticonvulsants posturing Time to full consciousness, hours 40 61 64 >120 52 72 12 Neurological sequelae at discharge Impaired speech Hemiparesis Impaired vision Quadriplegia Paraparesis Impaired speech Ataxia Impaired speech

consciousness suggesting that, an EEG during the acute phase might be a useful tool in predicting the outcome of children with cerebral malaria.

Transient periods of attenuations in the background EEG were associated multiple anticonvulsant drugs for recurrent seizures. The association between transient attenuations, multiple doses of CNS depressant anticonvulsants (e.g. diazepam) and death suggest that apart from the seizures, some deaths might have been due to the CNS depressant effects of multiple doses of anticonvulsant drugs. A burst suppression pattern was a pre-terminal event.

Proefschrift.indb 95 14-1-2008 13:09:15 Seizures on continuous EEG monitoring The majority of recurrent seizures (both electroclinical and electrographic) were detected in a few children. The patients were initially admitted with status epilepticus that appeared to respond to anticonvulsants. In the ward, they continued to have prolonged periods of epileptic discharges. The previous study of serial EEGs in children with cerebral malaria using a 12-lead EEG machine suggested that the majority of recurrent seizures were focal seizures that originated over the posterior temporal and parietal regions, a watershed area that lies in the cortex supplied by distal branches of the middle cerebral and posterior cerebral arteries and concluded that ischemia and hypoxia may be important in the causation of these seizures[8]. Compared to the previous study, the current study has two additional strengths: continuous rather than serial monitoring and more (16 and 21) EEG leads and therefore improved seizure detection and localization. Most seizures had a focal origin but among the seven patients with multiple seizures, discharges originating over a whole hemispheric were common. Focal seizures were observed to arise over the frontal lobes, and the anterior and posterior temporal regions. In addition, about one third were from the parieto-occipital area, a region served by the posterior cerebral artery whose flow is most at risk of compromise by raised ICP. Transcranial Doppler studies of blood flow in these vessels may be useful.

This study clearly demonstrates that clinical observation alone only detects a smaller proportion of seizures in children with cerebral malaria and in particular in children with status epilepticus. Without EEG monitoring, the frequency of seizures is grossly underestimated. Electrographic seizures formed 60% of the total number of seizures in this cohort and electrographic status was associated with poor outcome. Similar findings have been described in other settings in patients with convulsive status epilepticus on continuous EEG monitoring after the convulsive seizures had been ablated with anticonvulsant therapy[12]. Although the majority were observed in patients who had status epilepticus, in comatose patients, these electrographic or non-convulsive seizures can also develop after single seizures[10]. The study suggests that unrecognized electrographic status epilepticus might be a major cause of death and neurological deficits in children with cerebral malaria. Similar poor outcomes have been described in neonates with electrographic seizures in studies which suggested that electrographic seizures may have neuron damaging effects similar to electroclinical seizures[17, 18]. Physiological studies suggest that damage may arise from the failure to increase arterial blood pressure and flow to match cerebral metabolic needs[19]. Non-seizure events such as abnormal motor posturing (decorticate, decerebrate and opisthotonus posturing), rigidity, tremors and chewing movements with sudden changes in pulse, oxygen saturation or blood pressure were observed in some children. Although such events are not seizures, in the absence of EEG evidence, the clinical staff

Proefschrift.indb 96 14-1-2008 13:09:15 may document them as seizures. Anticonvulsant drugs with their attendant risks of respiratory depression might be administered to these children. Using the EEG, we were able to exclude 10 such episodes initially documented as seizures by the nursing team. We however acknowledge that at present, continuous EEG monitoring may be out of reach of most district hospitals in sub-Saharan Africa where the majority of children with cerebral malaria are treated. The costs involved and absence of personnel are major prohibitive factors.

Only seizures occurring after the initiation of the EEG recording were documented. In addition, the study was performed in the context of a trial of fosphenytoin for the prevention of recurrence of seizures. Pharmacokinetic studies suggest that fosphenytoin prevents over 50% of recurrent seizures in children with severe malaria[20]. The first 38 study subjects were part of this trial and had been randomized to receive either fosphenytoin or placebo. Put together, these factors could have led to an underestimate of the overall burden of seizures in these patients.

The WHO definition for cerebral malaria is not specific[21]. Recent studies suggest that cerebral malaria in African children is characterized by distinct changes in the retina (macular and peripheral retinal whitening, white and orange vessel changes, white centred retinal hemorrhages and different grades of papilloedema) on indirect ophthalmoscopy [22-24]. Future studies of the EEG in cerebral malaria should include detection of these signs in the definition of the disease.

CONCLUSION In conclusion, children with cerebral malaria and in particular those admitted with convulsive status epilepticus experience a big burden of electrographic seizures and electrographic status epilepticus is associated with poor outcome. Continuous EEG monitoring is a useful tool for the detection of such seizures and the acute EEG may be of prognostic value.

REFERENCES 1. Newton CR, Krishna S: Severe falciparum malaria in children: current understanding of pathophysiology and supportive treatment. Pharmacol Ther1998, 79:1-53. 2. Carter JA, Mung’ala-Odera V, Neville BG, Murira G, Mturi N, Musumba C, Newton CR: Persistent neurocognitive impairments associated with severe falciparum malaria in Kenyan children. J Neurol Neurosurg Psychiatry 2005, 76:476-481.

Proefschrift.indb 97 14-1-2008 13:09:15 3. Boivin MJ, Bangirana P, Byarugaba J, Opoka RO, Idro R, Jurek AM, John CC: Cognitive impairment after cerebral malaria in children: a prospective study. Pediatrics 2007, 119:e360-366. 4. Crawley J, Smith S, Kirkham F, Muthinji P, Waruiru C, Marsh K: Seizures and status epilepticus in childhood cerebral malaria. Qjm 1996, 89:591-597. 5. Idro R, Karamagi C, Tumwine J: Immediate outcome and prognostic factors for cerebral malaria among children admitted to Mulago Hospital, Uganda. Ann Trop Paediatr 2004, 24:17-24. 6. Molyneux ME, Taylor TE, Wirima JJ, Borgstein A: Clinical features and prognostic indicators in paediatric cerebral malaria: a study of 131 comatose Malawian children. Q J Med 1989, 71:441- 459. 7. Idro R, Jenkins NE, Newton CR: Pathogenesis, clinical features, and neurological outcome of cerebral malaria. Lancet Neurol 2005, 4:827-840. 8. Crawley J, Smith S, Muthinji P, Marsh K, Kirkham F: Electroencephalographic and clinical features of cerebral malaria. Arch Dis Child 2001, 84:247-253. 9. Saengpattrachai M, Sharma R, Hunjan A, Shroff M, Ochi A, Otsubo H, Cortez MA, Carter Snead O, 3rd: Nonconvulsive seizures in the pediatric intensive care unit: etiology, EEG, and brain imaging findings.Epilepsia 2006, 47:1510-1518. 10. Tay SK, Hirsch LJ, Leary L, Jette N, Wittman J, Akman CI: Nonconvulsive status epilepticus in children: clinical and EEG characteristics. Epilepsia 2006, 47:1504-1509. 11. Towne AR, Waterhouse EJ, Boggs JG, Garnett LK, Brown AJ, Smith JR, Jr., DeLorenzo RJ: Prevalence of nonconvulsive status epilepticus in comatose patients. Neurology 2000, 54:340-345. 12. DeLorenzo RJ, Waterhouse EJ, Towne AR, Boggs JG, Ko D, DeLorenzo GA, Brown A, Garnett L: Persistent nonconvulsive status epilepticus after the control of convulsive status epilepticus. Epilepsia 1998, 39:833-840. 13. Jaitly R, Sgro JA, Towne AR, Ko D, DeLorenzo RJ: Prognostic value of EEG monitoring after status epilepticus: a prospective adult study. J Clin Neurophysiol 1997, 14:326-334. 14. WHO: Severe falciparum malaria. Trans R Soc Trop Med Hyg 2000, 94 Suppl 1:S1-90. 15. Munday JA: Instrumentation and electrode placement. Respir Care Clin N Am 2005, 11:605-615, viii. 16. Idro R, Carter JA, Fegan G, Neville BG, Newton CR: Risk factors for persisting neurological and cognitive impairments following cerebral malaria. Arch Dis Child 2006, 91:142-148. 17. McBride MC, Laroia N, Guillet R: Electrographic seizures in neonates correlate with poor neurodevelopmental outcome. Neurology 2000, 55:506-513. 18. Boylan GB, Pressler RM, Rennie JM, Morton M, Leow PL, Hughes R, Binnie CD: Outcome of electroclinical, electrographic, and clinical seizures in the newborn infant. Dev Med Child Neurol 1999, 41:819-825. 19. Boylan GB, Panerai RB, Rennie JM, Evans DH, Rabe-Hesketh S, Binnie CD: Cerebral blood flow velocity during neonatal seizures. Arch Dis Child Fetal Neonatal Ed 1999, 80:F105-110. 20. Ogutu BR, Newton CR, Muchohi SN, Otieno GO, Edwards G, Watkins WM, Kokwaro GO: Pharmacokinetics and clinical effects of phenytoin and fosphenytoin in children with severe malaria and status epilepticus. Br J Clin Pharmacol 2003, 56:112-119. 21. Taylor TE, Fu WJ, Carr RA, Whitten RO, Mueller JS, Fosiko NG, Lewallen S, Liomba NG, Molyneux ME: Differentiating the pathologies of cerebral malaria by postmortem parasite counts. Nat Med 2004, 10:143-145. 22. Beare NA, Southern C, Chalira C, Taylor TE, Molyneux ME, Harding SP: Prognostic significance and course of retinopathy in children with severe malaria. Arch Ophthalmol 2004, 122:1141-1147.

Proefschrift.indb 98 14-1-2008 13:09:15 23. Hero M, Harding SP, Riva CE, Winstanley PA, Peshu N, Marsh K: Photographic and angiographic characterization of the retina of Kenyan children with severe malaria. Arch Ophthalmol 1997, 115:997-1003. 24. Lewallen S, White VA, Whitten RO, Gardiner J, Hoar B, Lindley J, Lochhead J, McCormick A, Wade K, Tembo M, et al: Clinical-histopathological correlation of the abnormal retinal vessels in cerebral malaria. Arch Ophthalmol 2000, 118:924-928.

Proefschrift.indb 99 14-1-2008 13:09:15 Proefschrift.indb 100 14-1-2008 13:09:15 Chapter 7

Pathogenesis, clinical features and neurological outcome of cerebral malaria

Richard Idro, Neil E Jenkins and Charles RJC Newton

Centre for Geographic Medicine Research - Coast, Kenya Medical Research Institute, Kilifi, Kenya; Department of Paediatrics and Child Health, Mulago Hospital/Makerere University Medical School, Kampala, Uganda; Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK; Neurosciences Unit, Institute of Child Health, University College London, UK

Lancet Neurology, 2005; 4(12): 827 – 40

Proefschrift.indb 101 14-1-2008 13:09:16 ABSTRACT Cerebral malaria is the most severe neurological complication of falciparum malaria. Although most cases occur in children living in sub-Saharan Africa, it should be considered in anybody with impaired consciousness who has recently travelled in a malaria endemic area. It has few specific features, but there are differences in clinical presentation between African children and non-immune adults. Subsequent neurological impairments are also more common and severe in children. Sequestration of infected erythrocytes appears to be an essential component of the pathogenesis. However, other factors such as convulsions, acidosis or hypoglycaemia may impair consciousness. In this review, we describe the clinical features and epidemiology of cerebral malaria. We highlight recent insights provided by ex-vivo work on sequestration and the examination of pathological specimens. We also summarise the recent studies of persisting neuro- cognitive impairments in children who survive and suggest areas for further research.

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Proefschrift.indb 102 14-1-2008 13:09:16 INTRODUCTION Cerebral malaria (CM) is the most severe neurological complication of infection with Plasmodium falciparum and is a major cause of acute non-traumatic encephalopathy in tropical countries (Panel I).

Mortality is high and over the last two decades, the extent of persistent neuro-cognitive deficits following survival has become apparent. In this paper, we review work that has provided further understanding of the pathogenesis, describe the long-term neuro- cognitive outcomes of CM and suggest areas for future studies.

EPIDEMIOLOGY AND IMMUNITY It is estimated that in 2002 there were 515 million clinical episodes of malaria in the world; 25% in South East (S.E) Asia and 70% in Africa, mostly in sub-Saharan Africa.1 In most Western countries, malaria is seen in immigrants or people returning from travels in malaria endemic areas. Thus, in the U.K, 1722 cases of malaria were seen in 2003.2 In sub-Saharan Africa, children are most commonly affected, such that malaria may account for 40% of paediatric admissions to some hospitals in malaria endemic areas, 10% of which may be due to CM.3 The annual burden of CM in malaria endemic areas of sub Saharan Africa is 1.12/1000 children per year4, with a mortality of 18.6%.5 Falciparum malaria may cause other complications such as severe anaemia, acidosis, or hypoglycaemia and, multiple complications can occur in a single patient.

The manifestations of severe malaria in young children in malaria endemic areas are dependent on age and level of transmission (i.e. number of infected mosquito bites per person per year). In areas of intense transmission, up until the age of 6 months, infection and clinical disease are rare, producing only mild symptoms as a result of passive immunity from maternal antibodies. The burden of disease falls within the first two years and by the age of four years children experience few clinical episodes which are usually mild.6 In areas with less intense transmission, the peak incidence of severe disease falls at a later age. Severe anaemia occurs much more commonly in infants less than two years of age and the peak incidence of CM is later, - the cause of this age related differences is unclear. Repeated infections over several years provide protection against disease. Immunity is effective but partial and declines in the absence of continuous exposure, although partial protective immunity was observed in Africans who had been resident in France for at least 4 years.7

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Proefschrift.indb 103 14-1-2008 13:09:16 Table I: Clinical features of cerebral malaria in African children and Southeast Asian adults Clinical features African children Adults Coma Develops rapidly often after a seizure9. Develops gradually following drowsiness, disorientation, delirium, and agitation over 2-3 days or may follow a generalized seizure10. Seizures Over 80% present with a history of seizures Occurs in up to 20%, mostly generalized and 60% have seizures during hospital tonic-clonic seizures. Status epilepticus is admission. Recurrent seizures are focal rare.10,12 motor in >50%, generalized tonic clonic in 34%, partial with secondary generalization in 14%, and subtle or electrographic in 15%. Status epilepticus is common.9,11 Other signs Pallor, respiratory distress, dehydration Jaundice (40-70%), Kussmaul’s breathing, and rarely jaundice. shock and spontaneous bleeding.8,13,14 Neurological signs Brainstem signs are present in >30% Patients typically have symmetrical and are associated with raised ICP. 15,16 upper motor neuron signs. Brainstem Retinal abnormalities are present in signs and retinal abnormalities are less >60%.17 Brain swelling on CT scan is seen common.10,19 in 40%.18 Major Complications Severe anaemia in 20-50%, of whom Part of a multi-system and organ and involvement of over 30% require a blood transfusion.8 (circulatory, hepatic, coagulation, renal other organs Severe metabolic acidosis (presents and pulmonary) dysfunction. Pulmonary as respiratory distress), often oedema, renal failure, lactic acidosis, associated with hyperlacteamia. haemoglobinuria may be observed.26-28 Others are hyponatraemia (>50%), Hypoglycaemia is present in only 8%.29 hypoglycaemia(30%) and changes in potassium. Renal failure and pulmonary oedema are rare.8,9,20-25 Outcome Recovery of Rapid, within 24-48 hrs8,30 Slower, occurs within 48 hours.31 consciousness Mortality 18.6%5,8, up to 75% of deaths occur within 20%8 about 50% occur within 24 hours13 24 hours of admission Neurological sequelae Occurs in 11%5. Common sequelae Few, occurs in <5%. Isolated cranial are ataxia (2.5%), hemiparesis (4.4%), nerve palsies, mononeuritis multiplex, quadriparesis (3.5%), hearing (1.9%), visual polyneuropathy, extrapyramidal tremor (2.3%) and speech (2.1%) impairments, and other cerebellar signs.10 behavioural difficulties (1.3%) and epilepsy.8,32,33

The World Health Organization (WHO) proposed a definition of CM as a clinical syndrome characterized by coma (inability to localise a painful stimulus) at least 1 hour after termination of a seizure or correction of hypoglycaemia, with asexual forms of P. falciparum malaria parasites on peripheral blood smears and exclusion of other causes of encephalopathy.8 This definition is particularly useful for comparisons between different areas and research studies. It is used in children and adults, however there are significant clinical differences (table I). It is not entirely clear if these differences are related to immunity or are dependent on age.

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Proefschrift.indb 104 14-1-2008 13:09:16 Clinical features of cerebral malaria in African children Children who are admitted with CM present with 1-3 day history of fever, anorexia, vomiting and sometimes cough. Coma, seizures and brainstem signs are the main neurological features.9,23,30

Coma Cerebral malaria is a diffuse encephalopathy characterized by coma and bilateral slowing on the electroencephalogram30,34 (figure 1a).

Figure 1 Electroencephalography recordings in children with cerebral malaria Figure 1a EEG recording in a Kenyan child with cerebral malaria showing diffuse high amplitude- slow wave activity more marked over the left hemisphere.

Figure 1b EEG recording in a Kenyan child with cerebral malaria showing electrical seizure activity most prominent over the left temporal region

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Proefschrift.indb 105 14-1-2008 13:09:16 It has many features similar to a metabolic encephalopathy such as presenting with abnormal pupillary signs and the coma being potentially reversible. The cause of impaired consciousness is unclear but is likely to be the result of a number of different interacting mechanisms. The depth of coma is an important prognostic factor.8,30

Seizures Seizures are commonly reported in African children with CM and occur in over 60% after admission11,23,34,35 (table I). Many patients with seizures are hypoxic and hypercarbic from hypoventilation and are at risk of aspiration.11,35-37 In 65 Kenyan children, 62% had seizures following admission; while subtle seizures occurred in 15%, manifesting as nystagmoid eye movements, irregular breathing, excessive salivation, and conjugate eye deviation.11 Seizures are often repetitive and prolonged, and 28% had an episode of status epilepticus. Multiple and prolonged seizures are associated with increased mortality33,38,39 and neuro-cognitive deficits.35,40

The causes of seizures are unclear. Most are not associated with fever at the time of the seizure.35 They do not appear to due to electrolyte disorders41 or antimalarial drugs in children.34 Electroencephalography show that many originate over the temporo-parietal regions (which is a watershed area) (figure 1b), suggesting that ischaemia and hypoxia may play a role.34 The seizures may be caused by sequestration of infected erythrocytes or parasite-derived toxins. Furthermore immune mechanisms may be important, since antibodies to voltage gated calcium channels are elevated in children with severe malaria and seizures.42

Brainstem signs Brainstem signs are common and are associated with other features of raised intracranial pressure (ICP) and with brain swelling (figure 2), but may occur after seizures.15,16 They do not appear to be associated with hypoglycaemia or electrolyte disorders.15,16 Common signs include changes in pupillary size and reaction, disorders of conjugate gaze and eye movements. Absence of corneal and oculocephalic reflexes are associated with increased mortality.9 Other signs include abnormal respiratory patterns (such as hyperventilation, ataxic and periodic breathing)36, posture (decerebrate, decorticate or opisthotonic posturing), and motor abnormalities of tone and reflexes.9,23 Abnormal motor posturing appears to be related to raised ICP rather than seizures.43

Malarial retinopathy Retinal abnormalities are common in children with CM and may reflect the pathology in the brain.17,44,45 Characteristic features include whitening of the macula (that spares

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Proefschrift.indb 106 14-1-2008 13:09:16 Figure 2: Radiological features of the brain in cerebral malaria

Figure 2a A CT scan of the brain in a Figure 2b A CT scan of the brain of a Kenyan Kenyan child with cerebral malaria showing child with cerebral malaria showing (A) brain (A) swelling of the brain with compressed swelling with diffuse hypodensity sparing the ventricles (arrow) and loss of sulci and (B) basal ganglia (arrows) and (B) Convalescent Resolution of the brain swelling scan showing cerebral atrophy with infarction (arrows) of the right frontal and parietal regions. (Newton et al 1994, with permission from the BMJ Publishing Group).31

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Proefschrift.indb 107 14-1-2008 13:09:17 Figure 3 Retinal changes in children with cerebral malaria (retinopathy of malaria) Figure 3a White-centred retinal haemorrhages and orange vessels in a Malawian child with cerebral malaria

Figure 3b Macular retinal whitening around the foveola (central dark disc) in a child with cerebral malaria. Cotton wool spots are also visible superio-temporal to the optic disc.

Figure 3c Vessel changes in a Malawian child with cerebral malaria - from red to pale orange

Figure 3d Vessel changes in a Malawian child with cerebral malaria - from red to white

(Photos Courtesy of Dr Nicholas Beare, Malawi- Liverpool-Wellcome Trust Clinical Research Programme College of Medicine, Malawi)

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Proefschrift.indb 108 14-1-2008 13:09:18 the central fovea), peripheral retina and retinal vessels; multiple retinal haemorrhages (often with pale centres) and papilloedema (figure 3). These signs are best seen by indirect ophthalmoscopy and have been found in over 60% of children with CM.45 Their specificity may help in the diagnosis of CM. In Malawian children, presence of retinopathy, particularly papilloedema, was associated with prolonged coma and death.17 In patients who recover, these features resolve over 1-4 weeks.

Concomitant complications Metabolic perturbations are common in African children with CM. Hypoglycaemia is present in up to one third of patients on admission and often recurs even after initial correction. Causes include depletion of glycogen stores, inadequate intake, impaired hepatic gluconeogenesis and quinine-induced hyperinsulinaemia.9,20,46 Metabolic acidosis manifests as deep breathing and is often associated with hyperlactaemia. It may be caused by hypovolaemia and inadequate tissue perfusion, anaemia, lactate production by parasites and cytokine-induced failure of oxygen utilization.3,36,37,47 Resuscitation with fluids or blood transfusion may improve outcome.48 Children with dehydration often have transient impairment of renal function but unlike adults, overt renal failure is rare. Hyponatraemia occurs in over 50% of patients21, but the cause is unclear.21,49 Concomitant bacterial infections occur in 5-8% of children with CM50,51 and leucocyte counts above 15,000/µl are associated with poor prognosis.9 Other features include hepatomegaly, splenomegaly and occasionally, jaundice.

Clinical features of cerebral malaria in adults Cerebral malaria in adults is part of a multi-organ disease.30 Patients usually present after a few days illness with fever, malaise, headache, joint and body aches, anorexia, delirium, and then develop coma. Seizures are less common than in African children and the incidence appears to be declining.30 The encephalopathy in adults is characterized by symmetrical upper neuron lesion signs. Dysconjugate eye deviation, extrapyramidal rigidity, trismus, decorticate and decerebrate rigidity may be observed.10 Papilloedema and retinal exudates are rare, but retinal haemorrhages occur in 15% and are associated with increased mortality.52 Recovery from coma is slower than in children.31 It has been suggested that thiamine deficiency may contribute to some of these neurological signs.53 In a few patients, abnormalities such as cortical infarcts, cerebral venous or dural sinus thrombosis (figures 4a54 and 4b55) may occur as a consequence of the hypercoagulable state.

In some patients, CM is complicated by pulmonary oedema or adult respiratory distress syndrome.13,56 Kussmaul’s breathing occurs with acute renal failure and

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Proefschrift.indb 109 14-1-2008 13:09:18 Figure 4: Cerebral infarcts in adults with cerebral malaria Figure 4a Infarcts in a 36-year old man with cerebral malaria Hyperintense cortical areas (infarcts) seen on a fast spin- echo T2 weighted MR image (arrow) (Cordoliani et al 2004, with permission © American Society of Neuroradiology).54

Figure 4b Contrast enhanced brain CT scan of a 48-year old man who presented with left focal becoming generalised seizures and left hemiparesis. A large area of hemorrhagic infarction is seen in the right frontal cortexwith surrounding oedema. Absence of contrast is seen as a hypodense area in the posterior aspect of the superior sagittal sinus. (Krishan et al. 2004, with permission © the British Infection Society).55

severe lactic acidosis.10,19 Other complications of falciparum malaria such as anaemia, haemoglobinuria, jaundice, shock and coagulation disorders may be observed.57-60 A higher incidence of multi-organ failure is observed among those admitted to intensive care units since mostly very ill patients who did not respond to earlier treatment are admitted to such units26. Bacterial co-infection is common, particularly in those with shock, and accounts for the majority of late deaths. Respiratory failure has the worst prognosis and develops late in the course of the illness.26 Chronic hepatitis B infection may be a risk factor for severe malaria, including CM in adults.61

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Proefschrift.indb 110 14-1-2008 13:09:21 DIAGNOSIS Cerebral malaria should be considered in the differential diagnosis of any patient who has a febrile illness with impaired consciousness in a malaria endemic area or one with recent travel to such areas. At least 3 negative blood smears (on microscopy) at 8-12 hourly intervals are required before the diagnosis can be excluded. Rapid tests such as the immunochromatographic test for P. falciparum histidine-rich protein 2 and lactate dehydrogenase may be helpful in the absence of positive blood smear although they do not estimate the parasite load and their sensitivity and specificity decreases at low parasitaemia.62 Polymerase chain reaction tests are more sensitive than microscopy but expensive and do not estimate the parasite load.63

In malaria endemic areas, CM is a diagnosis of exclusion. The high prevalence of asymptomatic parasitaemia in such populations makes accurate diagnosis less certain – almost any viral encephalopathy with incidental parasitaemia fulfils the diagnostic criteria for CM. Thus in a recent study, 24% of Malawian children who fulfilled the criteria for CM prior to death had evidence at post-mortem of an alternative cause for coma, including Reyes syndrome, hepatic necrosis and ruptured arterio-venous malformation.64 The presence of malarial retinopathy was the only clinical feature to distinguish patients with typical histopathological features of CM from those with alternative pathologies. A lumbar puncture must be performed to exclude other causes for the encephalopathy, although there are differences of opinion about the timing of this procedure16,65. There may be a mild pleocytosis and an increase in protein.66 Plasma and CSF lactate levels is of prognostic value since elevated levels are associated with increased mortality.9,46 Neuroimaging shows a swollen brain in over 40% of African children18(figure 2) although this finding is less common in adults.67

PATHOGENESIS In falciparum infections, consciousness can be impaired by a variety of mechanisms which may interact with each other30 (table II). The relative contributions of these mechanisms may differ in children and adults. Thus unlike adults, seizures appear to be an important cause of impairment of consciousness in African children.

Research strategies The main research strategies for studying pathogenesis have been clinical case series and case control studies, post-mortem surveys, in vitro or animal models. There is not a reliable animal model of CM. Many primates naturally suffer Plasmodium infections

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Proefschrift.indb 111 14-1-2008 13:09:21 Table II Postulated mechanisms for coma in cerebral malaria Obstruction of cerebral microvascular flow Parasite sequestration mediated through cytoadherence71, rosette formation72, autoagglutination72,73 and reduced red cell deformability.74 Seizures Overt seizures11,35,37 Subtle and electrographic seizures11,37 Post-ictal state37 Impaired delivery of substrate Hypoglycaemia9,23 Anaemia75 Hypoxia76 Impaired perfusion Hypovolaemia47,77 Shock78 Acidosis78 Raised intracranial pressure and brain swelling Disruption of the blood-brain barrier79,80 Raised intracranial pressure15,16,18 Cerebral oedema81-83 Cytotoxic odema18,81 Toxins Nitric oxide84 Reactive oxygen species85,86 Excitotoxins30,87-89 Malaria toxin90 Clotting Intravascular coagulation as a minor mechanism91

but rarely develop clinical features similar to human CM. P. falciparum will infect the new world monkeys but severe symptoms are common only in splenectomized animals. Some species do develop cerebral dysfunction associated with adherence of infected erythrocytes to cerebral endothelial cells.68,69 Although coma is not a typical finding, adherence of infected erythrocytes to cerebral endothelial cells has contributed to the understanding of parasite sequestration. Considerable research on CM has been carried out in mice. The characteristics of the infection are dependent on strains of mouse and Plasmodium. The most popular model is CBA-mice infected with P. bergei ANKA70. Coma, seizures and death occur, but unlike human CM, this cannot be reversed with treatment. The pathology is different in mice, in that infected erythrocytes do not commonly sequester; instead, monocytes occur in cerebral vessels, and inflammatory cytokines are essential for the pathogenesis.

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Proefschrift.indb 112 14-1-2008 13:09:21 However, monocytes are also seen in the cerebral vessels of some African children64, but the significance of this finding is still unclear. The use of murine models, particularly using gene ‘knock-out’ strains, has provided much information on the immune and inflammatory responses to Plasmodium infections.

Sequestration The consistent histological finding in CM in both children and adults is the presence of infected and non-infected erythrocytes packed within cerebral vessels (figure 5). It is thought that sequestration occurs as a consequence of cytoadherence of infected erythrocytes to endothelial cells, via P. falciparum derived proteins on the infected erythrocyte surface attaching to ligands upregulated in the venules. Sequestration may be increased when adherent infected erythrocytes bind other infected erythrocytes

Figure 5 Sequestration of infected erythrocytes in cerebral vessels

a b

5a: P. falciparum infected erythrocytes (IE) sequestered in a cerebral vessel of a Vietnamese adult with fatal cerebral malaria (H&E x400, courtesy of Gareth Turner). 5b: Electron microscopy showing the ultrastructural details of a P. falciparum IE adhering to an endothelial cell in vitro. P=parasite, En=endothelial cell and arrows point out the adhesion points at the electron dense knob proteins. Figure courtesy of Professor David Ferguson, Department of Clinical Laboratory Sciences, Oxford University. 5c: Freeze fracture electron micrograph of the IE surface revealing the symmetrical distribution of knob proteins on the surface. Figure courtesy of Professor c David Ferguson (as above).

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Proefschrift.indb 113 14-1-2008 13:09:21 (auto-agglutination), non-infected erythrocytes (rosetting) or use platelets to bind other infected erythrocytes (platelet-mediated clumping). Not all parasites display these adhesive properties, but these phenotypes are more commonly present in infected erythrocytes taken from children and adults with severe malaria.

Parasite binding is mediated by a group of variant surface antigens expressed at the red cell surface during development. The best described is P. falciparum erythrocyte membrane protein-1 (PfEMP-1) which is encoded by a family of approximately 60 var genes which are associated with different binding phenotypes. Each parasite expresses the transcript of only one var gene but, can switch to express a different var gene (about 2% per generation in vitro)92, and therefore display both a change in binding phenotype and antigen. Although the trigger for var gene switching is unknown this rapid switching in non immune volunteers does not support the role of immune pressure.93 Some variant surface antigens appear to be more common in young children with severe disease, and thus may be more capable of causing CM than others94 but whether this is due to adhesion phenotype or host response is unclear.

PfEMP-1 is able to bind to many host receptors on endothelial cells chief among which are CD36 and the intercellular adhesion molecule-1 (ICAM-1).95,96 The binding of infected erythrocytes to ICAM-1 has been implicated in the pathogenesis of CM.97 Postmortem studies have revealed upregulation of ICAM-1 expression on the cerebral vascular endothelium in CM98,79 which, in adults, was co-localised to areas of parasite sequestration.99 A common ICAM-1 polymorphism (ICAM-1Kilifi) that alters protein binding to infected erythrocytes100 was associated with susceptibility to CM in Kenyan children101 but not in the Gambia.102 In a study describing the binding affinities of parasites taken from Kenyan children with malaria, ICAM-1 binding was highest in CM cases.96 The inconsistency in the polymorphisms associated with CM in different sites illustrates a methodological problem of this ex-vivo work. However, we do not know how representative circulating parasites are of those sequestered within cerebral vessels and although ICAM-1 appears important, it is likely that a number of host receptors are involved in concert in the process resulting in CM.

Reduction in microvascular flow Sequestration of infected and non-infected erythrocytes within the cerebral vessels reduces the microvascular flow. In addition, the presence of parasites inside the erythrocyte decreases the ability of the erythrocyte to deform (reduced red cell deformability, RCD) so that erythrocytes have more difficulty in passing through the microvasculature. Studies of Thai adults103 and Kenyan children104 have found strong associations between reduced RCD and severe disease and in adults with outcome. The

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Proefschrift.indb 114 14-1-2008 13:09:22 rapid reversibility of clinical symptoms suggests that tissue necrosis is unlikely to occur. However, there may be a critical reduction in the supply of metabolic substrate to the brain. This will be exacerbated by increased demand during seizures and fever, and may be worse in patients with severe anaemia or hypoglycaemia.23,75 Cerebral blood flow may also be reduced by raised ICP. Inflammatory cytokines may result in inefficient use of substrates.

The inflammatory response P. falciparum infection results in increases in both pro- and anti-inflammatory cytokines. The balance of inflammatory mediators appears critical to parasite control, but their role in the pathogenesis of severe malaria is unclear. Thus in Malian children, both IL-6 (pro-inflammatory) and IL-10 (anti-inflammatory) were increased in CM compared to non-CM, but not IL-1β, IL-8, IL-12 or TNF.105 In Gambian and Ghanaian children, TNF and TNF receptor levels were higher in CM compared to non-severe malaria.106,107 Several polymorphisms in the TNF promoter region have also been associated with increased risk of CM and death (reviewed in Gimenez et al 2003)108 or neurological sequelae.109 In Vietnamese adults, levels of IL-6, IL-10 and TNF were raised in patients with multi-organ severe disease but were lower in patients with CM alone, suggesting their involvement in the process leading to severe malaria but not coma itself.110 Postmortem analysis of brains from Malawian children with CM suggest increased local production of TNF and IL-1β.111 However, there was no correlation between production or staining for these cytokines and parasite sequestration.

Nitric oxide (NO) has been suggested as a key effector for TNF in the pathogenesis of malaria. It is involved in host defence by killing intracellular organisms, maintaining vascular status and in neurotransmission. Cytokines may upregulate inducible NO synthase (iNOS) in brain endothelial cells, increasing NO production which diffuses into brain tissue and interferes with neuronal function.84 It may rapidly and reversibly reduce the level of consciousness84 since it is short-lived and can easily diffuse across the blood- brain barrier to interfere with neuronal function.

The associations between NO activity, iNOS or genetic polymorphisms in the iNOS promoter gene have not been consistent. Results have varied with age, endemicity and geographical location. Post-mortem staining of brain specimens in African children and SE Asian adults have revealed increased iNOS in vessel walls associated with sequestered parasites in CM112 while in other studies, NO is associated with protection.113,114 It is thought that TNF upregulation of iNOS sets off a negative feedback mechanism through NO to control the stimulatory action on iNOS. However, in some individuals, generation of NO occurs too slowly to downregulate the primary wave of TNF induction, so that a

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Proefschrift.indb 115 14-1-2008 13:09:22 relatively slow build up of iNOS-induced NO allows iNOS and NO to reach the harmful levels seen in CM.115

The blood-brain barrier function Since parasites are largely confined to the intravascular space, one major question regarding the pathogenesis of CM is how these parasites cause neuronal dysfunction.116 There is accumulating evidence that parasite sequestration changes blood brain barrier (BBB) function. In Thai adults, there was no increase in transfer of radioactively labelled albumin into CSF during unconsciousness when compared to convalescence.117 No significant changes were also observed in the albumin index (ratio of CSF to blood albumin levels) in Vietnamese adults.80 However, in Malawian children, albumin indices were significantly higher when compared to UK controls79 although there were no differences between children who died and those who survived.

Post mortem analysis has shown widespread cerebral vascular endothelial cell activation (increased ICAM-1 endothelial staining, reduction in cell junction staining and disruption of junctional proteins), particularly in vessels containing infected erythrocytes.118 Perivascular macrophages in these areas expressed scavenger receptor and sialoadhesin –normally only expressed after contact with plasma proteins. However, such disruption of intercellular junctions was not associated with evidence of significant leakage of plasma proteins (fibrinogen, IgG or C5b-9) into perivascular areas or CSF.79 Adams et al119 suggested that ICAM-1 binding by infected erythrocytes results in a cascade of intracellular signalling events that disrupt the cytoskeletal-cell junction structure and cause focal disruption to BBB. Focal disruptions in the barrier at sites of sequestration could result in the exposure of sensitive perivascular neurons to plasma proteins and the increased levels of cytokines and metabolites produced by abnormalities in the microcirculation. This may contribute to reduced consciousness and/or seizure activity.

Brain swelling In Kenyan children with deep coma, 40% had evidence of brain swelling on computerised tomography (CT) scan (figure 2), however during recovery, some children with severe encephalopathy had evidence of cytotoxic oedema which may contribute to the severe intracranial hypertension.18 Severe intracranial hypertension was associated with death or neurological sequelae.15 In a study of 21 Indian adults, abnormalities on CT scans were related to Glasgow Coma Score and mortality.120 Cerebral oedema was seen in eight patients, two of whom died. Other studies of Thai adults have found little evidence of cerebral oedema on CT scan121 or magnetic resonance imagining67 but documented

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Proefschrift.indb 116 14-1-2008 13:09:22 brain swelling. Although no significant leakage of plasma proteins has been observed79, the disruption of the BBB may contribute to the raised ICP found in African children. However the most likely cause of raised ICP is increased cerebral blood volume due to sequestration of infected erythrocytes and increased cerebral blood flow from seizures, hyperthermia or anaemia5,30.

Pathological findings Postmortem studies have provided a wealth of detailed information but they reflect, at best, pathology at a single point after death in the most severely ill patients. The recent surveys from Malawi and SE Asia have found a significant relationship between the amount of sequestration and antemortem diagnosis of cerebral malaria. Sequestration is extensive, occurring in all parts of the brain to a similar extent, but with considerable variability between individuals and between vessels in an individual.122 The brain is often swollen but evidence for frank herniation is unusual in adults, although more common in African children.83 The cut surface shows petechial haemorrhages (figure 6).123

Electron microscopy show knob-like protrusions on the surface of infected erythrocytes and at sites of attachment to vascular endothelium (figures 5).81 Studies in Malawian children show intravascular and perivascular pathologies (haemorrhages, accumulation of pigmented white blood cells and thrombi) in 75% of cases. These are associated with an increased extraerythrocytic haemazoin (a product haemoglobin metabolism by malaria parasites) inside cerebral vessels. Thus, rupture of infected erythrocytes may lead to an inflammatory process within and around brain capillaries.64 These findings have not been seen consistently in adults122,124 and may reflect a difference between adults and children.

Figure 6 Gross pathological appearance of the cut surface of the brain in fatal cerebral malaria Macroscopic section of the brain in a fatal case of CM showing petechial haemorrhages in the white matter, particularly in the subcortical rim and corpus callosum (Courtesy of Dr Peter King, South African Institute of Medical Research, Johannesburg, South Africa).

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Proefschrift.indb 117 14-1-2008 13:09:22 β-amyloid precursor protein staining (a marker of axonal injury) was found on postmortem brain specimens of adults with CM.125 Two patterns were observed; a diffuse increase or a predominance of axonal injury in one brain region, typically the internal capsule or pons. Axonal injury correlated with plasma lactate, CSF protein and Glasgow coma score. The increased levels of the microtubule-associated protein tau (from degenerated axons) but not neural cell body or astrocyte proteins in CSF suggested that most of the brain parenchymal damage is with axons.126 High levels of quinolinic acid was found in CSF87 but in Vietnamese adults, this was related to impaired renal function.88

OUTCOME OF CEREBRAL MALARIA The majority of patients with CM appear to make a full recovery, but neuro-cognitive sequelae have been increasingly recognised particularly in African children, in the last 20 years.

Mortality The mortality rate in adults and children is approximately 20%, and most deaths occur within 24 hours of admission before the anti-malarial drugs may have had time to work. The mechanisms of death appear to be different (figure 7). In African children, most deaths occur with brainstem signs following a respiratory arrest (initially with a good cardiac output) suggestive of transtentorial herniation or cardio-respiratory arrest in association with severe metabolic acidosis. Four out of seven children in Nigeria had cerebral oedema and/or features of herniation at autopsy.83 Mortality is high among children with shock, hypoglycaemia, multiple and prolonged seizures, deep coma, or severe acidosis.9,23,127

Many adults die with renal failure of pulmonary oedema. Mortality is particularly increased in pregnant patients or those with vital organ dysfunction.28,128 Patients may die with an acute respiratory arrest, often following a period of respiratory irregularity, but with a normal blood pressure. Others die with shock or hypoxia secondary to acute pulmonary oedema.

Neuro-cognitive deficits In African children, a higher incidence of neurological deficits (10.9%) was reported in a meta-analysis which used studies with a similar definition of CM.5 Some deficits are transient (e.g. ataxia), whilst others e.g. hemiparesis, improve over months, but may not resolve completely. Children living in Africa with severe neurological sequelae (spastic

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Proefschrift.indb 118 14-1-2008 13:09:22 Figure 7 Possible mechanisms for death and neuro-cognitive impairment in cerebral malaria and some areas for possible intervention

P. falciparum IE adhere to vascular endothelium sequestering in large numbers in the brain (A). Local and systemic changes produce significant vital organ dysfunction leading to severe metabolic derangement (B) which may quickly result in death unless urgent correction (e.g correction of blood glucose, dialysis or ventilation) is initiated. Sequestration of IE within the cerebral vessels increases the cerebral volume (C) which together with the increase in cerebral blood flow (from seizures, anaemia and hyperthermia - D), altered BBB function lead to brain swelling and raised ICP (E). This may cause death (through transtentorial herniation, brainstem compression or global cerebral ischaemia) or result in neuronal damage with consequent neuro- cognitive impairments. Sequestered parasites may also produce local toxins and ischaemia or influence the production of inflammatory products such as cytokines and result in multiple seizures and neuronal damage. Metabolic derangement is more common in adults while raised ICP and seizures are commoner in children. Possible areas for intervention are highlighted.

quadriparesis and vegetative states) often die within a few months of discharge.129 More recently, it has been demonstrated that epilepsy is associated with exposure to CM.130

Cognitive impairments have been described in some studies39, but not others.131 Impairment has been reported in a wide range of cognitive functions; memory, attention, executive functions and language.39,129,132-134 Neuro-cognitive impairments are associated with protracted seizures11,32,38, deep and prolonged coma33, hypoglycaemia and severe anaemia in some studies38 but not in others.32 The consistent association found between prolonged seizures or hypoglycaemia and neuro-cognitive impairment suggests hippocampal damage, which may manifest as memory impairment and complex

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Proefschrift.indb 119 14-1-2008 13:09:23 partial seizures at a later date. Other smaller studies have shown an association between the development of impairments and pathophysiological processes such as raised ICP.15 Most of these factors are also associated with death, and may simply reflect the severity of the underlying insult, rather than a specific neuropathogenic process. The fact that up to 24% of children have evidence of some impairment following CM, represents a significant burden in malaria endemic areas, suggesting that at least 250,000 children will develop neuro-cognitive impairments from malaria in sub Saharan Africa per year.134

In non-immune adults, the incidence (<5%) and severity of subsequent neurological impairments is less than in children. Impairments are not confined to CM, but may follow non-cerebral malaria.135 They include cranial nerve lesions, neuropathies and extrapyramidal disorders.10,136 Some patients develop a transient psychosis or delirium during recovery, whilst others develop focal epilepsy sometimes associated with transient tomographic opacities in the brain. In Vietnam, a self-limiting “post- malaria neurological syndrome” consisting of acute confusional state, acute psychosis, generalised convulsions or tremor occurred in 0.12% of patients with falciparum malaria.135

Cognitive deficits following malaria in adults are less well documented. There are case reports of impairment of memory and naming ability. Psychological tests did not detect any residual defects in a small group of American soldiers following CM137, although a recent retrospective study suggests that CM results in multiple, neuropsychiatric symptoms, including poor dichotic listening, “personality change,” depression, and in some cases, partial seizure-like symptoms.138 A study of Ghanaian adults suggested that subclinical, mixed anxiety-depression syndrome may occur after falciparum malaria.139

MANAGEMENT OF CEREBRAL MALARIA The World Health Organization has developed guidelines for the management of patients with CM8 and a new guidelines were recently proposed for the UK.140 Emergency management aims at rapidly correcting the severely deranged metabolic states, restoration of vital physiological functions (Panel II) and, the administration of an effective and rapidly active parasiticidal drug.

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Proefschrift.indb 120 14-1-2008 13:09:23 Resuscitation on admission Since most patients die within 24 hours of admission before the therapeutic benefits of anti-malaria drugs5, supportive therapy may improve outcome. Correction of hypoxaemia, hypoglycaemia, shock, severe metabolic acidosis and control of seizures are important. Urgent resuscitation with fluids may be required for those with hypovolaemia22,47,48,140, although fluids should be administered carefully. The administration of albumin reduced mortality in a small trial of children with CM 78, but confirmatory trials are still awaited. Whole blood or packed cell transfusions should be administered for severe anaemia. Recurrences of hypoglycaemia may be prevented by continuous infusion of glucose containing fluids until consciousness is regained.

Antimalarial therapy The cinchona alkaloids (quinine, quinidine and cinchonin) and artemisinin derivatives (artesunate, artemether and arteether) are recommended for CM (table III). The cinchona alkaloids act during the later stages of parasite development, whilst the artemisinins have a broader stage-spectrum of action. A loading dose of either drug should be given, to rapidly achieve antiparasiticidal concentrations.

Quinine is still used extensively and can be given intravenously or intramuscularly. A loading dose is associated with faster clearance of parasitaemia, resolution of fever and coma.141 A 12 hourly dose regimen may be used in younger children.142 Quinidine is more

Table III: Antimalarial treatment of cerebral malaria Drug Route Indicated for Loading dose Maintenance dose Quinine IV Children and adults 20 mg salt/kg over 2-4 hrs 10 mg salt/kg every 8 hrs, until dihydrochloride (max 600 mg) able to take orally8 Quinine IV Children 15-20 mg salt/kg over 2-4 10 mg salt/kg every 12 hrs, dihydrochloride hrs until able to take orally8,153 Quinine IM Children and adults 20 mg salt/kg (dilute iv 10 mg salt/kg every 8–12 hrs dihydrochloride formulation to 60mg/ml) until able to take orally142,154 given in 2 injection sites (anterior thigh) Quinidine gluconate IV Children and adults 10 mg salt/kg in normal 0.02 mg salt/kg/min saline over 1-2 hrs continuous infusion with ECG monitoring up to 72 hrs OR 10 mg salt/kg every 8-12 hrs143 Artemether IM Children and adults 3.2 mg/kg 1.6 mg/kg/day for a minimum of 5 days5 Artesunate IV/IM Children and adults 2.4 mg/kg 1.2 mg/kg after 12 and 24 hrs, then 1.2 mg/kg/day for 7 days. Change to oral route when possible5

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Proefschrift.indb 121 14-1-2008 13:09:23 toxic (especially cardiotoxicity) and expensive than quinine and, a dose reduction may be necessary if the corrected QT interval is prolonged.143 In some parts of Francophone Africa, Quinimax (a combination of quinine, quinidine, cinchonine and cinchonidine) is commonly used.144 The main side effects of the cinchona alkaloids are hyperinsulinaemic hypoglycaemia and cinchonism (giddiness, tinnitus, high tone deafness and colour aberrations). Although high doses of quinine may induce uterine contractions, normal therapeutic doses can be used safely in pregnancy.145 Doses of the cinchona alkaloids should be reduced by 30-50% if intravenous therapy is required beyond 3 days to avoid accumulation. The artemisinin derivatives clear circulating parasites faster than other antimalarial drugs146 and adults treated with artesunate have a lower mortality than those treated with quinine.147 The artemisinin derivatives should be used in combination with other antimalarial drugs to prevent resistance. Side effects are infrequent148 and they are easier to administer than the cinchoniods. Animal studies show that parenteral artemether and artheeter are associated with damage to brainstem nuclei149 but no evidence of such neurotoxic effects has been detected in humans.150 Rectal preparations may be useful in rural health facilities.151

Supportive therapy Ventilation and dialysis may be life saving in adults with pulmonary oedema or renal failure respectively. Children should receive antimicrobials to cover the possibility of bacterial infections until these can confidently be excluded by examination of CSF, blood and urine.8 Exchange transfusion has been recommended for non-immune adult patients with parasite densities >30% as it lowers parasitaemia and improves red cell rheology but there is no conclusive evidence that it reduces mortality.152

Therapies with deleterious or unproven value A number of other adjunct therapies have been tried, but as yet remain unproven.8 Steroids are deleterious, while acetyl salicylic acid, sodium bicarbonate and heparin may be harmful. Desferoxamine and dextran have unclear roles. Hyperimmune serum confers no benefit, while monoclonal antibodies to TNF were associated with a worse neurological outcome. Although pentoxifylline, was associated with earlier resolution of coma and lower mortality in Burundian children, no benefit was observed in other studies. Mannitol reduces intracranial hypertension but such decreases are neither sustained nor does it prevent the development of severe intracranial hypertension. Prophylactic Phenobarbital, 10 mg/kg did not control seizures155, 20 mg/Kg was associated with increased mortality in unventilated Kenyan children156 but in Thai adults a single intramuscular injection of 3.5 mg/kg prevented convulsions.157 Dicloroacetate,

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Proefschrift.indb 122 14-1-2008 13:09:23 an activator of pyruvate dehydrogenase, has been shown to reduce blood lactic acid levels, but clinical trials are awaited to evaluate its effects on outcome158.

AREAS FOR RESEARCH Prevention of malaria is clearly a priority and the widespread use of preventative measures such as insecticide-treated materials can reduce all childhood deaths by 20%.159 Together with the prompt treatment of fever with effective anti malarial drugs, these interventions may reverse the rising mortality due to malaria in Africa. Basic malaria research continues to explore vaccines as an ideal preventive tool. A vaccine that produces protection against infection has remained elusive, due to the complexity of parasite biology. Insights into the processes leading to CM might identify targets for a vaccine that allows infection and the acquisition of immunity, but prevents CM.

Further definition of the phenotype of CM would help provide insights into the pathogenesis, in particular the associations with genetic polymorphisms. A more robust exclusion of other causes of encephalopathies in patients presenting with coma and a peripheral parasitaemia in endemic areas would reduce the contamination effect of these conditions on the pathogenesis and outcome of studies of CM. More careful documentation of the retinal findings may be particularly important. There are technical difficulties in the study of subtle cerebral processes in comatose patients. The development of a reliable animal or in vitro model may provide further insights. The technology exists to refine the murine model by inserting human genes (transgenics) into the mouse genome to allow the replacement of murine proteins with human ones. Infection of these models with P. falciparum would recreate the key clinical and pathological processes. Most patients die before antimalarials have had time to kill the parasites. In addition to addressing the public health problems resulting in delayed presentation to hospital and ensuring children receive prompt and appropriate resuscitation, novel interventions that address the pathophysiological processes causing these early deaths is a priority.

The scale of neuro-cognitive impairment reflects an enormous socio-economic burden in resource-poor countries. Research is needed to more clearly define the patients at risk and identify risk factors for persistent impairments. Magnetic resonance imaging, particularly of African children during the acute illness and on recovery may provide insights into the pathogenesis of the neuro-cognitive damage. Interventions to prevent brain damage and rehabilitation programmes for those with neuro-cognitive impairments are needed. Development of neuro-protective agents, improvement in

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Proefschrift.indb 123 14-1-2008 13:09:23 prophylactic anticonvulsant regimes, treatment of raised ICP in children, or addressing alterations in brain biochemistry may be options (figure 7).

ACKNOWLEDGEMENTS Professor Charles Newton (070114) and Dr Neil Jenkins (GR066684MF) are supported by The Wellcome Trust, UK.

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Proefschrift.indb 128 14-1-2008 13:09:24 103. Dondorp AM, Angus BJ, Hardeman MR, et al. Prognostic significance of reduced red blood cell deformability in severe falciparum malaria. Am J Trop Med Hyg 1997;57(5):507-11. 104. Dondorp AM, Nyanoti M, Kager PA, Mithwani S, Vreeken J, Marsh K. The role of reduced red cell deformability in the pathogenesis of severe falciparum malaria and its restoration by blood transfusion. Trans R Soc Trop Med Hyg 2002;96(3):282-6. 105. Lyke KE, Burges R, Cissoko Y, et al. Serum levels of the proinflammatory cytokines interleukin-1 beta (IL-1beta), IL-6, IL-8, IL-10, tumor necrosis factor alpha, and IL-12(p70) in Malian children with severe Plasmodium falciparum malaria and matched uncomplicated malaria or healthy controls. Infect Immun 2004;72(10):5630-7. 106. Kwiatkowski D. Tumour necrosis factor, fever and fatality in falciparum malaria. Immunol Lett 1990;25(1-3):213-6. 107. Akanmori BD, Kurtzhals JA, Goka BQ, et al. Distinct patterns of cytokine regulation in discrete clinical forms of Plasmodium falciparum malaria. Eur Cytokine Netw 2000;11(1):113-8. 108. Gimenez F, Barraud de Lagerie S, Fernandez C, Pino P, Mazier D. Tumor necrosis factor alpha in the pathogenesis of cerebral malaria. Cell Mol Life Sci 2003;60(8):1623-35. 109. McGuire W, Hill AV, Allsopp CE, Greenwood BM, Kwiatkowski D. Variation in the TNF-alpha promoter region associated with susceptibility to cerebral malaria. Nature 1994;371(6497):508-10. 110. Day NP, Hien TT, Schollaardt T, et al. The prognostic and pathophysiologic role of pro- and antiinflammatory cytokines in severe malaria. J Infect Dis 1999;180(4):1288-97. 111. Brown H, Turner G, Rogerson S, et al. Cytokine expression in the brain in human cerebral malaria. J Infect Dis 1999;180(5):1742-6. 112. Clark IA, Awburn MM, Whitten RO, et al. Tissue distribution of migration inhibitory factor and inducible nitric oxide synthase in falciparum malaria and sepsis in African children. Malar J 2003;2(1):6. 113. Anstey NM, Weinberg JB, Hassanali MY, et al. Nitric oxide in Tanzanian children with malaria: inverse relationship between malaria severity and nitric oxide production/nitric oxide synthase type 2 expression. J Exp Med 1996;184(2):557-67. 114. Cramer JP, Nussler AK, Ehrhardt S, et al. Age-dependent effect of plasma nitric oxide on parasite density in Ghanaian children with severe malaria. Tropical Medicine and International Health 2005;10(7):672-680. 115. Clark IA, Alleva LM, Mills AC, Cowden WB. Pathogenesis of Malaria and Clinically Similar Conditions. Clin. Microbiol. Rev. 2004;17(3):509-539. 116. Gitau EN, Newton CR. Review Article: Blood-brain barrier in falciparum malaria. Trop Med Int Health 2005;10(3):285-92. 117. Warrell DA, Looareesuwan S, Phillips RE, et al. Function of the blood-cerebrospinal fluid barrier in human cerebral malaria: rejection of the permeability hypothesis. Am J Trop Med Hyg 1986;35(5):882-9. 118. Brown H, Hien TT, Day N, et al. Evidence of blood-brain barrier dysfunction in human cerebral malaria. Neuropathol Appl Neurobiol 1999;25(4):331-40. 119. Adams S, Brown H, Turner G. Breaking down the blood-brain barrier: signaling a path to cerebral malaria? Trends Parasitol 2002;18(8):360-6. 120. Patankar TF, Karnad DR, Shetty PG, Desai AP, Prasad SR. Adult cerebral malaria: prognostic importance of imaging findings and correlation with postmortem findings. Radiology 2002;224(3):811-6. 121. Looareesuwan S, Warrell DA, White NJ, et al. Do patients with cerebral malaria have cerebral oedema? A computed tomography study. Lancet 1983;1(8322):434-7.

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Proefschrift.indb 129 14-1-2008 13:09:24 122. Pongponratn E, Turner GD, Day NP, et al. An ultrastructural study of the brain in fatal Plasmodium falciparum malaria. Am J Trop Med Hyg 2003;69(4):345-59. 123. Turner G. Cerebral malaria. Brain Pathol 1997;7(1):569-82. 124. Patnaik JK, Das BS, Mishra SK, Mohanty S, Satpathy SK, Mohanty D. Vascular clogging, mononuclear cell margination, and enhanced vascular permeability in the pathogenesis of human cerebral malaria. Am J Trop Med Hyg 1994;51(5):642-7. 125. Medana IM, Day NP, Hien TT, et al. Axonal injury in cerebral malaria. Am J Pathol 2002;160(2):655-66. 126. Medana IM, Lindert RB, Wurster U, et al. Cerebrospinal fluid levels of markers of brain parenchymal damage in Vietnamese adults with severe malaria. Trans R Soc Trop Med Hyg 2005;99(8):610-7. 127. Jaffar S, Van Hensbroek MB, Palmer A, Schneider G, Greenwood B. Predictors of a fatal outcome following childhood cerebral malaria. Am J Trop Med Hyg 1997;57(1):20-4. 128. Phu NH, Hien TT, Mai NT, et al. Hemofiltration and peritoneal dialysis in infection-associated acute renal failure in Vietnam. N Engl J Med 2002;347(12):895-902. 129. Carter JA, Mung’ala-Odera V, Neville BG, et al. Persistent neurocognitive impairments associated with severe falciparum malaria in Kenyan children. J Neurol Neurosurg Psychiatry 2005;76(4):476-81. 130. Carter JA, Neville BG, White S, et al. Increased prevalence of epilepsy associated with severe falciparum malaria in children. Epilepsia 2004;45(8):978-81. 31. Muntendam AH, Jaffar S, Bleichrodt N, van Hensbroek MB. Absence of neuropsychological sequelae following cerebral malaria in Gambian children. Trans R Soc Trop Med Hyg 1996;90(4):391-4. 132. Boivin MJ. Effects of early cerebral malaria on cognitive ability in Senegalese children. J Dev Behav Pediatr 2002;23(5):353-64. 133. Dugbartey AT, Spellacy FJ, Dugbartey MT. Somatosensory discrimination deficits following pediatric cerebral malaria. Am J Trop Med Hyg 1998;59(3):393-6. 134. Carter JA, Ross AJ, Neville BG, et al. Developmental impairments following severe falciparum malaria in children. Trop Med Int Health 2005;10(1):3-10. 135. Nguyen TH, Day NP, Ly VC, et al. Post-malaria neurological syndrome. Lancet 1996;348(9032):917-21. 136. White NJ LS. Cerebral malaria. London: Butterworths, 1987. 137. Kastl AJ, Daroff RB, Blocker WW. Psychological testing of cerebral malaria patients. J Nerv Ment Dis 1968;147(6):553-61. 138. Varney NR, Roberts RJ, Springer JA, Connell SK, Wood PS. Neuropsychiatric sequelae of cerebral malaria in Vietnam veterans. J Nerv Ment Dis 1997;185(11):695-703. 139. Dugbartey AT, Dugbartey MT, Apedo MY. Delayed neuropsychiatric effects of malaria in Ghana. J Nerv Ment Dis 1998;186(3):183-6. 140. Maitland K, Nadel S, Pollard AJ, Williams TN, Newton CR, Levin M. Management of severe malaria in children: proposed guidelines for the United Kingdom. Bmj 2005;331(7512):337-43. 141. van der Torn M, Thuma PE, Mabeza GF, et al. Loading dose of quinine in African children with cerebral malaria. Trans R Soc Trop Med Hyg 1998;92(3):325-31. 142. Pasvol G, Newton CR, Winstanley PA, et al. Quinine treatment of severe falciparum malaria in African children: a randomized comparison of three regimens. Am J Trop Med Hyg 1991;45(6):702-13. 143. Stauffer W, Fischer PR. Diagnosis and treatment of malaria in children. Clin Infect Dis 2003;37(10):1340-8. 144. Barennes H, Munjakazi J, Verdier F, Clavier F, Pussard E. An open randomized clinical study of intrarectal versus infused Quinimax for the treatment of childhood cerebral malaria in Niger. Trans R Soc Trop Med Hyg 1998;92(4):437-40.

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Proefschrift.indb 130 14-1-2008 13:09:24 145. Looareesuwan S, Phillips RE, White NJ, et al. Quinine and severe falciparum malaria in late pregnancy. Lancet 1985;2(8445):4-8. 146. A meta-analysis using individual patient data of trials comparing artemether with quinine in the treatment of severe falciparum malaria. Trans R Soc Trop Med Hyg 2001;95(6):637-50. 147. Dondorp A, Nosten F, Stepniewska K, Day N, White N. Artesunate versus quinine for treatment of severe falciparum malaria: a randomised trial. Lancet 2005;366(9487):717-25. 148. Adjuik M, Babiker A, Garner P, Olliaro P, Taylor W, White N. Artesunate combinations for treatment of malaria: meta-analysis. Lancet 2004;363(9402):9-17. 149. Petras JM, Young GD, Bauman RA, et al. Arteether-induced brain injury in Macaca mulatta. I. The precerebellar nuclei: the lateral reticular nuclei, paramedian reticular nuclei, and perihypoglossal nuclei. Anat Embryol (Berl) 2000;201(5):383-97. 150. Hien TT, Turner GD, Mai NT, et al. Neuropathological assessment of artemether-treated severe malaria. Lancet 2003;362(9380):295-6. 151. Aceng JR, Byarugaba JS, Tumwine JK. Rectal artemether versus intravenous quinine for the treatment of cerebral malaria in children in Uganda: randomised clinical trial. BMJ 2005;330(7487):334-. 152. Riddle MS, Jackson JL, Sanders JW, Blazes DL. Exchange transfusion as an adjunct therapy in severe Plasmodium falciparum malaria: a meta-analysis. Clin Infect Dis 2002;34(9):1192-8. 153. Winstanley PA, Mberu EK, Watkins WM, Murphy SA, Lowe B, Marsh K. Towards optimal regimens of parenteral quinine for young African children with cerebral malaria: unbound quinine concentrations following a simple loading dose regimen. Trans R Soc Trop Med Hyg 1994;88(5):577-80. 154. Waller D, Krishna S, Craddock C, et al. The pharmacokinetic properties of intramuscular quinine in Gambian children with severe falciparum malaria. Trans R Soc Trop Med Hyg 1990;84(4):488-91. 155. Winstanley PA, Newton CR, Pasvol G, et al. Prophylactic phenobarbitone in young children with severe falciparum malaria: pharmacokinetics and clinical effects. Br J Clin Pharmacol 1992;33(2):149-54. 156. Crawley J, Waruiru C, Mithwani S, et al. Effect of phenobarbital on seizure frequency and mortality in childhood cerebral malaria: a randomised, controlled intervention study. Lancet 2000;355(9205):701-6. 157. White NJ, Looareesuwan S, Phillips RE, Chanthavanich P, Warrell DA. Single dose phenobarbitone prevents convulsions in cerebral malaria. Lancet 1988;2(8602):64-6. 158. Agbenyega T, Planche T, Bedu-Addo G, et al. Population kinetics, efficacy, and safety of dichloroacetate for lactic acidosis due to severe malaria in children. J Clin Pharmacol 2003;43(4):386-96. 159. Lengeler C. Insecticide-treated bed nets and curtains for preventing malaria. Cochrane Database Syst Rev 2004(2):CD000363.

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Proefschrift.indb 132 14-1-2008 13:09:24 Chapter 8

Risk factors for persisting neurological and cognitive impairments following cerebral malaria

Richard Idro, Julie A Carter, Greg Fegan, Brian G R Neville, Charles RJC Newton

Centre for Geographic Medicine Research–Coast, Kenya Medical Research Institute/ Wellcome Trust Research Labs, Kilifi – Kenya; Department of Paediatrics and Child Health, Mulago Hospital/Makerere University Medical School, Kampala – Uganda; Centre for International Child Health, Institute of Child Health (University College London), London – UK; Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London – UK; Neurosciences Unit, Institute of Child Health, University College London-UK

Archives of Disease of Childhood, 2006; 91 (2): 142-48

Proefschrift.indb 133 14-1-2008 13:09:24 ABSTRACT Objective Persisting neurological and cognitive impairments are common after cerebral malaria. Although risk factors for gross deficits on discharge have been described, few studies have examined those associated with persistent impairments.

Methods We determined the risk factors for impairments following cerebral malaria by examining hospital records of 143 children aged 6-9 years, previously admitted with cerebral malaria, who were assessed at least 20 months after discharge to detect motor, speech and language, and other cognitive (memory, attention and non-verbal functioning) impairments.

Results The median age on admission was 30 months (IQR 19-42) and the median time from discharge to assessment was 64 months (IQR 40-78). Thirty-four children (23.8%) were defined as having impairments: 14(9.8%) in motor, 16(11.2%) in speech and language and 20(14.0%) in other cognitive functions. Previous seizures (OR 5.6[95% CI 2.0-16.0], p=0.001); deep coma on admission (OR 28.8[95% CI 3.0-280], p=0.004); focal neurological signs observed during admission, (OR 4.6[95% CI 1.1-19.6], p=0.037) and neurological deficits on discharge (OR 4.5[95% CI 1.4-13.8], p=0.01) were independently associated with persisting impairments. In addition, multiple seizures were associated with motor impairment, age<3 years, severe malnutrition, features of intracranial hypertension and hypoglycaemia with language impairments while prolonged coma, severe malnutrition and hypoglycaemia were associated with impairments in other cognitive functions.

Conclusions Risk factors for persisting neurological and cognitive impairments following cerebral malaria include multiple seizures, deep/prolonged coma, hypoglycaemia, and clinical features of intra-cranial hypertension. Although there are overlaps in impaired functions and risk factors, the differences in risk factors for specific functions may suggest separate mechanisms for neuronal damage. These factors could form the basis of future preventive strategies for persisting impairments.

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Proefschrift.indb 134 14-1-2008 13:09:24 INTRODUCTION Worldwide, Plasmodium falciparum malaria is a leading cause of ill health. About 2 billion people are exposed to P. falciparum malaria annually resulting in over 500 million clinical attacks and about a million deaths, predominantly in children less than five years living in sub-Saharan Africa1.

Cerebral malaria (CM) is the most severe neurological complication of malaria. Even with appropriate anti-malarial treatment, 18.6% of patients die and 11% have gross neurological deficits detectable on discharge2. Due to the high mortality and limited means of detecting impairments in many resource-poor settings, the long-term effects of the disease have been poorly documented. However, given the magnitude of the problem, an enormous number of children may be at risk of impairments, with adverse educational and social consequences3-5. Indeed our recent studies in Kenyan children described persistent neurological and cognitive impairments in 24% of those who survived CM up to nine years after the episode6 7.

Risk factors for the development of neurological and cognitive deficits have been described3 8-12. However, these studies have generally not differentiated between impairments of different functions but classified patients as either having impairments or not. Analysis of risk factors for impairments has not examined the possibility of differences in impaired functions, whereas an alternative strategy of examining risk factors for individual functions may help elucidate the pathogenesis of impairments.

In this study, we used a cohort of Kenyan children (who had been exposed to CM and been assessed for long-term impairments) reported previously6 7 and examined clinical characteristics during hospital admission that are predictive of persistent impairments in motor, language and other cognitive functions.

METHODS Study design We compared a cohort of children who had survived the acute stage of CM to a similar group of children unexposed to severe malaria. This was part of a larger study investigating the effects of severe falciparum malaria on the neurological and cognitive functioning of children. Unlike earlier reports from this study6 7 13 we examined some of the antecedent events that may constitute risk factors for impairments.

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Proefschrift.indb 135 14-1-2008 13:09:24 Setting Participants were recruited from the Kilifi Demographic Surveillance area on the coast of Kenya. Kilifi District Hospital is the only hospital in the area, where the majority of very sick children are admitted for treatment. The characteristics of the study area have been described elsewhere14 15.

Participants Study participants were identified from the hospital’s admissions database, born between 1991 and 1995 and living in the study area. The study children had previously been exposed to CM and clinical data was collected prospectively during admission. Exposure to CM was defined as admission to hospital in coma with inability to localise a painful stimulus, a Blantyre coma score (BCS) ≤2 8, asexual forms of falciparum malaria on a blood smear and no evidence of pyogenic meningitis on examination of the cerebrospinal fluid16. As a comparison, we drew a random sample of children unexposed to severe malaria from a census database of children living in the study area.

At the time of assessment, all participants were 6-9 years old. This age group was chosen since the tests we used can assess cognition, speech and language more reliably above the age of 6 years. All spoke a Mijikenda language as their first language. Children were excluded if they refused verbal assent or their parents refused written informed consent. We obtained ethical permission for the study from the Kenya Medical Research Institute Scientific and Ethical Review Committees and from the Institute of Child Health, UK. Neurological and cognitive assessment The neurological and cognitive assessments have been described in detail elsewhere7. All children underwent the same battery of assessments for motor skills, behaviour, cognition, hearing and vision. Assessors were blinded to the participants’ exposure status. Cognitive assessment included tests of memory based on the Rivermead Behavioural Memory Test for Children17, attention (visual search), a parental rating of behaviour problems and non-verbal functioning (construction tasks such as copying shapes using drawings, blocks or sticks to assess the co-ordination of complex cognitive tasks)3. Speech and language assessments covered eight major areas of language: receptive grammar and vocabulary, lexical semantics (expressive vocabulary), syntax/ morphology, pragmatics (language use), phonology, higher level language and word finding7 18. The neurological assessment comprised a measure of motor skills based on a system for classifying motor function in children with cerebral palsy19. This is a five-level grading of motor deficits developed for use in clinical practice, research and teaching and comprises an assessment of cranial nerve function and motor function (using a hierarchical classification of spasticity, ataxia and fine motor dysfunction). Hearing was

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Proefschrift.indb 136 14-1-2008 13:09:24 tested with a Kamplex screening audiometer (P.C. Werth, London, UK) and vision, using a Sonksen-Silver chart20.

Nutritional status was assessed using weight for age z-scores (WAZ) calculated using the NutStat program in EpiInfo 2000 (CDC, Atlanta, USA). Socio-economic status was assessed using mother’s level of education, which is often regarded as a predictor of child health and development21 and father’s occupation, which has been found to be associated with income level in previous studies on the Kenyan coast22.

DATA MANAGEMENT Standard clinical classifications were used to ascertain levels of impairment in neurological, hearing and visual assessments171920. The scoring system of the behaviour questionnaire was used in a previous study3. Language, memory and non-verbal functioning assessments were not standardised on the local population, therefore impairment was classified using a commonly accepted classification of ability levels23. An estimate more than 2 standard deviations below the age-specific unexposed group mean or the 2.0 centile of the unexposed group results was adopted for normally distributed and skewed data respectively. A classification of ‘speech and language impairment’ was defined as an impairment level score on two or more of the language assessments, while a designation of ‘impairment in other cognitive functions’ described children with impairment level performance in any one of memory, attention or non- verbal functioning6 7.

Statistical Analysis Statistical analysis was carried out using SPSS version 11.5 (Chicago, USA). Patients were classified as impaired or non-impaired. For univariate analysis, admission clinical characteristics of children defined to have impairments were compared to those without impairments to identify risk factors for any impairment. Categorical variables were compared using Pearson’s Chi square or Fisher’s exact test (2-tailed) as appropriate. The means of normally distributed data were compared with the student’s t-test and the median used for skewed data. All variables with a p value <0.25 at univariate analysis were included in a logistic regression analysis to identify risk factors independently associated with persisting impairments and only those with a p value <0.05 were retained in the model. An a priori decision was made to include age in each model due to possible confounding effects of age at which the child was exposed to CM on the risk of neuronal damage: children were categorized as aged <3 or ≥3 years at the time of exposure. Separate regression analyses were then performed for motor, speech

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Proefschrift.indb 137 14-1-2008 13:09:24 and language, and impairments in other cognitive functions to identify risk factors independently associated with each function. Similar analyses were performed to identify risk factors for impairments in memory, attention or non-verbal functioning. The Bonferroni correction was performed to allow for multiple corrections.

RESULTS General description of study subjects Two hundred and forty-four children who fulfilled the criteria for CM and 273 children unexposed to severe malaria were identified from the databases. Of these, 20 children (14[5.7%] CM and 6[2.2%] unexposed, p=0.037) had died. Eighteen children (6 CM and 12 unexposed) did not fulfil the age requirements and were excluded (dates of birth reported at the time of admission were erroneous). One hundred and forty eight children (72 CM and 76 unexposed) had migrated out of the study area. A total of 331 (152 CM and 179 unexposed) children were eligible for recruitment.

Of the eligible children exposed to CM, those with incomplete records, who presented with evidence for other causes of the encephalopathy or significant developmental impairments before the admission with CM were excluded (a total of 9 children). These included one child with hemiplegia and delayed speech following birth asphyxia, four with incomplete records and four with evidence of other encephalopathies. Of the remaining 143 children, 74(51.7%) were male. The age and sex distribution of children in the two groups was similar. The median age at which the children were admitted with CM was 30 months (IQR 19-42) and the median time from discharge to assessment was 64 months (IQR 40-78).

Prevalence and types of impairments Thirty-four children exposed to CM (23.8%) and 18 (10.1%) unexposed children were defined to have impairments in at least one function. Of those functions measured, persisting impairments were most common in motor, speech and language and memory (table I).

Overlap of impaired functions in children exposed to cerebral malaria Some children with impaired functions had deficits in more than one function; 15 (42%) of the CM children with impairments had two or more impairments compared to 4 (22%) of the unexposed children. Multiple impairments were observed in clusters of language, memory and/or motor functions (figure 1). Non-verbal functioning, behaviour,

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Proefschrift.indb 138 14-1-2008 13:09:25 Table I Prevalence and types of persisting neurological and cognitive Type of neurological or cognitive Children Children Relative risk impairment exposed to unexposed to cerebral malaria severe malaria n=143 (%) N=179 (%) (95% CI) Any impairment 34 (23.8) 18 (10.1) 2.4 (1.4 - 4.0) Motor impairment 14 (9.8) 5 (2.8) 3.5 (1.3 - 9.5) Speech and language impairment 16 (11.2) 4 (2.2) 5.0 (1.7-14.5) Impairments in other cognitive functions 20 (14.0) 6 (3.4) 4.2 (1.7–10.1) Memory 14 (9.8) 5 (2.8) 5.0 (1.7-14.5) Attention 5 (3.5) 0 - Non-verbal functioning 4 (2.8) 1 (0.6) 5.0 (0.6-44.3) Behavioural difficulties 4 (2.8) 3 (1.7) 1.7 (0.4 - 7.3) Hearing and visual impairment 4 (2.8) 6 (3.4) 0.8 (0.2 – 2.9)

Figure 1 Overlap of impaired functions among 34 children defined to have persistent impairments following cerebral malaria Children with speech and language Children with motor impairments impairments (16) (14)

Children with impairments in other cognitive functions (20)

and attention were less often impaired relative to other functions in children with CM. Multiple impairments were particularly common among those who also had active epilepsy. Details of these impairments are described elsewhere.6 7 13

Risk factors for persistent impairments after cerebral malaria We compared the past medical history and the clinical characteristics at the time of admission of children exposed to CM defined to have impairments to those without impairments. In both groups, the median duration of illness was 3 days. There were no differences between children who developed impairments and those who did not in mother’s years of formal education, father’s income, sex or head circumference. Univariate analysis showed that a history of previous seizures, deep coma, hypoglycaemia, partial

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Proefschrift.indb 139 14-1-2008 13:09:25 Table II Clinical characteristics of study participants at the time of Patients’ characteristics on admission Patients without Patients with P impairments impairments Value n = 109 N = 34 Demographic characteristics and past medical history Sex, Males, (%) 60 (55.0) 14 (41.2) 0.17 Mean age at admission (SD) in months 31.3 (19.2) 30.2 (12.3) 0.74 Age < 3 years, (%) 65 (59.6) 22 (64.7) 0.69 Previous admissions, (%) 20 (18.3) 11 (32.4) 0.08 Previous history of seizures, (%) 13 (11.9) 11 (32.4) < 0.01 Clinical history Lack of history of fever, (%) 3 (2.8) 4 (11.8) 0.06 Duration of illness in days (median, IQR) 3 (2 – 4) 3 (2 – 3) - Anti malarial before admission, (%) 40 (37.4) 17 (50.0) 0.19 Seizures before admission, (%) 93 (85.3) 29 (85.3) 1.00 Type of seizures before admission Partial, (%) 27 (24.8) 14 (41.2) 0.07 Partial becoming secondarily generalized, (%) 14 (12.8) 6 (17.6) 0.48 Generalized, (%) 58 (53.2) 17 (50.0) 0.74 Number of seizures before admission (median, IQR) 3 (1 – 5) 4 (1 – 7) - Prolonged seizures before admission (seizures > 30 min, %) 30 (27.5) 13 (38.2) 0.23 Duration of coma before admission in hrs, (median, IQR) 4 (2 – 8) 5 (4 – 9) - Physical findings on admission Hyperpyrexia (axillary temp > 39.5 oC, %) 22 (20.2) 6 (17.6) 0.75 Severe malnutrition (WAZ score < - 3, %) 9 (8.3) 7 (20.6) 0.05 Head circumference in cm (mean, SD) 47.4 (2.7) 47.4 (1.5) 0.95 Deep coma at admission (BCS 0, %) 1 (0.9) 5 (14.7) <0.001 Abnormal motor posturing, (%) 22 (20.2) 8 (23.5) 0.68 Focal neurological signs observed during admission, (%) 6 (5.5) 5 (11.8) 0.21 Features of raised intra cranial pressure[1], (%) 28 (25.7) 13 (38.2) 0.16 Laboratory parameters Hypoglycaemia (blood sugar < 2.2 mmol/L) at or during 34 (31.2) 17 (50.0) 0.04 admission, (%) Red cell volume in fl, (mean, SD) 77.0 (9.8) 75.8 (11.3) 0.60 Severe anaemia (Hb < 5.0 g/dl) 29 (26.6) 9 (26.5) 0.99 Geometric mean parasite density /ml, 73,525 59,276 - Hyperparasitemia (Parasite density > 500,000/ml, %) 38 (34.9) 10 (29.4) 0.56 Hyponatraemia (admission serum Na+ < 135 mmol/L, %) 62 (56.9) 13 (38.2) < 0.01 CSF white blood cell count, (median, IQR) 2 (1 – 4) 2 (1 – 4) - CSF glucose in mmol/L, (mean, SD) 3.1 (1.1) 3.1 (0.9) 0.98 Features during hospital admission Seizures in the ward, (%) 43 (39.4) 19 (55.9) 0.09 Type of seizures in the ward Partial, (%) 27 (24.8) 16 (47.1) 0.01 Partial becoming secondarily generalized, (%) 14 (12.8) 4 (11.8) 0.86 Generalized, (%) 27 (24.8) 9 (26.5) 0.84 Multiple (3 or more) seizures in the ward, (%) 23 (21.1) 15 (44.1) < 0.01 Received a blood transfusion, (%) 39 (35.8) 15 (44.1) 0.38 Time to full consciousness in hrs, (median, IQR) 16 (8 –27) 24 (19 – 46) - Prolonged coma (> 24 hrs) in the ward, (%) 28 (25.7) 16 (47.1) 0.02 Discharged with neurological deficits or epileptic seizures (%) 12 (11.0) 8 (23.5) 0.07 [1] Sluggish, none reacting or unequal pupils, papilloedema, VI palsy

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Proefschrift.indb 140 14-1-2008 13:09:25 or multiple seizures in the ward, prolonged coma after admission or neurological deficits on discharge were significantly associated with impairments (table II).

Although the mean admission temperatures were similar, a higher proportion of children defined to have impairments were admitted without a history of fever. The risk of impairments was increased in those with deeper coma: 5/6 (83%) children with a BCS=0 had impairments compared to 5/18 (28%) of those with a BCS=1 and 24/119 (20%) of those with a BCS=2. The mean serum sodium was also higher in those with impairments, even though there was only one child with hypernatraemia. Children with impairments had a slower recovery from coma, with a median time to full consciousness about 8 hours longer than those without impairments.

Independent risk factors for any impairment In a logistic regression analysis, previous history of seizures, deep coma at admission, focal neurological signs, and neurological deficits at discharge were identified as factors independently predictive of any impairment (table III). i) Motor impairment Previous admissions, focal neurological signs and multiple seizures during the course of admission were independently associated with persisting motor impairments. ii) Speech and language impairment Age <3 years, previous history of seizures, brainstem and fundoscopic evidence of raised ICP, hypoglycaemia, and prolonged coma after admission were independently associated with persisting language impairments. iii) Impairment in other cognitive functions Previous seizures, deep coma on admission and prolonged coma after admission were independently associated with the development of impairment in other cognitive functions. When we examined risk factors for impairment in different functions, deep coma (adjusted OR 40.6[95% CI 2.8-583], p=0.006) and severe malnutrition (WAZ<-3.0, adjusted OR 23.0[95% CI 2.2-245], p = 0.009) were found to be independently associated with impaired attention. Lack of a history of fever (adjusted OR 7.9[95% CI 1.3-49.6], p=0.028), deep coma (adjusted OR 16.1[95% CI 2.3-112], p=0.005) and prolonged coma after admission (adjusted OR 6.4[95% CI 1.6-25.1], p=0.008) were associated with memory impairment. Hypoglycaemia was associated with impairment of non-verbal functioning (adjusted OR 1.1[95% CI 1.0- 1.2], p=0.015). No association was observed between impairment in these cognitive functions and either focal neurological signs or neurological deficits at discharge.

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Proefschrift.indb 141 14-1-2008 13:09:25 Table III Risk factors independently associated with persisting neurological and cognitive impairments following cerebral malaria Independent risk factors for impairment Any impairment Motor Speech and language Impairments in other cognitive (n=34) impairment impairment functions (memory, attention (n=14) (n=16) and non-verbal functioning) (n=20) Adjusted OR P Adjusted OR P Adjusted OR P Adjusted OR P (95% CI) Value (95% CI) Value (95% CI) Value (95% CI) Value Age <3 years * * * * 6.4 (1.2 – 36) 0.033 * * Previous history of seizures 5.6 (2.0 – 16.0) 0.001 * * 7.6 (1.7 – 33.6) 0.007 3.2 (1.0 – 10.0) 0.042 Previous admissions * * 4.8 (1.3 – 17.6) 0.018 * * * * Severe malnutrition (WAZ Score < - 3) * * * * 6.6 (1.4 – 30.6) 0.015 * * Deep coma at admission 28.8 (3.0 – 280) 0.004 * * * * 12.5 (2.0 – 78.8) 0.007 Focal neurological signs observed during 4.6 (1.1 – 19.6) 0.037 12.3 (1.4 – 110) 0.024 * * * * admission Multiple (3 or more) seizures in the ward * * 8.3 (2.3 – 29.5) 0.001 * * * * Features of raised intra cranial pressure * * * * 6.0 (1.4 – 30.6) 0.019 * * Hypoglycaemia at or during admission * * * * 6.1 (1.4 – 25.7) 0.014 * * Prolonged coma * * * * * * 3.3 (1.2 – 9.4) 0.027 Neurological deficits at discharge 4.5 (1.4 – 13.8) 0.010 * * 7.3 (1.4 – 36.8) 0.016 * * * Variables not included in the final model

When the Bonferroni correction was applied to adjust for multiple comparisons, motor impairment was still associated with multiple seizures, language impairment with previous seizures, severe malnutrition and hypoglycaemia while impairments in other cognitive functions were associated with deep coma.

DISCUSSION The aim of this study was to describe clinical risk factors during the acute phase of CM that are predictive of different types of persistent neurological and cognitive impairments. We found that previous seizures, deep coma on admission, focal neurological signs during hospital admission, and neurological deficits on discharge are predictive of the development of persistent impairments after CM. In addition, age <3 years, previous admissions, multiple seizures, hypoglycaemia, raised ICP and prolonged coma are associated with specific impairments. These risk factors differed according to the function impaired (figure 2).

Risk factors for long-term impairments following cerebral malaria We were unable to locate almost 30% of the children identified from the hospital’s database mostly as a result of migration. It is possible some died, especially those who had severe impairments5. Despite this, risk factors for impairments in specific functions could be identified.

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Proefschrift.indb 142 14-1-2008 13:09:25 Table III Risk factors independently associated with persisting neurological and cognitive impairments following cerebral malaria Independent risk factors for impairment Any impairment Motor Speech and language Impairments in other cognitive (n=34) impairment impairment functions (memory, attention (n=14) (n=16) and non-verbal functioning) (n=20) Adjusted OR P Adjusted OR P Adjusted OR P Adjusted OR P (95% CI) Value (95% CI) Value (95% CI) Value (95% CI) Value Age <3 years * * * * 6.4 (1.2 – 36) 0.033 * * Previous history of seizures 5.6 (2.0 – 16.0) 0.001 * * 7.6 (1.7 – 33.6) 0.007 3.2 (1.0 – 10.0) 0.042 Previous admissions * * 4.8 (1.3 – 17.6) 0.018 * * * * Severe malnutrition (WAZ Score < - 3) * * * * 6.6 (1.4 – 30.6) 0.015 * * Deep coma at admission 28.8 (3.0 – 280) 0.004 * * * * 12.5 (2.0 – 78.8) 0.007 Focal neurological signs observed during 4.6 (1.1 – 19.6) 0.037 12.3 (1.4 – 110) 0.024 * * * * admission Multiple (3 or more) seizures in the ward * * 8.3 (2.3 – 29.5) 0.001 * * * * Features of raised intra cranial pressure * * * * 6.0 (1.4 – 30.6) 0.019 * * Hypoglycaemia at or during admission * * * * 6.1 (1.4 – 25.7) 0.014 * * Prolonged coma * * * * * * 3.3 (1.2 – 9.4) 0.027 Neurological deficits at discharge 4.5 (1.4 – 13.8) 0.010 * * 7.3 (1.4 – 36.8) 0.016 * * * Variables not included in the final model

Figure 2: Risk factors associated with persisting motor, speech and language and other cognitive impairments Speech and Language impairments

Motor Impairments

Impairments in other cognitive functions

Risk factors for impaired motor function Children with motor impairments were more likely to have had previous admissions with seizures and focal neurological signs or multiple seizures during hospital

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Proefschrift.indb 143 14-1-2008 13:09:25 admission. Seizures are common precipitators of admission: it is probable that each episode cumulatively increases the risk of focal neurological damage and that on subsequent visits, patients present with multiple seizures, focal neurological signs and motor impairment10.

Risk factors for speech and language impairments Children who developed language impairments were more likely to have been young (<3 years), had hypoglycaemia, severe malnutrition, or raised ICP at the time of admission or neurological deficits on discharge. This would suggest that language impairment develops in children who suffer a more severe encephalopathy (patients with hypoglycaemia or raised ICP) at a young age and in those who had been exposed to long-term nutritional deprivation24. It is also possible however that, these deficits are a manifestation of global cerebral damage rather than injury to a specific area of the brain given the wide range of risk factors associated with language. Indeed, the overlap of language deficits with impairments in motor and other cognitive functions in some patients supports this suggestion. Impairment in one function may also worsen the outcome in another. Overlap of impairments in language and other cognitive functions (figure 1) therefore could have arisen from the secondary effects of impaired memory and attention (which had sizeable verbal components in terms of instructions, teaching elements or response formats), while some of the other language difficulties could have be due to impaired motor function25.

Impairments in other cognitive functions This study confirms previous reports of an association between deep coma on admission or prolonged coma after admission and cognitive impairment after CM3 26. In addition, it suggests that previous episodes of seizures increase the risk of cognitive impairment. Hypoglycaemia and absence of hyperpyrexia have been associated with impaired cognition3. Our findings appear to confirm this; hypoglycaemia was associated with impairment of non-verbal functioning but instead of absence of hyperpyrexia, we observed that children who failed to mount a febrile response (no history of fever before admission) had an increased risk of developing memory impairment. A recent study

found impaired prostaglandin E2 (PG E2) metabolism in some children with CM and this was associated with poor outcome27. Prostaglandins are important mediators of fever, macrophage activity and pro-inflammatory responses to infection. The failure to mount

a febrile response that we observed may be due to the impaired PG E2 metabolism in children with CM. It is however not clear which mechanisms are involved in the impaired

PG E2 metabolism and impaired cognitive function.

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Proefschrift.indb 144 14-1-2008 13:09:26 Risk factors for impairments and the pathogenesis of cerebral malaria Cerebral malaria is a diffuse encephalopathy characterized by sequestration of Plasmodium falciparum in cerebral capillaries28. Due to the diffuse nature, deficits are likely to involve more than one function3 and survivors of the more severe forms are likely to suffer greater neuronal damage. Children who develop CM in early childhood (before the age of 3 years), during the period of brain development and organization are likely to suffer more severe and lasting neurological damage26 29. Differences in risk factors for impairments in each function suggest separate pathogenic mechanisms. Each risk factor may influence different functions with differing degrees of severity (figure 2). In addition, some cognitive functions such as language may be affected by external factors such as the child’s nutritional state at the time of the encephalopathy30 31.

In a systematic review of neurological and cognitive impairments associated with common central nervous system infections, Carter et al32 found a pattern to suggest a low mortality and morbidity following viral meningitis. Both acute bacterial meningitis (ABM) and CM were associated with high mortality but those exposed to ABM had a higher prevalence of long-term impairments. The risk of impairments after ABM was greatest for, but not confined to, those who had acute neurologic complications such as coma. The results of our study suggest that some pre-existing factors, namely previous seizures and admissions, are related to outcome even though children with obvious impairments reported at the time of admission were excluded. However, just like ABM32 33, the data clearly demonstrated that markers of the severity of CM encephalopathy such as multiple seizures, deep or prolonged coma, hypoglycaemia or raised ICP, independently correlated with neurological and cognitive impairments, supporting a causative relationship between CM and these impairments. The risk factors we describe are similar to that found in earlier CM studies3 9 11. The prolonged time to full consciousness in children with impairments points to the severity of the encephalopathy they had been exposed to. Deep coma too was strongly associated with impairment even though few patients had this level of coma. Over 80% of the patients with deep coma developed cognitive impairment. Higher mortality rates have previously been described among children with deep coma8 10 16 34. Mortality and poor neurological and cognitive outcomes therefore seem to form a continuum with surviving patients suffering significant neurological damage leading to long-term deficits. The risk factors we have identified in this study may be used to select children at risk of developing impairments so that they are followed up to detect and possibly manage these impairments. The same risk factors may also be used to develop intervention programs.

The proportion of children with impairments in the unexposed group (10.1%) is comparable to that found in another resource poor country, Bangladesh (7% of 2-9 year

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Proefschrift.indb 145 14-1-2008 13:09:26 olds)35. Although this study cannot completely exclude the effects of insults such as iron deficiency on cognition, the differences we observed are unlikely to be due to iron deficiency at the time of admission since the mean red cell volumes in both groups were similar. There too was no association between haemoglobin level or severe anaemia at admission and long-term cognitive impairments.

CONCLUSION In conclusion, persisting impairments in neurological and cognitive functions are associated with childhood cerebral malaria. Although overlaps exist, specific risk factors can be identified for impairment in each function, suggesting that different mechanisms may be responsible. Children with such risk factors should be followed up to look for persistent impairments. The same risk factors may also be useful in identifying interventions to prevent their development during admission, for example prophylactic anticonvulsants.

ACKNOWLEDGEMENTS We thank the assessment team, in particular Joseph Gona, Gladys Murira, Elizabeth Obiero, Khamis Katana, Kenneth Rimba and Judy Tumaini; the records officers Ramos Kazungu and Josephene Kazungu. This paper is published with the permission of the director of KEMRI. Dr Carter and Professor Newton are supported by the Wellcome Trust (grants 059336 and 070114 respectively).

REFERENCES 1. Snow RW, Guerra CA, Noor AM, Myint HY, Hay SI. The global distribution of clinical episodes of Plasmodium falciparum malaria. Nature 2005;434(7030):214-7. 2. Newton CR, Krishna S. Severe falciparum malaria in children: current understanding of pathophysiology and supportive treatment. Pharmacol Ther 1998;79(1):1-53. 3. Holding PA, Stevenson J, Peshu N, Marsh K. Cognitive sequelae of severe malaria with impaired consciousness. Trans R Soc Trop Med Hyg 1999;93(5):529-34. 4. Dugbartey AT, Spellacy FJ, Dugbartey MT. Somatosensory discrimination deficits following pediatric cerebral malaria. Am J Trop Med Hyg 1998;59(3):393-6. 5. Mung’Ala-Odera V, Snow RW, Newton CR. The burden of the neurocognitive impairment associated with Plasmodium falciparum malaria in sub-saharan Africa. Am J Trop Med Hyg 2004;71(2 Suppl):64-70. 6. Carter JA, Mung’ala-Odera V, Neville BG, Murira G, Mturi N, Musumba C, et al. Persistent neurocognitive impairments associated with severe falciparum malaria in Kenyan children. J Neurol Neurosurg Psychiatry 2005;76(4):476-81.

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Proefschrift.indb 146 14-1-2008 13:09:26 7. Carter JA, Ross AJ, Neville BG, Obiero E, Katana K, Mung’ala-Odera V, et al. Developmental impairments following severe falciparum malaria in children. Trop Med Int Health 2005;10(1):3-10. 8. Molyneux ME, Taylor TE, Wirima JJ, Borgstein A. Clinical features and prognostic indicators in paediatric cerebral malaria: a study of 131 comatose Malawian children. Q J Med 1989;71(265):441-59. 9. van Hensbroek MB, Palmer A, Jaffar S, Schneider G, Kwiatkowski D. Residual neurologic sequelae after childhood cerebral malaria. J Pediatr 1997;131(1 Pt 1):125-9. 10. Idro R, Karamagi C, Tumwine J. Immediate outcome and prognostic factors for cerebral malaria among children admitted to Mulago Hospital, Uganda. Ann Trop Paediatr 2004;24(1):17-24. 11. Bondi FS. The incidence and outcome of neurological abnormalities in childhood cerebral malaria: a long-term follow-up of 62 survivors. Trans R Soc Trop Med Hyg 1992;86(1):17-9. 12. Schmutzhard E, Gerstenbrand F. Cerebral malaria in Tanzania. Its epidemiology, clinical symptoms and neurological long term sequelae in the light of 66 cases. Trans R Soc Trop Med Hyg 1984;78(3):351-3. 13. Carter JA, Neville BG, White S, Ross AJ, Otieno G, Mturi N, et al. Increased prevalence of epilepsy associated with severe falciparum malaria in children. Epilepsia 2004;45(8):978-81. 14. Mbogo CN, Snow RW, Khamala CP, Kabiru EW, Ouma JH, Githure JI, et al. Relationships between Plasmodium falciparum transmission by vector populations and the incidence of severe disease at nine sites on the Kenyan coast. Am J Trop Med Hyg 1995;52(3):201-6. 15. Schellenberg JA, Newell JN, Snow RW, Mung’ala V, Marsh K, Smith PG, et al. An analysis of the geographical distribution of severe malaria in children in Kilifi District, Kenya. Int J Epidemiol 1998;27(2):323-9. 16. HO. Severe falciparum malaria. Trans R Soc Trop Med Hyg 2000;94 Suppl 1:S1-90. 17. Wilson B I-CR, Aldrich F. The Rivermead Bahavioural Memory Test for Children. 2nd ed. Bury st Edmunds: Thames Valley Test Company, 1991. 18. Carter JA, Murira GM, Ross AJ, Mung’ala-Odera V, Newton CR. Speech and language sequelae of severe malaria in Kenyan children. Brain Inj 2003;17(3):217-24. 19. Palisano R, Rosenbaum P, Walter S, Russell D, Wood E, Galuppi B. Development and reliability of a system to classify gross motor function in children with cerebral palsy. Dev Med Child Neurol 1997;39(4):214-23. 20. Salt AT, Sonksen PM, Wade A, Jayatunga R. The maturation of linear acuity and compliance with the Sonksen-Silver Acuity System in young children. Dev Med Child Neurol 1995;37(6):505-14. 21. Connolly KJ, Kvalsvig JD. Infection, nutrition and cognitive performance in children. Parasitology 1993;107 Suppl:S187-200. 22. Hoorweg J FD, Klaver W. Seasons and nutrition at the Kenya Coast. Leiden: African Studies Centre, 1995. 23. Lezak MD. Neuropsychological assessment. 3rd ed. New York: Oxford University Press, 1995. 24. Walther FJ, Ramaekers LH. Language development at the age of 3 years of infants malnourished in utero. Neuropediatrics 1982;13(2):77-81. 25. Pennington L, Goldbart J, Marshall J. Speech and language therapy to improve the communication skills of children with cerebral palsy. Cochrane Database Syst Rev 2004(2):CD003466. 26. Boivin MJ. Effects of early cerebral malaria on cognitive ability in Senegalese children. J Dev Behav Pediatr 2002;23(5):353-64. 27. Perkins DJ, Hittner JB, Mwaikambo ED, Granger DL, Weinberg JB, Anstey NM. Impaired systemic production of prostaglandin E2 in children with cerebral malaria. J Infect Dis 2005;191(9):1548-57. 28. Phillips RE, Solomon T. Cerebral malaria in children. Lancet 1990;336(8727):1355-60.

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Proefschrift.indb 147 14-1-2008 13:09:26 29. Holding PA, Snow RW. Impact of Plasmodium falciparum malaria on performance and learning: review of the evidence. Am J Trop Med Hyg 2001;64(1-2 Suppl):68-75. 30. Neumann C, McDonald MA, Sigman M, Bwibo N. Medical illness in school-age Kenyans in relation to nutrition, cognition, and playground behaviors. J Dev Behav Pediatr 1992;13(6):392-8. 31. Johnston FE, Low SM, de Baessa Y, MacVean RB. Interaction of nutritional and socioeconomic status as determinants of cognitive development in disadvantaged urban Guatemalan children. Am J Phys Anthropol 1987;73(4):501-6. 32. Carter JA, Neville BG, Newton CR. Neuro-cognitive impairment following acquired central nervous system infections in childhood: a systematic review. Brain Res Brain Res Rev 2003;43(1):57-69. 33. Grimwood K, Anderson VA, Bond L, Catroppa C, Hore RL, Keir EH, et al. Adverse outcomes of bacterial meningitis in school-age survivors. Pediatrics 1995;95(5):646-56. 34. Jaffar S, Van Hensbroek MB, Palmer A, Schneider G, Greenwood B. Predictors of a fatal outcome following childhood cerebral malaria. Am J Trop Med Hyg 1997;57(1):20-4. 35. Khan N DM. Framework: prevalence. In: Disabled Children and Developing Countries. London: MacKeith Press, 1995.

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Proefschrift.indb 148 14-1-2008 13:09:26 Chapter 9

Axonal and astrocyte injury markers in the cerebrospinal fluid of Kenyan children with severe malaria

Isabelle M Medana, Richard Idro and Charles RJC Newton

Nuffield Department of Clinical Laboratory Sciences, University of Oxford, UK; Centre for Geographic Medicine Research – Coast, Kenya Medical Research Institute, Kilifi, Kenya; Department of Paediatrics and Child Health, Mulago Hospital/Makerere University, Kampala, Uganda; Neurosciences Unit, The Wolfson Centre, Institute of Child Health, University College London, UK

Journal of the Neurological Sciences, 2007; 258 (1-2): 93-8.

Proefschrift.indb 149 14-1-2008 13:09:26 ABSTRACT A retrospective study of cerebrospinal fluid (CSF) levels of markers of brain parenchymal damage was conducted in Kenyan children with severe falciparum malaria. Two markers were analysed by immunoassays: the microtubule-associated protein tau for degenerated axons; and S-100B for astrocytes. The level of tau proteins in the CSF was significantly elevated in children with cerebral malaria compared with either malaria with prostration or malaria with seizures but normal consciousness (p<0.001). Elevated tau was also found to be associated with impaired delivery of oxygen (severe anaemia), severe metabolic acidosis manifesting as respiratory distress (increased respiratory rate and deep acidotic breathing) and at higher parasite densities. Elevated S-100B in children was associated with an increased risk of repeated seizures. This study provides evidence that axonal injury is associated with malaria coma and identifies the potential role of severe anaemia, acidosis and hyperparasitaemia to causing brain parenchymal damage in children with malaria.

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Proefschrift.indb 150 14-1-2008 13:09:26 INTRODUCTION Falciparum malaria is arguably one of the most common causes of non-traumatic coma in children in the world. It kills over a million people each year, most of whom are children living in sub-Saharan Africa[1]. Cerebral malaria is the most severe neurological complication of falciparum malaria, manifesting as coma with frequent seizures[2]. It causes death in 18.6% of children, and 11% of the survivors have residual neurological deficits on discharge[3]. Furthermore up to 24% of children have neurological and cognitive impairments when carefully assessed more than 3 years after discharge[4]. Despite these findings, the pathophysiology of cerebral malaria, in particular the pathogenesis of neurocognitive sequelae is incompletely understood.

Biomarkers in cerebrospinal fluid (CSF) have been used to help delineate pathophysiological mechanisms, predict and monitor neurological outcomes and develop and evaluate new therapeutic strategies in human neurological disease (reviewed in [5]). Measurement of CSF biomarkers is a particularly useful tool for studies of pathological processes in malaria endemic countries, where post mortem brain tissue samples are rarely available and neuroimaging and neurophysiology facilities are limited. In this study we have examined the levels of two markers of brain damage, the tau protein (tau) and S-100B, in the CSF of Kenyan children with malaria.

Tau is a phosphorylated microtubule-associated protein, considered to be important for maintaining the stability of axonal microtubules involved in the mediation of fast axonal transport or synaptic constituents. S-100B is an acidic calcium-binding protein synthesised in astrocytes in all parts of the central nervous system. Increased CSF levels of these proteins have been observed in the CSF of patients with neurological symptoms and often reflect neurological outcome, disability and death[6, 7]. In a recent study[8], the levels of tau and S-100B were examined in the CSF of adult Vietnamese patients with severe malaria. Tau levels were found to be associated with duration of coma and S-100B was associated with seizures. Concentrations of these markers were also associated with vital organ dysfunction. In multivariate analysis, the correlations with coma and seizures were independent of vital organ dysfunction suggesting multiple mechanisms of CNS impairment in severe malaria. To gain further insight into pathogenesis of cerebral complications of malaria infection we studied a different population, African children, in whom the clinical and pathological presentation of severe and complicated malaria differs significantly with South East Asian adults (reviewed in [2, 9]).

We have examined CSF levels of tau and S-100B on admission in children from Kenya with differing levels of neurological severity of malaria. We have investigated the

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Proefschrift.indb 151 14-1-2008 13:09:26 systemic metabolic effects of malarial disease on the levels of tau and S-100B in CSF and compared the findings with those previously reported in the CSF of adult Vietnamese patients with severe malaria.

MATERIALS AND METHODS Case selection and sample collection A random sample of stored CSF from children admitted to Kilifi District Hospital located on coastal Kenya, between 1999 and 2004 was used to measure levels of tau and S-100B. Lumbar puncture was performed when clinically indicated as part of the investigation of children admitted with impaired consciousness prostration and/or seizures, to exclude infectious causes in particular bacterial meningitis[10].

The CSF was collected in plastic tubes and stored –20°C within 1 hour of being obtained and then frozen at –80°C and later transported to the UK on dry ice. All samples were obtained with permission from patients or relatives, and the National Ethical Research Committee of the Kenya Medical Research Institute approved sample collection protocols. CSF samples were used with ethical approval from the Oxford Tropical Research Ethics Committee (OXTREC).

A total of 143 CSF samples from children were studied. Patients were classified into 3 main groups according to the level of consciousness at admission and further subdivided on the basis of whether they had malaria or not:

a) Coma (Blantyre coma score [BCS] ≤2) [11] i. Cerebral malaria (CM): 38 children had a clinical diagnosis of CM (unable to localise a painful stimulus or BCS ≤2 on admission, asexual forms of P. falciparum parasitaemia, no improvement after treatment for hypoglycaemia and no evidence of meningitis in the CSF sample – CSF WBC<10/µl [12] ii. Non-cerebral malaria coma: - 7 children with coma due to other causes (no P. falciparum parasitaemia)

b) Prostration (inability to sit upright or breast feed) [12] but not comatose i. Malaria with prostration - this group was made up of 25 children with P. falciparum parasitaemia admitted either with mild impairment of consciousness (BCS 3 and 4 - able to localise pain) or with full consciousness (BCS 5) but unable to sit upright or breast feed.

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Proefschrift.indb 152 14-1-2008 13:09:26 ii. Non-malaria with prostration – This group included 7 children withoutP. falciparum parasitaemia and admitted with either mild impairment of consciousness or with full consciousness but unable to sit upright or breast feed.

c) Fully alert patients i. Children with malaria and seizures but no coma or prostration – 54 patients ii. Children with febrile seizures but no malaria, coma or prostration – 12 patients

Measurement of tau and S-100B using a solid phase sandwich ELISA Both markers are stable in CSF and do not degrade readily at room temperature or following multiple rounds of freeze-thaw [13, 14]. Total human tau (both phosphorylated and nonphosphorylated) and S-100B were measured using commercially available ELISA kits (BioSource International, Nivelles, Belgium and DAKO, Ely, UK, respectively) using a solid phase sandwich ELISA according to the manufacturer’s instructions. The reported intra-assay precision for tau is 2.7-5.2% and the inter-assay precision is 4.3-7.8%. The lower limit of sensitivity is 12pg/mL. There is no cross reactivity with human β-amyloid 1-42, α-synuclein, or β-synuclein. The reported intra-assay precision for S-100B is 1.3- 2.5% and the inter-assay precision is 1.5-2.2%. The lower limit of sensitivity is ≤10ng/L.

Clinicopathological correlations To investigate the significance of the CSF levels of markers of brain parenchymal damage in severe malaria, we performed a detailed clinicopathological correlation with a number of clinical and biochemical parameters. These included a history, number and duration of seizures, markers of metabolic acidosis, the level of consciousness, haemoglobin, blood glucose, the parasite density and CSF proteins.

STATISTICAL ANALYSES Statistical analysis was carried out using Stata 9 (StataCorp, College Station, TX) programme. Normally distributed continuous variables were compared between groups using unpaired Student’s t-test; data that were not normally distributed were compared using the Kruskall-Wallis test. Correlations between continuous variables were determined nonparametrically using Spearman’s rho. No adjustments for multiple comparisons were made, though for the purposes of interpretation and discussion p < 0.01 was regarded as significant.

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Proefschrift.indb 153 14-1-2008 13:09:26 RESULTS Of the 143 children, 72 were female and 71 male. The ages ranged from 1 - 98 months (median [IQR] 21 [12-36] months). One hundred and seventeen children had malaria and the remaining 26 had other diagnosis. These included 12 children with febrile seizures mainly associated with respiratory tract infections, 9 with suspected viral meningo- encephalitis, 2 with seizures and developmental delay, and a child each with septicaemia, pyogenic meningitis and epilepsy.

Tau and S100B levels in the CSF of Kenyan children admitted to hospital Cerebrospinal fluid Tau levels were available for 138 patients of who 54 (39.1%) had high (>300 pg/ml) levels while S100B levels were available for 142 patients 7 of who had high (>1ng/ml) levels. There was no significant difference between the median CSF levels of tau or S100B in the malaria group compared with the non-malaria group (p=0.87 and p=0.83 respectively). Within the malaria sub-groups, median tau levels were significantly higher in children with cerebral malaria (coma) compared with either malaria with prostration or malaria with seizures but normal consciousness, p<0.001 (Kruskall Wallis test, figure 1). A similar trend was observed with S100B but the differences were not statistically significant (p=0.089) (table I). Among children with non-malarial illnesses, the median CSF levels of tau did not vary with the level of consciousness (table I).

Table 1 Level of consciousness, manifestations of severe disease and markers of cerebral damage in children with and without malaria Clinicopathological Coma Prostration Normal consciousness correlations and markers of cerebral Malaria Non malaria Malaria Non malaria Malaria Non damage malaria N=38 n=7 n=25 n=7 n=54 n=12 Median (IQR) duration of 2 (1-3) 5 (3-7) 3 (2-4.5) 3 (2-5) 3 (2-3) 3 (2-4) illness, days History of seizures, (%) 36 (94.7) 6 (85.7) 19 (76.0) 2 (28.6) 47 (87.0) 12 (100) Median (IQR) number of 2 (1-3) 1 (0-2) 1 (0-3) 0 (0-1) 1 (1-2) 2 (1-2) seizures Deep breathing, (%) 15 (39.5) 3 (42.9) 8 (32.0) 1 (14.3) 1 (1.9) 0 (0) Severe anaemia, (%)* 4 (10.8) 2 (28.6) 7 (28.0) 1 (16.7) 2 (3.7) 0 (0) Hypoglycaemia, (%) 6 (15.8) 1 (14.3) 1 (4.0) 1 (14.3) 13 (24.1) 3 (25.0) Median (IQR) CSF Tau 346.8 218.9 266.1 219.8 132.3 222.3 levels, pg/ml‡ (204.5 - 793.9) (7.4 - 420.3) (159.6 – 494.8) (132.3-1159.3) (70.0-241.6) (68.4 – 676.2) Median (IQR) CSF S100 344.0 310.7 245.8 243.0 226.8 250.7 levels, pg/ml† (184.1-480.8) (266.8-1654.9) (174.0-361.7) (69.0-848.3) (138.9-340.0)(165.2-325.5) *n=140; ‡=138; †=142

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Proefschrift.indb 154 14-1-2008 13:09:27 Relationship between tau and S100 with neurological signs and other complications of faciparum malaria Cerebrospinal fluid levels of tau and S100B were categorized as normal or high and compared with complications of falciparum malaria that have been associated with poor outcomes. Tau was found to correlate with several complications of malarial infection (see Table II). In contrast S100B did not correlate with any of these markers.

Seizures Overall 116 (81%) patients were admitted with a history of seizures. The median (IQR) CSF levels of S100B was not different in patients who presented with and without seizures, (p=0.879). However, patients with high levels of S100B were more likely to have recurrence of seizures during the course of the admission, (5/7 [71.4%] vs 32/135 [23.7%], p=0.005). A similar but weak association was observed with high tau levels; (19/54 (35.2%) patients with high tau levels had seizure recurrence compared to 18/84 [21.4%], p=0.075). The median tau and S100B levels did not correlate with the number of seizures.

Severe anaemia The overall mean (SD) haemoglobin level was 6.7 (2.3) g/dl in patients with high tau and 8.1 (1.8) g/dl in those with normal levels, p<0.001. Thirteen children with malaria and 3 children with non-malarial illnesses had life threatening severe anaemia (haemoglobin levels <5g/dL). There was a strong correlation between levels of tau and severe anaemia:

Table II Levels of tau and features of disease severity Makers of disease severity Malaria P Non – malaria P High Tau Normal tau value High tau Normal tau value N=42 N=71 N=12 N=13 Age (SD) in months 21.8 (18.8) 26.8 (22.8) 0.147 20.9 (15.7) 29.0 (19.7) 0.272 Median (IQR) number of seizures 1 (0-2) 2(1-3) 0.180 1 (0-2) 1 (0-2) 0.870 Multiple seizures (>2 seizures, %) 5 (11.9) 23 (32.4) 0.015 2 (16.7) 1 (7.7) 0.490 Mean (SD) temperature, o C 37.8 (1.1) 38.2 (1.5) 0.104 37.4 (1.2) 37.9 (1.4) 0.416 Mean (SD) respiratory rate, per min 44.2 (13.3) 35.5 (11.7) <0.001 38 (10) 38 (10) 0.986 Deep breathing, (%) 14 (33.3) 8 (11.3) 0.004 2 (16.7) 2 (15.4) 0.930 Hypoglycaemia, (%) 4 (9.5) 16 (22.5) 0.080 1 (8.3) 4 (30.8) 0.161 Mean (SD) haemoglobin, g/L 64 (21) 81 (18) <0.001 77 (28) 84 (20) 0.461 Severe anaemia, (%) 10 (24.4) 2 (2.8) <0.001 3 (25.0) 0 (0) 0.075 Geometric (95% CI) mean parasite 83.3 32.9 0.017 - - - density x 103/ml (45.5.152.3) (20.7-52.3) Mean (SD) CSF glucose, mmol/L 2.7 (1.1) 2.8 (1.6) 0.832 3.3 (1.2) 2.5 (1.6) 0.182 Mean (SD) CSF protein, 0.40 (0.23) 0.20 (0.09) <0.001 0.29 (0.14) 0.74 (1.37) 0.266 Neurological deficits, (%) † 2 (4.8) 3 (4.2) 0.893 0 (0) 2 (15.4) 0.157 † Includes 3 children who had neurological deficits before admission and 4 with new onset neurological deficit

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Proefschrift.indb 155 14-1-2008 13:09:27 the median tau level in the cases with severe anaemia was 1135.8 (IQR 424.1 – 4416pg/ ml) compared to 192.8 (92.5 – 482.8 pg/ml in patients without severe anaemia. The association between high levels of tau and severe malaria anaemia (adjusted OR 9.4 [95%CI 1.9 – 50]) was independent of the presence of severe acidosis (deep breathing, adjusted OR 3.0 [95%CI 1.1 – 8.6]). The effect of severe anaemia on tau levels was also not limited to children with malaria since median levels in children with malaria (1085.3 [441.2-2901.9] pg/ml) and that in children with other non-malarial illnesses (1159.3 [383.7-3430.0] pg/ml) were similar.

Metabolic acidosis Respiratory distress, in particular deep breathing, is a feature of severe metabolic acidosis in African children with severe falciparum malaria [15, 16]. There was a strong correlation between CSF tau level and respiratory rate (p=0.0002, r=0.3) and with deep breathing. The median (IQR) of tau in children with malaria and deep breathing was 499.3 (153.2-1250.7) pg/ml compared to 190.2 (94.6-472.4) pg/ml in those without deep breathing (p=0.003). This relationship was not observed with S100B.

Level of consciousness Among children with malaria, the CSF levels of tau increased with decreasing level of consciousness (figure 1). Coma was particularly associated with high levels of CSF tau; 20/37 (54.1.6%) children with coma had high levels of tau compared to 22/76 (29.0%) of those who presented without coma, (p=0.01). This relationship was not observed with S100B (p=0.983).

Figure 1: Cerebrospinal fluid levels of tau and level of consciousness in children with malaria

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Proefschrift.indb 156 14-1-2008 13:09:27 In addition, coma was significantly associated with deep breathing, (39.5 vs 11.4%, p<0.001) but not severe anaemia (p=0.926). On logistic regression analysis (stepwise entry), the association between level of consciousness and high levels of tau (adjusted OR [95%CI], 2.4[1.1 – 5.3], p=0.026), was independent of deep breathing (adjusted OR [95%CI], 29.9 [3.9 – 230], p=0.001).

Hypoglycaemia Unlike patients with severe anaemia or severe metabolic acidosis, children presenting with hypoglycaemia (blood glucose <2.2 mmol/L) had lower median CSF tau levels compared to those who presented without hypoglycaemia (120.9 vs 217.2 pg/ml, p=0.005). Their median S100B level was also lower (154.5 vs 283.8 pg/ml, p=0.001).

Parasite density The geometric mean parasite density in children with high levels of tau was about two and half times that in patients with normal levels (table II). A similar association was not observed between parasite density and high S100B levels.

Outcome One child died and apart from the 3 children with neurological deficits on admission (one with epilepsy and two with delayed development), 4 other children had neurological deficits at discharge. This included one with continuing epileptic seizures discharged on phenytoin, a child with quadriparesis and 2 with generalised hypotonia. Three of the four children with deficits had malaria. The child who died had coma and a severe lower respiratory tract infection. She had a very high tau (2554 pg/ml) level. The median [IQR] CSF levels of tau in the 3 chidren with malaria who had neurological sequelae at discharge was higher (1010.4 [126.2-3580.6] pg/ml) compared to those discharged well (209.0 [109.0-539.4] pg/ml) although this difference was not statistically significant (p=0.197). Similarly, although the median levels of S100B in children with deficits were higher, the differences were not significant, (p=0.507).

DISCUSSION This study set out to examine the CSF levels of the proteins tau and S-100B, biomarkers of cerebral damage, to help understand the pathogenesis of neuronal involvement and damage in children with severe falciparum malaria. We found that CSF levels of tau inversely correlated with the level of consciousness and other complications of malaria infection such as severe anaemia and acidosis but not seizures. In contrast, children admitted with high levels of S-100B had an increased risk of repeated seizures.

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Proefschrift.indb 157 14-1-2008 13:09:27 There is a hierarchy of susceptibility of brain parenchymal elements to neurological insult with axons and astrocytes at either end of the spectrum. Axons are the most vulnerable because they often extend for great distances from their cell bodies of origin, and may possibly depend on local production of ATP to maintain ion gradients and sustain energy-consuming functions. They are therefore susceptible to ischaemic or toxic damage in several different vascular territories, which is not the case for neuronal cell bodies or glia. It was therefore not unexpected that we have found more significant elevations and clinical correlation with tau compared with S-100B. The differences in the findings between both markers may also reflect the timing of release of tau and S- 100B following injury. This can only be confirmed through the measurement of these biomarkers in serial CSF samples, a procedure that would be unethical in this group of patients.

The findings of this study suggest that in children with severe falciparum malaria, impaired consciousness and coma are associated with axonal injury. This is consistent with previous work showing that axonal damage is associated with clinically reversible coma or coma followed by a fatal outcome in both trauma and Vietnamese adults with severe malaria (reviewed in [17]). Post-mortem analysis of brain tissue from Vietnamese adults showed that frequency and extent of axonal injury was more severe in patients with cerebral malaria than in those with no cerebral involvement [18]. However, a CSF study showed that injury resulting in degeneration of axons did not correlate with coma depth but, rather, coma duration [19]. In the present study, elevated levels of CSF tau were associated with depth of coma in children with malaria. Multiple regression analysis was performed to determine if the association between tau and coma was due to systemic complications. Tau remained a significant independent predictor of coma. Median tau levels in children with cerebral malaria were 3-fold greater compared with our previously reported findings in adults although median tau levels do not differ between children and adults when all malaria patients are considered in the analysis (see [8]). These findings imply that axons are more susceptible to damage during malaria coma in children. Previous studies have also detected differences in the neurophysiological milieu of the CSF of African children and Vietnamese adults with severe malaria suggesting intrinsic age-related differences in the ability to regulate brain function in response to a pathological insult [20]. High quality magnetic resonance may provide further insights into the extent and distribution of axonal injury.

Systemic complications of severe malaria in adult South East Asian patients, such as severe anaemia, acidosis and renal failure[21], are recognised in paediatric African patients[22-24]. Whether systemic disease may cause or exacerbate damage to the brain, was investigated in this study. Axonal damage was not significantly associated

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Proefschrift.indb 158 14-1-2008 13:09:27 with seizures or hypoglycaemia, but tau was significantly elevated in patients with impaired delivery of oxygen (severe anaemia), those with severe metabolic acidosis manifesting as respiratory distress and at higher parasite densities. These systemic complications can alter cerebral blood flow, oxygen extraction or metabolite levels. Clearly, metabolic derangements such as acidosis could have a profound impact on the internal milieu of axons that are particularly susceptible to pH and oxygen stress. Although similar correlations have been found in adults with severe malaria[8, 18], this is the first finding of axonal injury in association with severe malaria anaemia. The brain is vulnerable to anaemia-induced injury (reviewed in [25]). Associations between neurological sequelae and severe malarial anaemia in children have been reported[26]. When compensatory mechanisms directed at maintaining cerebral oxygen delivery have become overwhelmed, hypoxia ensues. Markedly high levels of tau (>1000pg/ml) like those reported in children with progressive and lysosomal disease with abnormal white matter[27] were observed in children with severe anaemia and in the small group of patients with a bad outcome.

No clinical or biochemical parameter analysed correlated with S-100B levels so the mechanism for the elevation in the CSF of malaria patients is unclear. In our postmortem study of adults with severe malaria, the presence of astrocyte degeneration, clasmatodendrosis, did not correlate with coma, but there was a significant correlation with intracerebral haemorrhage[18]. Petechial haemorrhages are a pathological feature of severe malaria and in other clinical settings, intracerebral haemorrhage are associated with epileptic seizures in children[28]. Irrespective of the inducing factor there are numerous lines of evidence that implicate a role for astrocytes and S-100B with seizure activityThis data suggests that seizures after admission are a manifestation of brain damage, rather than a cause of brain damage. In severe falciparum malaria, multiple seizures are associated with increased mortality and neurological deficits[11, 29, 30]. The small number of children with a fatal outcome (n=1) and neurological deficits (n=4) in this study does not allow us to determine the potential usefulness of this marker as an indicator of poor neurological outcome.

CONCLUSIONS Our study provides evidence to support the view that several mechanisms may be involved in the causation of brain parenchymal damage in children with malarial infection. Axonal injury is associated with coma, hyperparasitaemia and metabolic disturbances both in paediatric and adult populations from different geographical regions. In addition, anaemia-induced impairment of oxygen delivery also plays a role in brain damage in children with malaria. Levels of CSF tau are greater in children

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Proefschrift.indb 159 14-1-2008 13:09:27 with coma compared with previous findings in adults and elevated levels of S-100B in children are associated with an increased risk of repeated seizures. Whether axonal and astrocyte dysfunction or degeneration are the pathological correlates of neurological sequelae and multiple seizures in children with malaria should be investigated further.

ACKNOWLEDGEMENTS This work was supported by grants from the Wellcome Trust (Advanced Training Fellowship in Tropical Medicine to Isabelle Medana, Grant number 67860). Prof C Newton is a Wellcome Trust Senior Fellow in Clinical Science (070114). This study is published with the permission of the Director of KEMRI.

REFERENCES [1] Snow RW, Guerra CA, Noor AM, Myint HY, Hay SI. The global distribution of clinical episodes of Plasmodium falciparum malaria.Nature, 2005; 434:214-7. [2] Idro R, Jenkins NE, Newton CR. Pathogenesis, clinical features, and neurological outcome of cerebral malaria. Lancet Neurol. 2005;4:827-40. [3] Newton CR, Krishna S. Severe falciparum malaria in children: current understanding of pathophysiology and supportive treatment. Pharmacol Ther. 1998;79:1-53. [4] Carter JA, Ross AJ, Neville BG, Obiero E, Katana K, Mung’ala-Odera V, et al. Developmental impairments following severe falciparum malaria in children. Trop Med Int Health. 2005;10:3-10. [5] Teunissen CE, Dijkstra C, Polman C. Biological markers in CSF and blood for axonal degeneration in multiple sclerosis. Lancet Neurol. 2005;4:32-41. [6] Martens P, Raabe A, Johnsson P. Serum S-100 and neuron-specific enolase for prediction of regaining consciousness after global cerebral ischemia. Stroke. 1998;29:2363-6. [7] Sussmuth SD, Reiber H, Tumani H. Tau protein in cerebrospinal fluid (CSF): a blood-CSF barrier related evaluation in patients with various neurological diseases. Neurosci Lett. 2001;300:95-8. [8] Medana IM, Lindert RB, Wurster U, Hien TT, Day NP, Phu NH, et al. Cerebrospinal fluid levels of markers of brain parenchymal damage in Vietnamese adults with severe malaria. Trans R Soc Trop Med Hyg. 2005;99:610-7. [9] Newton CR, Warrell DA. Neurological manifestations of falciparum malaria. Ann Neurol. 1998;43:695-702. [10] Berkley JA, Mwangi I, Ngetsa CJ, Mwarumba S, Lowe BS, Marsh K, et al. Diagnosis of acute bacterial meningitis in children at a district hospital in sub-Saharan Africa. Lancet. 2001;357:1753-7. [11] Molyneux ME, Taylor TE, Wirima JJ, Borgstein A. Clinical features and prognostic indicators in paediatric cerebral malaria: a study of 131 comatose Malawian children. Q J Med. 1989;71:441-59. [12] World Health Organization CDC. Severe falciparum malaria. . Trans R Soc Trop Med Hyg. 2000;94 Suppl 1:S1-90. [13] Aurell A, Rosengren LE, Wikkelso C, Nordberg G, Haglid KG. The S-100 protein in cerebrospinal fluid: a simple ELISA method. J Neurol Sci. 1989;89:157-64.

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Proefschrift.indb 160 14-1-2008 13:09:27 [14] Schoonenboom NS, Mulder C, Vanderstichele H, Van Elk EJ, Kok A, Van Kamp GJ, et al. Effects of processing and storage conditions on amyloid beta (1-42) and tau concentrations in cerebrospinal fluid: implications for use in clinical practice. Clin Chem. 2005;51:189-95. [15] English M, Waruiru C, Amukoye E, Murphy S, Crawley J, Mwangi I, et al. Deep breathing in children with severe malaria: indicator of metabolic acidosis and poor outcome. Am J Trop Med Hyg. 1996;55:521-4. [16] Marsh K, Forster D, Waruiru C, Mwangi I, Winstanley M, Marsh V, et al. Indicators of life- threatening malaria in African children. N Engl J Med. 1995;332:1399-404. [17] Medana IM, Esiri MM. Axonal damage: a key predictor of outcome in human CNS diseases. Brain. 2003;126:515-30. [18] Medana IM, Day NP, Hien TT, Mai NT, Bethell D, Phu NH, et al. Axonal injury in cerebral malaria. Am J Pathol. 2002;160:655-66. [19] Medana IM, Lindert RB, Wurster U, Hien TT, Day NP, Phu NH, et al. Cerebrospinal fluid levels of markers of brain parenchymal damage in Vietnamese adults with severe malaria. Trans R Soc Trop Med Hyg. 2005;99:610-7. [20] Medana IM, Day NP, Salahifar-Sabet H, Stocker R, Smythe G, Bwanaisa L, et al. Metabolites of the kynurenine pathway of tryptophan metabolism in the cerebrospinal fluid of Malawian children with malaria. J Infect Dis. 2003;188:844-9. [21] Day NP, Phu NH, Mai NT, Chau TT, Loc PP, Chuong LV, et al. The pathophysiologic and prognostic significance of acidosis in severe adult malaria. Crit Care Med. 2000;28:1833-40. [22] Mackintosh CL, Beeson JG, Marsh K. Clinical features and pathogenesis of severe malaria. Trends Parasitol. 2004;20:597-603. [23] Maitland K, Bejon P, Newton CR. Malaria. Curr Opin Infect Dis. 2003;16:389-95. [24] Olowu WA, Adelusola KA. Pediatric acute renal failure in southwestern Nigeria. Kidney Int. 2004;66:1541-8. [25] Hare GM. Anaemia and the brain. Curr Opin Anaesthesiol. 2004;17:363-9. [26] Brewster DR, Kwiatkowski D, White NJ. Neurological sequelae of cerebral malaria in children. Lancet. 1990;336:1039-43. [27] Kristjansdottir R, Uvebrant P, Lekman A, Mansson JE. Cerebrospinal fluid markers in children with cerebral white matter abnormalities. Neuropediatrics. 2001;32:176-82. [28] Masuhr F, Busch M, Amberger N, Ortwein H, Weih M, Neumann K, et al. Risk and predictors of early epileptic seizures in acute cerebral venous and sinus thrombosis. Eur J Neurol. 2006;13:852-6. [29] Idro R, Karamagi C, Tumwine J. Immediate outcome and prognostic factors for cerebral malaria among children admitted to Mulago Hospital, Uganda. Ann Trop Paediatr. 2004;24:17-24. [30] van Hensbroek MB, Palmer A, Jaffar S, Schneider G, Kwiatkowski D. Residual neurologic sequelae after childhood cerebral malaria. J Pediatr. 1997;131:125-9.

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Proefschrift.indb 161 14-1-2008 13:09:27 Proefschrift.indb 162 14-1-2008 13:09:27 Chapter10

High plasma levels of erythropoietin are associated with protection against neurological sequelae in african children with cerebral malaria

Climent Casals-Pascual, Richard Idro, Nimmo Gicheru, Samson Gwer, Barnes Kitsao, Evelyn Gitau, Robert Mwakesi, David J Roberts, Charles RJC Newton

Nuffield Department of Clinical Laboratory Sciences and National Blood Service Oxford Centre, Oxford, UK; Medical Research Council Laboratories, Fajara, PO Box 273, The Gambia; Centre for Geographical Medicine (Coast), Kenya Medical Research Institute, Kilifi, Kenya: Department of Paediatrics, Mulago Hospital/Makere University, Kampala, Uganda; Neurosciences Unit, The Wolfson Centre, University College London, Institute of Child Health, UK

Submitted

Proefschrift.indb 163 14-1-2008 13:09:27 ABSTRACT Background Cerebral malaria in children is associated with a high mortality and long-term neurological and cognitive sequelae. Both erythropoietin and vascular endothelial growth factor (VEGF) have been shown to be neuroprotective. We hypothesised that high plasma and cerebrospinal fluid (CSF) levels of these cytokines would prevent neurological sequelae in children with cerebral malaria.

Methods We measured erythropoietin, VEGF and tumour necrosis factor (TNF) in paired samples of plasma and CSF of Kenyan children admitted with cerebral malaria. Logistic regression models were used to identify risk and protective factors associated with the development of neurological sequelae.

Results Children with cerebral malaria (N=124) were categorized into 3 groups: 76 without sequelae, 32 with sequelae and 16 who died. The median (IQR) plasma concentrations of erythropoietin were 278 (96-1,852) U/L, 184 (23-694) U/L and 123 (29-1,726) U/L, respectively. Conditional logistic regression analysis matching the 32 patients with cerebral malaria and neurological sequelae to 64 patients with cerebral malaria without sequelae stratified for haemoglobin level estimated that plasma erythropoietin (>200 U/ L) was associated with greater than 80% reduction in the risk of developing neurological sequelae (adjusted OR 0.18, 95%CI 0.05-0.93, P=0.041). Both the level of erythropoietin in relation to haemoglobin level and the protective effect of erythropoietin for a given erythropoietin concentration were greater in younger children. Admission with profound coma (adjusted OR 5.47, 95%CI 1.45-20.67, P=0.012) and convulsions after admission (adjusted OR 16.35, 95%CI 2.94-90.79, P=0.001) were also independently associated with neurological sequelae. Plasma VEGF and TNF were not associated with sequelae.

Conclusions High levels of erythropoietin were associated with reduced risk of neurological sequelae in children with CM. The age-dependent erythropoietin response to anaemia and the age-dependent protective effect may influence the clinical epidemiology of CM. These data support further study of erythropoietin as an adjuvant therapy in CM.

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Proefschrift.indb 164 14-1-2008 13:09:28 INTRODUCTION Cerebral malaria is the most severe neurological complication of infection with Plasmodium falciparum. Even with appropriate anti-malarial treatment, 18.6% of children with CM die and 11% have neurological deficits detected on discharge1and 24% children have neurological and cognitive impairments 2,3 or epilepsy 4,5 when assessed many years later.

Over a wide range of endemic areas, severe malarial anaemia is the most common manifestation of severe malaria in younger children whereas the peak incidence of cerebral malaria occurs in the fourth year of life.6 The pathogenesis of cerebral malaria is not completely understood and the factors involved in the development of neurological sequelae remain unclear. A number of studies have consistently identified deep and prolonged coma, recurrent seizures and hypoglycaemia as independent risk factors associated with the development of neurological sequelae (reviewed in7). Protective factors are less well defined. Low levels of haemoglobin (Hb) were associated with neurological sequelae in the Gambia,8 but not in other African studies.9,10

We have hypothesized that the outcome of cerebral malaria is modified by the cytokine response to hypoxia. Erythropoietin, principally produced in the kidney in response to hypoxia, is crucial for sustained proliferation and differentiation of erythroid cells.11,12, 13 However, erythropoietin and erythropoietin receptor are also expressed in neurons and astrocytes.14,12 Recombinant human erythropoietin (rhEpo) is protective in animal models of brain injury 13,14 and reduces vasoconstriction and neuronal apoptosis.15-17 Indeed, preclinical studies in patients with stroke have supported a neuroprotective role for erythropoietin.18 High levels of erythropoietin have been found in African children with malaria anaemia.19-21 Furthermore, the administration of rhEpo in a murine model of cerebral malaria reduced mortality by 90 %.22

Vascular endothelial Growth Factor (VEGF) is also upregulated by hypoxia23 and is both neurotrophic and neuroprotective.24 It improves functional outcome in cerebral ischaemia in rats, reducing motor and cognitive defects.25 However, VEGF can also induce intercellular adhesion molecule-1 (ICAM-1) and macrophage inflammatory protein 1α (MIP1α) in endothelial and brain parenchymal cells26 and increase the permeability of the brain-blood barrier (BBB).27,28 Other studies show the levels of the pro-inflammatory cytokine, TNF, increase during acute stroke, 29 and in an animal models, intraventricular administration of TNF- enlarges infarct volume.30 In children with malaria, high TNF levels have been associated with poor outcome.31,32

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Proefschrift.indb 165 14-1-2008 13:09:28 We hypothesised that high levels of erythropoietin and VEGF protect children with cerebral malaria from developing neurological sequelae or death. We studied a well- defined group of children admitted with cerebral malaria who were assessed for neurological damage during admission and on discharge from hospital. We compared the levels of erythropoietin, VEGF and TNF in children who died and in those who survived with and without neurological deficits.

METHODS Study design The study was conducted in children admitted to Kilifi District Hospital (KDH), in which all children are seen by research clinicians from the Kenya Medical Research Institute /Wellcome Trust collaborative programme. This was a retrospective study of children admitted with cerebral malaria classified by outcome: children who survived without neurological deficits, children who survived with neurological deficits and children who died.

Study participants were identified from the hospital database admitted between 1999 and 2005. Cerebral malaria was defined as admission to hospital with coma (unable to localise a painful stimulus or Blantyre coma score [BCS] <2,9 at least 1 hour after termination of a seizure if present or correction of hypoglycaemia), with asexual forms of Plasmodium falciparum malaria parasites on a Giemsa stained blood smears, and no evidence of pyogenic meningitis on examination of the CSF.33 All patients had a detailed neurological examination on admission which included a fundoscopic examination of the retina for features of raised intracranial pressure. Lumbar punctures were performed to exclude pyogenic meningitis when the level of consciousness improved (BCS >2) or if the child did not have brainstem signs (within 48 hours of admission in almost all cases).33,34 A portion of the CSF was stored at -20oC and later frozen at -80oC together with the corresponding specimen of plasma obtained at the time of the lumbar puncture. Patients with epilepsy, cerebral palsy, sickle cell disease, those without stored paired samples or samples collected after a blood transfusion were excluded. The scientific and ethical committees of Kenya Medical Research Institute approved the study.

Procedures All patients had been treated with standard protocols developed for the management of severe falciparum malaria 35 and according to the WHO.36 All children were assessed for neurological sequelae at discharge from hospital. Neurological sequelae were classified as motor sequelae (cranial nerve palsies, spasticity and hypotonia), ataxia, movement

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Proefschrift.indb 166 14-1-2008 13:09:28 disorders (tremors, dystonia and choreoathetoid movements), speech (speech difficulties or aphasia), visual (blindness) and/or hearing impairments, epileptic seizures, and behavioural abnormalities (aggressive behaviour and hyperactivity).10

Laboratory procedures Plasma and CSF levels of erythropoietin, VEGF and TNF were measured by ELISA following the manufacturer’s instructions (R&D Systems, Abingdon, UK). The lower limit of detection for erythropoietin was 2.5 UI/L, VEGF was 15.6 pg/mL and TNF was 15.6 pg/mL. Ten percent of samples were tested in duplicate (the correlation of values was always r >0.90). Values above the standard detection range were diluted and re- assayed.

STATISTICAL ANALYSIS Data were analysed using STATA 9 (Stata Corporation, Texas, USA). Binary logistic regression analysis was used to identify risk/protective factors independently associated with the development of neurological sequelae in children with cerebral malaria. Goodness-of-fit was assessed by the Hosmer-Lemeshow test. Variables detected from previous studies in the unit and elsewhere,10,37 known to be associated with the dependent variable (neurological sequelae) were included in the analysis, namely, hypoglycaemia, depth of coma and seizures. The independent variables were checked for interaction. Conditional logistic regression was used to match 32 children with neurological sequelae to 64 children with good outcome. The subgroup of children not used in the matching (N=12) had similar age and Hb concentrations to those used in the regression. Matching was by age (within 18 months) and Hb level (within groups Hb<5, 5.1 to 7, 7.1 to 9 and >9.1 g/dL). Matching criteria were based on biologically meaningful cut-off points, but also on a value range that allowed inclusion of all children with sequelae. Ordinal logistic regression was used to identify risk factors associated with outcome (no sequelae at discharge, neurological sequelae and death). For ordinal logistic regression analysis deviance (chi-square test) was used to assess goodness-of-fit.

RESULTS Four hundred and twenty six children were admitted to Kilifi District Hospital with cerebral malaria from January 1999 through December 2001. Nine had incomplete admission data and were excluded. Of the remaining 417, paired plasma and CSF samples were available for 171 cases. Out of those, 65 received a blood transfusion. In 55 cases the samples had been obtained after a blood transfusion and were excluded from the study.

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Proefschrift.indb 167 14-1-2008 13:09:28 In addition, 8 other children were identified from among admissions of 2002 to 2005. Thus we included in this study 124 children with paired CSF and plasma samples. Table 1 is a description of the children by outcome.

Table 1: Description of the study population Patient characteristics Good outcome Neurological Dead (N= 76) sequelae (N=16) (N=32) Age (months), median (IQR) 27 (14.5-36.5) 28 (21-46) 36.5 (27-46) Gender, male N (%) 45 (59.2) 14 (43.7) 9 (56.2) Weight for age Z-score, mean (sd) -1.7 (1.1) - 2 (1.02) -1.7 (1.6) Fever, n (%) 69 (90.7) 29 (90.6) 15 (93.7)

Seizures before admission, n (%) 71 (93.4) 29 (90.6) 12 (75) Seizures during admission, n (%) 37 (48.6) 26 (81.2) 12 (75) Profound coma, BCS = 0, n (%) 14 (18.4) 13 (40.6) 6 (66.6) Abnormal motor posturing during admission, n (%) 21 (27.6) 17 (53.1) 8 (50) Features of raised intracranial pressure on 4 (7.8) 8 (34.7) 3 (27.2) fundoscopy a, n (%) Coma duration (hours), median (IQR) 8 (4-23) 78 (38-125) 17.5 (7.5-36.5)

Deep (acidotic) breathing, n (%) 15 (19.7) 8 (25) 12 (75) Hypoglycaemia, n (%) 8 (10.5) 6 (18.7) 5 (31.2) Parasite density / μL ,geometric mean, 42,328 28,439 60,495 (95% CI of mean) (25,870-69,256) (11,785-68,627) (16,207-225,801)

Hb (g/dL) , mean (sd) 8.0 (1.8) 8.6 (2.0) 8.3 (1.9) Severe anaemia (haemoglobin < 50 g/L), n (%) 3 (3.9) 2 (6.2) 0 (0) Transfused, n (%) 2 (3.8) 3 (6.4) 5 (31.2) Platelets (103μl-1), median (IQR) 101 (58-195) 192 (76-284) 93 (63-219)

Plasma Epo (mU/mL), median (IQR) 278.6 (96.7-1,852) 184.2 (23.9-694.4) 123.5 (29.5- 1,726.2) Plasma VEGF (ng/mL), median (IQR) 39.6 (23.2-77.2) 53.1 (25.9-118.1) 42.9 (18.1-122.7) Plasma TNF (pg/mL), median (IQR) 60.6 (24.8-137.4) 80.6 (13.9-187.80 92.1 (63.0-487.8)

CSF Epo (mU/mL), geom. mean (95%CI) 14.3(3.8-52.6) 14.3 (1.2-165.2) 29.8 (1.6-534.9) CSF VEGF (ng/mL), geom. mean (95%CI) 42.9 (21.1-87.2) 59.1(16.1-216.8) 37.7 (2.3-608.9) CSF TNF (pg/mL), geom. mean (95%CI) 361.3 (58.3-2,236.9) 199.8 (2.8-1,4165.6) 470.6* aN=85 (N=51, N=23, N=11, respectively) *TNF was detectable only in one case, hence the 95%CI of the geometric mean cannot be calculated.

The median age of the children was 28.5 (IQR 16-40) months and the mean (sd) haemoglobin concentration was 8.2 (1.91) g/dL. Fifteen out of 32 (46%) children discharged with neurological deficits had multiple neurological sequelae. The major sequelae were visual impairment (n=8), impairment of speech (n=14), motor impairment (paresis, hemiplegia, hemiparesis, quadriparesis and monoparesis) (n=9).

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Proefschrift.indb 168 14-1-2008 13:09:28 Figure 1 The variation of odds ratio for neurological sequelae with concentrations of plasma erythropoietin.

Odds ratios represent values after adjusting for hypoglycaemia, depth of coma, seizures during admission and age in a multivariate conditional logistic regression analysis. * P <0.05 # P 0.05-0.08

Erythropoietin levels and development of neurological sequelae To test our hypothesis that high erythropoietin levels were associated with protection from neurological sequelae in children with cerebral malaria, we first examined different concentrations of erythropoietin (ranging from 100 U/L to 2000 U/L) associated with development of neurological sequelae adjusting for confounders, namely hypoglycaemia, seizures during admission and depth of coma. A protective effect (odds ratio from 0.15 to 0.43) was found for erythropoietin concentrations ranging from 200 to 1000 U/L. We chose 200 U/L as a cut-off as this was the lowest value of erythropoietin associated with a significant protective effect (figure 1).

Logistic regression analysis identified plasma erythropoietin concentration (OR 0.28, 95%CI 0.09-0.79), depth of coma (OR 7.74, 95%CI 2.50-23.98) and seizures during admission (OR 3.42, 95%CI 1.11-10.48) as the main factors independently associated with development of neurological sequelae (table 2). The median level of erythropoietin in plasma was lower in patients with seizures during admission (204, IQR 39.8–1655U/L) compared to patients without recurrence of seizures (258, IQR 83.6–1779 U/L).

Age was associated with erythropoietin concentration (r = - 0.18, P=0.04) and BCS on admission (r = -0.22, P=0.01). We therefore performed the same analyses in a conditional logistic regression model matching by age. This analysis again identified plasma erythropoietin (OR 0.21, 95%CI 0.05-0.86), seizures (OR 6.9, 95%CI 1.37-34.92) and depth of coma (OR 18.6, 95% 2.91-118.8) as variables independently associated with neurological sequelae (table 2). The risk of developing sequelae given a certain concentration of erythropoietin was lower in the age-matched compared with the unadjusted model suggesting an age-dependent dose effect. Indeed, the odds ratio for neurological sequelae at different concentrations

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Proefschrift.indb 169 14-1-2008 13:09:28 Table 2. Factors associated with the development of neurological sequelae in Kenyan children with CM. Unmatched* Matched Agea Hb levelb OR P 95% CI OR P 95% CI OR P 95% CI EPO (>200 U/L) 0.28 0.01 0.09 0.79 0.21 0.030 0.05 0.86 0.18 0.041 0.03 0.93 Hypoglycaemia 3.11 0.11 0.76 12.71 4.51 0.152 0.57 35.65 9.27 0.057 0.93 91.73 Seizures 3.42 0.03 1.11 10.48 6.91 0.019 1.37 34.92 16.35 0.001 2.94 90.79 Depth of coma 7.74 0.00 2.50 23.98 18.6 0.002 2.91 118.80 5.47 0.012 1.45 20.67 Logistic regression analyses displays the odds ratio of high levels of plasma erythropoietin on the development of neurological sequelae (dependent variable) controlling for the effects of potentially confounding variables: depth of coma (BCS <2), seizures during admission and hypoglycaemia. To adjust for age and anaemia, a conditional (fixed-effects) logistic regression model was used to match each case of cerebral malaria with neurological sequelae and two controls (children with cerebral malaria and no neurological sequelae at discharge) by age and anaemia. Hosmer-Lemeshow = 5.54, P=0.476 a Cases of cerebral malaria with neurological sequelae (N=32) are matched by age (within 18 months) to 2 cases of CM discharged without neurological sequelae (N=64). b Cases of cerebral malaria with neurological sequelae (N=32) are matched by the Hb level (within groups Hb < 5, 5.1 to 7, 7.1 to 9 and >9.1 g/dL) to 2 cases of CM discharged without neurological sequelae (N=64). EPO=Erythropoietin.

of plasma erythropoietin, after adjusting for hypoglycaemia, depth of coma, seizures during admission and age in a multiple logistic regression analysis, was much lower in children under 2 years of age at concentrations of erythropoietin >500 U/L (figure 2).

Plasma erythropoietin was negatively associated with Hb concentrations (r = - 0.59, P< 0.001). We introduced Hb level as an ordinal independent variable (categorised as Hb<5, 5-7, 7-9 and >9 g/dL) in the logistic regression model to predict sequelae and found that children with Hb concentrations between 5 and 7 g/dL were associated with a significant reduction in the risk of developing neurological sequelae (OR 0.17, 95%CI 0.03-0.87). The median erythropoietin concentration for this subgroup was 2,097 (IQR 287-3,693). We therefore matched each case of cerebral with neurological sequelae and two cerebral malaria controls within the same Hb range. Plasma erythropoietin (OR 0.21, 95%CI 0.05-

Figure 2 Effect of age on the odds ration for neurological sequelae at different concentrations of plasma erythropoietin.

Odds ratio represents values after adjusting for hypoglycaemia, depth of coma, seizures during admission and age in a multiple logistic regression analysis using different cut-off values for age. * P <0.05 # P 0.05-0.08 (P values are the significance of the OR in the logistic regression analyses for erythropoietin>500 and erythropoietin>1,000 U/L).

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Proefschrift.indb 170 14-1-2008 13:09:28 0.86), seizures (OR 6.9, 95%CI 1.37-34.92) and depth of coma (OR 18.6, 95% 2.91-118.8) remained as variables independently associated with neurological sequelae (table 2).

Erythropoietin concentrations were also measured in CSF from the same patients. Levels of plasma erythropoietin correlated with erythropoietin levels in CSF (r = 0.40, P < 0.001) in children discharged without neurological sequelae (n = 76) but not in those who developed neurological sequelae (n = 32) (r = 0.21, P = 0.23). CSF erythropoietin levels were not associated with neurological sequelae.

Effect of plasma erythropoietin on mortality in cerebral malaria We performed an ordinal logistic regression to measure the impact of erythropoietin on poor outcome (death or neurological sequelae). In this model, mortality and discharge with and without sequelae were used as the dependent (ordinal) variable. In the subgroup of children with cerebral malaria who died (N=16) the median plasma concentration of erythropoietin was 124 (IQR 30-1,726) U/L. Blood transfusion, deep (acidotic) breathing and hyperparasitaemia were also more frequent in this group (Table 1). In addition to the independent variables used to identify risk factors for neurological sequelae, in this model we also adjusted for deep breathing and hyperparasitaemia as these factors may be associated with an increased fatality rate in severe malaria. Here, the logistic regression model identified the following variables to be independently associated with poor outcome: seizures during admission (OR 6.77, 95%CI 2.18-20.98), depth of coma (OR 5.65, 95%CI 2.24-14.21), deep breathing (OR 6.09, 95%CI 2.08-17.79) and hyperparasitaemia (OR 5.19, 95%CI 1.26-21.34). In this model, plasma erythropoietin was also independently associated with a better outcome (OR 0.21, 95%CI 0.08-0.54).

Effect of VEGF and TNF-α on neurological sequelae VEGF is both neuroprotective and pro-inflammatory in the brain. Plasma VEGF (>100 ng/mL) was not associated with development of neurological sequelae but was strongly associated with seizures during admission (OR 4.1, 95%CI 1.75-9.60). Similarly, plasma VEGF (>100 ng/ mL) was associated with a 4.5 fold increase in the risk signs of raised intracranial pressure by fundoscopy (95%CI 1.41-14.74) and a 12.1 fold increase in the risk of finding papilloedema (95%CI 1.84-79.37). VEGF concentrations in plasma were correlated with plasma TNF (r= 0.23, P<0.001) and inversely correlated with plasma erythropoietin (r = -0.17, P = 0.051). However, plasma VEGF was associated with higher concentrations of erythropoietin in CSF (r = 0.24, P = 0.007) and TNF in CSF (r = 0.38, P <0.001).

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Proefschrift.indb 171 14-1-2008 13:09:29 We measured the TNF in plasma and CSF to assess the role of inflammation in relation to the outcome of children with cerebral malaria. Plasma levels of TNF were higher in children with neurological sequelae compared with those healthy at discharge and were correlated with TNF concentration in CSF (r = 0.35, P < 0.001). High concentrations of plasma TNF (>100 pg/mL) and detectable TNF in CSF were associated with a 2.7- fold (95% CI 0.9-9.6) and a 3.4-fold (95% CI 0.72-16.0) increase in the risk of neurological sequelae, respectively.

DISCUSSION This study was designed to seek evidence for the neuroprotective role of erythropoietin and VEGF in children with cerebral malaria. Here, we report a strong association between high concentrations of plasma erythropoietin and a reduced risk of neurological sequelae in Kenyan children with cerebral malaria, and the association of VEGF with seizures and signs of raised intracranial pressure but not neuroprotection.

The potential neuroprotective mechanism(s) of erythropoietin and VEGF in response to hypoxia are related to their neurotrophic and pro-angiogenic activities.38 In the last decade a number of experimental and pre-clinical studies have investigated the tissue protective activities of erythropoietin (reviewed in 39) and VEGF (reviewed in 24).

In this study we have found that high erythropoietin levels in plasma are associated with a 70% reduction of the risk of being discharged with neurological sequelae, and 79% and 82% reduction when the analyses are matched by age or level of Hb, respectively. Erythropoietin is thought to prevent neuronal apoptosis16 and to down-regulate the inflammatory response in the brain40 which may explain differences in the clinical presentation and outcome in cerebral malaria. Neuronal apoptosis has been recently reported in a mouse model with experimental cerebral malaria.41 However, a recent trial in a similar model suggested that protection is due to the anti-inflammatory (rather than the anti-apoptotic) effect of erythropoietin.22

The neuroprotective activities of erythropoietin are time and dose-dependent. In a rodent model of stroke, neurons within the ischaemic penumbra undergo apoptosis unless exposed to erythropoietin within 3 hours.13 In our study it was impossible to ascertain if the patients were anaemic (and therefore had high levels of erythropoietin) prior to the malaria episode. We have shown that erythropoietin levels on admission above 200 U/L are independently associated with a reduced risk of sequelae. In vitro studies suggest that concentrations ranging from 100 U/L to 1,000 U/L are associated with the neuroprotective activities of erythropoietin.16 Although our data suggest

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Proefschrift.indb 172 14-1-2008 13:09:29 that erythropoietin concentrations up to 1,000 U/L were significantly associated with protection, a larger study would be required to establish the actual range of protection. The biological relevance of plasma erythropoietin is usually difficult to interpret in the absence of any direct measurement of erythropoietin in the brain. Erythropoietin is known to cross the blood-brain barrier by active translocation possibly via erythropoietin receptors expressed in the brain vasculature.13 We found CSF and plasma erythropoietin to be correlated in those cases discharged without neurological sequelae, but this association was moderately influenced by 5 cases with erythropoietin concentrations above 5,000 U/L. Moreover, erythropoietin in CSF was not associated with a reduced risk of neurological sequelae.

The association of younger age with higher erythropoietin concentrations and a greater protective effect of erythropoietin in children under 2 years of age are intriguing and there is no immediate biological explanation for these observations. However, these data do offer a possible reason for the widely observed and yet unexplained age-related presentation of severe malarial anaemia and cerebral malaria.6

We have previously shown that malaria infection per se is associated with an increased level of erythropoietin secretion for any haemoglobin concentration.19 The specific mechanisms responsible for the increased erythropoietin concentration in patients with acute malaria are unclear but may include tissue hypoxia, hypoglycaemia and possibly oxidative damage.7 These three mechanisms have been shown to induce the expression of hypoxia inducible factor (HIF-1), which up-regulates the production of erythropoietin and other hypoxia-related proteins.42 Similarly, iron deficiency may also contribute to the up-regulation of erythropoietin and other hypoxia-related proteins by inhibiting the function of HIF-prolyl 4-hydroxylases.43 The beneficial effect of iron chelation therapy in cerebral malaria is unclear 44 although one study suggested partial neuroprotection with desferoxamine.45 It is possible that up-regulation of HIF-1 and erythropoietin concentrations account for some of the tissue protective activities of iron chelation but these results may be partially obscured by the timing of the intervention.

The activation of VEGF has been reported in patients with cerebral malaria.46 VEGF is also up-regulated in response to tissue hypoxia47 and we initially predicted that the angiogenic and neurotrophic properties of VEGF would have a protective role in cerebral malaria. However, our data suggest that VEGF is not associated with neuroprotection but rather with features associated with a poor outcome. We have found that VEGF is associated with a 4-fold increase in the risk of seizures during admission. Pilocarpine- induced seizures in experimental animals are associated with a marked increase of VEGF levels in neuronal and glial cells and consequently vascular permeability and BBB

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Proefschrift.indb 173 14-1-2008 13:09:29 leakage.26 It is thus possible that increased VEGF concentrations may be the consequence rather than the cause of seizures. However, it is also likely that VEGF directly contributes to the pathophysiology of cerebral malaria by increasing the permeability of the BBB.27 Intracranial hypertension is a common finding in cerebral malaria in children.34 Here we report that concentrations of plasma VEGF (>100 pg/mL) are associated with a 4.5- fold increase in the risk signs of raised intracranial pressure and a 12-fold increase in the risk of papilloedema.

In this study, TNF was not identified as a risk factor for neurological sequelae. TNF (and other pro-inflammatory cytokines) have been shown to up-regulate VEGF, which may contribute to the development of neuropathological signs associated with inflammation in cerebral malaria.48 However, some of these effects may be partially antagonized by the TNF-induced up-regulation of erythropoietin receptor in the brain, 49 and may explain the lack of association of TNF with poor outcome in this study.

Our data suggest that in addition to the inflammatory response to the parasite, molecules that orchestrate adaptation to hypoxia may influence the clinical presentation and outcome of severe malaria syndromes. Moreover, this study supports the preliminary data from murine models of cerebral malaria to indicate a neuroprotective role for erythropoietin in cerebral malaria.22 While rapidly effective antimalarial drugs would be used to clear sequestered and non-sequestered parasites, the use of erythropoietin in cerebral malaria would aim to protect potentially viable brain tissue to prevent development of neurological sequelae. The potential use of erythropoietin (and non- erythropoietic erythropoietin derivatives50) as adjuvant treatment in cerebral malaria to prevent neurological sequelae should be investigated further.

ACKNOWLEDGEMENTS This study was supported by The Wellcome Trust-UK and Kenya Medical Research Institute. This paper is published with the permission of the director of KEMRI. Prof. David Roberts is supported by the Howard Hughes Medical Institute and the National Blood Service and this works benefits from NHS R&D funding. Prof. CRJC Newton holds a Wellcome Trust Career Post in Clinical Tropical Medicine (No. 070114).

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Proefschrift.indb 174 14-1-2008 13:09:29 3. Carter JA, Ross AJ, Neville BG, et al. Developmental impairments following severe falciparum malaria in children. Trop Med Int Health 2005;10(1):3-10. 4. Carter JA, Neville BG, White S, et al. Increased prevalence of epilepsy associated with severe falciparum malaria in children. Epilepsia 2004;45(8):978-81. 5. Ngoungou EB, Dulac O, Poudiougou B, et al. Epilepsy as a consequence of cerebral malaria in area in which malaria is endemic in Mali, West Africa. Epilepsia 2006;47(5):873-9. 6. Snow RW, Omumbo JA, Lowe B, et al. Relation between severe malaria morbidity in children and level of Plasmodium falciparum transmission in Africa. Lancet 1997;349(9066):1650-4. 7. Idro R, Jenkins NE, Newton CR. Pathogenesis, clinical features, and neurological outcome of cerebral malaria. Lancet Neurol 2005;4(12):827-40. 8. Brewster DR, Kwiatkowski D, White NJ. Neurological sequelae of cerebral malaria in children. Lancet 1990;336(8722):1039-43. 9. Molyneux ME, Taylor TE, Wirima JJ, Borgstein A. Clinical features and prognostic indicators in paediatric cerebral malaria: a study of 131 comatose Malawian children. Q J Med 1989;71(265):441-59. 10. Idro R, Carter JA, Fegan G, Neville BG, Newton CR. Risk factors for persisting neurological and cognitive impairments following cerebral malaria. Arch Dis Child 2006;91(2):142-8. 11 Carnot P. and Deflandre C. Sur l’activité hémopoiétique des différents organes au cours de la régénération du sang. C. R. Acad. Sci. Paris 1906;143:384-386. 12. Masuda S, Okano M, Yamagishi K, Nagao M, Ueda M, Sasaki R. A novel site of erythropoietin production. Oxygen-dependent production in cultured rat astrocytes. J Biol Chem 1994;269(30):19488-93. 13. Brines ML, Ghezzi P, Keenan S, et al. Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury. Proc Natl Acad Sci U S A 2000;97(19):10526-31. 14. Sakanaka M, Wen TC, Matsuda S, et al. In vivo evidence that erythropoietin protects neurons from ischemic damage. Proc Natl Acad Sci U S A 1998;95(8):4635-40. 15. Grasso G, Buemi M, Alafaci C, et al. Beneficial effects of systemic administration of recombinant human erythropoietin in rabbits subjected to subarachnoid hemorrhage. Proc Natl Acad Sci U S A 2002;99(8):5627-31. 16. Siren AL, Fratelli M, Brines M, et al. Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress. Proc Natl Acad Sci U S A 2001;98(7):4044-9. 17. Wen TC, Sadamoto Y, Tanaka J, et al. Erythropoietin protects neurons against chemical hypoxia and cerebral ischemic injury by up-regulating Bcl-xL expression. J Neurosci Res 2002;67(6):795-803. 18. Ehrenreich H, Hasselblatt M, Dembowski C, et al. Erythropoietin therapy for acute stroke is both safe and beneficial. Mol Med 2002;8(8):495-505. 19. Casals-Pascual C, Kai O, Cheung JO, et al. Suppression of erythropoiesis in malarial anemia is associated with hemozoin in vitro and in vivo. Blood 2006;108(8):2569-77. 20. Kurtzhals JA, Rodrigues O, Addae M, Commey JO, Nkrumah FK, Hviid L. Reversible suppression of bone marrow response to erythropoietin in Plasmodium falciparum malaria. Br J Haematol 1997;97(1):169-74. 21. Newton CR, Warn PA, Winstanley PA, et al. Severe anaemia in children living in a malaria endemic area of Kenya. Trop Med Int Health 1997;2(2):165-78. 22. Kaiser K, Texier A, Ferrandiz J, et al. Recombinant human erythropoietin prevents the death of mice during cerebral malaria. J Infect Dis 2006;193(7):987-95. 23. Wenger RH. Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia-inducible transcription factors, and O2-regulated gene expression. Faseb J 2002;16(10):1151-62. 24. Greenberg DA, Jin K. From angiogenesis to neuropathology. Nature 2005;438(7070):954-9.

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Proefschrift.indb 175 14-1-2008 13:09:29 25. Wang Y, Galvan V, Gorostiza O, Ataie M, Jin K, Greenberg DA. Vascular endothelial growth factor improves recovery of sensorimotor and cognitive deficits after focal cerebral ischemia in the rat. Brain Res 2006;1115(1):186-93. 26. Croll SD, Goodman JH, Scharfman HE. Vascular endothelial growth factor (VEGF) in seizures: a double-edged sword. Adv Exp Med Biol 2004;548:57-68. 27. Zhang ZG, Zhang L, Jiang Q, et al. VEGF enhances angiogenesis and promotes blood-brain barrier leakage in the ischemic brain. J Clin Invest 2000;106(7):829-38. 28. Chi OZ, Hunter C, Liu X, Weiss HR. Effects of VEGF and nitric oxide synthase inhibition on blood-brain barrier disruption in the ischemic and non-ischemic cerebral cortex. Neurol Res 2005;27(8):864-8. 29. Intiso D, Zarrelli MM, Lagioia G, et al. Tumor necrosis factor alpha serum levels and inflammatory response in acute ischemic stroke patients. Neurol Sci 2004;24(6):390-6. 30. Barone FC, Arvin B, White RF, et al. Tumor necrosis factor-alpha. A mediator of focal ischemic brain injury. Stroke 1997;28(6):1233-44. 31. Grau GE, Taylor TE, Molyneux ME, et al. Tumor necrosis factor and disease severity in children with falciparum malaria. N Engl J Med 1989;320(24):1586-91. 32. Kwiatkowski D, Hill AV, Sambou I, et al. TNF concentration in fatal cerebral, non-fatal cerebral, and uncomplicated Plasmodium falciparum malaria. Lancet 1990;336(8725):1201-4. 33. Berkley JA, Mwangi I, Ngetsa CJ, et al. Diagnosis of acute bacterial meningitis in children at a district hospital in sub-Saharan Africa. Lancet 2001;357(9270):1753-7. 34. Newton CR, Kirkham FJ, Winstanley PA, et al. Intracranial pressure in African children with cerebral malaria. Lancet 1991;337(8741):573-6. 35. Njuguna P, Newton C. Management of severe falciparum malaria. J Postgrad Med 2004;50(1):45-50. 36. WHO. WHO Expert Committee on Malaria: twentieth report., 2000. 37. Idro R, Karamagi C, Tumwine J. Immediate outcome and prognostic factors for cerebral malaria among children admitted to Mulago Hospital, Uganda. Ann Trop Paediatr 2004;24(1):17-24. 38. Marti HH. Erythropoietin and the hypoxic brain. J Exp Biol 2004;207(Pt 18):3233-42. 39. Brines M, Cerami A. Emerging biological roles for erythropoietin in the nervous system. Nat Rev Neurosci 2005;6(6):484-94. 40. Villa P, Bigini P, Mennini T, et al. Erythropoietin selectively attenuates cytokine production and inflammation in cerebral ischemia by targeting neuronal apoptosis. J Exp Med 2003;198(6):971-5. 41. Wiese L, Kurtzhals JA, Penkowa M. Neuronal apoptosis, metallothionein expression and proinflammatory responses during cerebral malaria in mice. Exp Neurol 2006;200(1):216-26. 42. Fandrey J, Frede S, Ehleben W, Porwol T, Acker H, Jelkmann W. Cobalt chloride and desferrioxamine antagonize the inhibition of erythropoietin production by reactive oxygen species. Kidney Int 1997;51(2):492-6. 43. Siddiq A, Ayoub IA, Chavez JC, et al. Hypoxia-inducible factor prolyl 4-hydroxylase inhibition. A target for neuroprotection in the central nervous system. J Biol Chem 2005;280(50):41732-43. 44. Smith HJ, Meremikwu M. Iron chelating agents for treating malaria. Cochrane Database Syst Rev 2003(2):CD001474. 45. Gordeuk V, Thuma P, Brittenham G, et al. Effect of iron chelation therapy on recovery from deep coma in children with cerebral malaria. N Engl J Med 1992;327(21):1473-7. 46. Deininger MH, Winkler S, Kremsner PG, Meyermann R, Schluesener HJ. Angiogenic proteins in brains of patients who died with cerebral malaria. J Neuroimmunol 2003;142(1-2):101-11. 47. Levy AP, Levy NS, Goldberg MA. Post-transcriptional regulation of vascular endothelial growth factor by hypoxia. J Biol Chem 1996;271(5):2746-53.

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Proefschrift.indb 176 14-1-2008 13:09:29 48. Ryuto M, Ono M, Izumi H, et al. Induction of vascular endothelial growth factor by tumor necrosis factor alpha in human glioma cells. Possible roles of SP-1. J Biol Chem 1996;271(45):28220-8. 49. Nagai A, Nakagawa E, Choi HB, Hatori K, Kobayashi S, Kim SU. Erythropoietin and erythropoietin receptors in human CNS neurons, astrocytes, microglia, and oligodendrocytes grown in culture. J Neuropathol Exp Neurol 2001;60(4):386-92. 50. Leist M, Ghezzi P, Grasso G, et al. Derivatives of erythropoietin that are tissue protective but not erythropoietic. Science 2004;305(5681):239-42.

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Proefschrift.indb 177 14-1-2008 13:09:29 Proefschrift.indb 178 14-1-2008 13:09:29 Summary

Proefschrift.indb 179 14-1-2008 13:09:29 Proefschrift.indb 180 14-1-2008 13:09:29 SUMMARY Malaria is a leading cause of ill health in tropical countries. Infection with Plasmodium falciparum is the most severe and mainly affects children <5 years in sub-Saharan Africa. In this age group, falciparum malaria may be complicated by severe anaemia, prostration, impaired consciousness, hypoglycaemia, metabolic acidosis and seizures, with cerebral malaria the most severe neurological presentation[1].

Worldwide, febrile seizures are the commonest seizure disorder. In susceptible children, most are induced by infections such as respiratory tract and ear infections or gastroenteritis. The majority are short-lived single seizures that rarely progress to status epilepticus. The outcome is good. In tropical countries however, acute symptomatic seizures may be more common and falciparum malaria is an important cause of acute symptomatic seizures. Infection with Plasmodium falciparum has been associated with over 50% of seizures in children admitted to hospital. Although malaria-related seizures also occur in the context of a febrile illness, unlike simple febrile seizures, the pattern and outcomes are different. Mortality is higher and neuronal damage manifesting as neurological deficits at discharge, epilepsy or long-term neurological and cognitive impairments has been described in up to 24% of exposed children. These poor outcomes are not limited to cerebral malaria, but extend to other less severe forms such as malaria with multiple or complicated seizures[2]. Chapter 1 is a summary of the clinical features of falciparum malaria in African children and studies of malaria related seizures.

The risk factors and precipitants of seizures in acute falciparum malaria, the public health burden and the mechanisms by which neuronal damage develops are poorly understood. In this thesis, I estimated the contribution of falciparum malaria to the burden of acute seizures in children living in a well-defined area in Kilifi in coastal Kenya. I examined some of the risk factors for seizures and long-term neurological and cognitive impairments, and looked at possible mechanisms by which neuronal damage develops. Lastly, I examined the possibility of offering neuro-protection to prevent the development of neuronal damage.

Incidence of acute seizures and malaria related seizures in children To determine the incidence of acute seizures in the study area, we recruited all children aged 0-13 years presenting with incident admissions and acute seizures to Kilifi district hospital over a 2-year period. The population denominator was estimated from the census data of the study area. Seizures were reported in 900/4,921(18.3%) children and at least 98 had status epilepticus. The incidence of acute seizures in children 0-13 years was 425/100,000/year and was 879/100,000/year in children <5 years. Over 80% of the

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Proefschrift.indb 181 14-1-2008 13:09:29 seizures were associated with infections. Neonatal sepsis (28/43[65.1%]) and falciparum malaria (476/821[58.0%]) were the main diseases associated with seizures, in neonates and in children 6 months or older respectively. Falciparum malaria was also the main illness associated with status epilepticus. Other illnesses associated with seizures included pyogenic meningitis, respiratory tract infections and gastroenteritis. The study concluded that malaria is the leading cause of acute seizures in children living in this area and that most of the important causes of the seizures are diseases that are preventable with available public health programs (chapter 2).

Using a retrospective data set of all admissions of children from the study area over a 13- year period from 1992–2004, we estimated that at a minimum, 1,156 children <5 years per 100,000 were exposed to brain insults from malaria annually. Involvement of the central nervous system was mostly characterised by seizures (incidence 911 per 100,000 per year). The incidence was lower in children older than 5 years[3]. This incidence data is an absolute minimum, since it does not account for children from the study area that did not attend Kilifi district hospital during the study period. From previous estimates, two thirds of deaths in children <5 years in this study area occurred outside the hospital. Although not investigated, the difference in incidence between the two studies suggests that there has been a decline in the incidence of acute seizures. This decline may partly be due to a reduction in the number of cases with malaria: although malaria was associated with 66.5% of seizures in Kilifi district hospital in 1996, over the years 2005– 6, this had declined to 53.7%.

Risk factors and precipitants of seizures in children with malaria The causes of seizures in children with falciparum malaria are not well understood. Apart from the epileptogenic nature of the parasite, my colleagues and I hypothesized that seizures in children with falciparum malaria occur in those with a genetic susceptibility to seizures and in particular children with either some common genetic traits in the tropics or single nucleotide polymorphisms in ion channels. The seizures may be precipitated by metabolic derangements or if co-infected with viruses and that the threshold for seizures is lowered by micronutrient deficiencies such as iron or zinc.

To examine these hypotheses, I compared the children with seizures described in chapter 2 to those admitted without seizures. In addition I used the large retrospective cohort of children with malaria admitted between 1992-2004 (chapter 4) to examine some risk factors for seizures. Malaria was the most common illness associated with acute seizures in children 6 months or older and was responsible for over 50% of all acute seizures in children. The median number of seizures in children with malaria was twice that in patients with other causes of seizures. In addition, seizures in children infected

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Proefschrift.indb 182 14-1-2008 13:09:29 with malaria were of a longer duration and status epilepticus was more common. Although seizures were observed even at low parasitaemia, the proportion of patients with seizures increased with parasitaemia. Children with seizures were older and were more likely to have had seizures in the past (chapters 2 and 4).

Genetic susceptibility to seizures is well recognised and a number of polymorphisms have been associated with the development of febrile seizures[4]. There are a number of polymorphisms that are common in tropical countries, which influence the presentation of falciparum malaria[5]. To determine the association between two very common genetic traits in tropical countries and the high incidence of acute seizures in this region, I examined the polymorphisms associated with the haptoglobin and α-globin genes. The HP2-2 polymorphism of the haptoglobin gene has been associated with epilepsy and individuals with the α-thalassaemia are thought to have an altered iron metabolism that may increase the risk of seizures. The proportion of cases and controls with the HP2-2 genotype was similar. Similarly, I found no difference between cases and controls in the proportions of children with deletions in the α-globin gene. Among cases, the HP2-2 polymorphism and deletions in the α-globin gene were neither associated with a change in the type, number or duration of the seizures nor did they affect the outcome of treatment (chapter 3). This study suggests that, unlike the idiopathic generalized epilepsies in which the HP2-2 genotype may be involved in the inheritance of the chronic seizure disorder, neither the HP2-2 genotype nor α-thalassaemia are risk factors for acute seizure disorders.

Seizures and abnormal motor posturing in children with cerebral malaria Cerebral malaria is the most severe neurological complication of falciparum malaria. Posturing is a common feature but the aetiology and pathogenesis of this sign is poorly understood. Raised intracranial pressure (ICP) is a recognized cause of posturing and raised ICP has been described in cerebral malaria in African children[6]. Seizures have also been described in over 60% of patients with acute non-traumatic encephalopathies and posturing. The hypothesis that posturing may be caused by seizures or is a manifestation of a seizure has led to the use of anticonvulsants in their management. There is little evidence to support this management. In chapter 5, we examined records of children with cerebral malaria to determine risk factors for posturing. Posturing was observed in 163(39.1%) patients and was associated with features of raised ICP on fundoscopy. In addition, decerebrate and opisthotonic postures were associated with recurrence of seizures. Among patients who died, the majority of deaths (19/31 [61.3%]) were associated with features suggestive of transtentorial herniation and in survivors, neurological sequelae were more common. The study suggested that raised ICP might

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Proefschrift.indb 183 14-1-2008 13:09:29 be the primary cause of posturing in children with cerebral malaria and the association with seizures may be due to raised ICP worsening perfusion to areas with already a critically compromised blood flow[7].

Continuous electroencephalographic monitoring and seizures in cerebral malaria Seizures are a common feature of cerebral malaria in children and multiple seizures are a risk factor for poor outcome. Continuous EEG recordings in comatose patients in intensive care units have demonstrated a high incidence of non-convulsive seizures that may damage neurons. In this study, I examined the electroencephalographic (EEG) characteristics of 52 children with cerebral malaria on continuous EEG monitoring and related specific EEG features to outcome. The background EEG was characterized by very slow generalised high amplitude waves. One hundred and forty nine clinical seizures (40%) and 223 (60%) electrographic seizures were detected in 20 (38.5%) children. The majority had a focal origin. A higher frequency of the background EEG was associated with a shorter duration of coma while an asymmetrical background EEG, rhythmic runs and electrical status epilepticus were associated with death or neurological sequelae. The study concluded that children with cerebral malaria and convulsive status epilepticus experience frequent electrographic seizures and that electrographic status epilepticus is associated with poor outcome. Continuous EEG monitoring is a useful tool for the detection of such seizures and the acute EEG may be of prognostic value. This section ends with chapter 7, a detailed review of the published literature on cerebral malaria examining the clinical features, pathogenesis and neurological outcome.

Risk factors for long-term impairments after cerebral malaria and mechanisms of neuronal damage in malaria In children who survive cerebral malaria, gross neurological deficits are detectable in 11% on discharge. Persistent neuro-cognitive impairments have been documented in 24% several years after exposure. In chapter 8, I determined the risk factors for these persisting impairments by examining hospital records of 143 exposed children assessed at least 20 months after discharge to detect motor, speech and language, and other cognitive impairments. I found that previous seizures, deep coma on admission, focal neurological signs observed during admission, and neurological deficits on discharge were independently associated with persisting impairments. In addition, multiple seizures were associated with motor impairment, age<3 years, severe malnutrition, features of raised ICP and hypoglycaemia were associated with language impairments while prolonged coma, severe malnutrition and hypoglycaemia were associated with impairments in other cognitive functions[8]. I concluded that although there are overlaps in impaired functions and in the risk factors for such impairments, the differences in risk

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Proefschrift.indb 184 14-1-2008 13:09:29 factors for specific functions might suggest separate mechanisms for neuronal damage. These factors could form the basis for future preventive strategies.

Biomarkers in cerebrospinal fluid (CSF) have been used to help delineate pathophysiological mechanisms, predict and monitor neurological outcomes and develop and evaluate new therapeutic strategies in human neurological disease. My colleagues and I determined levels of two markers of brain damage, the tau protein (as a marker of axonal injury) and S-100B (a marker of astrocyte injury), in the CSF of 143 Kenyan children with and without malaria and with different levels of consciousness (chapter 9). The level of tau in the CSF was significantly elevated in children with cerebral malaria compared with either malaria with prostration or malaria with seizures but normal consciousness[9]. Median levels of tau in children with cerebral malaria were 3-fold greater than previous reports in adults and may explain the higher prevalence of neurological sequelae in children[10]. Elevated S-100B in children was associated with an increased risk of recurrent seizures suggesting that the recurrent seizures after admission may be a manifestation of brain damage, rather than a cause of brain damage.

Neuro-protection in children with cerebral malaria Although several poor prognostic factors have been identified for cerebral malaria in African children, protective factors are less well defined. Erythropoietin is protective in animal models of brain injury and administration of this compound in a murine model of cerebral malaria reduced mortality by 90%[11]. My colleagues and I hypothesized that the outcome of cerebral malaria is modified by the cytokine response to hypoxia, in particular to erythropoietin and that high plasma and CSF levels of erythropoietin protect children with cerebral malaria from developing neurological sequelae or death. In chapter 10, we retrospectively compared plasma and CSF levels of erythropoietin in 3 groups of children with cerebral malaria: children who died, those who survived with and without neurological deficits (n=124). The median plasma levels of erythropoietin were 123, 184 and 278 U/L respectively. Plasma erythropoietin >200U/L was associated with greater than 80% reduction in the risk of developing neurological sequelae. These data would support further study of erythropoietin as adjuvant therapy for children with cerebral malaria.

Ongoing and future studies Some of our initial hypotheses were not tested and others are in the process of being examined in ongoing studies. I plan to investigate the association between micronutrient deficiencies in particular, iron deficiency and acute seizures in children. I will also investigate if viral co-infection of children with malaria increases the risk of acute seizures. In addition, I also intend to examine the relationship between acute

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Proefschrift.indb 185 14-1-2008 13:09:30 seizures and single nucleotide polymorphisms in ligand and voltage gated ion channels. Completion of these studies will provide a better understanding of the genetics of acute seizure disorders in the tropics and the interaction between genetics and environmental factors and the risk of acute seizure disorders.

Several observational and retrospective studies have documented recurrent seizures in children with cerebral malaria as a risk factor for neurological sequelae in surviving patients. What is not clear is whether the recurrent seizures cause the neurological damage or both the recurrent seizures and the neurological sequelae are manifestations of neuronal damage in patients with a more severe illness. If indeed it is the recurrent seizures that cause the sequelae, preventing seizure recurrence should reduce the number of children developing neurological sequelae. We hypothesized that a prophylactic dose of fosphenytoin given on admission to children with cerebral malaria will prevent both the recurrence of seizures and prevent neurological and cognitive impairments when compared to placebo. Recruitment into the trial is in progress. This trial has the potential to improve our understanding of the role of seizures in the causation of neuronal damage.

CONCLUSIONS In conclusion, falciparum malaria is the leading cause of seizures in children living in malaria endemic areas. The characteristics of the seizures differ from simple febrile seizures to complex, focal, prolonged or multiple seizures and they are a major risk factor for neurological and cognitive impairments in children. The risk of seizures increases with parasitaemia but the role of genetic susceptibility in the development of malaria- related seizures is still unclear. Future studies examining the relationship between acute seizures and polymorphisms in ion channels and the role of prophylactic anticonvulsants in the prevention of seizure recurrences and neuro-cognitive impairments will aid our understanding of pathogenesis and the design of intervention strategies. It may also be possible in future to improve outcome with neuro-protective adjuvant therapies with drugs such as erythropoietin or its analogues.

REFERENCES 1. WHO: Severe falciparum malaria. Trans R Soc Trop Med Hyg 2000, 94 Suppl 1:S1-90. 2. Carter JA, Mung’ala-Odera V, Neville BG, Murira G, Mturi N, Musumba C, Newton CR: Persistent neurocognitive impairments associated with severe falciparum malaria in Kenyan children. J Neurol Neurosurg Psychiatry 2005, 76:476-481.

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Proefschrift.indb 186 14-1-2008 13:09:30 3. Idro R, Ndiritu M, Ogutu B, Mithwani S, Maitland K, Berkley J, Crawley J, Fegan G, Bauni E, Peshu N, et al: Burden, features, and outcome of neurological involvement in acute falciparum malaria in Kenyan children. Jama 2007, 297:2232-2240. 4. Audenaert D, Van Broeckhoven C, De Jonghe P: Genes and loci involved in febrile seizures and related epilepsy syndromes. Hum Mutat 2006, 27:391-401. 5. May J, Evans JA, Timmann C, Ehmen C, Busch W, Thye T, Agbenyega T, Horstmann RD: Hemoglobin variants and disease manifestations in severe falciparum malaria. Jama 2007, 297:2220-2226. 6. Newton CR, Crawley J, Sowumni A, Waruiru C, Mwangi I, English M, Murphy S, Winstanley PA, Marsh K, Kirkham FJ: Intracranial hypertension in Africans with cerebral malaria. Arch Dis Child 1997, 76:219-226. 7. Idro R, Otieno G, White S, Kahindi A, Fegan G, Mithwani S, Maitland K, Neville BG, Ogutu B, Newton CR: Decorticate, decerebrate and opisthotonic posturing and seizures in Kenyan children with cerebral malaria. Malar J 2005, 4:57. 8. Idro R, Carter JA, Fegan G, Neville BG, Newton CR: Risk factors for persisting neurological and cognitive impairments following cerebral malaria. Arch Dis Child 2006, 91:142-148. 9. Medana IM, Idro R, Newton CR: Axonal and astrocyte injury markers in the cerebrospinal fluid of Kenyan children with severe malaria. J Neurol Sci 2007. 10. Medana IM, Lindert RB, Wurster U, Hien TT, Day NP, Phu NH, Mai NT, Chuong LV, Chau TT, Turner GD, et al: Cerebrospinal fluid levels of markers of brain parenchymal damage in Vietnamese adults with severe malaria. Trans R Soc Trop Med Hyg 2005, 99:610-617. 11. Kaiser K, Texier A, Ferrandiz J, Buguet A, Meiller A, Latour C, Peyron F, Cespuglio R, Picot S: Recombinant human erythropoietin prevents the death of mice during cerebral malaria. J Infect Dis 2006, 193:987-995.

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Proefschrift.indb 187 14-1-2008 13:09:30 Proefschrift.indb 188 14-1-2008 13:09:30 Samenvatting

Proefschrift.indb 189 14-1-2008 13:09:30 Proefschrift.indb 190 14-1-2008 13:09:30 Samenvatting Malaria is een van de leidende oorzaken van ziekte in tropische landen. Infectie met Plasmodium falciparum is de ernstigste vorm van malaria. Deze vorm van malaria treft voornamelijk kinderen jonger dan 5 jaar in Afrika ten zuiden van de Sahara. In deze leeftijdsgroep kan malaria gecompliceerd worden door ernstige bloedarmoede, uitputting, vermindering van het bewustzijn, lage bloedsuikerspiegels, verzuring (metabole acidose) en toevallen. Hersenmalaria is de ernstigste neurologische uitingsvorm van malaria.

Wereldwijd gezien zijn koortsstuipen de meest voorkomende vorm van toevallen. Bij daarvoor gevoelige kinderen worden de meeste koortsstuipen veroorzaakt door ontstekingen zoals die van de luchtwegen, van de oren of van de maag of de darmen. Merendeels zijn het kortdurende, enkelvoudige toevallen die zelden overgaan in een zogeheten “status epilepticus”. De uiteindelijke afloop is gunstig. Maar in tropische landen komen plotselinge toevallen wellicht vaker voor en daar is malaria tropica (infectie met P. falciparum) een belangrijke oorzaak. Malaria tropica is in verband gebracht met meer dan 50% van de toevallen van kinderen die in een ziekenhuis worden opgenomen. Hoewel toevallen die met malaria verband houden zich voordoen in de context van een koortsende ziekte, zijn het patroon en de afloop anders dan die van eenvoudige koortsstuipen. De sterfte is hoger en beschadiging van zenuwcellen zich uitend als neurologische uitval, epilepsie of langdurige neurologische en cognitieve beperkingen is beschreven in percentages oplopend tot 24 % van de kinderen. Een slechte afloop doet zich voor bij kinderen met cerebrale malaria maar ook bij kinderen met minder ernstige vormen van malaria, bijvoorbeeld met veelvuldige of gecompliceerde toevallen. Hoofdstuk 1 betreft een samenvatting van de klinische manifestaties van malaria bij Afrikaanse kinderen en onderzoeken van met malaria samenhangende toevallen.

Er is onvoldoende kennis van de risicofactoren en uitlokkende momenten van toevallen bij acute P. falciparum-infectie Wij weten niet hoe belangrijk dit is voor de openbare gezondheidszorg en we kennen de mechanismen niet waardoor schade aan zenuwcellen ontstaat. In dit proefschrift heb ik een schatting gemaakt van de bijdrage van malaria tropica aan het totaal van de acute toevallen bij kinderen die leven in een goed omschreven gebied in Kilifi aan de Keniaanse kust. Ik onderzocht enkele van de risicofactoren voor toevallen en neurologische en cognitieve beperkingen zoals gezien na lange termijn. Ik keek naar mogelijke mechanismen van schade aan zenuwcellen. Tenslotte onderzocht ik de mogelijkheid van neuro-protectie om de ontwikkeling van schade aan de zenuwcellen te voorkomen.

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Proefschrift.indb 191 14-1-2008 13:09:30 Incidentie van acute toevallen en van met malaria samenhangende toevallen bij kinderen Om de incidentie van acute toevallen in het onderzoeksgebied te bepalen, bestudeerden we de gegevens van alle kinderen van 0 tot 13 jaar die in een periode van 2 jaar werden opgenomen in het Kilifi District Ziekenhuis met acute toevallen. De noemer van de bevolking werd bepaald uit de bevolkingstelling van het onderzoeksgebied. Toevallen werden gemeld bij 900/4 921 (18,3%) kinderen en tenminste 98 hadden een status epilepticus. De incidentie van acute toevallen bij kinderen van 0 tot 13 jaar was 425/100 000/jaar en 879/100 000/jaar bij kinderen jonger dan 5 jaar. Meer dan 80% van de toevallen hielden verband met ontstekingen. Neonatale sepsis (28/43 [65,1%]) en malaria tropica (476/821 [58,0%]) waren de belangrijkste ziekten die verband hielden met toevallen, respectievelijk bij neonaten en kinderen vanaf de leeftijd van 6 maanden. P. falciparum- infectie was ook de belangrijkste ziekte die verband hield met status epilepticus. Andere, met toevallen geassocieerde ziekten waren pyogene meningitis, luchtweginfecties en gastroenteritis. De conclusie van het onderzoek luidde dat malaria de belangrijkste oorzaak is van acute toevallen bij kinderen levend in dit gebied en dat de meeste van de belangrijke oorzaken van toevallen ziekten zijn die voorkómen kunnen worden binnen bestaande volksgezondheidsprogramma’s (hoofdstuk 2).

Gebruikmakend van een gegevensbestand van alle opnames van kinderen uit het onderzoeksgebied van 1992 to 2004, een periode van 13 jaar, schatten wij dat minimaal 1 156 kinderen jonger dan 5 jaar per 100 000 jaarlijks een hersenbeschadiging ondergingen door malaria. Aantasting van het centrale zenuwstelsel uitte zich meestal door toevallen (incidentie 911 per 100 000 per jaar). De incidentie was lager bij kinderen ouder dan 5 jaar [3]. Omdat geen rekening is gehouden met kinderen in het onderzoeksgebied die het district ziekenhuis niet bezochten, zijn deze incidentiegegevens een absoluut minimum. Uit eerdere schattingen weten wij dat twee derde van de sterfte van kinderen jonger dan 5 jaar in het onderzoeksgebied zich voordoet buiten het ziekenhuis. Het verschil in incidentie tussen de twee studies suggereert dat er een daling is opgetreden in incidentie van acute toevallen. Deze daling kan deels verklaard worden door een daling van het aantal gevallen van malaria. Malaria was in 1996 geassocieerd met 66,5% van de toevallen in Kilifi District Ziekenhuis; in de jaren 2005-6 daalde dit tot 53,7%.

Risicofactoren en uitlokkende momenten van toevallen bij kinderen met malaria De oorzaken van toevallen bij kinderen met malaria tropica zijn niet goed bekend. Wij vormden de hypothese dat naast een factor in de parasiet die aanleiding geeft tot toevallen, een genetische aanleg voor toevallen een rol zou kunnen spelen. Deze aanleg zou kunnen bestaan uit in de tropen gewoonlijk voorkomende kenmerken of

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Proefschrift.indb 192 14-1-2008 13:09:30 polymorphismen van enkelvoudige nucleotiden in ionen kanalen. Toevallen zouden kunnen worden veroorzaakt door metabole stoornissen of co-infecties met virussen. Bovendien zou de drempel voor toevallen verlaagd kunnen zijn door tekorten aan micronutriënten als ijzer en zink.

Om deze hypothesen te onderzoeken vergeleek ik de kinderen met toevallen die beschreven zijn in hoofdstuk 2 met in het ziekenhuis opgenomen kinderen zonder toevallen. Bovendien maakte ik gebruik van een groot retrospectief cohort kinderen met malaria, opgenomen tussen 1992 en 2004 (hoofdstuk 4) om enkele risicofactoren voor toevallen te onderzoeken. Van alle ziekten was malaria het vaakst geassocieerd met acute toevallen bij kinderen van 6 maanden en ouder en malaria was verantwoordelijk voor meer dan 50% van alle toevallen. Het mediane aantal toevallen bij kinderen met malaria was twee maal hoger dan het aantal bij kinderen met andere oorzaken. De toevallen bij kinderen met malaria duurden langer en deze kinderen hadden vaker een status epilepticus. Hoewel toevallen ook bij een lage parasitemie werden gezien nam het aantal patiënten met toevallen toe in relatie tot de parasitemie. Kinderen met toevallen waren ouder en hadden vaker toevallen gehad in het verleden (hoofdstukken 2 en 4).

Het is bekend dat er een genetische gevoeligheid bestaat voor toevallen en een aantal polymorphismen is in verband gebracht met het ontstaan van koortsstuipen [4]. Enkele vaak in de tropen voorkomende polymorphismen beïnvloeden de klinische presentatie van malaria tropica [5]. Om het verband tussen twee zeer vaak voorkomende genetische kenmerken en de hoge incidentie van acute toevallen in dit gebied te bepalen, onderzocht ik polymorphismen in het haptoglobine en α-globine gen. Het HP2-2 polymorphisme van het haptoglobine gen is geassocieerd met epilepsie en gedacht wordt dat individuen met α-thalassaemie een ander metabolisme van ijzer hebben waardoor het risico op toevallen is toegenomen. De proporties van kinderen met toevallen (cases) en van kinderen zonder toevallen (controles) met het HP2-2 genotype waren gelijk en ook vond ik geen verschil tussen cases en controles wat betreft deleties in het α-globine gen. Bij de kinderen met toevallen waren HP2-2 polymorphisme en deleties in hetα-globine gen niet geassocieerd met een verandering in het type, het aantal of de duur van de toevallen en ook niet met het resultaat van de behandeling (hoofdstuk 3). Dit onderzoek suggereert dat HP2-2 genotype en α-thalasaemie geen risicofactoren vormen voor acute toevallen, dit in tegenstelling tot idiopathische gegeneraliseerde epilepsie waarbij HP2-2 genotype betrokken kan zijn bij de chronische vorm van toevallen,.

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Proefschrift.indb 193 14-1-2008 13:09:30 Toevallen en abnormale, verkrampte lichaamshouding (o.a. opisthotonus) bij kinderen met cerebrale malaria Cerebrale malaria is de ernstigste neurologische complicatie van malaria. Een verkrampte lichaamshouding, bijvoorbeeld opisthotonus (met het hoofd en de hielen gespannen, achterovergebogen) komt vaak voor maar de etiologie en pathogenese zijn niet bekend. Verhoogde intracraniële druk is een bekende oorzaak van opisthotonus en deze is beschreven bij cerebrale malaria bij Afrikaanse kinderen [6]. Toevallen zijn ook beschreven bij meer dan 60% van de patiënten met acute niet-traumatische encephalopathieën en opisthotonus. The hypothese dat opisthotonus veroorzaakt zou kunnen worden door toevallen of een uitingsvorm is van toevallen heeft geleid tot gebruik van anticonvulsiva als behandeling. Er is weinig bewijs ter ondersteuning van deze behandeling. In hoofdstuk 5 beschrijven wij retrospectief statusonderzoek naar risicofactoren voor verkrampte lichaamshouding bij kinderen met cerebrale malaria. Deze werd gezien bij 163 (39,1%) patiënten en was geassocieerd met tekenen van toegenomen intracraniële druk zoals gezien bij fundoscopie. Bovendien waren ernstige uitingen van verkrampte lichaamshouding, met name opisthotonus geassocieerd met terugkeer van toevallen. Bij 19 van de 31 patiënten (61,3%) die stierven waren er tekenen die herseninklemming suggereerden en bij degenen die overleefden waren er vaker neurologische restverschijnselen. Het onderzoek duidt erop dat verhoogde intracraniële druk de belangrijkste oorzaak zou kunnen zijn voor abnormale, verkrampte lichaamshouding bij kinderen met cerebrale malaria en het verband met toevallen zou kunnen berusten op verhoogde intracraniële druk die de bloedtoevoer naar gebieden waar de toevoer al beperkt is, nog verder doet afnemen [7].

Continue elektro-encefalografische monitoring en toevallen bij cerebrale malaria. Toevallen komen vaak voor bij kinderen met cerebrale malaria en multipele toevallen zijn een risicofactor voor een slechte afloop .Continue registratie van het elektro-encefalogram (EEG) bij patiënten in coma, opgenomen op intensive care units heeft getoond dat er een hoge incidentie is van niet-convulsieve toevallen die neuronen kunnen beschadigen. In dit onderzoek bestudeerde ik de elektro-encefalografische kenmerken van 52 kinderen met cerebrale malaria bij wie het EEG continu werd geregistreerd. Ik bestudeerde of deze kenmerken verband hielden met de afloop. Het achtergrond-EEG werd gekarakteriseerd door zeer langzame golven met hoge amplitude. Honderd negen en veertig klinisch manifeste toevallen (40%) en 223 (60%) elektrografische toevallen werden ontdekt bij 20 (38,5%) kinderen. De meerderheid kwam voort uit een focus. Een hogere frequentie van het achtergrond-EEG was geassocieerd met een kortere duur van het coma terwijl een asymmetrisch achtergrond-EEG, rhytmische runs en een elektrische status epilepticus geassocieerd waren met sterfte of neurologische restverschijnselen. Het onderzoek leidt

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Proefschrift.indb 194 14-1-2008 13:09:30 tot de conclusie dat kinderen met cerebrale malaria en convulsieve status epilepticus frequente elektrografische toevallen ondergaan en dat een elektrografische status epilepticus geassocieerd is met een slechte afloop. Continue EEG registratie is een nuttig instrument om dergelijke toevallen vast te stellen en het EEG bij opname kan van prognostische waarde zijn. Dit onderdeel eindigt met hoofdstuk 7, een gedetailleerd overzicht van de gepubliceerde literatuur over cerebrale malaria betreffende klinische kenmerken, pathogenese en neurologische afloop.

Risicofactoren voor beperkingen op de lange termijn na cerebrale malaria en mechanismen van beschadiging van neuronen bij malaria. Bij kinderen die cerebrale malaria hebben overleefd zijn bij ontslag uit het ziekenhuis bij 11% belangrijke neurologische defecten aantoonbaar. Blijvende neuro-cognitieve beperkingen zijn aangetoond bij 24%, verscheidene jaren na de aandoening. In hoofdstuk 8 onderzocht ik risicofactoren voor deze blijvende beperkingen door de ziekenhuisgegevens te bestuderen van 143 kinderen die ten minste 20 maanden na ontslag werden onderzocht op het bestaan van beperkingen in motorische functies, spraak en taal en andere cognitieve functies. Ik ontdekte dat eerdere toevallen, diep coma bij opname, focale neurologische tekenen tijdens opname en neurologische defecten bij ontslag onafhankelijk van elkaar geassocieerd waren met blijvende beperkingen. Bovendien bleken multipele toevallen geassocieerd met beperking van motorische functies, bleken leeftijd beneden 3 jaar, ernstige ondervoeding, tekenen van verhoogde intracraniële druk en hypoglycemie geassocieerd met taalbeperkingen terwijl langdurig coma, ernstige ondervoeding en hypoglycemie geassocieerd waren met andere cognitieve functies [8]. Ik kwam tot de conclusie dat de verschillen in risicofactoren voor specifieke functies verschillende mechanismen voor beschadiging van neuronen zouden kunnen suggereren hoewel er nogal wat overlap is betreffende de aangetaste functies en de risicofactoren voor de beperkingen. Deze factoren zouden de basis kunnen vormen voor toekomstige preventie strategieën.

Biomarkers in de liquor cerebrospinalis , kortweg liquor (hersen- en ruggenmergvloeistof) zijn toegepast om pathofysiologische mechanismen te beschrijven, neurologische uitkomsten te voorspellen en vervolgen en om nieuwe therapeutische benaderingen te evalueren. Wij hebben de spiegels van 2 parameters van hersenbeschadiging bepaald, het tau eiwit (parameter voor axonale schade) en S-100B (parameter voor schade aan astrocyten), in de liquor van 143 Keniaanse kinderen met en zonder malaria en verschillende stadia van bewustzijn (hoofdstuk 9). De tau spiegel was significant hoger bij kinderen met cerebrale malaria vergeleken met kinderen met malaria en uitputting of met toevallen maar met normaal bewustzijn [9]. Mediane spiegels van tau bij kinderen met cerebrale malaria waren 3 maal hoger dan gemeld in eerdere rapporten betreffende

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Proefschrift.indb 195 14-1-2008 13:09:30 volwassenen; zij zouden een verklaring kunnen vormen voor de hogere prevalentie van neurologische restverschijnselen bij kinderen [10]. Verhoogde spiegels aan S-100B bij kinderen waren geassocieerd met toegenomen risico van recidiverende toevallen hetgeen de suggestie wekt dat de recidiverende toevallen na opname een uiting zouden kunnen zijn van hersenschade, in plaats van oorzaak van deze schade.

Neuro-protectie bij kinderen met cerebrale malaria Diverse prognostische factoren voor het ontstaan van cerebrale malaria bij Afrikaanse kinderen zijn geïdentificeerd maar beschermende factoren zijn niet goed omschreven. Erythropoietine heeft een beschermende werking bij diermodellen met hersenschade en toediening van dit middel bij een muizenmodel van cerebrale malaria verminderde de sterfte met 90% [11]. Wij ontwikkelden de hypothese dat de afloop van cerebrale malaria gemodificeerd wordt door de reactie van cytokinen op hypoxie en in het bijzonder op erythropoietine en dat hoge spiegels aan erythropoietine in plasma en liquor kinderen met cerebrale malaria beschermen tegen het ontwikkelen van neurologische restverschijnselen of sterfte. In hoofdstuk 10 vergeleken wij, achteraf, spiegels aan erythropoietine in plasma en liquor in 3 groepen kinderen met cerebrale malaria: kinderen die stierven en kinderen die overleefden met en zonder neurologische schade (n=124). De mediane plasmaspiegels aan erythropoietine waren respectievelijk 123, 184 en 278 U/L. Een erythropoietine spiegel in het plasma > 200 U/L was geassocieerd met meer dan 80% reductie van risico voor ontwikkelen van neurologische restverschijnselen. Deze gegevens ondersteunen verder onderzoek van erythropoietine als adjuvante behandeling bij kinderen met cerebrale malaria.

Lopende en toekomstige studies Sommige van onze aanvankelijk geformuleerde hypothesen werden nog niet onderzocht en andere zijn momenteel onderwerp van studie. Ik ben van plan onderzoek te doen naar de associatie tussen deficiënties van micronutriënten, in het bijzonder van ijzer, en acute toevallen bij kinderen. Ik zal ook onderzoeken of co-infectie met virussen het risico van acute toevallen verhoogt. Bovendien ben ik van plan onderzoek te doen naar het verband tussen acute toevallen en single nucleotide polymorphismen in “ligand en voltage gated” ionen kanalen. Dit zal leiden tot een beter begrip van de genetische achtergrond van aandoeningen met acute toevallen in de tropen en van de interactie tussen genetica en omgevingsfactoren en het risico op acute toevallen.

Verschillende observationele en retrospectieve studies hebben aangetoond dat recidiverende toevallen bij kinderen met cerebrale malaria een risicofactor vormen voor neurologische restverschijnselen. Het is evenwel niet duidelijk of de recidiverende toevallen de neurologische schade veroorzaken of dat de recidiverende toevallen en de

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Proefschrift.indb 196 14-1-2008 13:09:30 neurologische restverschijnselen uitingen zijn van de neuronale schade bij patiënten met ernstige ziekte. Als de recidiverende toevallen de restverschijnselen veroorzaken zou voorkómen van recidieven van toevallen vermindering moeten geven van het aantal kinderen met restverschijnselen. Een profylactische dosis fosphenytoine, gegeven bij opname van kinderen met cerebrale malaria zou heroptreden van toevallen en neurologische en cognitieve beperkingen moeten voorkomen. Dit placebo-gecontroleerde onderzoek is gaande. Door dit onderzoek zou ons inzicht in de rol van toevallen als oorzaak van neuronale schade kunnen verbeteren.

Conclusies P.falciparum-infectie is de leidende oorzaak van toevallen bij kinderen in landen waar malaria endemisch is. De toevallen variëren van eenvoudige koortsstuipen tot complexe, focale, langdurige of multipele toevallen en zij zijn een belangrijke risicofactor voor het ontstaan van neurologische en cognitieve beperkingen bij kinderen. Het risico op toevallen neemt toe met toenemende parasitemie maar de rol van genetische gevoeligheid voor toevallen die aan malaria te wijten zijn, is nog onduidelijk. Toekomstige studies naar het verband tussen acute toevallen en polymorphismen in ionen kanalen en de rol van profylactisch toegediende anticonvulsiva ter voorkoming van recidiverende toevallen en van neuro-cognitieve beperkingen, zullen ons begrip van de pathogenese vergroten en helpen bij het ontwerpen van interventies. Mogelijk kan in de toekomst de uiteindelijke uitkomst verbeterd worden door neuro-protectieve, adjuvante therapie met geneesmiddelen als erythropoietine of vergelijkbare middelen.

Referenties

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Proefschrift.indb 197 14-1-2008 13:09:30 Proefschrift.indb 198 14-1-2008 13:09:30 ACKNOWLEDGMENTS I am extremely grateful to Prof Charles Newton, my supervisor and mentor, who initiated these projects and through him, the Wellcome Trust. His fellowship from the Trust funded most of my training and the studies in this thesis. His insight, availability, generosity and commitment have been total. Through his efforts, I have not only been able to undertake this PhD but was also able to start training in Paediatric Neurology. I am also very grateful to my other two supervisors; Prof Brian Neville and Prof Piet Kager whose insight into the pathogenesis of disease and using data to understand it and the speed and detail with which they have helped improve my drafts has been amazing. Prof Kager has also had to take on all the administrative challenges of the PhD. To you all, I say thank you very much.

I would like to thank the Kenya Medical Research Institute and the Wellcome Trust Collaborative Research Programme in Kenya for providing me with the opportunity to undertake this PhD. In particular, I am grateful to the Centre Director Dr Norbert Peshu, the Scientific Director Prof Kevin Marsh and the clinical, scientific, administrative and support staff in the Centre for Geographic Medicine Research for making me feel so welcome and at home in Kenya. Kilifi has been a wonderful place and time well spent. I want to particularly thank the children and the parents/guardians who took part in these studies. May the souls of those who died rest in peace. In addition, I am grateful to the Director, Mulago Hospital, Uganda and the Head, Department of Paediatrics for giving me time off to undertake this work. I would also like to thank the International Brain Research Organization that provided me with travel fellowships to attend specialized “IBRO Schools” and present some of this work.

Apart from my supervisors, there are other people who worked hard to bring this work to what it is today. Chief among them is Dr Samson Gwer who spent many sleepless nights hooking up patients on monitors, dopplers and following them up, keeping so many records and fighting with machine failures together with Godfrey Otieno and Edwin Chengo. In the labs, Dr Thomas Williams and his team, Sophie Uyoga and Alex Macharia; Robert Mwakesi, Barnes Kitsao, Oscar Kai and Nimmo Gicheru; in the wards Anderson Kahindi, Mwanamvua Boga, Siti Ndaa, Mercy Mwaboza, Sidi Kazungu, Esther Kivaya, Japhet Karisa and the rest of the nursing team in KEMRI Ward; the fieldworkers both in KEMRI ward and in Ward one; the Social and Behavioural team and the assessment centre team led by Judy Tumaini and Ken Rimba; the records staff Ramos and Josephine Kazungu who spent hundreds of hours storing and retrieving files; the data management team Tony Kazungu, Michael Kahindi and Rachael Odhiambo; and the men and women who deal with numbers Greg Fegan, Hellen Gatakaa, Benedict Orindi and Lazarus

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Proefschrift.indb 199 14-1-2008 13:09:30 Mramba. Prof Kevin Marsh was insightful in his talks to PhD students while Dr Steve White of the Department of Neurophysiology, Great Ormond Street Hospital for Sick Children, was instrumental in teaching me the fundamentals of EEG.

I shared many moments with colleagues Drs Sadik Mithwani and Kathryn Maitland, my fellow PhD students in particular, Michael Kihara, Cleopatra Mugyenyi, Simon Muchohi and Rachael Opiyo, medical Officers, Moses Ndiritu, Ismail Ahmed, James Ignas, Samuel Aketch, Michael Mwaniki and Mohammed Shebbe, my office mates Faith, Margaret and Vandana, Reuben Orwaru and many friends with whom we watched football, got thrilled as we won and agonized when we lost matches and, the community of St Patrick’s Catholic Church, Kilifi.

I want to thank my wife Catherine for standing in for me and playing daddy’s role when our children Ignatius, Philomena, Priscilla and Victoria could not understand where daddy was, for bearing my absence with them even during the very difficult events at home and supporting me through the four years.

Lastly to HIM who never fails and is more than able to accomplish in us more than we can ever think, I am very grateful.

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Proefschrift.indb 200 14-1-2008 13:09:30 CURRICULUM VITAE Dr Richard Idro was born in Moyo district on 9th May 1970, in North Western Uganda. He attended Moyo Boys Primary School from 1976–83. His ordinary (1984-87) and advanced (1988-90) level Secondary School education was in St Joseph’s College Ombaci in neighbouring Arua district. In 1990, he enrolled at Makerere University Medical School for a 5-year Bachelor of Medicine and Bachelor of Surgery Degree course, which he completed in 1995. After 1 year in internship and 2 years in a district hospital, he studied in Makerere University Medical School for a 3-year Master of Medicine (Paediatrics) degree course from 1998-2001. He currently works with the Kenya Medical Research Institute Centre for Geographic Medicine Research – Kilifi in Coastal Kenya where he undertook the PhD Research projects and with Mulago Hospital/Makerere University Department of Paediatrics and Child Health, Kampala, Uganda.

Other papers Idro R, Bitarakwate E, Tumwesigire S, John CC. Clinical manifestations of severe malaria in the highlands of South Western Uganda. American J Tropical Medicine and Hygiene, 2005; 72(5): 561–67. Idro R, Aloyo J, Mayende L, Bitarakwate E, John CC, Kivumbi GW. Severe malaria in children in areas with low, moderate and high transmission intensity in Uganda. Tropical Medicine and International Health, 2006; 11(1): 115 – 24. John CC, Opika-Opoka R, Byarugaba J, Idro R, Boivin MJ. Low levels of RANTES are associated with mortality in children with cerebral Malaria. Journal of Infectious Diseases 2006 Sep 15; 194(6): 837-45. Akech S, Gwer S, Idro R, Fegan G, Eziefula AC, Newton CRJC, Levin M, Maitland K. Volume expansion with albumin compared to Gelofusine in children with severe malaria: Results of a controlled trial. PLoS Clinical Trials 1(5):e21. DOI: 10.1371/journal. pctr. 0010021. Idro R, Aketch S, Gwer S, Newton CR and Maitland K. Research priorities in the management of severe malaria in children. Annals of Tropical Medicine and Parasitology, 2006; 100 (2):95-108. Bangirana P, Idro R, John CC, Boivin MJ. Rehabilitation for cognitive impairments after cerebral malaria in African children: strategies and limitations. Tropical Medicine and International Health 2006 Sep; 11(9): 1341-9. Boivin MJ, Bangirana P, Byarugaba J, Opoka RO, Idro R, Jurek AM, John CC. Cognitive impairment after cerebral malaria in children: a prospective study. Pediatrics 2007 Feb; 119 (2): e360-6. Epub 2007 Jan 15.

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