SEIZURES IN CHILDHOOD CEREBRAL MALARIA

A thesis submitted to the University of London for the degree of Doctor of Medicine

June 2001

Jane Margaret Stewart Crawley MB BS, MRCP

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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT

Every year, more than one million children in sub-Saharan Africa die or are disabled as a result of cerebral malaria. Seizures complicate a high proportion of cases, and are associated with an increased risk of death and neurological sequelae. This thesis examines the role of seizures in the pathogenesis of childhood cerebral malaria.

The clinical and electrophysiological data presented suggest that seizures may contribute to the pathogenesis of coma in children with cerebral malaria. Approximately one quarter of the patients studied had recovered consciousness within 6 hours of prolonged or multiple seizures, or had seizures with extremely subtle clinical manifestations. EEG recording also demonstrated that electrical seizure activity arose consistently from the posterior temporo-parietal region, a “watershed” area of the brain that is particularly vulnerable to hypoxia. Cerebral computerised tomography (CT) scans from children who had multiple seizures during their clinical course, and who subsequently developed a hemiplegia, revealed infarction in this area.

The thesis discusses the aetiology of seizures in cerebral malaria, and explores the hypotheses that chloroquine may precipitate seizures, and that some of the neurological manifestations of cerebral malaria may be explained by an excitotoxic mechanism.

If anticonvulsant prophylaxis can reduce the incidence of seizures complicating cerebral malaria, this may in turn reduce the risk of neurological sequelae and death. The thesis describes a randomised, controlled intervention study, in which a single intramuscular dose of phenobarbitone 20mg/kg reduced the incidence of seizures by 50% but was associated with an unacceptable doubling of mortality. Post hoc analysis revealed that mortality was highest among those who had been treated with phenobarbitone and multiple doses of diazepam, raising the possibility that respiratory depression was responsible for the increased mortality in the phenobarbitone-treated group. The thesis concludes with a discussion of alternative strategies for anticonvulsant prophylaxis. TABLE OF CONTENTS

Chapter and contents Page

List of tables 7 List of figures 8 Acknowledgements 9 Dedication 11

1. BACKGROUND 12 Introduction 13 Human malaria parasites 13 Epidemiology of malaria caused by P. falciparum 15 The burden of disease 15 The natural history of P. falciparum infection 18 The clinical features of falciparum malaria 20 Uncomplicated malaria 20 Severe malaria 21 Impaired consciousness 23 Cerebral malaria 23 The pathophysiology of cerebral malaria 26 Sequestration of parasitised erythrocytes 27 Cytokines 28 Nitric oxide 29 Raised intracranial pressure 30 Seizures and status epilepticus 32 Definitions 32 Classification 32 Neurobiology of seizures and status epilepticus 33 The emergency treatment of status epilepticus 40 Page

Outcome of status epilepticus in children 42 Seizures as a complication of childhood malaria 44 Local perception of seizures 44 Seizures in uncomplicated malaria 45 Seizures and cerebral malaria 45 Aims of this thesis 46

2. METHODS 47 Introduction 48 Study site 48 Patients 51 Clinical assessment 52 Laboratory investigations 53 Antimalarial and antibiotic chemotherapy 53 Supportive care 54 Ethical permission 54 Data collection and management 54 Statistical analysis 55

3. CLINICAL and ELECTROPHYSIOLOGICAL 56 FINDINGS Introduction 57 Methods 57 Electrophysiology 57 Intracranial pressure monitoring 58 Anticonvulsant treatment 58 Follow up 58 Page

Results 60 Admission characteristics 60 Clinical course 60 Electrophysiology 63 Intracranial pressure monitoring 69 Outcome 71 Discussion 75

4. AETIOLOGY: Is chloroquine a risk factor? 79 Introduction 80 Methods 81 Results 85 Discussion 87

5. AN EXCITOTOXIC MECHANISM for 89 CHILDHOOD CEREBRAL MALARIA? Introduction 90 Methods 93 Results 97 Discussion 98

6. PHENOBARBITONE PROPHYLAXIS: 101 A randomised, controlled intervention study Introduction 102 Methods 103 Results 107 Recruitment 107 Clinical tolerance 107 Page Pharmacokinetics 107 Admission characteristics 108 Clinical course 111 Seizures 111 Death 111 Neurological sequelae 116 Discussion 116

7. CONCLUSIONS 119 Introduction 120 The role of seizures in the pathogenesis of cerebral malaria 120 Seizures and neurological sequelae 121 The aetiology of seizures in cerebral malaria 122 Intervention studies 124 In conclusion 125

REFERENCES 126

APPENDIX 160 Declaration 161 Collaborators 161 Ethical approval and consent 162 List of publications 164 Study forms 165 Reprints of 5 selected publications

MATERIAL NOT BOUND WITH THESIS CD-ROM: Seizures in childhood cerebral malaria LIST OF TABLES

Table Page

Table 1. Frequency of severe manifestations of 19 P. falciparum malaria

Table 2. Practical classification of severe malaria 22 in children

Table 3. The Blantyre coma scale 24

Table 4. Admission clinical and laboratory features 59 by outcome

Table 5. EEG findings from children who died 72

Table 6. EEG and CT scan results from children 73 with sequelae

Table 7. Comparison of baseline admission characteristics 83

Table 8. Baseline drug levels, status epilepticus 84

Table 9. Cerebrospinal fluid metabolite concentrations 95

Table 10. Cerebrospinal fluid metabolite concentrations 96 by outcome

Table 11. Admission clinical features by treatment group 109

Table 12. Admission laboratory features by treatment group 110

Table 13. Clinical outcome according to treatment group 112

Table 14. Mortality according to number of doses of 115 diazepam LIST OF FIGURES

Figure Page

Figure 1. Life cycle of malaria parasite 14

Figure 2. Global distribution of malaria 16

Figure 3. Map of Kenya showing Kilifi district 49

Figure 4. Left posterior temporo-parietal discharge 62

Figure 5a. Background slow activity 65 5b. Normal EEG for comparison 65

Figure 6. Asymmetric EEG 66

Figure 7a. Recruiting rhythm 67 7b. Onset of generalised seizure 67

Figure 8. CFAM tracing showing multiple short 68 generalised seizures

Figure 9. Burst suppression 68

Figure 10a. Slow-wave activity in association with opisthotonic 70 posturing 10b. EEG 10 hours later 70

Figure 11 Cerebral CT scan showing infarction 74

Figure 12. Metabolic inter-relationships 92

Figure 13. Phenobarbitone concentrations 106

Figure 14a. Kaplan-Meier plot of coma resolution 113 Figure 14b. Kaplan Meier plot of overall survival 113 ACKNOWLEDGEMENTS

The work presented here would not have been possible without the help of a large number of people. I am enormously grateful to all of them, but would particularly like to thank the following individuals:

I have been exceptionally fortunate with my scientific advisors. I could not have had a better supervisor than Professor Kevin Marsh, the Scientific Director of the KEMRI- Wellcome Trust Clinical Research Unit in Kilifi. His considerable scientific expertise has been invaluable throughout the planning, execution, and presentation of these studies. In addition, over the past eight years he has consistently provided me with practical support and fi’iendship. It is largely because of his leadership that the Kilifi Clinical Research Unit is such a productive and happy place. Dr Fenella Kirkham, my University of London advisor, has also been enormously generous with her time and expertise, for which I am extremely gratefiil.

All of the studies were performed on the KEMRI Research Ward at Kilifi District Hospital, and I am hugely grateful to all the nursing staff, whose dedication and skill made the studies possible, and whose sense of humour made them enjoyable: Gladys Binns, Anderson Kahindi, Steve Wale, Boniface Mwambi, Naomi Lewa, Elizabeth Mwangi, Mercy Mwabonje, Susan Mwangi, Simon Kenga, Janet Mwaro, Judith Peshu, Ann Muhoro, and the ward assistant, Connie Dama. In addition. Sister Kabibu and her team on Ward One continue to provide an excellent clinical service for the children of Kilifi District.

I have received a great deal of support fi-om my clinical colleagues in Kilifi, who have taken on the extra work of these studies with professionalism and humour. I would particularly like to thank Drs Catherine Waruiru and Michael English for all their support and fi’iendship over the years. With great patience. Dr Charles Newton taught me how to insert a subarachnoid catheter, which made it possible to monitor intracranial pressure. Dr Penny Holding used her skills as an educational psychologist to set up the developmental follow-up for the children in the phenobarbitone prophylaxis study. Brett Lowe ran an extremely efficient laboratory service, while Tom Oluoch and his staff provided excellent computing support. Drug assays were carried out at the Wellcome Trust Laboratories in Nairobi, under the leadership of Dr Bill Watkins and Professor Gilbert Kokwaro. I am extremely grateful to Helen Watkins and Pauline Lowe, for coping so efficiently with the vital but often thankless task of unit administration in Nairobi and Kilifi respectively. None of the work would have been possible without the negotiating skills of Dr Norbert Peshu, the Head of the Kilifi Research Unit, who has ensured that the relationship between the Ministry of Health and the Kilifi Research Unit remains good.

Several other people provided invaluable help. I was fortunate to have expert advice from Dr Shelagh Smith, of the Institute of Neurology in London, when purchasing an EEG machine. Dr Smith subsequently spent a considerable amount of time interpreting all of the EEG recordings. Peter Muthinji, Chief Technician at the EEG Department of the Kenyatta National Hospital, Nairobi, provided me with excellent practical training in electroencephalography. Dr Robert Surtees of the Institute of Child Health in London kindly arranged for the assays of quinolinic acid, pterins, and nitric oxide metabolites to be carried out in his laboratory. Professor Peter Winstanley provided much helpful advice during the design of the phenobarbitone prophylaxis trial, and Dr Nick Day and Professor Tim Peto subsequently provided invaluable statistical support. Professor Nick White is responsible for my initial, and continuing, interest in tropical medicine. The Kilifi Clinical Research Unit receives financial support from the Wellcome Trust, and I have received much helpful advice and encouragement from Professor Robert Howells and Dr Catherine Davies.

Finally, I would like to thank my parents, who have provided me with so much encouragement and help during the writing of this thesis, and who continue to support me in everything I do.

10 DEDICATION

This thesis is dedicated to the memory of my brother Patrick, who explored so much of Kenya with me

11 1. BACKGROUND

12 Introduction

Almost all of the 1 to 2 million deaths from severe malaria that occur worldwide each year are caused by Plasmodium falciparum (WHO 1999a). Cerebral malaria, one of the most serious clinical manifestations, is associated with a mortality of up to 30%, while approximately 10% of survivors are left with permanent neurological sequelae (Molyneux 1989; Bondi 1992; Waller 1995). Seizures complicate a high proportion of cases and, when prolonged and multiple, are associated with an increased risk of death or neurological sequelae (Brewster 1990; Jaffar 1997; van Hensbroek 1997). This thesis examines the role of seizures in the pathogenesis of childhood cerebral malaria.

The introductory chapter will provide a broad overview of the epidemiology and clinical features of Plasmodium falciparum, with particular emphasis on cerebral malaria. A review of the classification and neurobiology of seizures will be followed by an outline of the treatment and outcome of status epilepticus. The chapter will end with a description of the prevalence and outcome of seizures in both uncomplicated and severe malaria, and an outline of the central questions that this thesis will address.

Human malaria parasites

Malaria results from infection with one or more of the four species of protozoal plasmodia {Plasmodium falciparum, P. vivax, P. ovale, and P. malariae) that infect humans. Transmission follows the bite of a female Anopheles mosquito, and the inoculation into the bloodstream of saliva containing infectious sporozoites (Figure 1). Sporozoites circulate in the blood for approximately 30 minutes, before entering hepatocytes or being removed by phagocytic cells. Pre-erythrocytiç schizogony in the liver culminates in the release of thousands of merozoites into the bloodstream. Unlike Plasmodium falciparum and P. malariae, P. vivax and ovale have the ability to differentiate into hypnozoites, which lie dormant in the liver, and may therefore cause clinical relapses of malaria many years after the initial infection.

13 Figure 1. Life cycle of the malaria parasite

Oocyst Ookinete Zygote

Sporogony Sporozoites ^ I Fertilization

r O" Gamete & Idevelopment l^squfto

Human Gametocyte formation Liver y ^ trophozoite Merozoites^ Schizont

Blood cycle ^ Merozoites

Schizont (^Trophozoite

14 Once released from the liver, the merozoites invade red blood cells, where they develop into ring forms (trophozoites). Trophozoites are able to metabolise haemoglobin within the red blood cell, producing malaria pigment (haemozoin). Mature trophozoites (schizonts) undergo a second stage of asexual multiplication, producing a species-specific number of merozoites. Although schizonts of P. vivax, P. ovale, and P. malariae may be seen in the peripheral blood, in P. falciparum infections they are preferentially sequestered within the microvascular beds of internal organs. Subsequent rupture of the red blood cell causes the release of merozoites into the bloodstream, and the cycle is repeated (approximately every 48 hours for P. falciparum) until increasing parasitaemia is inhibited by immunity, chemotherapy, or by the death of the host.

After several cycles of erythrocytic schizogony, some merozoites differentiate into sexual forms (gametocytes), which are ingested by the female Anopheles mosquito at the time of a blood meal. The male and female gametocytes fuse to form a zygote, which penetrates the stomach wall of the mosquito. Here, a further stage of asexual reproduction results in the release of approximately 1,000 sporozoites, which migrate through the body cavity of the mosquito to the salivary gland. The entire life cycle is repeated when the mosquito takes a further human blood meal.

Epidemiology of malaria caused by Plasmodium falciparum

The burden of disease Over 40% of the world’s population live where there is a risk of malaria (Figure 2), and approximately 300 million clinical cases occur worldwide each year (WHO 1999b). At least 90% of the 1.2 million deaths caused by severe falciparum malaria occur in sub- Saharan Africa, where malaria accounts for one in five of all deaths in children below the ] age of five years. The populations most at risk are those for whom poverty, isolation, and i inadequate health services mean that there is considerable delay in obtaining appropriate treatment. Malaria is a major impediment to the development and economic advancement

15 Figure 2. Global distribution of malaria

■ High nsk of malana □ Low risk of malaria

16 of countries within tropical Africa, and recent research suggests that the economic burden of malaria in Africa is greater than 1% of gross domestic product (GDP) (Nchinda 1998).

The recent resurgence in malaria-related deaths in Africa contrasts dramatically with the global decline in mortality from malaria since 1900. The most important contributory factor is the rapid spread of antimalarial drug resistance (Trapo 1998). Resistance to chloroquine is now widespread throughout East and Southern Africa, and is becoming an increasing problem in parts of West Africa. Several countries in East and Southern Africa have been forced to change from chloroquine to sulphadoxine-pyrimethamine (SP) as their first-line treatment for falciparum malaria, yet resistance to SP is increasing fast. Recent data from the East African Network for the Monitoring of Antimalarial Treatment (EANMAT) shows that in Kenya, for example, up to 55% of patients treated with chloroquine and 5-15% of those treated with sulphadoxine-pyrimethamine (SP) have evidence of treatment failure (B.Watkins personal communication). This is extremely worrying, since the cost of any second-line alternatives far exceeds the health budget of most African countries (White 1999b; White 1999d).

But the situation is not hopeless. Malaria is now a major health priority for many countries and international agencies, and considerable amounts of public and private money have been committed to research and control. The World Health Organisation (WHO) has responded to this current crisis with its “Roll Back Malaria” initiative and, at the recent summit in Japan, G8 countries made a commitment to help WHO halve the burden of disease associated with malaria by the year 2010. Insecticide-impregnated bed nets have been shown to reduce all-cause mortality in children below the age of five years by up to 30% (D'Alessandro 1995; Nevill 1996; Lengeler 2000), although it is not clear what the long-term effects of reducing transmission will be (Snow 1997). Over the last decade, advances in molecular technology have resulted in considerable progress towards the development of a malaria vaccine (Riley 1997; Miller 1998).

In response to the drug resistance crisis, the Medicines for Malaria Venture, a joint public-private sector initiative which forms part of the Multilateral Initiative on Malaria

17 in Africa (Davies 1999), is planning to develop new anti-malarial drugs and drug combinations and to make them available in poor countries. Current research is being directed at the development of new anti-folate combinations, including chlorproguanil- dapsone (Amukoye 1997), which has a shorter elimination half-life than SP and is therefore less likely to induce resistance. There is also considerable interest in the development of combination therapy, using the rapidly acting artemisinin derivatives in combination with other antimalarial drugs (White 1999a; White 1999c).

The natural history of Plasmodium falciparum infection: parasitaemia versus disease In contrast to other infectious diseases, complete immunity to malaria (aparasitaemia plus clinical protection) is probably never achieved, even in settings where individuals have been exposed since birth to continuous, intense malaria transmission (Trape 1994). Clinically “useful” malarial immunity therefore constitutes protection from severe clinical illness, and not the absolute absence of parasites.

Intensity of malaria transmission appears to affect the pattern of both clinical disease (Snow 1994a; Snow 1997) and mortality (Greenwood 1987; Brinkmann 1991; Marsh 1992). In areas of stable transmission, children bear the brunt of malarial morbidity and mortality, with attacks of clinical malaria evident from the age of about four months, and the highest mortality occurring between the ages of one and three years. In areas of very high transmission, the majority of severe cases presenting to hospital are young infants with malarial anaemia, while in lower transmission settings there appears to be an increased proportion of older children with cerebral malaria (Snow 1994a). The subsequent gradual acquisition of acquired immunity means that most individuals above the age of five years will no longer be at risk of death. Infected adults may present with mild symptoms (headache, fever, arthralgia), or remain asymptomatic, and therefore provide a reservoir of infection for subsequent transmission to non-immune individuals. Although non-immune adults do develop life-threatening complications of malaria, the clinical presentation varies in some respects from that of African children (see Table 1).

18 Table 1. Frequency of severe manifestations of Plasmodium falciparum malaria in children and adults

* See Table 3

Frequency Clinical or laboratory Definition African SE Asian feature children adults

Prostration Inability to sit unassisted, or to drink in +++ -H- the case of children too young to sit

Impaired *Blantyre coma score 3 or 4, but able + + + ++ consciousness to localise painful stimulus

Coma *Blantyre coma score 2 or less; unable -t-H- ++ to localise painful stimulus

Multiple seizures 2 or more seizures within 24 hours ++ + +

Deep breathing (Kussmaul respiration) Respiratory distress +-H- + and indrawing of lower chest wall

Severe anaemia Haemoglobin <5 g/dl -HH- +

Hypoglycaemia Blood glucose <2.2 mmol/1 + + + Yellow colouration of sclerae and Jaundice + ++ + buccal mucosae Renal failure Urine output of <12 ml/kg/24 hours + ++ +

Chest x-ray: increased interstitial Pulmonary oedema markings, hazy peri-hilar interstitial +/- ++ shadowing

Dark red/black urine, positive for blood Haemoglobinuria on urinanalysis dipstick. Absence of + + microscopic haematuria

Abnormal bleeding From gums, nose, venepuncture sites +/- +

Shock Hypotension, cool peripheries + +

19 In situations of high transmission intensity, where a significant proportion of the population may have eisymptomatic P. falciparum parasitaemia, it becomes difficult to make a definitive diagnosis of malaria. In Kilifi, Kenya, where the studies described in this thesis were carried out, community parasite prevalence rates in children aged one to nine years range from 49% to 74% (Snow 1997). In a setting such as this, therefore, it is not appropriate to base a diagnosis of falciparum malaria solely on the results of a blood smear demonstrating malaria parasites. Although diagnostic specificity may be increased by the use of additional criteria, such as measured fever, this may be at the cost of reducing sensitivity (Armstrong Schellenberg 1994). Diagnostic specificity is, however, dramatically increased by the presence of clinical symptoms and signs known to be associated with severe malaria. It is, therefore, reasonable to diagnose severe malaria in any child with parasitaemia who fulfils the appropriate diagnostic criteria, and in whom other possible diagnoses have been excluded.

The clinical features of falciparum malaria

Uncomplicated malaria The clinical features of uncomplicated malaria are non-specific. Symptoms include fever, vomiting, diarrhoea, and headache, while the presence of persistent vomiting, convulsions, or severe anaemia indicates the need for hospital referral. The key factor is to always consider the diagnosis of malaria in any patient from an endemic area, and to institute prompt treatment with an appropriate dose of an effective antimalarial drug. The danger of delayed or inadequate treatment of uncomplicated malaria is the risk of progression to severe, life-threatening disease.

It is important to remember that, in Africa, the vast majority of patients suffering from febrile illnesses are treated at home with medication bought from a shop or local dispensary. In Kilifi district, a community-based training programme for shopkeepers significantly increased the number of transactions involving the sale of an appropriate dose of chloroquine for the treatment of childhood fevers (Marsh 1999). This approach clearly has considerable potential as an innovative malaria control strategy. Presumptive treatment for

20 malaria also occurs in health clinics throughout Africa, since the lack of equipment and personnel precludes the use of microscopy.

Severe malaria Any patient with malaria who is unable to tolerate oral medication, has evidence of vital organ dysfunction, or a high parasite count, is at an increased risk of dying (WHO 2000). The exact risk depends upon the age and background immunity of the patient, and on access to appropriate treatment. Since over 50% of hospital deaths from severe malaria occur within the first six hours of admission, immediate assessment and treatment are mandatory.

Table 1 lists the clinical features that are indicative of severe malaria in patients with P. falciparum asexual parasitaemia and no other clearly defined cause for their symptoms. It is clear that the clinical manifestations of severe malaria vary considerably between adults and children. Jaundice and renal failure are prominent features of severe malaria among Southeast Asian adults (Trang 1992; Wilairatana 1994), while convulsions, respiratory distress, and severe anaemia are characteristic of severe disease in children (Molyneux 1989; Marsh 1995; Waller 1995; Allen 1996). Since African children account for over 90% of all deaths from severe malaria, it is useful to classify this group further according to prognosis, in order to identify those at highest risk of dying. Recent research has identified two clinical features, namely respiratory distress (deep breathing, shown to be strongly associated with metabolic acidosis (English 1996a)) and impaired consciousness, that are associated with a greatly increased risk of death (Marsh 1995). Table 2 (WHO 2000) incorporates these findings into a practical classification, likely to be of particular value in situations where limited resources need to be directed at the most severely ill children.

21 Table 2. Practical classification of severe malaria in children

* See Table 3

Group 1

Children at increased risk of dying who require immediate parenteral antimalarial drugs and supportive therapy

a. Prostrated children: Three subgroups of severity should be distinguished

Prostrate but fully conscious Prostrate with impaired consciousness but not in coma (*Blantyre coma score 3 or 4) Coma (the inability to localise a painftil stimulus)

b. Respiratory distress (acidotic breathing)

Mild: sustained nasal flaring and mild intercostal indrawing (recession) Severe: the presence of either marked indrawing (recession) of the lower chest wall or deep (acidotic) breathing

Group 2

Children who show none of the above features, but who require supervised management because of the risk of clinical deterioration

a. Children with a haemoglobin of <5 g/dl

b. Children with 2 or more seizures within a 24-hour period

Group 3

Children who require parenteral treatment because of persistent vomiting, but who lack any specific clinical or laboratory features of groups 1 or 2 (above)

22 Impaired consciousness The conscious level of children with severe malaria is most conveniently assessed using the Blantyre Coma Scale (BCS) (Molyneux 1989). This scale (Table 3), a modified version of the Glasgow Coma Scale used for assessing level of consciousness in adults (Teasdale 1974), is based on the verbal, motor, and ocular responses to a painful stimulus. The BCS is simple, easy to leam, does not depend upon a child’s ability to speak, and gives a good overall assessment of disease severity (Newton 1997a). It is, however, of limited use in children below the age of 8 months, who have not yet acquired the developmental skills necessary to localise a painful stimulus (Newton 1997a). Furthermore, parts of the assessment (such as the decision as to whether a cry is “appropriate” or not) are essentially subjective, and the BCS requires local standardisation in order to minimise the effect of inter-observer variation.

Children who score 4 or less on the BCS are said to have impaired consciousness, while those who are unable to localise a painful stimulus (in whom the BCS will generally be 3 or less) fulfil the definition of unrousable coma. Children in unrousable coma who have P. falciparum parasitaemia and no other apparent cause for their encephalopathy are considered to have cerebral malaria. It is important to appreciate, however, that “impaired consciousness” and “cerebral malaria” merely describe the spectrum of a clinical syndrome that may have many causes. Consciousness is usually impaired after a convulsion or following the administration of anticonvulsant drugs, and in these situations it is conventional to allow one hour to elapse before assessing the BCS. Similarly, it is not appropriate to apply the term cerebral malaria to children in whom normal consciousness is restored by the administration of glucose.

Cerebral malaria Afi*ican children with cerebral malaria typically present with a short (1-3 day) history of fever, accompanied in approximately 50% of cases by vomiting, and in up to 80% of cases by seizures (Molyneux 1989; Steele 1995; Waller 1995; Crawley 1996; Olumese 1999). In these series, children were unconscious for a median of 8 hours (range 1-72

23 Table 3. A coma scale for children (Blantyre coma score) (Molyneux et al. Quarterly Journal o f Medicine (1989); 71 (265): 441-459)

Clinical response Score

Best motor response

Localises painful stimulus* 2 Withdraws limb from pain** 1 Non-specific or absent response 0

Verbal response

Appropriate cry 2 Moan or inappropriate cry 1 None 0

Eye movements

Directed (follows mother’s face) 1 Not directed 0

TOTAL Maximum 5 (frilly conscious) Minimum 0 (deep coma)

* Painful stimulus: rub knuckles on patient’s sternum

** Painful stimulus: firm pressure on thumbnail bed with horizontal pencil

24 hours) prior to hospital admission, and in over 50% of cases the onset of coma followed one or more seizures (Crawley 1996).

Clinical examination reveals the presence of brain stem signs (dysconjugate eye movements, decerebrate posturing) in approximately one third of cases (Molyneux 1989; Newton 1991; Newton 1997b; Olumese 1999). The studies described in this thesis have also led to the realisation that children with “cerebral malaria” who present with conjugate eye deviation, nystagmus, salivation, and hypoventilation may be in subtle status epilepticus. Treatment with anticonvulsant drugs may restore consciousness (Crawley 1996; Crawley 1998).

Abnormalities of the ocular fundus have been demonstrated by direct and indirect opthalmoscopy in over 50% of patients with cerebral malaria (Lewallen 1993; Lewallen 1999). Haemorrhages are the commonest finding, and occur in over one third of cases. These may range in number from 1 to more than 100 and in children, unlike adults, (Looareesuwan 1983a) are not associated with a poor outcome (Lewallen 1996). Papilloedema, found in only 6% of cases (Lewallen 1999), is associated with a relative risk of an adverse outcome (death or neurological sequelae) that is 5.2 times that of children without this finding (Lewallen 1993).

Most children with cerebral malaria recover consciousness within 24 hours of the start of treatment, and the majority go on to make a complete recovery (Molyneux 1989; Steele 1995; Waller 1995). Mortality in different series ranges from between 15-27% (Molyneux 1989; Marsh 1995; Waller 1995; van Hensbroek 1996a). Clinical features consistently shown to be associated with an increased risk of death include deep coma, respiratory distress, and multiple convulsions (Molyneux 1989; Marsh 1995; Jaffar 1997). Laboratory findings of poor prognostic significance include hypoglycaemia (blood glucose below 2.2 mmol/1), acidosis (capillary pH < 7.300), raised plasma lactate (venous lactate 5 mmol/1 or more), hyperparasitaemia (parasite density greater than 1 million/pl), and 20% or more of the total parasite count comprising mature trophozoites and schizonts (Taylor 1988; Krishna 1994; Marsh 1995; Waller 1995; Jaffar 1997).

25 Neurological sequelae occur in a median of 14% (range 11-23%) of survivors at the time of discharge from hospital (Molyneux 1989; Brewster 1990; Bondi 1992; van Hensbroek j 1997). Many of these children improve over the ensuing weeks, however, and 7-9% have \ sequelae that are still present one month after discharge. The commonest problems are hemiplegia, spastic quadriplegia, ataxia, blindness, and speech problems. Factors consistently associated with an increased risk of neurological sequelae are prolonged coma, multiple or prolonged convulsions, and young age. Two recent studies have addressed the possibility that children surviving cerebral malaria may have cognitive problems that could adversely affect their subsequent educational attainments (Muntendam 1996; Holding 1999). Both studies tested a group of children approximately 42 months after an episode of cerebral malaria, and compared their performance with a group of age-matched controls. One study (Holding 1999) demonstrated significant impairment in the ability to initiate, plan, and carry out tasks (the executive functions) among children who had previously had cerebral malaria, while the other study (Muntendam 1996) found no significant difference between the two groups. Further studies are needed to clarify this issue.

The pathophysiology of cerebral malaria

Although the pathological features of cerebral malaria have been described in numerous studies (Edington 1967; MacPherson 1985; Pongponratn 1991; Patnaik 1994), marked heterogeneity between these studies has made their interpretation difficult. The introduction of standardised WHO clinical diagnostic criteria, and recent developments in electron microscopy (EM), immunohistochemistry, and molecular biology have now made it possible to address specific questions regarding the pathogenesis of cerebral malaria (Turner 1997; Newton 1998a). One should note, however, that although the pathological hallmark of cerebral malaria is sequestration of parasitised erythrocytes in the deep microvasculature of the brain (see below), the cause of coma remains unknown. In addition, any pathogenic mechanism needs to explain how, in the vast majority of cases, coma resolves spontaneously, without residual neurological sequelae.

26 Sequestration of parasitised erythrocytes Post-mortem histology and electron microscopy of brain tissue from patients with cerebral malaria shows that the cerebral capillaries and post-capillary venules are distended by numerous parasitised erythrocytes, attached by electron dense “knob proteins” to cerebral endothelial cells. This process, sequestration, is mediated through cytoadherence of parasitised erythrocytes to the endothelial lining of the cerebral micro vasculature (Berendt 1994b). A number of host molecules, including thrombospondin, intracellular adhesion molecule-1 (ICAM-1), CD36, and E-selectin, are able to bind parasitised erythrocytes, and therefore act as endothelial ligands. Immunohistochemical studies on brain tissue from adults dying of cerebral malaria have demonstrated increased expression of ICAM-1 and E-selectin, with significant co­ localisation of sequestered parasitised erythrocytes (Turner 1994). In a recent post­ mortem study on 50 Vietnamese adults, evidence of cerebral sequestration was observed in all patients with cerebral malaria (G Turner personal communication).

Adhesion of infected erythrocytes to endothelial ligands is mediated by parasite derived antigens expressed on the red cell surface, of which one of the most important is Pfempl {P. falciparum erythrocyte membrane protein 1) (Newbold 1997b). These antigens are encoded by a large family of Var genes, which have the ability to undergo clonal antigenic variation (Kyes 1997). It is hoped that future studies of ligand-binding var gene sequences will help elucidate the mechanisms underlying the variation in cytoadherence phenotype between parasite strains. The increased capacity of parasites causing cerebral malaria to bind to ICAM-1 (Newbold 1997a) supports the hypothesis that severity of clinical disease is associated with specific parasite cytoadherence phenotypes. Further evidence comes from the recently demonstrated association between severe malaria and specific receptor-binding phenotypes, namely those capable of binding immunoglobulin, heparin, and soluble CD31 (K Marsh personal communication), and those involved in rosette formation (the adherence of parasitised erythrocytes to two or more uninfected erythrocytes in vitro) (Carlson 1990; Treutiger 1992; Rowe 1995).

27 Although sequestration appears to be a central event in the pathogenesis of cerebral malaria, the clinical picture cannot be explained satisfactorily by a single mechanism (Berendt 1994a; Clark 1994). Some authors propose that sequestration is merely an epiphenomenon, and that the rapidly reversible symptoms of cerebral malaria may be mediated by soluble circulating factors such as cytokines or nitric oxide (Clark 1991; Clark 1992). It seems more likely that sequestration of parasitised erythrocytes initiates a chain of events that includes the release of soluble mediators from parasitised erythrocytes or host cells in the brain, leading ultimately to coma.

Cytokines The concept that severe malaria may be mediated by the products of activated macrophages (Clark 1981) arose from the observation that clinical similarities exist between severe malaria and endotoxaemia. There is now a considerable body of clinical and laboratory evidence to suggest that cytokines play an important role in the pathogenesis of severe malaria. Soluble antigens of P. falciparum, which share many of the properties of bacterial lipopolysaccharide, are potent inducers of TNF release from monocytes in vitro (Kwiatkowski 1989; Taverne 1990). Several clinical studies have now confirmed the positive association between plasma concentrations of TNF and mortality from severe malaria (Grau 1989; Kern 1989; Kwiatkowski 1990; Krishna 1994). More recently, a large case-control study demonstrated that Gambian children with a genetic polymorphism in the TNF promoter region had a seven-fold increase in the risk of death or neurological sequelae from cerebral malaria (McGuire 1994). Plasma concentrations of the pro-inflammatoiy cytokines interleukin (IL)-l (Kwiatkowski 1990), IL-6 (Kern 1989), and IL-8 (Friedland 1993) have been shown to be correlated with disease severity, although there is considerable overlap between different patient groups. Severe malaria may also be associated with an inadequate negative feedback response to XL-10, which has been shown to counter-regulate the pro-inflammatory response to P. falciparum malaria (Ho 1995; Ho 1998).

There are a number of limitations to the hypothesis that cytokines per se play a major role in the pathogenesis of severe malaria. Firstly, plasma concentrations of pro-inflammatory

28 cytokines are very high in P. vivax malaria, particularly following schizogony in synchronous infeçtions (Karunaweera 1992), yet P. vivax is not lethal and does not cause coma. If the pro-inflammatory cytokines do play a central role in severe malaria, this is likely to be at a local level, and may not be reflected in systemic cytokine measurements. Finally, although anti-TNF antibody has been shown to be anti-pyretic (Kwiatkowski 1993), there was no evidence of clinical benefit when it was administered to Gambian children with severe malaria in a prospective, randomised trial (van Hensbroek 1996b).

Nitric oxide TNF is a potent inducer of nitric oxide synthase type 2 (N0S2), and investigators have hypothesised that, in cerebral malaria, nitric oxide (NO) may play a central role in the pathogenesis of coma (Clark 1992). High local concentrations of TNF may induce excessive synthesis of nitric oxide (NO) within cerebrovascular endothelial cells. NO diffuses across the blood-brain barrier, and may inhibit the glutamate-induced entry of calcium into post-synaptic neurones, so reducing the activity of calcium-dependent constitutive NO synthase. The consequent reduction in the level of NO within post- synaptic neurones could result in profound but reversible coma. The same mechanism has been postulated to underlie coma induced by general anaesthesia or ethanol (Clark 1992).

The testing of this hypothesis has centred round the measurement in plasma of the stable NO metabolites nitrate and nitrite (collectively termed reactive nitrogen intermediates, RNI). Results from studies adopting this approach have been contradictory. Some studies have found a positive correlation between plasma RNI and disease severity (Cot 1994; A1 Yaman 1996; Kremsner 1996), while others have shown either no association (Agbenyega 1997; Taylor 1998) or an inverse relationship (Anstey 1996). Discussion has focussed on the role of two potential confounding factors, namely dietary nitrogen and reduced clearance of RNI due to renal impairment. Anstey, who controlled for both in his carefully designed study, also measured N0S2, and found that levels were undetectable in children with cerebral malaria (Anstey 1996). He went on to demonstrate an age- related pattern of NO production among asymptomatic, malaria-exposed Tanzanian children that was the reverse of the age-related pattern of mortality from cerebral malaria

29 among that group (Anstey 1999). The elevation of NO production that was observed among infants and older children suggests that this may be a mediator of anti-disease immunity.

Raised intracranial pressure Raised intracranial pressure (ICP), which is a feature of many paediatric encephalopathies (Minns 1991), appears to play a more significant role in the pathogenesis of cerebral malaria in children compared to adults. At lumbar puncture, opening cerebrospinal fluid (CSF) pressures were found to be normal in non-immune adults with cerebral malaria (Warrell 1986), while cerebral computerised tomography (CT) (Looareesuwan 1983b) and magnetic resonance imaging (Looareesuwan 1995) suggested that, in this group of patients, cerebral oedema is a rare, agonal event. In a controlled study on adult patients with cerebral malaria, dexamethasone was associated with prolongation of coma and an increased complication rate (Warrell 1982). These findings suggest that, in adults, raised ICP does not appear to play an important role in the pathogenesis of cerebral malaria.

But the situation appears different in children. Cerebrospinal fluid opening pressure at lumbar puncture was raised in two studies on African children with cerebral malaria (Newton 1991; Waller 1991). In a subsequent study on 23 Kenyan children, ICP was directly measured by means of a subarachnoid catheter (Newton 1997b). Concurrent measurement of intra-arterial pressure meant that it was also possible to calculate cerebral perfusion pressure (CPP). All children had raised ICP, though in 10 cases this was mild (maximum ICP 10-20 mm Hg, minimum CPP > 50 mm Hg). Thirteen children had intracranial hypertension of a degree that would normally merit specific intervention. Of these, 9 had intermediate intracranial hypertension (maximum ICP > 20 mm Hg, minimum CPP 40 - 50 mm Hg for >15 minutes continuously), and in 4 cases this was severe (maximum ICP >40 mm Hg, minimum CPP < 40 mm Hg for >15 minutes continuously). Although intravenous mannitol controlled ICP in children with intermediate intracranial hypertension, it failed to prevent progression to an intractable state in children with severe intracranial hypertension, of whom two died (one with

30 clinical signs compatible with the medullary stage of transtentorial herniation), and two were left with severe neurological sequelae. Ten of these children were included in a study that compared the findings fi*om transcranial Doppler ultrasonography on 50 children with cerebral malaria with those fi'om 115 conscious Kenyan children (Newton 1996). In children with severe intracranial hypertension, a linear relationship was found between CPP and blood flow velocity, suggesting that cerebral autoregulation may have been impaired. Of four children who died, three had sonographic features compatible with progressive intracranial hypertension.

Intracranial pressure rises if there is an increase in the volume of cerebrospinal fluid, blood, or brain tissue. CT scans fi*om both adults and children with cerebral malaria showed no evidence of acute hydrocephalus (Looareesuwan 1983b; Newton 1994). Brain swelling (defined as the loss of cerebrospinal fluid spaces) was, however, documented on cerebral CT scans fi’om 6/14 Kenyan children recovering fi-om cerebral malaria, while conspicuously small ventricles were seen in two fiirther patients (Newton 1994). In two cases this appearance resolved completely, and the children went on to make a full recovery. In the remaining four cases, difftise brain swelling was associated with the presence of hypodense lesions and loss of grey/white differentiation, appearances consistent with the presence of cytotoxic oedema secondary to cellular damage. Severe intracranial hypertension had been documented in two of these children, and all four developed severe neurological sequelae. These findings suggest that, for the majority of children with cerebral malaria in whom intracranial hypertension is only mild or moderate, increased cerebral blood volume is likely to account for the brain swelling. Increased blood volume may result fi'om impaired venous drainage (secondary to sequestration), vasodilatation (secondary to acidosis or nitric oxide), reduced blood viscosity (secondary to anaemia) or increased cerebral blood flow (secondary to seizures). In the few children who develop severe intracranial hypertension, cellular damage and secondary cytotoxic oedema may result from reduced cerebral perfusion, hypoglycaemia, or uncontrolled seizure activity.

31 Seizures and status epilepticus: definitions and classification

Definitions The following definitions, formulated in 1993 by the International League against Epilepsy (ILAE), will be used throughout this thesis:

Epileptic seizure'. A transitory clinical manifestation of sudden onset, presumed to result from an abnormal and excessive discharge of a set of neurons in the brain. Clinical features, perceived by either the patient or an observer, can include an alteration of consciousness, and motor, sensory, autonomic, or psychic events.

Status epilepticus'. A single epileptic seizure of more than 30 minutes duration, or a series of epileptic seizures over a period of more than 30 minutes, with failure to regain normal function between each seizure.

Classification In 1993, the ILAE produced the following classification of epileptic seizures, which is based predominantly on clinical criteria:

1. Classification by type: Generalised'. A seizure is considered generalised when there is no evidence of a focal onset, and clinical symptomatology provides no information on anatomical localization. Generalised seizures may be subdivided into convulsive seizures with predominantly tonic, clonic, or tonic-clonic features, non-convulsive (absence) seizures, and myclonic seizures.

Partial: A seizure is considered partial when there is clinical evidence of a focal onset. The initial clinical features are of great localising value, since they arise from neuronal activation of part of one cerebral hemisphere. Seizures are described as simple partial when alertness and the ability to interact appropriately with the environment are maintained. When impairment of consciousness, amnesia, or

32 confusion during or after a seizure is reported, the seizure is classified as complex partial. Partial seizures may become secondarily generalised.

2. Classification by cause: Provoked (acute symptomatic): These seizures arise from known or suspected cerebral dysfunction. They occur in association with an acute systemic toxic or metabolic insult, or in association with an acute insult (infectious, metabolic, toxic, or traumatic) to the central nervous system. Seizures complicating malaria clearly fall within this category.

Unprovoked (seizures o f unknown aetiology): include idiopathic epilepsy syndromes with specific clinical and electroencephalographic characteristics, and cryptogenic seizures for which no risk factors have been identified.

Neurobiology of seizures and status epilepticus

Neurochemistry Seizures reflect an imbalance between excitation and inhibition in the brain (Mody 1992), and computer models of neuronal networks suggest that only a small reduction in inhibition will cause the system to discharge excessively (Traub 1982). In the brain, the most important excitatory process is the excitatory postsynaptic potential (EPS?), while inhibition is mediated through the inhibitory postsynaptic potential (IPSP) (Fisher 1995). Over 100 neurotransmitters or neuromodulators are involved in neuronal transmission, of which the amino acid glutamate is probably the most important excitatory neurotransmitter, and gamma-aminobutyric acid (GABA) the most important inhibitory neurotransmitter (Fisher 1991).

The glutamate receptor has a complex macromolecular structure, and to date at least five subtypes have been demonstrated (Wasterlain 1993). Of these, the most studied is the N- methyl-aspartic acid (NMDA) receptor (Dingledine 1983). While other glutamate receptor subtypes open sodium channels and generate depolarizing EPSPs, the NMDA

33 receptor comes into play once the neuron has been partially depolarized, and amplifies the response. Depolarization of the neuron causes magnesium ions to be extruded from the ion channel within the NMDA receptor, so opening the channel to an influx of calcium ions. These depolarize the neuron further and initiate a multiplicity of changes that are critical to learning and memory (Collingridge 1987; Worley 1987). Excessive calcium within the neuron can, however, lead to cell injury and death (Sloviter 1991). Because it is inactivated at normal resting potentials, and becomes active in neurons that are already depolarized, the NMDA receptor is believed to play a significant role in the generation of epileptiform discharges. NMDA antagonists and channel blockers are now under study as potential anticonvulsant drugs, and several are being assessed for their possible neuroprotective action after stroke. The special role of magnesium as a “guardian” of the NMDA channel has led to its use in the treatment of seizures, and in a multicentre, randomised study, magnesium sulphate was shown to be highly effective at controlling eclamptic seizures (Duley 1995).

The GABA receptor is also a macromolecular complex, comprising sites for GABA binding, regulatory sites, and ionic channels (Costa 1981). When GABA, or a pharmacological GABA agonist, binds to the receptor it initiates a change in its protein configuration. This opens a channel for the influx of chloride and, in the mature brain, produces the hyperpolarization of the IPSP. In the first week of postnatal life, however, when excitatory inputs are still poorly developed, stimulation of GABA receptors results in depolarisation (Ben-Ari 1994). During this period, therefore, GABA receptors provide the excitatory drive necessary for the outgrowth of pyramidal cells. Benzodiazepines and barbiturates, which interact with regulatory sites on the GABA complex and augment the inhibitory activity of GABA, are effective anticonvulsants (MacDonald 1979).

Neurophysiology of status epilepticus Since ethical constraints prevent detailed recording from the cerebral neurons of living human subjects, much of our current knowledge of the neurophysiology of seizures and status epilepticus has come from animal models. Unfortunately, no ideal animal model exists, and since physiological changes at a systems (as opposed to a cellular) level show

34 marked inter-species variation, caution is needed before extrapolating experimental findings from animals to the human condition (Loscher 1988; Fisher 1989).

Although the electroclinical features of isolated seizures are similar to those seen in the initial stages of status epilepticus, there is evidence to suggest that the precipitating factors are not the same (Shorvon 1994b). In animal experimentation, the intensity of an epileptic stimulus (duration of electrical stimulation or dose of a chemical convulsant) determines whether isolated seizures or status occur. In humans, there is an increased propensity for status following drug withdrawal, fever, or metabolic disturbance. Seizure initiation requires the presence of cells with intrinsic burst-generating properties (pacemaker cells), loss of postsynaptic inhibitory control around these cells, and synchronisation of the epileptic discharges (Schwartzkroin 1986). Cells with burst potential are particularly evident in layers CAl and CA3 of the hippocampus, and in layers IV and V of the neocortex. Inhibition is largely mediated by GABA receptors, as described above, while synchrony is thought to result from both glutamate-mediated excitatory neurotransmission and non-synaptic mechanisms. Epileptogenesis may result from a number of processes, including alteration in the properties of neuronal membranes leading to hyperexcitability, selective impairment of inhibitory processes, and gliosis with the resultant accumulation of ionic potassium (Prince 1985). The activity of individual cells forms the basis for subsequent synchronised discharges from large populations of neurones (hypersynchrony) (Wong 1986).

Propagation of epileptic discharges occurs largely along existing pathways, but may also utilise non-synaptic mechanisms (Meldrum 1988). It is now clear that, as status progresses, specific physiological and chemical changes occur which perpetuate the seizure activity. Experimental induction of limbic status epilepticus in the rat, a useful model of focal status, suggests that seizure activity is maintained within a feedback loop linking hippocampal and parahippocampal structures (Lothman 1989). Hippocampal slices from these animals show a decrease in GABA-mediated inhibition and an altered sensitivity to extracellular ions, suggesting that these are the biochemical changes that might underlie chronic focal epilepsy (Lothman 1990). Cycling of electrographic activity

35 is also a common feature of complex partial status in humans, and the identification of a similar underlying mechanism may provide a target for future therapy.

The increased incidence of status epilepticus in children compared to adults is probably due to a combination of increased seizure susceptibility and decreased ability to mount an adequate inhibitory response. The seizure threshold in the immature brain appears to be lower than in the mature brain, although the mechanisms underlying this susceptibility remain unclear. Excitatory synapses mature earlier than inhibitory synapses, increasing the likelihood that excitation-inhibition imbalance may occur. In addition, the immature cerebral cortex has a high synaptic density, and this may contribute to the development of hypersynchrony among neural groups (Huttenlocher 1987; Schwartzkroin 1993).

Detailed neurophysio logical data on generalised status epilepticus has come from the experiments of Medrum and Horton on bicuculline-induced seizures in the adolescent baboon (Meldrum 1973a). At the onset of status, myoclonic jerks are associated vsdth irregular widespread cortical spike-wave activity on electroencephalogram (EEG). A tonic spasm, lasting for 10-30 seconds, is then followed by continuous clonic activity, with rhythmic poly-spikes and waves on EEG. This clinical and electrographic pattern can be maintained for approximately one hour, after which the limb jerking starts to wax and wane and to become asymmetrical, and EEG activity becomes irregular in both rhythm and distribution. At the end of status, postictal electrographic silence is followed by the appearance of isolated biphasic waves, which gradually become more rhythmic and generalised, evolving into difftise high-voltage delta activity. A similar sequence of electrographic events has been observed during convulsive status in both humans and in three different rat models of status (Treiman 1990), and presumably reflects evolving neurochemical changes or possible neuronal damage secondary to prolonged seizure activity.

Meldrum and Horton were the first to point out that prolonged status results in physiological decompensation, the shift from phase I (compensation) to phase II (decompensation) occurring after about 20-30 minutes of continuous seizure activity in

36 baboons, and after approximately 30-60 minutes in humans. During phase 1 cerebral blood flow is greatly increased, and is sufficient to compensate for the great increase in cerebral metabolic activity. Blood pressure and cardiac output both rise, and autonomic over-activity results in sweating, hyperpyrexia, bronchial secretion, and salivation. Arteriovenous differences for oxygen and carbon dioxide both fall, and glucose and lactate levels rise. In phase II, the physiological mechanisms compensating for the increase in cerebral metabolism begin to fail. Cerebral autoregulation breaks down progressively, and cerebral blood flow becomes increasingly dependent on systemic blood pressure. Hypotension develops and, in the terminal stages, may be profound. Falling blood pressure reduces cerebral blood flow and metabolism. The high metabolic demands of the epileptic cerebral tissue now cannot be met, and there is a risk of ischaemic or metabolic damage. Blood glucose falls and severe hypoglycaemia can develop. Hypokalaemia and acidosis may induce cardiac arrythmias, which are a common cause of death. Intracranial pressure can rise precipitously during the late stages of status, and the combined effects of systemic hypotension and intracranial hypertension can compromise the cerebral circulation and cause cerebral oedema. Other late complications are acute tubular necrosis secondary to myoglobinuria or dehydration, acute hepatic failure, rhabdomyolysis, and disseminated intravascular coagulation.

Neuropathology of status epilepticus Assessment of the neuropathological features of status epilepticus is complicated by the fact that pathological changes within the brain may represent the cause or the result of either status or its systemic complications. Whilst there is no doubt that status can induce cerebral damage, the degree to which this occurs in humans, and the nature of the changes, are both still highly controversial (Shorvon 1994a). The situation has been complicated by differing definitions of status epilepticus, and by the fact that propensity to cerebral damage varies with the age of the patient and with the cortical site, duration, and type of status.

37 Human pathological studies Gross postmortem examination of the brain of patients dying in status epilepticus is often normal, although cerebral swelling, congestion, and scattered haemorrhages are observed in some patients. The hippocampus may be swollen during the acute phase of status, especially in small children, and atrophic changes may subsequently develop in both the hippocampus and the cerebellum. Hippocampal damage was first described in a series of patients with chronic epilepsy (Sommer 1880). The gliosis and pyramidal cell loss, occurring predominantly in the CAl region of the hippocampus, is now referred to as Ammon’s horn sclerosis, hippocampal sclerosis, or mesial temporal sclerosis. Further studies of the acute cerebral changes in status epilepticus have confirmed these findings, and have also documented damage in the C A3 region of the hippocampus, certain regions of the cerebral neocortex, and in the cerebellum (Norman 1964; Margerison 1966; Ounsted 1966). The relative vulnerability of the CAl and CA3 regions to damage in human status has recently been confirmed by the quantitative assessment of neuronal density (DeGiorgio 1992), and may be due to the high concentration of NMDA and kainate receptors in this region. Sano and Malamud (Sano 1953) originally described the association between hippocampal sclerosis and anterior temporal spikes-wave discharges in patients with epilepsy. Demonstration of the increased vulnerability of the childhood brain to status-induced ischaemic damage (Margerison 1966; Ounsted 1966; Corsellis 1983) subsequently led to the hypothesis that status epilepticus in early childhood may cause ischaemic brain damage and hippocampal sclerosis, which in turn may cause temporal lobe epilepsy.

The mechanism of the cerebral changes in status remains speculative. A number of hypotheses have been proposed in the past, including cerebral vascular spasm, hypotension, systemic hypoxia, and cerebral oedema causing mesial temporal tentorial herniation and anterior choroidal and posterior cerebral artery compression (Shorvon 1994b). The absence of either oedema or tentorial herniation in pathological specimens makes the latter theory unlikely. Other workers have suggested that the seizure discharges themselves may damage the brain through ischaemic or metabohc injury (Falconer 1964; Ounsted 1966). There is now a considerable body of evidence to suggest

38 that excitotoxic amino acids play an important role in the development of structural brain damage secondary to status epilepticus. Direct application of glutamate onto hippocampal cultures causes neuronal death, similar to that seen in experimental animal models of status epilepticus (see below) (Vomov 1995). Neuronal death appears to be mediated by co-activation of NMDA and AMP A (alpha-amino-3-hydroxy-5-methylisoxazole) receptors, since blockade of either receptor type prevents neuronal injury. Binding of glutamate to the NMDA receptor causes an influx of calcium, and high intracellular concentrations of calcium initiate a large number of processes that may damage the cell (Choi 1994). These include the activation of protein C kinase which damages the cell wall, the formation of nitric oxide which inhibits mitochondrial respiration both directly and indirectly through the formation of cytotoxic peroxynitrite free radicals, and the activation of phospholipase A 2, which breaks down membrane lipids (Dawson 1992; Bruno 1993). Glutamate receptor stimulation also regulates the expression of a number of effector genes, some of which activate brain repair mechanisms and are therefore neuroprotective, while others induce cell death (Akins 1996).

Animal pathological studies The clearest evidence that status epilepticus can result in cerebral damage has come from animal experimentation, of which the classic studies were on adolescent baboons (Meldrum1973a; Meldrum 1973c). Meldrum and Brierley used bicuculline, a GABA- antagonist, to induce generalized seizures, lasting for between 82 and 299 minutes, in 10 baboons. The systemic manifestations, namely hypertension, acidosis and hyperglycaemia followed by increasing hyperpyrexia, hypotension and hypoglycaemia, were strikingly similar to those observed in human status. Neuronal damage was incurred from about 25 minutes, and consisted of ischaemic changes in the CAl region of the hippocampus, layers III, V, and VI of the neocortex, the arterial watershed zone of the cerebellum, and portions of the basal ganglia. This pattern corresponded to the selective pattern of vulnerability seen in the human brain after status epilepticus. The authors considered that, had the animal survived, the dead nerve cells would have been removed by phagocytosis and replaced by glial proliferation, resulting in cerebral and cerebellar atrophy and sclerosis of the hippocampus.

39 The experiments were then repeated in eight paralysed and artificially ventilated baboons, in whom hypotension, acidosis, hypoxia, and hypoglycaemia were prevented (Meldrum 1973c). Neuropathological examination revealed a similar pattern of neocortical and hippocampal damage, although the pathological changes were generally less severe. Only the cerebellar damage was totally prevented by paralysis, suggesting that was related to hyperpyrexia and hypotension. It was concluded that the cerebral changes seen in status were not of vascular origin, but resulted from impaired cellular metabolism.

Work on other animal models of generalised convulsive status has largely confirmed the findings of Meldrum and colleagues, although more recent research has concentrated on focal status, especially that involving the limbic or mesial temporal lobe structures. Systemic or intra-amygdaloid injection of kainic acid, a glutamic acid analogue, will induce limbic status epilepticus in the rat. This results in a disseminated pattern of acute brain damage, particularly affecting the CA3 and CA4 regions of the hippocampus. The extent of damage correlates with the severity and duration of seizure activity (Olney 1974; Olney 1979). Other convincing experimental models are those that show “remote” damage resulting from seizure spread, so differentiating “epileptic” damage from intrinsic damage produced by the experimental lesion (Sloviter 1983). Differences in the distribution, degree, and timing of cerebral damage caused by seizure activity, hypoglycaemia, and hypoxia have also been the subject of intensive study (Siesjo 1986).

The emergency treatment of generalised tonic-clonic status epilepticus

Generalised tonic-clonic status epilepticus is a medical emergency. Immediate treatment is required because outcome is directly related to the duration of status. The three main aims of treatment are to stop seizure activity as quickly as possible, to maintain adequate cardiovascular and respiratory function (with particular emphasis on the avoidance of hypotension and hypoxia), and to minimise other metabolic, systemic, and medical complications. The following modified treatment regimen is based on the facilities and

40 drugs that were available at the Wellcome Trust-KEMRI Clinical Research Unit at the time the studies described in this thesis were carried out (Crawley 1996; Crawley 2001):

General measures

0-10 minutes Assess cardiorespiratory function, secure airway, give oxygen Establish intravenous access Emergency anticonvulsant drug therapy (see below)

10-30 minutes Monitor neurological status, vital signs, electrocardiogram (ECG), oximetry Measurement of blood gases, electrolytes, glucose, full blood count Emergency treatment of hypoglycaemia if indicated (glucose 0.5 g/kg i/v over 15 minutes)

30-90 minutes Specialized monitoring, as indicated: intra-arterial blood pressure, central venous pressure, intracranial pressure (ICP) monitoring, electroencephalography (EEG) or cerebral function monitor Pressor therapy, mannitol as required: there were no facilities for mechanical ventilation at the Clinical Research Unit, but patients could be intubated and hand ventilated in an emergency Active treatment of any additional complications: hyperpyrexia, hypoglycaemia, electrolyte disturbance, lactic acidosis, acute renal failure

Emergency anticonvulsant drug therapy

0-30 minutes Emergency anticonvulsant therapy is started at 5 minutes Diazepam: 0.3 mg/kg i/v over 2 minutes or 0.5 mg/kg of intravenous solution per rectum. If seizure activity continues, a maximum of 3 further doses of diazepam can be given at 10 to 15-minute intervals

41 Then Paraldehyde: 0.2 ml/kg i/m or per rectum. The dose can be repeated after 10 minutes, and the risk of accumulation is low. Paraldehyde has a longer duration of action than diazepam, and blood levels are virtually constant between 20 minutes (time of peak concentration) and 120 minutes of a single i/m injection. In contrast, blood levels of diazepam fall from high peak to subtherapeutic levels (< 200ng/ml) within 20 minutes of i/v injection (Shorvon 1994b))

30-90 minutes Phenytoin: 15-20 mg/kg i/v over 30 minutes Or Phenobarbitone: 15-20 mg/kg i/m or i/v over 30 minutes

Refractory status (> 90 minutes) Thiopentone: 3 - 5 mg/kg i/v over 30 seconds, followed by 1 - 5 mg/kg/hour Maintenance of long-term anti-epileptic therapy in tandem with emergency treatment

Outcome of status epilepticus in children

There are several points that should be remembered when considering the large number of studies describing the outcome of status epilepticus in children. Most studies are confined to convulsive status, are retrospective, and are based on patients within a hospital setting. This introduces an obvious source of bias, since milder cases are more likely to have been missed, thereby magnifying the apparent overall mortality and morbidity from status (Verity 1998). In retrospective series, it is often difficult to determine whether deaths should be attributed to status or to a wide variety of underlying conditions. Changing definitions of status pose additional obstacles to accurate risk assessment.

In children, there are considerable differences between the outcome of febrile status epilepticus (status epilepticus associated vsdth fever in a neurologically normal child

42 between the ages of 6 months and 5 years) and non-febrile convulsive status. The outcome from non-febrile status is primarily dependent on the underlying aetiology, which in turn is dependent on the age of the child (Shorvon 1994a). The prognosis of febrile status is generally good, with a very low risk of neurological or cognitive impairment, but a significant risk of subsequent epilepsy (21%, compared to 0.5-1% for the population as a whole) if seizures are prolonged (Verity 1993). About half of these children will go on to have complex partial seizures, of whom many will have mesial temporal sclerosis. Although the relationship between convulsive status epilepticus and mesial temporal sclerosis is presumed to be causative, this has not been proved. Approximately 50% of adults with temporal lobe epilepsy secondary to mesial temporal sclerosis have a history of prolonged febrile convulsions in childhood (Shorvon 1994b).

There has been an apparent decrease in mortality and morbidity from convulsive status epilepticus since 1970, when Aicardi and Chevrie published their retrospective review of 239 hospitalised cases (Aicardi 1970). In this series, mortality was 11%, while 53% of patients had a poor neurological or cognitive outcome. The hemiconvulsion, hemiplegia, epilepsy syndrome documented in this series is now a rare complication of convulsive status, occurring only in children with seizures lasting for more than one hour. By 1997, mortality had decreased to between 0% and 6% (Maytal 1989; Lacroix 1994; Eriksson 1997), while between 15% and 33% had neurological sequelae. There are a number of possible explanations for this observation. Benzodiazepines had not been introduced into clinical practice in 1970, and the incidence of prolonged seizures was consequently likely to be higher. Aicardi and Chevrie also required that seizures lasted for at least one hour to fulfil their definition of status epilepticus, while recent studies have used a cut-off of only 30 minutes. The longer a seizure lasts, the more difficult it becomes to treat, increasing the likelihood of a poor outcome (Shorvon 1994a).

Prospective, community-based epidemiological studies that attempt to define the incidence and outcome of convulsive status epilepticus avoid the bias that is inevitably introduced by the study of hospital-based populations. Unfortunately, such studies are difficult to perform, since they are time-consuming and depend upon the existence of a

43 network between all hospitals in a delineated area. Using this approach, DeLorenzo and colleagues in Richmond, Virginia, found a total incidence of convulsive status epilepticus of 41/100,000 residents, this figure rising to 147/100,000 in infants aged 1 month to 1 year (DeLorenzo 1995). The mortality in children was only 2.5%, with all the deaths being caused by non-central nervous system infections. Among a cohort of 14,676 children who were bom during one week in 1970 and followed for 10 years as part of the Child Health and Education Survey, 32 had one or more episodes of convulsive status epilepticus (Verity 1993). Two of these children died, both of whom had presented with non-febrile status epilepticus. One child developed neurological sequelae, while non- febrile seizures developed in 21% of children following prolonged febrile convulsions.

Seizures as a complication of childhood malaria

Local perception of seizures Seizures are well recognised by the rural community of Mijikenda farmers living in the Kilifi district of Kenya. (Further details of this population are given in Chapter 2). Seizures, or nyuni, are perceived as a serious childhood illness, requiring immediate attention (Mwenesi 1995a; Molyneux 1999b). The recognised symptoms include high fever, rolling up of the eyes, “looking terrified”, “chewing the teeth”, unconsciousness, jerking of the arms and legs, and frothing at the mouth. Within the local community, seizures are interpreted from both a biocultural and a biomedical standpoint (Molyneux 1999a). The biocultural view emphasises the importance of spirits or witchcraft, the local words nyama and ndege being used to describe the animal or bird-like spirits that are thought to have entered the child’s brain and caused the seizures. In this instance, help is usually obtained from a local healer, or mganga. In contrast, the biomedical view is that seizures are caused by fever, usually secondary to malaria, and that appropriate treatment should obtained from a clinic or hospital. Consequently, a child with seizures may be subjected to a wide variety of possible treatments, ranging from the administration of antimalarial and/or antipyretic drugs, to local herbs, being washed with urine, or hung in a pit latrine (since the pungent smell is thought to drive out evil spirits). The choice of biomedical or biocultural treatment is affected by a mother’s previous experience of each

44 health system, the views of her husband and family elders, and financial and logistic considerations. In many instances a mother will try both approaches: “It’s like betting, because you cannot be sure where the problem can be solved. You can start with the hospital first, and in case the hospital fails you can visit the healer” (interview with Mijikenda mother in Kilifi District. (Molyneux 1999a)). It is clear that, to be effective, any malaria control strategy must take into account the prevailing beliefs and behavioural patterns of the local community.

Seizures in uncomplicated malaria The true prevalence of seizures in African children is largely unknown, due to the absence of accurate community-based data (Snow 1994b). A recent community survey of childhood fevers in Kilifi district found that between 2% and 5% of fever episodes in children under the age of 5 years were associated with seizures (V. Marsh, personal communication). Seizures, however, account for a large proportion of the children presenting to hospitals and clinics in sub-Saharan Africa, of which malaria is the single most important reported cause (Hendrickse 1971; Asindi 1993).

Recent inpatient studies from Kenya (Waruiru, 1996; J Berkley personal communication) have reported seizures in at least 25% of children admitted with non-cerebral malaria. Seizures were commonest in children below the age of 3 years and, in a study from Thailand, were significantly associated with infections caused by P. falciparum compared to P. vivax (Wattanagoon 1994).

Seizures and cerebral malaria Seizures complicate the clinical course of cerebral malaria in approximately 60% to 80% of cases (Lemercier 1969; Molyneux 1989; Marsh 1995; Steele 1995; Waller 1995; Olumese 1999). When prolonged or multiple, they are associated with an increased risk of death (Jaffar 1997), gross neurological sequelae (Molyneux 1989; Brewster 1990; Bondi 1992; van Hensbroek 1997), and subsequent cognitive difficulties (Holding 1999). It is therefore surprising that, despite their high prevalence and potential pathogenic

45 importance, there have been few detailed studies of seizures in childhood cerebral malaria.

Aims of this thesis

1. To provide a detailed description of the clinical and electrophysio logical features of seizures in African children with cerebral malaria.

2. To explore two hypotheses regarding the aetiology of seizures in cerebral malaria, namely that: a. That the ingestion of chloroquine may be a precipitating factor and b. They may be caused by the release of the endogenous excitotoxin quinolinic acid.

3. To conduct a randomised, controlled intervention study to assess whether a single intramuscular dose of phénobarbital 20 mg/kg given on admission can reduce the frequency of seizures complicating the clinical course of childhood cerebral malaria.

46 2. METHODS

47 Introduction

The studies described in this thesis form part of a collaborative research programme, run jointly by the Kenyan Institute of Medical Research (KEMRI) and the University of Oxford, and funded by the Wellcome Trust. The studies were carried out between January 1994 and January 1998 at the KEMRI Clinical Research Unit, based at Kilifi District Hospital. The first part of this chapter contains background information on the demography, geography, and health services of the study site and surrounding area. A review of the routine clinical management of children admitted to the paediatric ward, with particular reference to the management of severe malaria, is then followed by a description of the methods used for the collection, management, and statistical analysis of clinical data.

Study site

The town of Kilifi is located 60 kilometres north of Mombasa, on the coast of Kenya (Figure 3). The area, situated 4° south of the Equator, has mean daily temperatures of between 22°C and 30°C, with relative humidity of about 70%. Approximately 1000 mm of rain falls each year, mostly between the months of April to July and October to November. Malaria transmission (of which over 95% is Plasmodium falciparum) follows the seasonal pattern of rainfall, with peak periods occurring between June to August and December to January. The principal malaria vectors are of the Anopheles gambiae complex (Mbogo 1993). There is marked local heterogeneity in entomological inoculation rate (EIR), with rates varying between approximately 2 and 200 infected bites per person per year. However, most residents of Kilifi district are exposed to an average of 4 infective bites per person per year (Mbogo 1995).

Kilfi town has a population of approximately 13,000, and is the administrative centre for Kilifi district. This predominantly rural area has an estimated population of 500,000, with most individuals belonging to the Mijikenda ethnic group (Snow 1993a). The Mijikenda family system is patriarchal and polygamous (Parkin 1991). Subsistence

48 Figure 3. Map of Kenya showing Kilifi district

/

\ ^ Indian Ocean /Mombasa J

Indian Ocean Klffi

LEGEND

Primary and secondary roads Tracks, trails. orfootpaUrs Urban Centres

m 49 farming is widespread, the main cash crops being coconuts and cashews, while maize is grown for home consumption. Household incomes are frequently supplemented by remittances from family members working in Kilifi, Mombasa, and Malindi (Molyneux 1999a). Part of this rural population, living in a geographically defined study area to the north and south of the town, has been the subject of extensive demographic surveillance (Snow 1993a). The 1998 Kenya Demographic Health Survey revealed an infant mortality rate of approximately 45 per 1000 live births (compared to 6 per 1000 live births in the UK).

Formal healthcare services for this population are provided by Kilifi District Hospital, and by 12 government dispensaries and private health clinics. Dispensaries operate on a cost-retrieval basis, charging approximately 20 to 40 KSH (20 to 40 pence) to treat a child with fever. Private clinics, charging approximately 150 KSH to treat a febrile child, are relatively expensive (Molyneux 1999a). There are in addition approximately 22 Community Health Workers (CHW), trained to supply a limited range of subsidised drugs from their homes, and to refer patients to other facilities when necessary. Access to these services is, however, a problem for the majority of rural dwellers. One third of all rural households are located more than 2 kilometres from a bus-stage, which makes access to the district hospital and clinics in Kilifi both difficult and time-consuming. In addition, over 60% of rural households are more than 2 kilometres from the nearest clinic or dispensary, and approximately half are more than 2 kilometres from a CHW (Molyneux 1999a). Consequently, it is not surprising that the majority of people turn to the informal sector for first-line healthcare. Over 80% of rural households are within one kilometre of a shop, of which the majority stock antipyretics and antimalarials. Approximately 80% of mothers in rural areas initially purchase drugs from a local shop when their child develops fever (Mwenesi 1995b; Marsh 1999; Molyneux 1999b). The other source of informal healthcare to which mothers have easy access is from waganga, or traditional healers. There is considerable variation in the reputation, “speciality”, and charges of different waganga (Molyneux 1999a).

50 Kilifi District Hospital has a forty-bed paediatric ward, to which approximately 5000 children (about 10% of those seen in the paediatric outpatient department) are admitted each year. Malaria, acute respiratory tract infections, and gastroenteritis account for the majority of paediatric admissions, while malaria contributes to most of the deaths (Snow 1994c). The services offered by Kilifi District Hospital are typical of many government hospitals in sub-Saharan Africa. During periods of intense malaria transmission, more than 120 children may be inpatients on the paediatric ward at any one time, and may be cared for by as few as 2 to 3 nurses. At the same time of year, individual clinical officers in the outpatient department may see up to 100 patients each day. Drugs are often in short supply, and facilities for laboratory investigation are limited to microscopy (of blood smears, urine, and faeces), determination of packed cell volume, and a crossmatch service. Biochemical analyses and measurement of blood gases are not available, while radiological services are intermittent. Consequently, the Clinical Research Unit operates its own laboratory service, which offers a full range of haematological, biochemical, and microbiological investigations.

The work presented in this thesis was performed in a six-bedded high dependency research ward, which was supported by the KEMRI Clinical Research Unit, and situated adjacent to the main paediatric ward.

Patients

Children were admitted to hospital through the outpatient department, which was staffed by Ministry of Health clinical officers. These health workers, who were answerable to the Medical Superintendent for the hospital, cared for all acute medical and surgical emergencies. The staff of the Research Unit did not, therefore, determine hospital admission policy, and consequently the pattern of paediatric admissions reflected that of other district hospitals in coastal Kenya.

Shortly after admission, all children were reviewed by a clinician from the Research Unit, and a fingerprick blood sample was taken for measurement of haemoglobin and

51 preparation of a malaria parasite slide. Quantitative parasite counts were perfomed on thick and thin blood films that had been stained in 10% Giemsa for 10 minutes. The number of asexual forms of Plasmodium falciparum were then counted per 100 white blood cells (thick film) or per 500 red blood cells (thin film). Children with pre-defined signs of severe malaria (see Table 2) (Marsh 1995) were then transferred to the high dependency research ward. Use of this definition made it possible to accurately identify more than 90% of the patients who had a high risk of dying from malaria in hospital.

All children recruited to the studies described in this thesis were aged 9 months and above, and had a diagnosis of cerebral malaria. Cerebral malaria was defined as unrousable coma (inability to localise a painful stimulus, equivalent to a Blantyre coma score of 3 or less, see Table 3 (Molyneux 1989)), in the presence of asexual Plasmodium falciparum parasitaemia. Children with seizures were assessed a minimum of 30 minutes after cessation of clinical seizure activity. Children who were hypoglycaemic (blood glucose below 2.2 mmol/1) at the time of admission, and who recovered consciousness following the administration of intravenous glucose, were also excluded from the studies.

Clinical assessment

A hill history and examination were performed on admission to the research ward, and results recorded onto a standard proforma (See Appendix). Children subsequently underwent frequent clinical review, with 4-hourly monitoring of vital signs (temperature, pulse, respiratory rate), Blantyre coma score (Molyneux 1989), transcutaneous oxygen saturation (N180 pulse oximeter, Nellcor, USA), and blood glucose (using indicator sticks (BM 1-44 test strips) and Refloflux II reflectance meters (Boehringer-Mannheim, UK)). All children underwent neurological examination at the time of discharge from hospital, and all were asked to return after one month (following the clinical and electrophysiological studies) or after 3 months (following the phenobarbitone prophylaxis study) for neurodevelopmental assessment.

52 Laboratory investigations

On admission to the research ward, venous blood was taken for full blood count (M 530 Coulter Counter, Luton UK), glucose (glucose oxidase method, Analox GM6), sodium and potassium (Coming 614 Na^/K^ autoanalyser. Coming Diagnostics Ltd, Halstead, UK), urea (Beckman BUN analyzer 2, Beckman, Fullerton, USA), and creatinine (Beckman creatinine analyzer 2, Beckman, USA). Blood gases were measured with a Coming 178 pH/blood gas analyser (Coming, Halstead, UK), with all measurements being individually corrected for the patient’s haemoglobin and temperature. Blood cultures were performed on all children, while those that were unconscious underwent lumbar puncture. Since intracranial pressure is a feature of cerebral malaria (Newton 1997b), lumbar puncture was delayed until the neurological status of the child had improved, or was done post-mortem for those who died. Cerebrospinal fluid was assessed by microscopy and culture, and all samples with a white cell count of above 10 cells per pi were screened for antigens of Haemophilus influenzae and Streptococcus pneumoniae.

Antimalarial and antibiotic chemotherapy

Children were treated with a loading dose of intravenous quinine dihydrochloride (15 mg/kg), with subsequent doses of 10 mg/kg every 12 hours (Winstanley 1994). During 1994, children were randomised to receive intravenous quinine or intramuscular artemether, 3.2 mg/kg loading dose and 1.6 mg/kg every 24 hours, as part of a multicentre study comparing the efficacy of the two treatment regimes (Murphy 1996). Parenteral therapy was replaced by a single oral dose of sulphadoxine-pyrimethamine (SP) once the child had received at least 3 doses of parenteral treatment and was able to tolerate oral dmgs.

All children were treated with intravenous benzylpenicillin (60 mg/kg every 6 hours) and chloramphenicol (25 mg/kg every 6 hours) until the results of lumbar puncture and blood culture were available.

53 Supportive care

Intravenous fluids and blood were given as clinically indicated (Crawley 1999). Hypoglycaemia (blood glucose below 2.2 mmol/1) was corrected with a slow intravenous bolus of glucose 0.5 g/kg. Children with rectal temperatures of above 38.5®C were treated with paracetamol (15 mg/kg per rectum or via nasogastric tube every 6 hours). Anticonvulsant treatment was described in Chapter 1. Oxygen was available if required, but there were no facilities for artificial ventilation.

Ethical permission

Approval for all studies was obtained from the Kilifi Scientific Coordinating Committee, the KEMRI Scientific Coordinating Committee, and the Kenyan National Ethical Review Committee. Informed, written consent was also obtained from the parent or guardian of each child enrolled into a study. An explanation, written in the caretaker’s first language (KiSwahih or KiGiriama), was read aloud by a trained fieldworker, and caretakers were encouraged to ask questions. It was stressed that failure to grant permission would not in any way compromise the clinical care given to the child, and that consent could be withdrawn at any time without penalty.

Data collection and management

All clinical and laboratory data were double entered in dBase IV (Ashton Tate, USA) onto IBM personal computers. Range checking and verification were carried out using Foxpro 2 software (Fox Holdings, USA). Each patient was given a unique study number.

Statistical analyses

Data were analysed with SPSS version 5.0 (SPSS Corporation, Gorinchem, The Netherlands) and Stata version 5.0 (Stata Corporation, College Station, Texas, USA).

54 Descriptive statistics are presented as means with 95% confidence intervals, or medians with interquartile ranges.

Univariate analysis Analysis of variance (ANOVA) and Student’s t test were used for comparisons of normally distributed quantitative data, with logarithmic transformation if the distribution was skewed. The Mann-Whitney test and Spearman’s rank correlation coefficient were used for data without a normal distribution. Categorical data were compared with Fisher’s exact test.

Multivariate analysis Multivariate logistic regression was used to examine the relationship between admission characteristics and subsequent outcome.

Survival analysis In the randomised controlled trial of phenobarbitone prophylaxis, recovery times in the two groups were compared by survival analysis, using the log-r^nk (proportional hazards) and Peto-Peto-Wilcoxon (non-proportional hazards) (Peto 1972) tests for equality of survivor function.

55 3. CLINICAL and ELECTROPHYSIOLOGICAL FINDINGS

56 Introduction

Cerebral malaria is a major cause of death and disability in sub-Saharan Africa (WHO 1999b), yet its pathophysiology remains poorly understood (Marsh 1996; Enghsh 1997a). Prolonged, multiple seizures are a prominent clinical feature (Molyneux 1989; Marsh 1995; Waller 1995), and are associated with an increased risk of death (Jaffar 1997) and neurological sequelae (Brewster 1990; Jaffar 1997; van Hensbroek 1997). Despite their importance, and in common with many other causes of childhood coma, there have been few descriptions of the clinical and electroencephalographic features of seizures in cerebral malaria (Lemercier 1969). Electroencephalography (EEG) can supply the clinician with information on the origin and distribution of seizure activity, detect subtle or sub- clinical seizures, and may reveal abnormal electrical activity of prognostic significance (Hughes 1994). It is therefore of potential value in the clinical management of cerebral malaria, and may improve understanding of the underlying pathophysiology. This chapter describes the clinical and EEG findings from a detailed study of 65 children with cerebral malaria.

Methods

Principles of management are described in Chapter 2. Children were eligible for this study if they were aged 9 months or above, and fulfilled the WHO criteria for cerebral malaria (see Chapter 1). During the study period of January to September 1994, 110 inpatient children fulfilled these entry criteria. Of these 65 were recruited since, for technical reasons, it was only possible to study 2 children at any one time.

Electrophysiology Electroencephalographic (EEG) recordings were made on a 14-channel Medelec 1A94 EEG machine. Silver/silver chloride electrodes were fixed with Elefix and tape to the child's shaved head, and the International 10-20 system used for electrode placement. Recordings were taken within 6 hours of admission, and at 12 hourly intervals until recovery of consciousness. Continuous recordings using a CFAM (cerebral function

57 analysing monitor, Medaid Ltd, UK) were obtained from selected children who had been unconscious for more than 24 hours or who had prolonged or subtle seizure activity. All EEGs were subsequently analysed by a consultant neurophysiologist, who knew the age of each child and what drugs they had been given, but was blind to any other clinical information.

Intracranial pressure monitoring As part of a separate study, which aimed to document the pattern of intracranial pressure (TCP) in children with cerebral malaria, and to determine the efficacy of mannitol (Newton 1997b), TCP was monitored in selected deeply comatose children using a subarachnoid catheter (Camino 110-4B).

Anticonvulsant treatment All clinical seizures were timed and recorded by medical or nursing staff, and classified as generalised tonic-clonic, partial motor, or partial with secondary generalisation (Hopkins et al. 1995). Seizures lasting for more than 5 minutes were treated with diazepam 0.3 mg/kg, given as a slow intravenous injection over 2 minutes. Children with recurrent (more than 3) seizures or status epilepticus (continuous clinical seizure activity lasting for 30 minutes or more) were treated with a loading dose of intravenous phenytoin 18 mg/kg and, if that foiled, with intramuscular phenobarbitone 18 mg/kg. One child with intractable generalised status epilepticus required treatment with intravenous thiopentone 4 mg/kg.

Follow up All survivors were seen one month after discharge for neurological examination and repeat EEG. Cerebral computerised tomography (CT) was performed on children with neurological sequelae.

58 Table 4. Admission clinical and laboratory features by outcome

Clinical or Normal recovery (1) Sequelae (2) Died (3) P valuesî laboratory feature N = 50 N = 8 N -7

Age (months)* 33.1 (27.6-39.7) 16.1(9.1-28.5) 38.7(22.2-67.5) 0.02 for 2 V 1

0.04 for 2 V 3

Duration of coma 6.3 (4.7-8.3) 11.5 (5.0-26.4) 13.3 (6.9-25.8) 0.07 before admission (hours)*

Status epilepticust 28/50 (56%) 6/8 (75%) 2/7 (29%) 0.24 before or after admission

Blantyre score+ 35/50 (76%) 5/8 (63%) 7/7 (100%) 0.45 2 or less

Opisthotonic 2/50 (4%) 1/8 (12%) 4/7 (57%) 0.003 for 3 V 1 posturing

Median (IQR) 85200 11854 81600 0.27 parasitaemia (990-1017600) (1264-220000) (420-352800) (per pi)

Haemoglobin (g/dl) 6.5 (5.8 - 7.2) 8.0(5.4-10.6) 7.8(5.0-10.6) 0.20

Median (IQR) 5.9 (0.7-11.3) 5.0 (0.7 - 8.3) 3.4 (0.4-7.8) 0.17 glucose (mmol/1)

Median (IQR) 134(127-142) 139(128-168) 139(127-147) 0.12 sodium (mmol/1)

Potassium 4.3 (4.0-4.5) 4.7 (4.2-5.3) 4.3 (3.2-5.9) 0.5 (mmol/1)*

Urea (mmol/1)* 4.1 (3.3-5.2) 8.6 (3.0-24.7) 6.5 (3.6-11.8) 0.06

Creatinine (pmol/1)* 5 2 (4 5 -6 1 ) 104 (55 -197) 72 (38-137) 0.009 for 2 V 1

Base excess -5 (-8 to -3) -11 (-17 to -5) -13 (-21 to -5) 0.04 for 3 V 1

Data are mean (95% Cl) or number (%) unless stated; * Geometric mean t Any clinical seizure reported (before admission) or observed (after admission) to last for 30 minutes or more + 0 = deep coma 5 = full consciousness J Where p< 0.05, results of multiple comparison tests (Scheffe or Bonferroni) are given

59 Results

Admission characteristics Sixty-five children, of age range 9 months to 11 years (median 30 months), were recruited to the study. All children had previous normal development. Prior to hospital admission, 68% (44/65) had seizures which, in 27 cases, were reported to have lasted for at least 30 minutes. Thirteen children (20%) had received diazepam on or up to 6 hours prior to admission, in doses of between 0.2 - 1 mg/kg. In 5 cases, diazepam had been administered by intramuscular injection. Children had been comatose for between 1 and 72 hours (median 7 hours) prior to admission and, in over 50% of cases, the onset of coma had been heralded by seizure activity. At the time of admission, 75% of the children were deeply unconscious with a Blantyre coma score of 2 or less. Admission clinical and laboratory findings are shown in Table 4.

Clinical course Of the 65 children studied, 14/65 (22%) recovered consciousness within six hours of multiple or prolonged seizures that had occurred on or immediately prior to hospital admission. Following admission, 62% (40/65) had at least one seizure, while 19 children had more than 5 seizures. Seventy five percent of all seizures occurred within 24 hours of admission. Twenty-eight percent (18/65) of the children had an episode of clinical status epilepticus (continuous clinical seizure activity lasting for 30 minutes or more), while 3 fiirther children had continuous electrical discharges lasting for 30 minutes or more with no apparent clinical correlates (electrographic status epilepticus). Twenty three percent of the children (15/65) had seizures that were either clinically subtle or electro graphic (see Subtle seizures, below). Fifly-two percent of all seizures were partial motor, 14% partial with secondary generalization, and 34% generalized tonic-clonic. Children with generalised seizures were significantly older than those with partial motor seizures (mean age 64.0 months (95% Cl 44.7 - 83.5) versus 26.7 months (95% Cl 20.6 - 32.9) for partial motor; p < 0.001). Median duration of seizure activity was 3 minutes (range 0.5 to 600

60 minutes). Of 310 seizures documented, only 130 (42%) were associated with a rectal temperature of 38 ®C or above, and 2 with hypoglycaemia (blood glucose <2.2 mmoVl).

Subtle seizures Fifteen children had continuous electrical seizure discharges on EEG, but clinical features that were either extremely subtle (subtle seizures) or not discernible (electrographic seizures). Children with subtle seizures typically presented in coma, with a history of a prolonged seizure that had either terminated spontaneously, or as a result of treatment with diazepam. Tonic eye deviation, nystagmus, salivation, and hypoventilation were the most distinctive clinical features. The nystagmus was of large amphtude, with a slow phase of movement that crossed the midline. Respiration was shallow and irregular, and the children were hypoxaemic (arterial oxygen saturation below 80%) and hypercarbic (pCOi above 6.5 kPa). EEG showed continuous spike-wave discharges over the posterior temporo-parietal region contralateral to the direction of eye deviation (Figure 4). These children recovered consciousness, as judged by their ability to localise a painful stimulus, a median of 5 hours (range 1 to 8) after anticonvulsant treatment. The clinical features of subtle seizures are illustrated in the enclosed video.

Clinical features also became increasingly subtle during prolonged generalised seizures. One child was admitted following a generalised seizure at home that had lasted for six hours. Tonic-clonic movements had ceased one hour before admission, and the child was deeply comatose, with irregular respiration, salivation, and priapism. Despite the paucity of clinical features, EEG revealed continuous, generalised seizure activity.

61 Figure 4. Left posterior temporo-parietal discharge

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01 - Av

100 HW K»Hi M* ~n«f Lff 0 5 Pi

62 Electrophysiology Background activity Admission EEGs were dominated by high amplitude (>100 pv) slow wave activity with frequencies of between 0.5-7 Hz (Figure 5). Admission recordings of very slow frequency (0.5-3 Hz) were obtained from 17/65 (26%) children, and were associated with an increased risk of death (odds ratio 25.6 (95% Cl 2.8 to 235; p=0.004) (Table 5). This association remained significant despite adjustment for the possible confounding effects of age, glucose, and base excess. Recordings of very slow frequency were not significantly associated with duration of coma, administration of diazepam in the 12 hours prior to admission EEG, age, parasitaemia, glucose, or base excess (p >0.1 for all comparisons). Ten children had admission recordings that were asymmetric (Figure 6), with increased slow wave activity over all or part of one hemisphere. Nine of these children had a history of multiple partial motor seizures or status epilepticus in the six- hour period prior to admission, and 3 developed neurological sequelae.

Ictal activity A striking feature of the EEGs in this series was the consistent localisation of ictal discharges over the posterior temporo-parietal regions (Figure 4). Over 75% of ictal or inter- ictal discharges occurred in this region, sometimes spreading to involve both temporo­ parietal regions, or one or both cerebral hemispheres. In 8 cases, electrical seizure activity persisted for between 2 and 140 minutes (median 45 minutes) after successful treatment of a clinical seizure with diazepam.

The onset of generalised electrical and clinical seizure activity usually followed an increased frequency of bilateral, predominantly posterior, interictal spike-wave discharges (Figure 7). Runs of up to 40 short, generalised seizures, each lasting for 0.5 to 1 minute (Figure 8), were observed in four children and were followed by marked post-ictal flattening.

Inter ictal epileptic activity Interictal spikes and sharp waves were observed on admission EEGs from 3 children, all with a history of prolonged (seizure activity lasting for more than one hour) partial motor

63 status epilepticus. The discharges were bilateral and multifocal, and located predominantly in the temporo-parietal regions (Table 6). All of these children developed neurological sequelae.

Episodic low amplitude events (ELAE) Periods of relative attenuation of background activity lasting 3-5 seconds were seen on admission EEGs from 5 children. In 2 cases, these followed the administration of intravenous diazepam 0.3 mg/kg for the treatment of seizures. Four of these children recovered normally, while one child died 7 hours after admission.

Burst suppression A burst suppression pattern, consisting of bursts of polymorphous complexes occurring synchronously over both hemispheres, and alternating with periods of relative quiescence, was observed on the initial EEGs from 2 children (Figure 9). One child had been hypotensive and hypoglycaemic on admission, and the other had a history of multiple, prolonged generalised seizures. Both children died.

64 Figure 5a. Background slow-wave activity in cerebral malaria

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Figure 5b. Normal EEG (age 12 months) for comparison

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65 Figure 6. Postictal EEG asymmetry

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66 Figure 7a. Recruiting rhythm: increased frequency of bilateral interictal spike wave discharges

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hfC 100 Mi l_Çf Ut

Figure 7b. Onset of generalised seizure

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67 Figure 8. CFAM tracing showing multiple short generalised seizures

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Figure 9. Burst suppression

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68 Intracranial pressure monitoring Ten children had simultaneous EEG and intracranial pressure (ICP) monitoring. Four children had clinical seizures during ICP monitoring. Intracranial pressure rose by a median of 164% (range 108-285) during the 18 generalised seizures, and by 50% (range 0-186) during the 14 partial seizures recorded. Concomitant rise in mean arterial pressure meant that cerebral perfusion pressure was maintained above 50 mmHg during clinical seizures. There was no significant rise in ICP during two 90-minute periods of electrographic seizure activity confined to the posterior temporo-parietal regions. At intracranial pressures of below 20 mmHg, fluctuations in ICP appeared to have no effect on the background frequency of the EEG. EEG recording from one child with severe intracranial hypertension, however, showed widespread slow wave activity (frequencies of 0.5 - 1 Hz) in association with episodes of opisthotonic posturing, during which ICP rose to peaks of 30 to 40 mmHg (Figure 10a). Within 10 hours, EEG showed attenuation of activity in the right temporo­ parietal region (Figure 10b), and the right pupil became dilated with sluggish reaction to light. Over the next five hours, despite regular doses of mannitol 0.5 g/kg, the ICP rose to above 80 mmHg, the cerebral perfusion pressure fell below 30 mmHg, and the entire EEG became flat and featureless. ICP rose to a peak of 125 mmHg, and the child died following a respiratory arrest.

A fiirther child of 3 years was admitted with a 12-hour history of opisthotonic posturing. The initial EEG was reactive, with a background frequency of 2-6 Hz. ICP monitoring was commenced at 7 hours, and the child also started continuous CFAM recording. Initial intracranial pressures ranged from 25-30 mmHg, with peaks of 40 mmHg during episodes of opisthotonus. Over the next 4 hours, despite inotropic support, the mean arterial pressure remained below 75 mmHg, and rising intracranial pressure consequently caused a progressive decline in cerebral perfusion pressure. Subsequent infusion of mannitol was accompanied by a marked deterioration in clinical condition, and CFAM recording showed a sharp drop in background frequency. The child died shortly afterwards following a cardio-respiratory arrest.

69 Figure 10a. Slow wave activity associated with onset of opisthotonic posturing

Figure 10b. EEG 10 hours later (Note marked attenuation of electrical activity in right temporo-parietal region: T4-C4, C4-Cz, T6-P4, P4-Pz)

70 Outcome Seventy-seven percent (50/65) of the children made a hill recovery, 7/65 (11%) died, and 8/65 (12%) were left with neurological sequelae (4 hemiplegia, 2 spastic quadriplegia, 1 cognitive and speech problems, 1 grand mal epilepsy) that were still present one month after discharge. Among survivors, median time to recovery of consciousness was 20 hours (range 4-112 hours). Serial EEGs showed a gradual increase in background frequency over the period from admission to recovery of consciousness. The fifty children who made a frill recovery had normal EEGs one month after discharge. Details of the children who died or developed neurological sequelae are shown in Tables 4, 5, and 6. Children with sequelae were significantly younger (p = 0.02) and had a significantly higher creatinine (p = 0.009) compared to those who recovered fully. Admission base excess was significantly greater (p = 0.04) in children who died compared to those who recovered fully, while four of the seven children who died had sustained opisthotonic posturing during their clinical course in hospital. There were otherwise no significant differences between the three outcome groups in the admission variables, but numbers are small. Follow-up EEGs from children with hemiplegia showed decreased activity in the contralateral parieto-temporal region, and cerebral computerised tomography (CT) scans showed infarction in the same region (Figure 11). Follow-up EEGs from two children with spastic quadriplegia and one child with severe cognitive and speech problems were of low amplitude, with a paucity of normal cerebral rhythms, while CT scans from these children showed global cerebral atrophy.

71 Age (months) 21 50 32 132 31 35 27

Any status No No No No No Yes Yes epilepticus*

Number of 2 0 0 7 0 0 15 seizures after admission

Background EEG 2Hz 0.5-2HZ 3-7HZ 0.5-3HZ 0.5-2HZ 2-6Hz Burst activity Unreactive, low End-stage Widespread Low amplitude Episodic low suppression amplitude electrographic slowing to IHz amplitude status with opisthotonus events

Localisation of None Continous ictal None None None None None ictal or inter-ictal discharges L discharges hemisphere

Time of death 15 25 63 11 16 (hours from admission)

Mode of death Cardio- Cardio­ Respiratory Respiratory Respiratory Respiratory Respiratory respiratory arrest respiratory arrest Arrest arrest arrest arrest arrest

Table 5. EEG findings from children who died

* Clinical seizure reported (before admission) or observed (after admission) to last for 30 minutes or more

72 Age (months) 18 9 11 11 9 13 56 36 Any status Yes Yes Yes No Yes Yes Yes No epilepticus*

Number of 2 14 0 0 0 >20 1 1 clinical seizures after admission Background 3-5 Hz R 2-4 Hz 2-5 Hz 1-4 Hz L 2-6 Hz 3-7HzR 3-7 Hz 1-4 Hz EEG activity 2H zL 0.5-2 Hz R 2H zL

Localisation of L temporo­ L temporal ictal None None None L parieto­ Bilateral None ictal or inter­ parietal inter­ discharges occipital parieto­ ictal discharges ictal sharp slow R mid temporal occipital inter­ waves Ictal and inter­ ictal ictal discharges discharges Type of R hemiplegia R hemiplegia R hemiplegia L hemiplegia Spastic Spastic Attention and Grand mal sequelae quadriplegia quadriplegia speech deficits epilepsy

Follow-up EEG Asymmetric Asymmetry of Asymmetry of Asymmetry of Low Movement Abnormal Normal EEG one monfti post Increased slow sleep spindles sleep spindles sleep spindles amplitude, artifact sleep activity with hyper­ discharge activity on L Low amplitude, Increased slow Increased slow featureless ventilation featureless over L activity L activity R posterior quadrant posterior posterior quadrant quadrant

CT scan one Infarction L Infarction L Failed to attend Infarction R Generalised Generalised Generalised Normal month post parieto-occipital parieto-occipital parieto-occipital cerebral cerebral atrophy cerebral discharge region region region atrophy atrophy

Table 6. EEG and CT scan results from children with neurological sequelae

* Clinical seizure reported (before admission) or observed (after admission) to last for 30 minutes or more

73 Figure 11. Cerebral CT scan in child with right hemiplegia (Shows extensive area of infarction in left temporo-occipital region (I and 2) plus associated cerebral atrophy)

74 Discussion

Traditionally, it has been assumed that sequestration of parasitised red blood cells occurs within the cerebral micro vasculature of all comatose patients with falciparum malaria (MacPherson 1985; WHO 2000). Clinical and experimental work has shown, however, that the pathophysiology of cerebral malaria is highly complex, and that a number of different pathological mechanisms may cause coma (Marsh 1996). Reduced cerebral perfusion (secondary to raised intracranial pressure and systemic hypotension), severe anaemia, hypoglycaemia, and the local release of both cytokines and nitric oxide, may all be contributory factors (White 1987; Clark 1991; Marsh 1996; English 1997a; Newton 1997b). Our study suggests that an additional pathophysiological factor, seizures, may play an important role in the pathogenesis of coma.

Over one third of the children in this study had more than 5 seizures or an episode of status epilepticus during their clinical course in hospital. Twenty-two percent recovered consciousness within six hours of prolonged or multiple seizures, suggesting that their coma may have been a postictal phenomenon, directly related to seizures. Following a prolonged seizure, it is thought that the brain enters a phase of “cortical exhaustion”, with depletion of adenosine triphosphate, glucose, oxygen, and the accumulation of lactic acid (Shorvon 1994b). The EEG may be flattened or show difihise slow wave activity during this postictal period, the duration of which may be prolonged after prolonged or multiple seizures. Some children in this study received large (up to 1 mg/kg) doses of diazepam before admission, which may have depressed consciousness further. In several cases, diazepam was administered by intramuscular injection and, since the absorption of diazepam from this site is extremely variable, this may have led to high blood concentrations and secondary sensorineural and respiratory depression (Gamble 1975; Langslet 1978).

This study has also shown that, in a proportion of children with cerebral malaria, coma is due to continuing subtle seizure activity, which is likely to go undetected, but which is responsive to anticonvulsant drugs. EEG was essential for identifying the fifteen children in whom seizures were either clinically subtle (tonic eye deviation, nystagmus.

75 hypoventilation) or electrographic. Although tonic deviation of the head and eyes commonly occurs during epileptic seizures, nystagmoid eye movements are an unusual ictal manifestation. The clinical characteristics of the nystagmus in these patients were consistent with epileptic nystagmus type two (EN-2), which typically has a seizure focus in the temporo-parietal-occipital cortex, contralateral to the direction of the nystagmus beats (Kaplan 1993; Harris 1997; Newton 1998a). This area overlaps the putative cortical region of visual motor sensitivity (V5), which is important for the generation of ipsiversive smooth pursuit (Lekwuwa 1996). Subtle seizures have not been described in previous studies of childhood cerebral malaria but are of great clinical importance, since several of these children recovered consciousness within a few hours of anticonvulsant treatment. In addition, epileptic inhibition of respiration may prevent some of these children from compensating for a profound metabolic acidosis (English 1996a; Crawley 1998). In a busy, understaffed hospital in the developing world such seizures are easily missed, yet their clinical features are sufficiently distinct for them to be recognised by medical or nursing staff with appropriate training, without the need for EEG.

The pathogenesis of seizures in childhood cerebral malaria is likely to be complex and multifactorial, and is discussed in more detail in Chapter 4. Although fever is known to precipitate seizures in young children (Wallace 1988) it is of note that, in this series, over 50% of all seizures occurred when the rectal temperature was below 38 °C. In addition, only two seizures were associated with blood glucose of below 2.2 mmol/1. Instead, this study suggests that cerebral hypoxia may be one of the factors predisposing to seizures. EEG recordings demonstrated that electrical seizure activity consistently arose from the posterior tenporo-parietal region, which is a "watershed" area, lying between territories supplied by the middle cerebral (carotid circulation) and posterior cerebral (vertebro-basilar circulation) arteries. Consequently, it is particularly vulnerable to hypoxia when oxygen delivery to the brain is compromised as a result of sequestration, severe anaemia, or inadequate cerebral perfusion due to hypotension or raised intracranial pressure (MacPherson 1985; Newton 1994; English 1997a; Newton 1997b). Because of their anatomical position close to the tençoral lobes in the tentorial notch, the posterior cerebral arteries may be particularly vulnerable to compromise in those patients who have acute intracranial hypertension and

76 incipient transtentorial herniation, as illustrated by the EEGs in Figure 10. Raised intracranial pressure, reduced cerebral perfusion pressure, and transtentorial herniation have been documented in cerebral malaria (Newton 1994; Newton 1997b), and in other paediatric encephalopathies (Minns 1991). By initiating the release of excitotoxic mediators such as glutamate or quinolinic acid (Dobbie 2000), local hypoxia may precipitate seizures, which, by raising intracranial pressure and increasing the demand for oxygen and glucose, may exacerbate the situation further. Although there is no evidence for failure of cerebral autoregulation in the majority of these patients, relatively poor collateral circulation in some patients may mean that cerebral blood flow in the border-zone between the carotid and vertebro-basilar territories is insufficient to compensate for increased local demand. In these patients, a vicious cycle might then be generated, leading to intractable partial seizures and eventually to focal infarction (Figure 11).

Diffuse background slow wave activity on EEG occurs in many conditions (metabolic, toxic, and infectious) that have a generalised effect on the brain (Plum 1982; Hughes 1994). Background slow wave activity in cerebral malaria has been described in adults (Stotka 1973; Chen 1991; Thumasupapong 1995), and in one previous study on children (Lemercier 1969). The mechanism for the increased slow wave activity seen during episodes of intracranial hypertension and opisthotonus is unknown, but may have been due to concurrent hyperventilation, leading to hypocapnia and cerebral vasoconstriction (Kiloh 1981). Intracranial pressure rose acutely during seizures, and was accompanied by a concurrent rise in mean arterial pressure, suggesting that cerebral autoregulation was intact. In one case, however, mean arterial pressure was insufficient to maintain adequate cerebral perfusion, despite treatment with pressor agents. Infiision of mannitol was followed by clinical and electrophysio logical deterioration, suggesting that the integrity of the blood- brain barrier had been breached.

EEGs have been used to predict prognosis in a number of childhood encephalopathies (Pampiglione 1968; Kuroiwa 1980; Tasker 1988). In this study, burst suppression or background activity of very slow fi*equency was associated with an increased risk of death. Of five children with episodic low amplitude events (ELAE) on initial EEG, four made a

77 full recovery. In two of these cases, ELAE were associated with the administration of diazepam, which may partly explain why their prognosis appears better here than in other types of encephalopathy (Brenner 1975; Rae-Grant 1991). Children with a history of status epilepticus prior to admission, and an initial EEG showing asymmetric background activity or continuous inter ictal multifocal spike waves, had an increased incidence of neurological sequelae. Overall, almost 70% of the children with an adverse outcome (death or neurological sequelae) had one or more poor prognostic features on initial EEG. Although this suggests that in some cases significant cerebral pathology must have had occurred prior to hospital admission, anticonvulsant prophylaxis given at the time of admission may prevent further cerebral damage. There is an urgent need to find a prophylactic anticonvulsant drug that is both safe and effective in children with cerebral malaria. This will be discussed further in Chapter 6.

Electrophysio logical monitoring using serial EEG and CFAM has made it possible to identify a wide variety of seizures (clinical, subtle, and electrographic) in children with cerebral malaria, and has emphasised their importance in the pathogenesis of coma. We have been able to correlate the electroencephalographic and clinical findings from this group of unconscious children, something that is not possible in most parts of the developed world where the majority of unconscious children are ventilated. The information obtained from this study may, therefore, provide some valuable insights into a wide variety of other paediatric encephalopathies.

78 4. AETIOLOGY Is chloroquine a risk factor?

79 Introduction

What accounts for the high prevalence of seizures in childhood cerebral malaria? This chapter discusses various aetiological hypotheses, and then considers in further detail the possible role of chloroquine and its active metabolite desethylchloroquine.

Since fever is known to precipitate seizures in young children (Verity 1985; Wallace 1988), and the majority of children with cerebral malaria have fever as a presenting feature, it is not unreasonable to consider that the seizures of cerebral malaria may be “febrile seizures”. However, among the 65 children with cerebral malaria described in Chapter 3, and for 582 children with malaria whose case notes were reviewed retrospectively (Waruiru 1996), more than 50% of all seizures occurred at rectal temperatures of below 38®C. In addition, “febrile seizures” are usually generalised and of short duration (Wallace 1988), while the majority of seizures associated with malaria are partial motor, with a high proportion being prolonged (lasting for more than 5 minutes), or multiple (2 or more seizures) (Molyneux 1989; Akpede 1993).

These factors, plus the observation that, despite similar peak temperatures, seizures complicate twice as many cases of malaria caused by Plasmodium falciparum compared to Plasmodium vivax (Wattanagoon 1994), suggest that the pathological processes induced by Plasmodium falciparum may be specifically epileptogenic. Plasmodium falciparum is the only species of malaria parasite to undergo sequestration in deep capillary beds, and the characteristic histopathological feature of cerebral malaria is intense sequestration of parasitized red blood cells in the cerebral microvasculature (MacPherson 1985). This suggests that local interference with blood flow could lead to seizures, either directly as a result of hypoxia, as discussed in Chapter 3, or by initiating the release of excitotoxic mediators such as glutamate or quinoIonic acid (see Chapter 5).

Seizures can also result from metabolic disturbances, such as hypoglycaemia or electrolyte imbalance. However, no association between hypoglycaemia and seizures was found during a study of severe malaria in Gambian children (White 1987), or in the study

80 described in Chapter 3. Mild hyponatraemia occurred in a small proportion of children in this study, but was not significantly associated with the occurrence of seizures.

Despite the relentless spread of drug-resistant Plasmodium falciparum, chloroquine continues to be widely used throughout Africa for the treatment and prevention of malaria. In addition, the drug is used in many parts of the world as second-line treatment for hepatic amoebiasis and connective tissue disease. A number of case reports in the medical literature have described an association between seizures and the ingestion of chloroquine, both at therapeutic concentrations (Torrey, 1968; Fish 1988; Luijckx 1992) and following overdose (Cann 1961; Kiel 1964; Jaffe 1988; Riou 1988). A large proportion of patients admitted to hospital in Kenya with malaria have taken chloroquine, obtained from shops or local dispensaries, prior to attending hospital (Snow 1992; Marsh 1999). The purpose of the following study, therefore, was to investigate the possibility that seizures in childhood cerebral malaria may be caused or exacerbated by chloroquine or its active metabolite desethylchloroquine.

Methods

The study formed part of a randomised, double-blind, placebo-controlled trial of phenobarbitone prophylaxis in childhood cerebral malaria, which will be described in detail in Chapter 6. Children aged 9 months to 13 years were recruited if they had cerebral malaria (see Chapter 2). As soon as possible after admission, children were randomised to receive a single intramuscular dose of phenobarbitone 20 mg/kg or identical placebo. Blood was taken at baseline, 2, 4, 8, 12 and 24 hours for measurement of phenobarbitone, chloroquine (CQ), and desethylchloroquine (DCQ). Analysis of the relationship between blood CQ and DCQ concentrations and subsequent seizure activity was confined to the placebo group.

Treatment Children received standard treatment for cerebral malaria (Crawley 1999). The number and duration of all seizures were recorded using timers pre-set to alarm at 5, 15, and 30

81 minutes. Seizures lasting for 5 minutes or more were treated in a standardised manner, with intravenous diazepam 0.3 mg/kg and, as second-line therapy, intramuscular paraldehyde 0.2 ml/kg.

Chloroquine assay Chloroquine (CQ) and desethylchloroquine (DCQ) concentrations were measured in whole blood using a modification of the method of Patchen et al (Patchen 1983). Blood samples (lOOpl) were extracted with a combination of methyl-rer/-butyl-ether and hexane (1:1), centrifuged (1000 x g; 10 minutes), and the organic layer separated and evaporated in a water bath (37 °C) under a gentle stream of nitrogen. The residue was reconstituted in mobile phase (lOOpl) and analyzed by high-performance liquid chromatography, using ultraviolet detection at 340nm. The lower limit of detection for both chloroquine and desethylchloroquine was 5pg/l.

82 Table 7. Comparison of baseline admission characteristics in children with cerebral malaria

(CQ: chloroquine; DCQ: desethylchloroquine; values are median (interquartile range) unless stated)

SEIZURES AFTER ADMISSION

No Yes P value (n = 50) (n = 59)

Age (months) 32.7 (28.0-38.1) 28.7 (25.1 -32.7) 0.19 Geometric mean (95% Cl)

Seizures before 39/50 (78%) 56/59 (95%) 0.01 admission No. (%)

Anticonvulsants 7/50 (14%) 17/59 (29%) 0.07 before admission No. (%)

Baseline CQ (|ig/l) 227.5 (79.4-430.2) 169.4 (75.1 -374.9) 0.53

Baseline DCQ (pg/1) 364.0(131.3-709.4) 352.3 (81.9-580.1) 0.53

AUC 0-12 CQ (pg.hr/1) (n = 43) 2058.9(1014.1 -5469.4) 2057.6 (802.8-2536.8) 0.37

AUC 0-12 DCQ 4534.8 (2420.3-6416.6) 4285.2(1985.0-5740.6) 0.53 (pg.hr/1) (n = 39)

Parasitaemia (/p.1) 64805 (29987 -140049) 42293 (22473 - 79598) 0.39 Geometric mean (95% Cl)

Glucose (mmol/1) 4.0 (3.0-5.3) 4.1 (3.3-5.3) 0.83 Geometric mean (95% Cl)

Base excess -7 (-12 to -4) -8 (-13 to -3) 0.90

83 Table 8. Baseline drug levels for children with and without status epilepticus

STATUS EPILEPTICUS AFTER ADMISSION

No Yes P (n = 100) (n = 9) value

Baseline CQ (pg/1) 227.5 (85.6-441.2) 75.1 (7.4-116.5) 0.02

Baseline DCQ (pg/1) 375.9(134.8-701.2) 81.9 (0-322.9) 0.07

AUC 0-12 CQ (pg.hr/1) 2170.1 (1089.5-4053.6) 470.3 (211.3-659.7) 0.009 (n = 43)

AUC 0-12 DCQ (pg.hr/1) 4769.4(2420.3-6416.6) 1188.7 (392.5-1985.0) 0.06 (n = 39)

Chloroquine (CQ); Desethylchloroquine (DCQ); Area under the concentration curve for 0-12 hours (AUC 0-12)

Values expressed as median (interquartile range)

84 Results

Samples Of 340 patients recruited to the main phenobarbitone prophylaxis study, 170 received placebo. Sufficient blood was available for measurement of CQ and DCQ on 109 (64%) of these patients. There was no difference in the frequency or duration of seizures between patients who had levels measured and those who did not (p > 0.1 for all comparisons). Chloroquine was detected in 100/109 (92%) of the baseline blood samples. Median (interquartile range) baseline concentrations were 214.9 pg/1 (79.4 - 427.3) for CQ and 349.3 pg/1 (90.3 - 594.7) for DCQ. Of 80 children who had 3 or more CQ levels taken during their hospital course, 29 (36%) were thought to have received CQ in the period immediately (< 24 hours) before hospital admission, since the concentration of CQ in their blood fell by more than 50% during the first 24 hours of admission (White 1988b). These children had median (interquartile range) baseline levels of CQ and DCQ that were significantly higher than those given chloroquine more than 24 hours prior to admission (CQ 374.9 pg/1 (278.6 - 888.3) versus 114.8 pg/1 (41.0 - 185.8), p = 0.0001; DCQ 580.1 pg/1 (312.3 - 1156.2) versus 284.1 pg/1 (52.6 - 518.9), p = 0.001).

Seizures Table 7 compares the baseline characteristics of those who had seizures with those who did not. Overall, fifty four percent (59/109) of the children had one or more witnessed seizures after admission. Of the children with undetectable baseline blood levels of chloroquine, 6/9 (67%) had between 1 and 27 (median 5) seizures. Forty one percent (45/109) had a seizure lasting for 5 minutes or more, while 8% (9/109) had an episode of status epilepticus (seizure lasting for 30 minutes or more). Children who had received chloroquine less than 24 hours prior to admission had a similar frequency and duration of seizures to those who had received CQ earlier (p > 0.4 for all comparisons). There was no significant correlation between the total number of seizures after admission and median baseline concentrations of CQ (Spearman’s rho -0.089, p = 0.36) or DCQ (Spearman’s

85 rho -0,039, p = 0.69). Baseline levels of CQ and DCQ and the corresponding area under the concentration curves (AUCo-12) were not significantly associated with the occurrence of seizures lasting for 5 minutes or more (p > 0.5 for CQ, DCQ, and AUCo-12). Baseline CQ levels and AUC0.12 were, however, significantly reduced in the nine children who had an episode of status epilepticus (Table 8). Multivariate logistic regression analysis, taking into account factors likely to affect the risk of seizures in hospital (Table 7) failed to change the significance of these results.

86 Discussion

There are many possible explanations for the high incidence of seizures in childhood cerebral malaria. Fever, known to precipitate seizures in young children (Wallace 1988), is an almost universal feature of cerebral malaria, although many of the seizures in cerebral malaria occur at temperatures of below 38 degrees Centigrade (Crawley 1996). Hypoglycaemia (White, 1987) and hyponatraemia (English 1996b), both complications of severe malaria, may also induce seizures. Sequestration of parasitised red blood cells in the cerebral microvasculature, the histopathological hallmark of cerebral malaria, may cause seizures through hypoxia, or by initiating the release of excitotoxic mediators, such as quinolinic acid (Dobbie 2000).

Throughout Africa, chloroquine continues to be widely used for the treatment of febrile illnesses at the community level, as illustrated by this and by previous studies (Snow 1992; Marsh 1999). Baseline chloroquine concentrations in this study were generally at the low end of the therapeutic range, and were well below concentrations (2000pg/l and above) considered toxic (Riou 1988). The association between chloroquine and seizures has been described in both adults and children, at therapeutic concentrations (Torrey 1968; Fish 1988; Luijckx 1992) and following overdose (Cann 1961; Kiel 1964; Jaffe 1988; Riou 1988). Seizures may occur within a few hours of overdose (Cann 1961; Kiel 1964; Jaffe 1988), or between one day and several weeks after the start of treatment with therapeutic doses of chloroquine (Luijckx 1992). The concentration of chloroquine within the brain is approximately four times that of plasma (Titus 1989), and experimental evidence suggests that chloroquine may precipitate seizures by attenuation of gamma aminobutyric acid (GABA) pathways (Amabeoku 1992), and by the enhancement of dopaminergic neurotransmission (Amabeoku 1993).

In this study, however, there was no association between baseline levels, area under the concentration curve (which reflects both absorption and subsequent elimination) of chloroquine and its active metabolite desethylchloroquine, and the number of subsequent seizures in children with cerebral malaria. In the small sub-group of children who had

87 one or more episodes of status epilepticus, baseline levels of chloroquine and desethylchloroquine were reduced.

There are a number of limitations to this study. The dose and route of chloroquine administration were not known, and it was only possible to obtain a rough estimate of the time of administration in 73% of the patients. Information on seizures that occurred prior to admission was based on clinical history, which can be unreliable, and analysis was therefore confined to seizures that were witnessed by clinical staff in hospital. In addition, it is not known whether the reported association between chloroquine ingestion and seizures is a dose-related or idiosyncratic phenomenon. Although this study suggests that the seizures of childhood cerebral malaria are unlikely to be caused by chloroquine alone, more definitive evidence would come from prospective longitudinal studies. Such studies could assess the prevalence of seizures complicating cerebral malaria in relation to changing patterns of chloroquine useage. The rapid spread of chloroquine resistance, and the urgent need to change to alternative antimalarial drugs, is likely to provide such an opportunity in the near future. 5. AN EXCITOTOXIC MECHANISM for CHILDHOOD CEREBRAL MALARIA?

89 Introduction

Despite many years of clinical and experimental investigation, the pathogenesis of cerebral malaria remains poorly understood. This chapter explores the possibility that some of the clinical features may be explained by an excitotoxic mechanism.

The adherence of parasitised erythrocytes to endothelial cells of the cerebral vasculature initiates a cascade of events that cause intense endothelial activation on the luminal side of the blood-brain barrier (Turner 1994). Much less is known about events occurring on the abluminal side of the blood-brain barrier. Experimental stimulation of cells of the macophage/monocyte lineage by cytokines causes the parallel induction of indoleamine- pyrrole 2,3-dioxygenase, GTP cyclohydrolase I, and nitric oxide synthase (Werner 1989; Sakai 1995). These enzymes catalyse the first step of pathways leading to the formation of quinolinic acid, neopterin, tetrahydrobiopterin, and nitric oxide (see Figure 12). In the brain, microglia constitute the resident macrophage/monocyte cells. Experimental stimulation of microgUa with cytokines also causes induction of these enzymes (Sakai 1993; Heyes 1996).

Quinolinic acid is an endogenous excitotoxin that is a selective agonist of N-methyl-D- aspartate (NMDA) glutamate receptors (Stone 1993). When apphed to the central nervous system of experimental animals or to cerebral tissue culture, it causes seizures, sodium influx with reversible neuronal swelling, and calcium influx with delayed neuronal disintegration (Lapin 1982; Choi 1992; Giulian 1993). Thus, the toxic action of quinolinic acid could explain the neurological complications of childhood cerebral malaria, namely seizures, reversible cerebral oedema, and permanent neurological damage.

Nitric oxide synthases catalyse the formation of the gas nitric oxide from arginine (Figure 12) (Nathan 1994). NMD A receptors are modulated by endogenous nitric oxide, with increasing concentrations of nitric oxide causing receptor blockade, and decreasing concentrations potentiating NMD A-induced calcium influx (Manzoni, 1992; Manzoni,

90 1993). Nitric oxide synthases require tetrahydrobiopterin as an essential co-factor. GTP- cyclohydrolase catalyses the first step in the tetrahydrobiopterin synthesis pathway (Figure 12), in which dihydroneopterin acts as an intermediary. Activation of human macrophage/monocyte cells results in the accumulation of the breakdown product neopterin, and the concentration of neopterin in biological fluids is widely used as a marker of immune activation (Wachter 1989). 5-hydoxyindoleacetic acid (5-HIAA) is a metabolite of serotonin, whose concentration in cerebrospinal fluid (CSF) reflects serotonin turnover.

We hypothesised that intense cerebrovascular endothelial activation in cerebral malaria causes activation of microglia within the brain. These activated cells then produce quinolinic acid, which may be a cause of the cerebral symptoms. In order to test this hypothesis, we have measured CSF concentrations of quinolinic acid, its precursor tryptophan, 5-HIAA, individual pterin species, and nitrate plus nitrite (stable breakdown products of nitric oxide) in CSF from children recovering fi*om cerebral malaria.

91 Figure 12. Metabolic inter-relationships between quinolinic acid, neopterin, tetrahydrobiopterin, and nitric oxide

1 2 Quinolinic acid A Tryptophan — —► Serotonin—► 5-HIAA

BH2

NMDA BH4 ^ NEO GTP

NO Arginine

Citrulline

1 : Indoleamine-2,3-dioxygenase

2: Tryptophan monooxygenase

3: GTP-cyclohydrolase I

4: Nitric oxide synthase

5-HIAA: 5-hydroxyindoldeacetic acid

BH2: Dihydrobioterin BH4: Tetrahydrobioterin

NEO: Dihydroneopterin triphosphate GTP: Guanosine triphosphate

NO: Nitric oxide

NMDA: N-methyi-D-aspartate glutamate receptor

92 Methods

Children aged 8 months and above were recruited if they fulfilled the definition for:

• Cerebral malaria, namely failure to locahse pain in the presence of Plasmodium falciparum parasitaemia. Or • Malaria with impaired consciousness, defined as a Blantyre coma score (Table 3) of 4 or less in the presence of Plasmodium falciparum parasitaemia

Children received standard treatment for cerebral malaria, as described in Chapter 2. CSF was obtained by lumbar puncture once conscious level had started to improve. In 11 children who died, CSF was obtained within 15 minutes of death. The first 1ml aliquot of CSF was used for diagnostic purposes, while the second and third 1 ml aliquots were used for this study. The third 1ml was collected into a bottle containing 1ml each of dithioerythritol (DTE) and diethylenetetraaminopentaacetic acid (DETAPAC). The second and third 1ml aliquots were frozen at the bedside on dry ice, and stored at -70 °C until analysis. All CSF analysed had a normal cell count.

The concentrations of the following compounds were measured in CSF: quinolinic acid, tryptophan, 5-HIAA, total neopterin, tetrahydrobiopterin, dihydrobiopterin, and nitrate plus nitrite. Quinolinic acid was measured by gas chromatography electron impact mass spectrometry after tert-butyldimethylsilyl derivitization (Dobbie 1997). Tryptophan was measured by high-performance liquid chromatography (HPLC) with fluorometric detection after precolumn derivatization with o-phthaladialdehyde- 2 -mercaptoethanol (Dobbie 1997). 5HIAA was measured by HPLC with electrochemical detection (Hyland 1993). Total neopterin, tetrahydrobiopterin, and dihydrobiopterin were measured by HPLC with dual electrochemical and fluorometric detection (Hyland 1993). Nitrate plus nitrite were measured by spectrophotometry (Clelland 1996). Reference ranges for the metabolite concentrations (n = 24 to 78 depending on the metabolite) were constructed from children and young adults with a variety of neurological or metabolic diseases in

93 whom no disturbance of the biochemical pathways was expected, who were living in the United Kingdom. In these children, CSF was sampled after a 4-hour fast. There was an age-related decrement in CSF concentration for all metabolites except neopterin, and therefore all children with cerebral malaria or malaria with impaired consciousness were exactly age-matched to the reference group. We were also able to collect an age-matched local reference group of 9 children who were being investigated for seizures, or were undergoing staging for Burkitt’s lymphoma. CSF fi*om these children was used for comparison of nitrate plus nitrite concentrations, but was not suitable for measurement of quinolinic acid or the pterin species, because the majority of these patients were febrile.

94 Table 9. Cerebrospinal fluid metabolite concentrations in cerebral malaria

Geometric mean (95% Cl) Geometric P value Reference Cerebral mean ratio range malaria (95% Cl)

Quinolinic acid 16.2 229 14.1 < 0 .0 0 1 (nmol/1) (12.3-21.5) (188-278) (9.8-20.4)

Nitrate plus nitrite 1 2 .6 5.7 0.45* <0 .0 0 1 (pmol/1) (10.5-15.1) (5.1-6.5) (0.35-0.59)

Total neopterin 17.0 186 10.9 <0 .0 0 1 (nmol/1) (15.2-19.1) (162-213) (9.1-13.0)

Tetrahydrobiopterin 33.9 119 3.5 <0 .0 0 1 (nmol/1) (30.2-38.0) (106-134) (3.0-4.2)

Dihydrobiopterin 3.8 12.7 3.3 <0 .0 0 1 (nmol/1) (2.9-4.9) (11.2-14.4) (2.6-4.5)

5-Hydro xyindoleacetic 194 233 1 .2 0 .0 2 acid (nmol/1) (172-218) (210-259) (1.0-1.4)

2.7 2.5 0.93 Tryptophan (pmol/1) 0.48 (2.4-3.0) (2.1-3.0) (0.77-1.1)

* Geometric mean ratio (95% Cl) for local reference range: 0.55 (0.45 - 0.68), p <0.001

95 Table 10. Cerebrospinal fluid metabolite concentrations in the different outcome groups

Geometric mean (95% Cl)

Normal Neurological Died Analysis of recovery sequelae (n = 1 2 ) variance (n = 74) (n = 1 1 ) statistics

Quinolinic acid 194 359* 429* Fi,95 — 5.35 (nmol/ 1) (161 -234) (170-759) (188-978) P = 0.006

Nitrate plus nitrite 5.4 6.3 8 .1 p 2,93 — 2.64 (pmol/ 1) (4.7-6.1) (4.3-9.2) (5.1-12.9) P = 0.076

Total neopterin 180 169 243 P2,9 5~ 1 .1 2 (nmol/ 1) (154-251) (114-251) (141 -419) P = 0.33

Tetrahydrobiopterin 130 10 1 114 P2,9 3“ 1.67 (nmol/ 1) (116-144) (74.8-134) (62.1-210) P = 0.19

Dihydrobiopterin 1 1 .0 14.5 28.2* P2,9 5~ 15.7 (nmol/ 1) (9.8 -12.3) (9.9-21.2) (16.9-47.1) P < 0.001

5-Hydroxyindoleacetic 216 242 335* P2,95 — 3.94 acid (nmol/ 1) (194-241) (173-338) (220-511) P = 0.023

Tryptophan (pmol/ 1) 2.4 2.4 3.5 p 2,93 " 1.18 (2 .0 - 2 .8 ) (1.6-3.7) (1.6-7.8) P = 0.31

* Significantly increased (analysis of variance with Duncan’s new multiple range test)

96 Results

Ninety-seven children were studied, of whom 79 had cerebral malaria, and 18 had malaria with impaired consciousness. Median age was 2.2 years (95% Cl 0.7 to 6.9 years). Eighty-two children (85%) had CSF taken within 48 hours of admission. There were no differences in any of the metabolite concentrations between CSF taken within 48 hours of admission (n = 82) compared with that taken after 48 hours (n = 15). There were also no significant differences in metabolite concentrations between the children with cerebral malaria and those with impaired consciousness. Geometric mean ratios (cerebral malaria/malaria with impaired consciousness) for each metabolite were as follows. Quinolinic acid 1.1 (95% Cl 0.5-1.9), neopterin 0.9 (95% Cl 0.6 - 1.2), tetrahydrobiopterin 0.8 (95% Cl 0.6 - 1.1), dihydrobiopterin 1.1 (95% Cl 0.8 - 1.5), 5HIAA 1.0 (95% Cl 0.8 - 1.4), tryptophan 1.2 (95% Cl 0.8 - 1.8), nitrate plus nitrite 1.2 (95% Cl 0.9 - 1.5). Therefore, for subsequent analysis, children with cerebral malaria and those with malaria and impaired consciousness were grouped together as “cerebral malaria” for simplicity (Table 9).

In comparison with the reference range, children with cerebral malaria had highly significant increases in the CSF concentrations of quinolinic acid, neopterin, tetrahydrobiopterin, and dihydrobiopterin (Table 9). There was a significant but small increase in 5HIAA, and no change in tryptophan concentration. There was no significant difference or obvious trend in metabolite concentrations between children who had seizures or status epilepticus and those who did not. In contrast, CSF nitrate plus nitrite concentrations were significantly reduced in cerebral malaria compared to both the United Kingdom reference range (Table 9) and the local reference range. There was no difference in nitrate plus nitrite concentrations between the United Kingdom and local reference ranges (geometric mean ratio 0.82 (95% Cl 0.60 - 1.11), p = 0.19).

In children with cerebral malaria, concentrations of CSF quinolinic acid, dihydrobiopterin, and 5HIAA were significantly different across the three outcome groups (Table 10). CSF quinolinic acid was significantly increased in survivors with

97 neurological sequelae and those who died compared with those who recovered normally. CSF dihydrobiopterin and 5HIAA concentrations were significantly increased in children who died compared to those who survived.

Discussion

Our results show that Kenyan children with cerebral malaria have a characteristic neurochemical profile, with increased CSF concentrations of quinolinic acid and neopterin, and a reduced concentration of nitrate plus nitrite. Increased concentrations of quinolinic acid (Brouwers 1993; Hagberg 1993; Heyes 1995) and neopterin (Weiss 1998) within the central nervous system (CNS) have been demonstrated in a number of CNS infections, and in a murine model of cerebral malaria (Sanni 1998). There is evidence that this results from induction of indoleamine 2,3-dioxygenase and GTP cyclohydrolase I, the first enzymes in the pathways leading to quinolinic acid and neopterin synthesis respectively (Wachter 1989; Heyes 1993; Nathan 1994).

Experimental support for our findings comes from the work of Sanni and others, who demonstrated a 3-fold increase in brain quinolinic acid concentrations in mice with fatal cerebral malaria due to experimental infection with Plasmodium berghei ANKA (Sanni 1998). In a previous study of children with cerebral malaria, Weiss and co-workers found CSF concentrations of total neopterin and biopterin that were considerably lower than those detected in this study (Weiss 1998). The clinical and epidemiological characteristics of cerebral malaria are similar in Zambia and Kenya, and methodological differences may account for the variation between the two studies.

Conditions that cause induction of indoleamine 2,3-dioxygenase and GTP cyclohydrolase I have also been shown experimentally to cause induction of nitric oxide synthase, leading to increased synthesis of nitric oxide (Sakai 1995). Nitrate and nitrite are stable breakdown products of nitric oxide, and clinical and experimental evidence suggests that concentrations of nitrate plus nitrite in lumbar CSF reflect nitric oxide synthase activity within the brain (Salter 1996; Dobbie 1997). In contrast to the modest elevation of CSF

98 nitrate and nitrite concentrations previously found in cerebral malaria (Weiss 1998), we found decreased concentrations of nitrate plus nitrite in cerebral malaria, compared to both United Kingdom and local reference populations. Our findings are similar to those found in Tanzanian children with Plasmodium falciparum infection (Anstey 1996). In this study, reduced nitrite plus nitrate levels in both plasma and urine were found in children with cerebral malaria, compared with controls and with children with sub- clinical or uncomplicated malaria. Reduction in concentration of nitrate plus nitrite can only occur as a result of reduced activity of constitutive nitric oxide synthase, or by increased removal of nitric oxide. Within the brain, the activity of both constitutive iso forms of nitric oxide synthase is calcium-dependent and requires tetrahydrobiopterin as a co-factor. (Nathan 1994). We have shown that tetrahydrobiopterin concentrations are increased in cerebral malaria, and co-factor deficiency does not, therefore, seem a likely explanation. It is possible that, in cerebral malaria, the activity of nitric oxide synthase within the brain is limited by reduced concentrations of fi-ee intracellular calcium or of the substrate arginine. Both possibilities require further investigation.

The combination of increased quinolinic acid and reduced nitric oxide concentrations within the brain may provide an excitotoxic mechanism that accounts for the symptoms of cerebral malaria. When quinolinic acid is synthesised in excess, it readhy enters the extracellular compartment (Spéciale 1993). There is neither extracellular metabohsm nor active transport of quinolinic acid in cerebral tissue, and toxic quantities of quinolinic acid may therefore accumulate within the synaptic cleft (Foster 1983). Nitric oxide has been shown to protect against NMDA-mediated neurotoxicity by reducing conductance of calcium through the receptor pore (Manzoni 1992; Manzoni 1993). Reduction in nitric oxide production could therefore enhance the neurotoxicity of quinolinic acid (Manzoni 1992; Manzoni 1993).

The mechanisms by which NMDA-toxicity is mediated can be separated into two components, distinguishable on the basis of time course and ionic dependence. An initial influx of sodium causes reversible neuronal swelling, while subsequent influx of calcium leads to delayed neuronal disintegration (Choi 1992). Excitotoxic mechanisms can thus

99 explain the neurological symptoms of cerebral oedema, namely coma and cerebral oedema that usually resolve and, in a proportion of cases, neurological damage that may be permanent. The graded increment in quinolinic acid concentrations across the three outcome groups lends support to this hypothesis. Although it is possible that the increased concentrations of quinolinic acid found in children who died was a post­ mortem artefact, this is unlikely because intracellular accumulation of quinolinic acid does not occur in the central nervous system (Spéciale 1993), and the CSF was sampled within 15 minutes of death.

In conclusion, we have found biochemical evidence for an excitotoxic mechanism that may account for the symptoms of cerebral malaria. This is of potential importance because selective NMDA-receptor antagonists are becoming more widely used in medicine, and could provide a novel approach to the treatment of cerebral malaria (Lipton 1993).

100 6. PHENOBARBITONE PROPHYLAXIS A randomised, controlled intervention study

101 Introduction

If anticonvulsant prophylaxis can reduce the incidence of seizures complicating cerebral malaria, this may in turn reduce the risk of death and neurological sequelae (Molyneux 1989; Brewster 1990; Jaffar 1997; van Hensbroek 1997), and have an important impact on the educational potential of children in sub-Saharan Africa (Holding 1999). This chapter describes a randomised, controlled intervention study of anticonvulsant prophylaxis in childhood cerebral malaria.

Phenobarbitone has been used as an anticonvulsant for many years, and is highly effective in the treatment of both partial and generalised seizures (Shorvon 1994b). It is cheap and, unlike the majority of anticonvulsant drugs used for seizure prophylaxis, widely available throughout Africa. It may be given by intramuscular injection, which is a further advantage, since intravenous therapy is not possible in many health facilities throughout the continent. The loading dose recommended for children is 10-20 mg/kg (Rylance 1990; Bone 1993).

The three published studies of phenobarbitone prophylaxis in cerebral malaria have yielded disparate results. In a small, randomised, controlled study on Thai adults, a single intramuscular dose of phenobarbitone 3.5 mg/kg reduced the incidence of seizures by 40% (White 1988a). A single intramuscular dose of phenobarbitone 10 mg/kg given to Indian adults in an open study reduced the incidence of seizures by 20% (Kochar 1997) while, in an open study on Kenyan children, the same dose failed to produce therapeutic blood concentrations, and had no effect on seizure frequency (Winstanley 1992). The randomised, placebo controlled study presented here was designed to assess whether a single intramuscular dose of phenobarbitone 20 mg/kg given on admission to Kenyan children with cerebral malaria could reduce the incidence of seizures complicating the clinical course in hospital. The safety and clinical tolerance of this dose was assessed at the start of the trial.

102 Methods

Patients Children aged 9 months to 13 years with cerebral malaria (see Chapter 2) were eligible for the study. Those who had pre-existing afebrile epilepsy or significant neurodevelopmental problems, and those who had received treatment with phenobarbitone or phenytoin during the current illness were excluded.

Sample size A sample size calculation, assuming 90% power and a significance level of 5%, suggested that a total of 320 children would be required to detect a 50% reduction in seizures occurring prior to recovery of consciousness in the phenobarbitone-treated group. The following seizure categories were considered to be of particular clinical importance:

• Three or more seizures of any duration • Any seizures lasting for 5 minutes or more • Any episodes of status epilepticus (defined as seizure activity lasting for 30

minutes or more, or 6 or more seizures within a period of 2 hours).

Randomisation Using a sequentially numbered register, children were randomised as soon as possible after admission to receive a single intramuscular injection of phenobarbitone 2 0 mg/kg, or the same volume of identical placebo (90% propylene glycol, the normal vehicle for parenteral preparations of phenobarbitone). Numbered 5ml ampoules of phenobarbitone and placebo were prepared by the pharmacy department of Torbay Hospital, UK. The code identifying drug and placebo was kept at Torbay Hospital, and therefore none of the clinical or scientific staff involved in the study knew which patients had received phenobarbitone. Since previous work (Crawley 1996) had shown an increased frequency

103 of seizures among younger children with cerebral malaria, randomisation was stratified into two age groups of 24 months or below and above 24 months.

Clinical tolerance The clinical tolerance of phenobarbitone 20 mg/kg was assessed at the start of the trial. Twenty three children received the study drug (phenobarbitone or placebo) by constant rate intravenous infusion over 4 hours instead of by intramuscular injection. The intravenous route was chosen because the infusion could have been stopped should any adverse events have occurred. As in the main study, the clinical investigators were unaware of which patients had received phenobarbitone. Pulse, respiratory rate, blood pressure, and transcutaneous oxygen saturation were measured at baseline, and at thirty- minute intervals for 5 hours. Blood was taken at the same times for phenobarbitone level, and at hourly intervals for venous gas and lactate. The trial was then unblinded for these 23 patients only, and the phenobarbitone and placebo groups compared with respect to all clinical and biochemical findings.

Investigations Baseline blood samples were taken for parasite count, full blood count, glucose, electrolytes, blood gas, blood culture, and phenobarbitone level. Further blood samples were taken for phenobarbitone level at 1,2, 4, 8 , 12, 24, 36, and 48 hours. Parasite counts were repeated every 8 hours until discharge, death, or clearance of parasitaemia.

Treatment Patients received standard therapy for cerebral malaria, as described in Chapter 2. The number and duration of all seizures were recorded using timers pre-set to alarm at 5, 15, and 30 minutes. Seizures lasting for 5 minutes or more were treated in a standardised manner, with intravenous diazepam 0.3 mg/kg, and, as second line therapy, intramuscular paraldehyde 0.2 ml/kg. Anticonvulsants were administered at 5, 15, 30, and 45 minutes, if seizure activity persisted. Intravenous phenytoin 20 mg/kg was given to children who, following randomisation, had received 2 doses of both diazepam and paraldehyde, or had experienced 6 or more seizures within a period of 2 hours.

104 Follow up A neurological examination was performed on all patients at the time of discharge from hospital. All patients were asked to return 3 months later for full neurodevelopmental assessment.

Pharmacokinetics Serial blood samples for phenobarbitone concentration were taken from all patients. Whole blood phenobarbitone concentrations were subsequently assayed by reversed- phase high performance liquid chromatography (Winstanley 1992) in Nairobi. Derived pharmacokinetic parameters, namely maximum concentration (Cmax), time to maximum concentration (Tmax), and area under the concentration / time curve (AUCo -12 hours) were obtained after every 1 0 profiles on patients who had received phenobarbitone by intramuscular injection. Since there was very little variability in the data, with no significant change in the mean derived pharmacokinetic profiles as numbers increased, it was decided that profiles on 50 patients would provide a good representation of the phenobarbitone group as a whole.

Stastisticai analysis This is described in Chapter 2. An interim analysis was performed by an independent statistician in January 1997, at which stage 170 patients had been recruited. The results of the analysis were reviewed by the trial monitor, but were not made available to the clinical investigators. The trial was continued until the calculated sample size had been achieved.

105 Figure 13. Whole-blood phenobarbitone concentrations according to mode of administration

30 1

2 0-

o 10-

0 10 20 100 140 160 Time (hours)

Circles denote intravenous and squares intramuscular administration

Error bars: 1 SD

106 Results

Recruitment Between June 1995 and January 1998, 440 children fulfilling the criteria for entry to the study were admitted to the research ward. One hundred were not recruited, for the following reasons. Relatives refused consent (63), death considered imminent (23), clinical pressures made recruitment impossible (14). The remaining 340 children were randomised, and all are included in the main analysis. Eleven children were recruited to the study but were subsequently (before the study code was broken) recognised as fiilfilling the criteria for exclusion. Repeat analysis, following exclusion of these cases, did not change the statistical significance of any of the study endpoints.

Clinical tolerance Twenty three children (10 phenobarbitone, 13 placebo) were assessed for clinical tolerance. Baseline clinical (pulse, systolic blood pressure, respiratory rate, and transcutaneous oxygen saturation) and biochemical (venous blood gas and lactate) characteristics were similar in both groups (p > 0.40 for all comparisons). There were no significant differences between the groups in the mean change between baseline and 5- hour or baseline and maximum / minimum values for any of the parameters measured (p = 0.07 for fall in lactate (0-5 hours) and maximum rise in pH, p > 0.10 for all other comparisons).

Pharmacokinetics Whole blood concentrations of phenobarbitone obtained by intramuscular injection and intravenous infusion of 20 mg/kg are shown in Figure 13. Intramuscular injection (n = 50) resulted in a median maximum concentration (Cmax) of 25.6 pg/ml (range 10.0 to 53.7 pg/ml, interquartile range 23.6 to 30.2pg/ml). Median time to maximum concentration (Tmax) was 4.0 hours (range 1.0 to 48.0 hours, interquartile range 2.0 to 12.0 hours). Concentrations were maintained above 15 pg/ml, which is within the range (10-30 pg/ml) thought to provide effective seizure prophylaxis (Faero et al. 1979), (Dodson 1984), (Shorvon 1994b), for at least 48 hours. The mean area under the

107 concentration-time curve for the period 0 - 1 2 hours (AUCo- 12) following intramuscular injection was 250 pg.hr/ml (95% Cl 232.3 to 268.2). There were no significant differences in Cmax, Tmax, and AUCO-12 between children who survived and those who died (p > 0.4 for all comparisons).

Due to technical problems, it was only possible to obtain profiles on 4 of the 10 children who had received phenobarbitone by intravenous infusion. The mean AUCo -12 following intravenous infusion was 196.2 pg.hr/ml (95% Cl 154.1 to 238.4). The difference in

AUCo -12 between the intravenous and intramuscular routes of administration was of borderline statistical significance (p = 0.05).

Admission characteristics Clinical and laboratory features of all patients in the study are shown in Tables 11 and 12. The placebo and phenobarbitone groups were well matched with regard to the majority of clinical and laboratory parameters. Twenty eight percent of the children (49 placebo, 45 phenobarbitone), had a previous febrile illness that had been complicated by seizures. During the current illness, a significantly higher proportion of children randomized to placebo had a history of seizures prior to admission (148 (87%) versus 133 (78%), p=0.04). The difference in the proportion of children in each treatment group who had been given a dose of diazepam in the 6 -hour period prior to admission failed, however, to reach statistical significance (43 (25%) placebo, 32 (19%) phenobarbitone, p = 0.19).

108 Table 11. Admission clinical features by treatment group

Placebo Phenobarbitone Clinical feature P value (n=170) (n=170)

Age (months) Mean (95% Cl) 35.4 (32.4-38.5) 35.4 (32.7-38.0) 0.97

Male sex Number (%) 93 (55) 91(54) 0.92

Seizures before admission Number (%) Any seizures 148 (87) 133 (78) 0.04 Status epilepticus^ 82 (48) 63 (37) 0.05

Duration of coma prior to Admission (hours) Median 5 5 0.95 Range 1-72 1-72 Interquartile range 2 - 1 0 2 - 1 0

Blantyre coma score^ Number (%) 0 or 1 45 (26) 38(22) 0.45 2 or 3 125 (74) 132 (78)

Rectal temperature (®C) Mean (95% Cl) 38.9 (38.8-39.1) 38.8 (38.6-38.9) 0.16

Pulse (per minute) Mean (95% Cl) 149(146-152) 148(144-152) 0.60

Respiratory rate (per minute) Geometric mean (95% Cl) 41 (39-43) 42 (40-44) 0.69

Respiratory distress* Number (%) 50 (29) 49 (29) >0.99

^ History of multiple seizures (“too many to count”), or of any seizure lasting for 30 minutes ot more ^ See Tables * Increased depth of respiration (Kussmaul’s respiration)

109 Table 12. Admission laboratory investigations by treatment group

Investigation Placebo Phenobarbitone P value (n=170) (n=170) Mean (95% Cl) Mean (95% Cl)

Parasitaemia (per pi) Median 79620 89600 0.96 Range 85-1441800 184-1980000 Interquartile range 6448-351120 4294-422400

Haemoglobin (g/dl) 7.2 (6.8-7.5) 7.2 (6.9-7.6) 0.78

Sodium (mmol/1) 135 (134-136) 134(133-135) 0.29

Potassium (mmol/1) Median 4.3 4.3 >0.99 Range 2.8-8.3 2 .0 -8 .0 Interquartile range 3.9-4.8 3.8-4.9

Urea (mmol/1) Median 4.2 4.9 0.14 Range 0.2-25.4 1.0-36.0 Interquartile range 3.1-6.7 3.2-7.7

Creatinine* (pmol/1) 63.8 (58.9-69.1) 64.5 (59.6-69.7) 0.85

Glucose (mmol/1) 5.4 (4.9-5.9) 5.3 (4.8-5.8) 0.70 pH 7.30 (7.28-7.32) 7.31 (7.28-7.33) 0 .6 6

PCO2 (kPa) Median 4.1 3.8 0.06 Range 1.2-11.5 1.1-13.3 Interquartile range 3.2-4.8 3.0-4.4

Bicarbonate (mmol/1) 15.6(14.8-16.5) 15.1 (14.2-15.9) 0.34

Base excess -9.2 (-10.3 to -8.2) -9.5 (-10.6 to -8.4) 0.73

Lactate (mmol/1)* 3.7 (3.4 -4.2) 4.0 (3.5- 4.5) 0.44

* Geometric mean

110 Clinical course Sixty-three percent (213/340) of the patients achieved parasite clearance before discharge or death. Median (interquartile range) times to parasite clearance were 40.0 (31.5 to 52.8) hours for the phenobarbitone group, and 45.7 (30.6 to 63.0) hours for placebo (p = 0.21, Peto-Peto-Wilcoxon test for equality of survivor function).

There was no difference between the two groups in the time taken to recover consciousness (the ability to localise a painful stimulus (Molyneux 1989)), as shown in Figure 14 (p = 0.39, Log Rank test).

Seizures Phenobarbitone provided effective seizure prophylaxis, reducing the frequency and duration of seizures by over 50% (Table 13). Multivariate logistic regression analysis, allowing for the slight excess of children in the placebo group who had a history of seizures prior to admission, failed to change the significance of this result (Table 13). Significantly fewer children treated with phenobarbitone subsequently required treatment with phenytoin (14/170 (8.2%), versus placebo 27/170 (15.9%) p = 0.04).

Death Mortality among children treated with phenobarbitone was more than double that of the placebo-treated group. Of the forty-four deaths that occurred in total (Table 13), 30/170

(18%) were in children given phenobarbitone, compared to 14/170 ( 8 %) children given placebo (odds ratio 2.39, 95% Cl 1.28-4.46, p = 0.01). Using multivariate logistic regression analysis, the significance of this result could not be accounted for by minor imbalances in risk factors between the two groups (Table 13). Median time from drug administration to death was 22.5 hours (range 0.42 to 92.7) for children treated with phenobarbitone and 24.2 hours (range 0.4 to 66.7) for those who received placebo (p= 0.72). Kaplan-Meier curves, comparing overall survival in the two groups, are shown in Figure 14.

I l l Table 13. Clinical outcome after treatment with placebo or phenobarbitone

Placebo Phenobarbitone Odds ratio P (n=170) (n=170) value Number (%) Number (%)

SEIZURES

Three or more seizures 46 (27) 18(11) 0.32 (0.18-0.58) <0 .0 0 1 of any duration *0.34 (0.19-0.62) <0.001

Any seizures lasting 5 43 (25) 2 0 (1 2 ) 0.39 (0.22-0.70) 0 .0 0 2 minutes or longer *0.42(0.24-0.76) 0.004

Any episodes of status 23 (14) 9(5) 0.36 (0.16-0.78) 0 .0 1 epilepticus** *0.38 (0.17-0.85) 0.02

DEATH / NEUROLOGICAL SEQUELAE

Died 14(8) 30(18) 2.39 (1.28-4.64) 0 .0 1

^2.49 (1.19-5.23) 0.02

Neurological sequelae 33/156 (21) 18/140(13) 0.55 (0.30-1.02) 0.06 at discharge *0.56 (0.30-1.05) 0.07

Neurological sequelae 3 15/144(10) 9/131 (7) 0.63 (0.27-1.47) 0.39 months post discharge *0.69 (0.29-1.65) 0.40

** Seizure lasting 30 minutes or longer, or a minimum of 6 seizures within a 2-hour period

Figures in italics represent adjusted odds ratios and p values * Adjusted for seizures prior to admission ^Adjusted for risk factors associated with increased mortality (Blantyre score, respiratory distress, base excess, glucose, urea, creatinine)

112 Figure 14a. Kaplan-Meier plot of coma resolution (Circles denote phenobarbitone, and squares placebo)

1

.8 6 - tù p=0.39 O3 o m c .6 8 6-

O t .4 & 2 O. 2

0

0 20 40 60 80 100 120 140 160 Time (hours)

Figure 14b. Kaplan-Meier plot of overall survival

.95

D)

p=0.01

.75

20 40 60 80 100 Time (hours) 113 Children who died were, on admission, more deeply comatose, and significantly more dehydrated, acidotic, and hypoglycaemic compared to those who survived (p < 0.01 for all parameters). Among those who died, mean admission respiratory rate was higher in the placebo group (57 (95%CI 50-64) compared to those given phenobarbitone (47 (95%CI 41-52), p = 0.03). However, respiratory rate at 4 hours (maximum phenobarbitone concentration) was comparable for the two groups (placebo 47 (95%CI 41-43), phenobarbitone 48 (95%CI 43-53) p = 0.76). There were no other significant differences between the groups in admission clinical or laboratory characteristics.

Thirty-three children had a respiratory arrest (cessation of breathing in the presence of normal cardiac function) during their clinical course in hospital, of whom thirty died. Of the 33 who had a respiratory arrest, 22 (66.7%) had received phenobarbitone, and 11 (33.3%) placebo (odds ratio 2.1, 95%CI 1.0-4.5, p = 0.06). When total diazepam dose (which included doses given in the 6 hours prior to admission, plus all doses given in hospital, both before and after randomisation) was divided into four categories, the odds of death for children treated with phenobarbitone rose sharply from less than 0.2 for those given 0, 1, or 2 doses of diazepam to 1.68 (95% Cl 0.40 to 6.97, p = 0.004) for those given 3 or more doses. The relationship between death and the interaction between drug group (phenobarbitone or placebo) and diazepam category (less than 3 doses/ 3 or more doses) was then examined using logistic regression and the Likelihood Ratio test. Diazepam category alone was not significantly associated with an increased risk of death (odds ratio 0.55 95% Cl 0.07 to 4.48, p = 0.5). The interaction between diazepam category and drug group (phenobarbitone or placebo) was, however, associated Avith a greatly increased risk of death (odds ratio 16.47, 95% Cl 1.26 to 214.84, p = 0.03). Table 14 compares the difference in mortality between the phenobarbitone and placebo groups following less than 3 and 3 or more doses of diazepam. In this post hoc analysis, a substantial part of the excess mortality in the phenobarbitone group can therefore be accounted for by the interaction between phenobarbitone and multiple (3 or more) doses of diazepam.

114 Table 14. Mortality in phenobarbitone and placebo groups, according to number of doses of diazepam

0,1, OR 2 DOSES OF DIAZEPAM

Placebo Phenobarbitone Odds ratio P value N=150 N=162 (95% Cl)

13 (9%) 25 (15%) 1.9 (0.9-3.9) 0.07

3 OR MORE DOSES OF DIAZEPAM

Placebo Phenobarbitone Odds ratio P value N=20 N=8 (95% Cl)

1 (5%) 5 (62%) 31.7(1.2-814.3) 0.001

115 Neurological sequelae Fifty-one children (17% of those who survived) had neurological sequelae at the time of discharge from hospital. The commonest problems were hemiplegia, spastic quadriplegia, blindness, speech delay, epilepsy, or a combination of these features. Although the proportion of children with sequelae was reduced in the phenobarbitone group (Table 13), the difference was of borderline statistical significance (placebo 33/156 (21%), phenobarbitone 18/140 (13%), p = 0.06). Of 275 children seen 3 months after discharge, 9% had persistent sequelae, and the difference between the two groups was not significant (p = 0.39, see Table 13).

Discussion

There are many reasons why anticonvulsant prophylaxis is likely to be beneficial in childhood cerebral malaria. Clinical and experimental evidence suggests that prolonged, uncontrolled seizure activity can damage the brain (Aicardi 1983; Meldrum 1973a; Corselhs 1983; Stafstrom 1993). Seizures complicating cerebral malaria are associated with an increased risk of neurological sequelae (Molyneux 1989; Brewster 1990; Crawley 1996) and survivors may, in addition, have significant cognitive problems.

The antiepileptic properties of phenobarbitone were first recognised in 1912, and there is now extensive experience of its use in both adults and children (Treiman, 1998; Shorvon 1994b; Bone 1993; Shaner 1988). Experimental evidence suggests that phenobarbitone can prevent structural damage resulting from uncontrolled seizure activity within the developing brain (Sutula 1992; Mikati 1994). This study shows that a single intramuscular dose of phenobarbitone 20 mg/kg is effective at preventing seizures in children with cerebral malaria. Neurological sequelae were reduced in the phenobarbitone treated group, although the difference failed to reach statistical significance. It is important to remember, however, that the clinical conditions of this study, with rapid treatment and close observation of all children with seizures, were very different from those that prevail in many hospitals throughout Africa. There, inadequate

116 staffing and paucity of drugs and equipment mean that children may fit for many hours without adequate treatment, so increasing the morbidity and mortality that may be attributed to seizures.

In this study, the unacceptable price of effective seizure prophylaxis was the doubling of mortality in the phenobarbitone treated group. The most plausible explanation is that phenobarbitone-induced respiratory depression has precipitated respiratory arrest in a group of unventilated children who are critically dependent on their respiratory drive. Support for this hypothesis comes from the greater proportion of children treated with phenobarbitone who had a respiratory arrest, and the increased mortality among children treated with both phenobarbitone and multiple (3 or more) doses of diazepam. There was, however, no difference in respiratory rate at 4 hours (the time of peak phenobarbitone concentrations) between the phenobarbitone and placebo groups. Children who continue to have seizures despite phenobarbitone prophylaxis may represent a sub-group with particularly refractory cerebral pathology, who could be unusually susceptible to further sedation with diazepam.

Intravenous diazepam is the drug of first choice for the immediate treatment of status epilepticus, because of its ease of administration and rapid onset of action (Shorvon 1994b). The drug is highly lipid soluble, and is rapidly distributed to cerebral tissue and lipid stores. Following repeated administration, redistribution ceases as tissue stores equilibrate, and further doses result in high, persistent concentrations within the brain, and the consequent risk of sudden respiratory arrest and hypotension. In contrast, phenobarbitone has been shown in two controlled studies (Treiman, 1998; Shaner 1988) to be highly effective in the treatment of status epilepticus, with no associated increase in respiratory depression or hypotension. A retrospective review of children with refractory status epilepticus, who had been treated with phenobarbitone in the dose range 30 to 120 mg/kg (Crawford 1988), demonstrated a striking lack of respiratory depression. Despite case reports in the literature suggesting that the combination of phenobarbitone and diazepam may result in respiratory depression and hypotension (Prensky 1967; Sawyer

117 1968; Browne, 1973), this is the first study to document an increase in mortality resulting from concomitant use of the two drugs.

Is there a dose of phenobarbitone that is both safe and effective in childhood cerebral malaria? Phenobarbitone is said to provide effective seizure prophylaxis at plasma levels of between 10 and 30 pg/ml. Plasma levels may, however, vary widely between individuals given the same dose (Treiman 1992), a finding confirmed in this study. In addition, phenobarbitone has a pKa of 7.2, and changes in body pH will, therefore, affect the distribution and excretion of the drug. If the pH of the blood is lower than that of the brain, the gradient will favour movement of phenobarbitone into the brain (Simon 1987; Dodson 1984). Status epilepticus disrupts the blood-brain barrier, causing cerebral uptake of phenobarbitone to be enhanced even further (Walton 1989; Ramsay 1979; Brzakovic 1997). The relationship between plasma concentration and effect in cerebral malaria could be different from other conditions without generalised cerebral pathology. It is possible, therefore, that effective anticonvulsant prophylaxis could result from plasma concentrations of phenobarbitone usually considered “sub-therapeutic” (Goldberg 1983). In adults with cerebral malaria, phenobarbitone at doses of both 3.5 mg/kg and 10 mg/kg provided effective seizure prophylaxis (White 1988a; Kochar 1997). Might phenobarbitone 10 mg/kg, in a larger study, prove both safe and effective in childhood cerebral malaria, despite blood concentrations at the lower limit of the “therapeutic range” (Winstanley 1992)7

We have demonstrated that phenobarbitone 20 mg/kg is highly effective at preventing seizures in childhood cerebral malaria, but is associated with an unacceptable increase in mortality. It is not yet clear whether a lower dose may provide effective anticonvulsant prophylaxis without the increased mortality. Other therapeutic options, such as magnesium sulphate (Duley 1995), should be explored. Having found a drug that is both safe and effective, it would then be important to establish, in a sufficiently large study, whether seizure prophylaxis can reduce the incidence of neurological sequelae complicating this important and devastating disease.

118 7. CONCLUSIONS

119 Introduction

This chapter summarises the studies presented in this thesis, discusses their limitations, and indicates possible areas for future research.

The role of seizures in the pathogenesis of cerebral malaria

The studies described in this thesis were confined to hospitalised children with cerebral malaria, a group that represents one end of the broad spectrum of clinical disease caused by Plasmodium falciparum. In order to gain a better understanding of seizures in cerebral malaria, it is important to consider the role of seizures within the broader context of uncomplicated malaria, in both community and hospital settings.

Community surveys in Kilifi District (where malaria accounts for the majority of fevers in young children) reveal that less than 5% of all febrile episodes in children below the age of 5 years are associated with seizures (V. Marsh, personal communication). Within this rural community, seizures are regarded as a serious condition requiring immediate attention, and social and economic factors determine whether help is sought from the biocultural (local healers) or biomedical (shop-bought drugs, community health workers, clinics, or hospital) sector (Mwenesi 1995a; Molyneux 1999a). Since seizures are a recognised indication for admission to hospital, the proportion of hospitalised children with uncomplicated malaria who have a history of seizures (approximately 25%) is much higher than that observed among febrile children in the community (Waruiru 1996). The proportion of cases of cerebral malaria that are complicated by seizures is, however, even higher. Seizures occur in up to 80% of cases, are often prolonged or multiple, and are associated with an increased risk of neurological sequelae and death (Molyneux 1989; Brewster 1990; Jaffar 1997; van Hensbroek 1997). Although it is possible that all malaria-related seizures have the same pathological basis (sequestration of parasitised erythrocytes within the cerebral vasculature), the work presented here suggests that, in cerebral malaria, seizures themselves play a significant role in the pathogenesis of coma.

120 Approximately one quarter of the patients recruited to the electroencephalography (EEG) study had recovered consciousness within 6 hours of prolonged or multiple seizures, suggesting that their coma may have been a postictal phenomenon, directly related to seizures. In a similar proportion of patients (and there is overlap between the two groups), coma was associated with continuing subtle seizure activity. In a busy, understaffed hospital in the developing world, subtle seizures could easily be missed, yet their clinical features (tonic eye deviation, nystagmus, hypoventilation, salivation) are sufficiently distinct for them to be recognised by clinical or nursing staff with appropriate training. In most hospital settings in the developed world, the majority of unconscious children are artificially ventilated and paralysed, making it impossible to correlate electroencephalographic findings with clinical signs. The information derived from this observational study may, therefore, provide some valuable insights into a variety of other paediatric encephalopathies.

Seizures and neurological sequelae

The studies presented here also confirm the relationship between multiple, prolonged seizures and poor prognosis among patients with cerebral malaria. For the 65 children in the electrophysio logical (EEG) study, and the 170 children recruited to the placebo arm of the phenobarbitone study, there was a significant association between the number and duration of seizures and subsequent neurological sequelae at the time of discharge from hospital. A similar trend was observed between seizures and subsequent death, but the smaller number of patients in this category meant that the observed differences failed to reach statistical significance. EEG recordings demonstrated that electrical seizure activity consistently arose from the posterior temporo-parietal region, which is a "watershed" area, lying between territories supplied by the middle cerebral (carotid circulation) and posterior cerebral (vertebro-basilar circulation) arteries. Consequently, it is particularly vulnerable to hypoxia when oxygen delivery to the brain is compromised as a result of sequestration, severe anaemia, or inadequate cerebral perfusion due to hypotension or raised intracranial pressure (MacPherson 1985; English 1997a; Newton 1997b). Because of their anatomical position close to the temporal lobes in the tentorial notch, the posterior cerebral arteries may

121 be particularly vulnerable to conqjromise in those patients who have acute intracranial hypertension and incipient transtentorial herniation, as illustrated by Figure 10 in Chapter 3. By initiating the release of excitotoxic mediators such as glutamate or quinolinic acid (Dobbie 2000), local hypoxia may precipitate seizures, which, by raising intracranial pressure and increasing the demand for oxygen and glucose, may exacerbate the situation further. Although there is no evidence for failure of cerebral autoregulation in the majority of these patients, relatively poor collateral circulation in some patients may mean that cerebral blood flow in the border-zone between the carotid and vertebro-basilar territories is insufficient to compensate for increased local demand. In these patients, a vicious cycle might then be generated, leading to intractable partial seizures and eventually to focal infarction. It is of note, therefore, that of the three children in the EEG study who developed a hemiplegia and who subsequently returned for follow-up cerebral computerised tomography (CT), all had evidence of infarction in the contralateral posterior quadrant. Although this hypothesis suggests that seizures may cause infarction, it is equally possible that an area of infarction could act as a focus for subsequent seizure activity. To demonstrate causation, it would be necessary to show, by means of a sufficiently large randomised intervention study, that effective anticonvulsant prophylaxis reduces the incidence of neurological sequelae in children with cerebral malaria.

The aetiology of seizures in cerebral malaria

In addition to hypoxia, there are several other factors that may trigger seizure activity in children with cerebral malaria. Fever is an obvious candidate, yet the majority of seizures in both this and other studies (Waruiru 1996) occurred at temperatures of below 38®C. In addition, many seizures were partial motor, multiple, or prolonged (lasting for more than 30 minutes), unlike the short-lived generalised seizures characteristic of “febrile seizures” commonly observed in young children. Metabolic disturbances (hypoglycaemia, hyponatraemia, hypocalcaemia, hypomagnesaemia) can also precipitate seizures but, with the exception of two children admitted with hypematraemic dehydration (Na

>150mmol/l, urea > 2 0 mmol/l) and status epilepticus, no association between metabolic derangement and subsequent seizure activity was found in the studies presented here. At

122 the time that these studies were carried out, chloroquine was widely used throughout Kenya for the treatment of uncomplicated malaria. A number of case reports (Fish 1988; Jaffe 1988; Luijckx 1992) have suggested an association between seizures and the ingestion of chloroquine yet, in children recruited to the placebo arm of the phenobarbitone prophylaxis study, no association was found between blood chloroquine concentrations on admission and subsequent seizures. Similarly, there was no obvious relationship between the occurrence of seizures and cerebrospinal fluid (CSF) concentrations of quinolinic acid, an endogenous excitotoxin. Compared to a reference UK population, however, children with cerebral malaria had CSF concentrations of quinolinic acid that were significantly raised, and CSF concentrations of nitrate and nitrite, stable breakdown products of nitric oxide, that were significantly reduced. In addition, there was a graded increment in the concentration of quinolinic acid across outcome groups of increasing severity. These studies need to be repeated on a larger number of children, and should include a control group derived from the local population. The mechanism underlying the unexpected reduction in CSF concentrations of nitric oxide metabolites could be explored further by measuring CSF concentrations of arginine (a substrate for nitric oxide synthase) and citrulline (a product of the same enzyme). Quinolinic acid, an N-methyl-D-aspartate (NMDA) receptor agonist, induces the influx of both sodium and calcium into neurones, leading to reversible neuronal swelling and subsequent neuronal disintegration. Nitric oxide is an NMDA receptor blocker, and may provide cerebral protection. The increased CSF concentrations of quinolinic acid and reduced concentrations of nitric oxide metabolites observed in these patients with cerebral malaria therefore lends support to the hypothesis that some of the neurological manifestations of cerebral malaria may be explained by an excitotoxic mechanism. In addition, the advent of NMDA-receptor antagonists, such as magnesium sulphate (Duley 1995), raises the possibility of novel approaches to treatment.

123 Intervention studies

The best way of proving a causative link between seizures and adverse outcome in children with cerebral malaria is to demonstrate, by means of a randomised, controlled intervention study, that effective anticonvulsant therapy reduces the subsequent incidence of neurological sequelae and death. Phenobarbitone is a highly effective anticonvulsant drug that has been used for years in the treatment of both partial and generalised seizures. It is cheap, widely available throughout Africa, and may be given by intramuscular injection. In children with cerebral malaria, a single intramuscular dose of phenobarbitone 2 0 mg/kg produced blood concentrations above the therapeutic level of 15pg/ml within 2 hours of administration. These levels were subsequently maintained for a minimum of 48 hours. An initial clinical tolerance study showed that, at this dose, phenobarbitone had no adverse effects on pulse, respiratory rate, oxygen saturation, venous blood gas or lactate, when compared to placebo. Phenobarbitone halved the proportion of children with prolonged or multiple seizures, and reduced from 2 1 % to 13% the proportion of children with neurological sequelae at discharge. In many hospitals throughout Africa, prolonged seizures may go untreated, due to shortages of both staff and equipment. The morbidity and mortahty of seizures under such “real world” conditions may therefore be higher than that observed in this study. The unexpected and disturbing finding of this study was, however, that the mortality of children treated Avith phenobarbitone 2 0 mg/kg was double that of the children given placebo. Post hoc analysis demonstrated that mortality was highest among children treated with both phenobarbitone and multiple (3 or more) doses of diazepam, raising the possibility that phenobarbitone had increased mortality by depressing respiratory drive. It is also possible that, if mortality in the phenobarbitone group had been less, the incidence of neurological sequelae among this group might have been higher. These findings pose a dilemma for clinicians treating children with cerebral malaria in Africa. Throughout the continent, phenobarbitone remains the single most widely available prophylactic anticonvulsant drug. In children with cerebral malaria, a dose of 20mg/kg is clearly contraindicated. But is there a lower dose of phenobarbitone that is both effective and safe, or should the assessment of other, more expensive anticonvulsant drugs become a

124 priority? There is an urgent need for studies that address these questions but, at this stage, it would seem pragmatic to treat children with recurrent, prolonged convulsions with a single dose of phenobarbitone 10-15mg/kg. Under these circumstances, it would also seem advisable to limit the administration of diazepam to a maximum of two doses. Paraldehyde, another cheap and effective drug for the rapid termination of seizures, could be used as an alternative.

In conclusion

Prolonged and multiple seizures, one of the most dramatic characteristics of childhood cerebral malaria, are associated with an increased risk of neurological sequelae and death. Further work needs to be done to clarify the nature of the cognitive problems (Holding 1999) that may also result from uncontrolled seizure activity, and which could have a major impact on the educational potential of children in sub-Saharan Africa. There is a an urgent need to find a safe and effective anticonvulsant drug for seizure prophylaxis and to assess, by means of a randomised, controlled intervention study, whether anticonvulsant prophylaxis reduces the incidence of neurological sequelae and death. Fortunately, there is now considerable political and economic interest in attempts to “Roll Back Malaria” (Nabarro 2000), and a renewed urgency to reduce the unacceptable burden of mortality from malaria in Africa. The provision of safe and effective drugs for the prevention and treatment of seizures in childhood cerebral malaria must be a priority for all future case management strategies.

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loading dose regimen. Trans R Soc Trop Med Hyg 8 8 : 577-80.

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159 APPENDIX

y

160 Declaration

I was personally responsible for the design of all the studies presented in this thesis. This included the production of written protocols for subsequent submission to the local and national scientific and ethical co-ordinating committees.

During the clinical and electrophysio logical study (chapter 3), I performed all of the electroencephalographic (EEG) recordings. For all ten children who underwent intracranial pressure monitoring, I inserted the subarachnoid catheter and undertook the majority of their clinical care. I filmed all of the video footage in the enclosed CD-ROM.

For the phenobarbitone prophylaxis intervention study, which required the prospective recruitment of a large number of subjects, the majority of patients were recruited and managed by my clinical colleagues, with whom I shared clinical duties.

I was responsible for the compilation and cleaning of all data prior to computer entry, and for subsequent statistical analysis. I wrote all of the papers listed in this Appendix.

Supervisors/Collaborators

Supervisors

Professor Kevin Marsh Scientific Director, KEMRI Clinical Research Unit, Kilifi, Kenya

Dr Fenella Kirkham Neurosciences Unit, Institute of Child Health, London, UK (London Advisor)

Collaborators

Dr Shelagh Smith Institute of Neurology, London, UK

Mr Peter Muthinji EEG Department, Kenyatta National Hospital, Nairobi, Kenya

Dr Robert Surtees Neurosciences Unit, Institute of Child Health, London, UK

161 Ethical approval and consent

Approval for all studies was obtained from the Kilifi Scientific Coordinating Committee, the KEMRI Scientific Coordinating Committee, and the Kenyan National Ethical Review Committee (see covering letter in this Appendix). Ethical approval for intracranial pressure monitoring was also obtained from the Ethics Committee of the Hospital for Sick Children, Great Ormond Street, London.

Informed, written consent was obtained from the parent or guardian of each child enrolled into a study. An explanation, written in the caretaker’s first language (KiSwahili or KiOiriama), was read aloud by a trained fieldworker who was fluent in the appropriate local language, and caretakers were encouraged to ask questions. It was stressed that failure to grant permission would not in any way compromise the clinical care given to the child, and that consent could be withdrawn at any time without penalty. Prior to the start of all studies, seminars were held for fieldworkers and nurses in order to ensure that they fully understood and approved of the studies, and were competent at answering any questions that might arise.

All children with severe malaria were treated in the same way on the research ward, irrespective of whether they had been recruited into a study, and all medical care was provided free of charge. The presence of the research unit meant that children admitted to Kilifi District Hospital received a higher standard of clinical care than is usually possible in most district hospitals in Africa. The unit had an adequate number of appropriately trained staff, and could provide a regular supply of drugs and equipment. Consequently, many parents would travel a considerable distance from neighbouring districts in order to bring their child to Kihfi District Hospital.

162 KENYA MEDICAL RESEARCH INSTITUTE

Address all correspondence Centre for Geographic Medicine Research • Coast To the Director of the Centre P. O. Box 426 Telephone: (0125)22063 KILIFI Fax: (0125) 22390

1

TO WHOM IT MAY CONCERN

Dr Jane Crawley - Research for MD Thesis

I confirm that the research studies described in Dr Crawley’s thesis received full approval from the Kilifi Unit Scientific Coordinating Committee, the KEMRI Scientific Coordinating Committee, and the Kenyan National Ethical Review Committee.

Yours sincerely

Professor Kevin Marsh MBchB FRCP Scientific Team Leader KEMRI Centre for Geographic Medicine Research (Coast), Kilifi

20"’ April 2001

163 List of publications arising from this research

1. Crawley J, Smith S, Kirkham F, Muthinji P, Waruiru C, Marsh K. Seizures and status epilepticus in childhood cerebral malaria. Quarterly Journal o f Medicine 1996; 89:591-597

2. Newton CRJC, Crawley J, Sowumni A, et al. Intracranial hypertension in Africans with cerebral malaria. Archives of Disease in Childhood 1997; 76(3): 219-226

3. Crawley J, English M, Waruiru C, Mwangi I, Marsh K. Abnormal respiratory patterns in childhood cerebral malaria. Transactions o f the Royal Society of Tropical Medicine and Hygiene 1998; 92: 305-308

4. Crawley J. Malaria; new challenges, new treatments. Current Paediatrics 1999; 9: 34- 41

5. Crawley J, Waruiru C, Mithwani S, et al. Effect of phénobarbital on seizure frequency and mortality in childhood cerebral malaria: a randomised, controlled intervention study. Lancet 2000; 355: 701-706

6 . Crawley J, Kokwaro G, Ouma D, Watkins W, Marsh K. Chloroquine is not a risk factor for seizures in childhood cerebral malaria. Tropical Medicine and International Health 2000; 5(12): 860-864

7. Dobbie M, Crawley J, Waruiru C, Marsh K, Surtees R. Cerebrospinal fluid studies in children with cerebral malaria: an excitotoxic mechanism? American Journal of Tropical Medicine and Hygiene 2000; 62(2): 284-90

8 . Crawley J, Smith S, Muthinji P, Marsh K, Kirkham F. Electroencephalographic and clinical features of cerebral malaria. Archives of Disease in Childhood 2001; 84: 247-253

164 SEIZURE SHEET

Name:[ ][ KEMRI No.:

W eigh t: kg

Drug doses (Clinician to prescribe): Clinician^ signature

Diazepam 0.3mg/kg i/v: mg

Paraldehyde 0.2ml/kg i/m: ml

Phenytoin 20mg/kg i/v: mg

Date

Time of onset PRE STUDY DRUG

Type'

Duration^ T1,T2, T3 or T4

Type: P: Partial seizure G: Generalised seizure PG: Partial seizure with secondary generalisation

^Duration: Tl: <5 minutes T2: 5-14 minutes T3: 15-29 minutes T4: 30 minutes plus

165 KILIFI UNIT

FRONT SUMMARY SHEET

7b be filled in by clinician

1. KEMRI num ber...... [__ |___|___|___|___]

2. First name......

S u rn am e......

3. Date of birth (day/mth/yr) ...... [__|___|__| ___|___|__]

4. Sex (M/F) ______[__]

5. Date admitted to hospital (day/mth/yr) ...... [__|___|__|___|___|__]

6. Date admitted to KE^dRI ward (day/mth/yr)...... [__|___|__ |___|___|__ ]

To be filled in by ward clerk

7. Inpatient surveillance number ______[__ |___|___|____|__|.

Hospital number...... [__ |___|___|___|___]/[___[.

8. Has the child been admitted to Kilifi District Hospital previously? (Y/N)...... [. When was the last admission?______[__ |___|___|___|___|.

If records for last admission traced, Inpatient surveillance number______[__ |___|___|___|. KEMRI number...... [__j__ j__ j.

9. Date discha^ed from hospital______[__ |___|___|___|___|. Alive or dead on discharge? (A/D)...... [.

10. Lab sample saved? (Y/N)...... [.

166 HISTORY (Please record as complete a history as possible, please write legibly) 11. How long has this illness episode lasted? (in days) (If more than 30 days, write 99)...... [__ |___]

CURRENT PROBLEMS

DRUGS IN LAST 7 DAYS

PAST MEDICAL HISTORY

167 HISTORY (continued) (Please record as complete a history as possible, please write legibly) FAMILY HISTORY

OTHER SIGNIFICANT HISTORY

CNS BOX (Fill in for all children with malaria and any other children with disturbed consciousness)

12. How long has the child been comatosed? (in hours) ...... [__ |___|___]

13. Has the child ever had seizures prior to (but notincluding)

this illness? (Y/N)...... [__] Were these associated with fever? (Y/N)...... [__]

14. How many seizures did s/he have this illness?...... [___|__ ]

How many seizures did s/he have in the past 24 hours?...... [___j__ ] How long ago was the last seizure (round up to the

nearest hour)...... [___| __ ] If the child has had a fit, ask informant to demonstrate the fit.

15. What type of seizure; ...... [___|__ ] G.Generalised P.Partial PG.Partial becoming generalised If partial, which side was involved? R.Right L.Left...... [__]

16. Did the seizure last more than 30 minutes? (Y/N)______[__ ]

17. Before the child was Ul, could s/he :

sit unsupported? (Y/N)...... [__] stand unsupported? (Y/N)...... [__]

walk unsupported? (Y/N)...... [__]

18. Is speech normal for age? (Y/N) ...... [__]

168 CLINICAL EXAMINATION FORM

1. Date of examination (day/mth/yr) ...... [__ |___|___|___|___|. 2. Time of examination (24 hour clock) ...... [__ |___]:[___|. (NOTE: midnight is 00:00 of the following day)

3. Temperature: Axillary (centigrade) ______[__ |___].[_ Rectal (centigrade) ______[__ j___].[.

4. Naked weight (kg) ______[__ |___].[__ ]

5. General condition J au n d ice?(Y/N) ...... [_ Pallor? (Y/N)...... [_

Record other general comments below.

CARDIOVASCULAR

6. Pulse: rate / min

7. Blood Pressure S y s to lic ...... [___I____I. D ia s to lic ...... [___|___j. C uff s iz e (cm)______

8. Auscultation: 1.Normal 2.Abnormal ...... [ If ABNORMAL, circle whether Gallop Murmur Other (specify) ______

Record other cardiovascular observations below.

169 RESPIRATORY

9. Cough: (Y/N)...... [. Nasal flaring? (Y/N)...... [. Respiratory rate: (bpm) ...... [__ |___|.

Deep breathing? (Y/N)...... Indrawing (inward movement of bony chest wall)? (Y/N), If indrawing present, is it severe? (Y/r^......

10. Oxygen saturation % (off oxygen for 5 m inutes) ...... [__

11. C hest: 1.Normal 2.Abnormal ...... If ABNORMAL (including crackles, wheeze, asymmetry), describe in box below.

GIT

12. L iven cm below costal margin in MCL ...... [__ |. Spleen: cm below costal margin in MCL ...... [__ j.

Record other GIT observations below.

NERVOUS SYSTEM EXAMINATION

13. The child is: 1.Awake 2.Drow^ S.Unconscious 4.Agitated ...... Fontanelle 1.Normal 2.Sunken 3.Bulging 4.Not present ...... Neck stiffliess? (Y/N)......

14. Can the child sit unsupported? (Y/N/D)...... If less than 1 year old, is the child able to breastfeed? (Y/N/D).

170 15. Any relevant sedative medication given in last 6 hours? (Y/N). If YES, how many hours ago was it given? ...... State type, route and dose in box below.

16. Respiratory pattern: 1.Normal 2.Abnormal If ABNORMAL, describe in box below.

17. P osition: Decerebrate (Y/N)______[__ ]

Decorticate (Y/N) ______(__ ]

Opisthotonic (Y/N) ______[__ ]

COMA SCALES

18. Motor response to painful stimulus (sternal pressure) 4.LocaHses pain 3.Flexion 2.Extension l.None ...... [__]

19. Blantyre scale Verbal response: 2.Appropriate ciy l.Moan/inappr. O.None ...... [_ Motor response: 2.Localises pain 1.Withdraws O.Nil/non-spec ...... [_ E^es: 1.Directed O.Not directed ...... [_

20. Is RIGHT fundus adequately seen? (Y/N)...... If YES, Haemorrhage (Y/N)...... R: Paphloedema (Y/N)...... R:

Is LEFT fundus adequately seen? (Y/N)...... If YES, Haemorrhage (Y/N)...... L: Paphloedema (Y/N)...... L:

171 CNS BOX (Complete for children with suspected CNS disease)

21. Head circumference (cm)...... [ 1 -].[_]

22. Pupil reaction (using strong torch) 1 .Brisk 2.Slu^sh 3.None ...... R :l_ ] L:[_] 23. Pupil size 1.Dilated 2.Constricted 3.Midpoint ...... R:[ ] L :[_ l

24. Spontaneous eye movements 1.Orienting 2.Roving conjugate 3.Roving dysconjugate 4.None ...... [__] 25. Comeal reflex 1.Brisk 2.Weak 3.Absent...... R:[ 1 L:[ ]

26. Horizontal oculocephalic (rotate head side to side) 1.Normal or full deviation 2.Minimal or none ...... [ ]

27. Limb to n e Arms: 1.Normal 2.Increased 3.Decreased ...... R :[_ ] L:[_J Legs: 1.Normal 2.1ncreased 3.Decreased ...... R:[ ] L:[__)

28. R eflexes Arms: 1.Normal 2.1ncreased 3.Decreased ...... R :[_ ] L:[_J Legs: 1.Normal 2.1ncreased 3.Decreased ...... R:[ 1 L:[_]

29. Plantars l.Downgoing 2.Equivocal 3.Upgoing ...... R:[_] L:[_]

Record other CNS observations in box below.

172 EYES AND ENT

Record observations in box below.

MISCELLANEOUS FINDINGS

30. Other clinical abnormalities? (Y/N). If YES, please describe in box below.

173 WORKING DIAGNOSIS ______

Problems:

MANAGEMENT PLAN (Please write as clear and complete a plan as possible and include your initials).

Investigations ______

Medications:

Fluids:

Observations to be made:

Other comments:

Chest Xray ordered (Y/N) [__]

Clinician's initials [__ |___|__ ]

174 DISCHARGE FORM To be completed on discharge from KEMRI unit

KEMRI number [ I I I I 1 Date [__l. I I I I 1

Naked weight (kg)...... f | __ l.f__ ] Height (cm) ...... \ \ \__ ].[__ ]

Is the child fully conscious? (Y/N) ______[__ ] Is the child's speech back to normal? (Y/N/D) ______[__ ] Can the child see a small object? (Y/N) ______[__ ] Can the child hear? 1.Definitely 2.Probably 3.No ......

9. Does the child have any abnormal movements? (Y/N)______[__ ] If YES, specify in box

10. Can the child: sit unsupported? (Y/N/D)...... [__] stand unsupported? (Y/N/D)...... [__] walk unsupported? (Y/N//D)...... [__]

11. Any abnormalities of gait? (Y/N)______[___] If YES, specify in box

12. Does the child have hemiparesis (weakness of one side of the body)? (Y/N) ...... [__]

13. Further neurol%ical review needed prior to discharge from Ward I? (Y/N)...... [__] If YES, arrange fix discharge throng KEMRI unit. Inform mother and recwd on transfer notes.

14. On final discharge from hospital, are there any significant neurological sequelae? (Y/N) ...... [__] If YES, specify in box

Appointmoit date for followup / /

175 Dis Child 2001j84:247-253 247 Electroencephalographic and clinical features of cerebral malaria

J Crawley, S Smith, P Muthinji, K Marsh, F Kirkham

Abstract sequelae.® ® Despite their importance, and in Background^SeAxures are a prominent common with many other causes of childhood feature of childhood cerebral malaria, and coma, there have been few descriptions of the are associated with an increased risk of clinical and electroencephalographic features death and neurological sequelae. We of seizures in cerebral malaria. Electroencepha­ present the electroencephalographic lography (EEG) can supply the clinician with (EEG) findings hrom a detailed clinical information on the origin and distribution of and electrophysiological study. seizure activity, detect subtle or subclinical sei­ Methods—ChBdren with cerebral malaria zures, and may reveal abnormal electrical had EEGs recorded within six hours of activity of prognostic significance.’ It is there­ admission, and at 12 houHy intervals until fore of potential value in the clinical manage­ recovery of consciousness. Ten deeply ment of cerebral malaria, and may improve comatose children imderwent intracranial understanding of the underlying pathophysiol­ pressure monitoring. Children were not ogy. Here we present the EEG findings from a mechanically ventilated, which made it detailed clinical and electrophysiological study possible to directly correlate the clinical of 65 children with cerebral malaria.* and EEG findings. Results—O f 65 children aged 9 months and above, 40 had one or more seizures, and 18 had an episode of status epilepti- Methods cus. Most seizures were partial motor, and STUDY SITE spike wave activity consistently arose from The study was conducted at the Kenya the posterior temporo-^arietal region, a Medical Research Institute (KEMRI) unit, border zone area lying between territories located at Kilifi District Hospital, on the coast supplied by the carotid and vertebrobasi­ of Kenya. The hospital serves a predominantly rural population, and approximately 5000 chil­ lar circulations. Fifteen children had sei­ dren are admitted to the 35 bed paediatric zures that were clinically subtle or electrographic. Clinical seizures were as­ ward annually. Malaria transmission (of which sociated with an abrupt rise in intracra­ over 95% is Plasmodium falciparum) occurs throughout the year, with peak transmission nial pressure. Fifty children recovered following die rainy seasons of April-May and fully, seven died, and eight had persistent October-November. “* neurological sequelae. Initial EEG re­ cordings of very slow frequency, or with IKEMRI C entre for background asymmetry, burst suppres­ PATIENTS jkographic Medicine sion, or interictal discharges, were associ­ Children aged 9 months and above were eligi­ Ikearch (Coast), ated with an adverse outcome. ble for enrolment in the study if they fulfilled llilifi, Kenya the World Health Organisation definition of 'ICrawley Conc/ustons—Serial EEG recording has ;i; Marsh uncovered a range of clinical, subtle, and cerebral malaria, namely unrousable coma not electrographic seizures complicating attributable to any other cause in the presence Institute o f Neurology, childhood cerebral malaria, and has em­ of asexual Plasmodium falciparum parasitae- London, UK phasised their importance in the patho­ mia." To fulfil the definition of cerebral S Smith genesis of coma. Further work is required malaria, coma had to persist for at least one EEG Departm ent, to determine the most appropriate regi­ hour after a seizure and/or after the administra­ lenyatta N ational men for the prophylaxis and treatment of tion of diazepam. Consent was obtained once Hospital, N airobi, seizures in cerebral malaria, in order to all procedures had been fully explained to the fenya improve outcome. child’s parent or guardian in their first P Muthinji (Arch Dis Child 2001;84:247-253) language. institute o f Child Keywords: cerebral malaria; coma; seizures; Health, London, UK electroencephalogram INVESTIGATIONS F Kirkham On admission, baseline blood was taken for Correspondence to: parasite count, fuU blood count, electrolytes, DrJ Crawley, Centre for Cerebral malaria, a diffuse encephalopathy glucose, and venous gas. Blood glucose was Fropical Medicine, Nuffield caused by Plasmodium falciparumy remains a subsequently monitored every four hours, and Department of Clinical major cause of death and disability in sub- parasite count eight hourly. Lumbar puncture lledicine. Level 7, John lidcliffe Hospital, Saharan Africa/ The pathophysiology of the was performed once the level of consciousness Headington, Oxford disease is complex and poorly understood/ ^ was starting to improve. In deeply comatose 0X3 9DU, UK Prolonged, multiple seizures are a prominent children, intracranial pressure was monitored [email protected] clinical feature,^ ’ and are associated with an using a subarachnoid catheter (Camino 110- Accepted 9 October 2000 increased risk of death and neurological 4B)."

wv3V}.archdischUcLcom CrawUy, Smith, Muthinji, Marsh, Kirkham

Mie 1 Clinical and laboratory features by outcome

Normal recovery Sequelae Died n = 50 n = 8 n = 7 pvcduef

Ip(mlh)* 33.1 (27.6 to 39.7) 16.1 (9.1 to 28.5) 38.7 (22.2 to 67.5) 0.02 for 2 1 0.04 for 2 0 3 Dusdan of coma before admission Qi)* 6.3 (4.7 to 8.3) 11.5 (5.0 to 26.4) 13.3 (6.9 to 25.8) 0.07 Siius epilepticus before or after admission^: 28/50 (56%) 6/8 (75%) 2/7 (29%) 0.24 Sutyre score 2 or less$ 35/50 (76%) 5/8 (63%) 7/7 (100%) 0.45 Opisthotcnic posturing 2/50 (4%) 1/8 (12%) 4/7 (57%) 0.003 for 3 c 1 iedian (IQR) parasitaemia (per |d) 85 200 (990 to I 017 600) 11 854 (1264 to 220 000) 81 600 (420 to 352 800) 0.27 Biemoglobin (g/1) 65 (58 to 72) 80 (54 to 106) 78 (50 to 106) 0.20 ü(dian (IQR) glucose (mmol/1) 5.9 (0.7 to 11.3) 5.0 (0.7 to 8.3) 3.4 (0.4 to 7.8) 0.17 Uedian (IQR) sodium (mmol/1) 134 (127 to 142) 139 (128 to 168) 139 (127 to 147) 0.12 hitassium (mmol/1)* 4.3 (4.0 to 4.5) 4.7 (4.2 to 5.3) 4.3 (3.2 to 5.9) 0.5 BtM (mmol/1)* 4.1 (3.3 to 5.2) 8.6 (3.0 to 24.7) 6.5 (3.6 to 11.8) 0.06 totinine (pmol/1)* 52 (45 to 61) 104 (55 to 197) 72 (38 to 137) 0.009 for 2 » 1 lise excess -5 (-8 to -3 ) -11 (-17 M -5) — 13 (—21 to —5) 0.04 for 3 p 1

Diq aie mean (95% Cl) or num ber (%) unless stated. 'Geometric mean. |Wbeie p < 0.05, results of multÿle comparison tests (Sdteffe or Bonferroni) are given. jk; clinical seizure reported (before admission) or observed (after admission) to last for 30 minutes or more. jO: deep coma, 5 = full consciousness.

ELECTROPHYSIOLOGY STATISTICS EEG recordings were made on a 14 channel Analyses were carried out using Stata version Medelec 1A94 EEG machine. Silver/silver 5.0. Student’s t test and analysis of variance, chloride electrodes were fixed with Elefix and with the Scheffe or Bonferroni correction for tape to the child’s shaved head, and the multiple comparisons, were used for normally International 10-20 system used for electrode distributed quantitative data. Data were log placement. Recordings were taken within six tranformed if the distribution was skewed. The hours of admission, and at 12 hourly intervals Mann-Whimey test was used for data failing to until recovery of consciousness. Continuous conform to abnormal distribution, and Fisher’s recordings using a cerebral function analysing exact test for categorical data. Multivariate monitor (CFAM, Medaid Ltd, UK) were logistic regression analysis was used to examine obtained from selected children who had been the relation between specific admission charac­ unconscious for more than 24 hours or who teristics and subsequent outcome. had prolonged or subtle seizure activity. All EEGs were subsequently analysed by SS, who Results knew the age of each child and what drugs they ADMISSION CHARACTERISTICS had been given, but was blind to any other Sixty five children (age range 9 months to 11 clinical information. years, median 30 months), were recruited to the study. All children had previous normal development. Prior to hospital admission, 68% CLINICAL MANAGEMENT (44/65) had seizures which, in 27 cases, were Children received standard treatment for reported to have lasted for at least 30 minutes. cerebral malaria, as detailed previously.® Oxy­ In more than half the cases, seizure activity gen was available if required, but there were no heralded the onset of coma. Children had been facilities for mechanical ventilation, a situation comatose for between one and 72 hours common to most hospitals throughout Africa. (median seven hours) prior to admission and, All clinical seizures were timed and recorded at the time of admission, 75% were deeply by medical or nursing staff, and classified as unconscious with a Blantyre coma score of 2 or generalised tonic-clonic, partial motor, or par­ less.* Table 1 details clinical and laboratory tial with secondary generalisation." Seizures features of the children. lasting for more than five minutes were treated with a maximum of three doses of diazepam CLINICAL COURSE 0.3 mg/kg, given as a slow intravenous injection Fourteen children (22%) recovered conscious­ over two minutes. Children with recurrent ness within six hours of multiple or prolonged (more than three) seizures or status epilepticus seizures that had occurred on or immediately (continuous clinical seizure activity lasting for prior to hospital admission. Following admis­ 30 minutes or more) were treated with a load­ sion, 62% (40/65) had at least one seizure, ing dose of intravenous phenytoin 18 mg/kg while 19 children had more than five seizures. and, if that failed, with intramuscular pheno- Eighteen children (28%) had an episode of barbitone 18 mg/kg. One child with intractable clinical status epilepticus (continuous clinical generalised status epilepticus required treat­ seizure activity lasting for 30 minutes or more), ment with intravenous thiopentone 4 mg/kg. while three fiirther children had continuous electrical discharges lasting for 30 minutes or more with no apparent clinical correlates (elec­ FOLLOWUP trographic status epilepticus). Fifty two per All survivors were seen one month after cent of the seizures were partial motor, 14% discharge for neurological examination and partial with secondary generalisation, and 34% repeat EEG. Cerebral computerised tomogra­ generalised tonic-clonic. Children with gener­ phy (CT) was performed on children with alised seizures were significantly older than neurological sequelae. those with partial motor seizures (mean age

V3V3V).archdischUd. com hroencephalographic and dinical features o f cerebral malaria 249

64.0 months (95% confidence interval (Cl) 44.7 to 83.5) versus 26.7 months (95% Cl 20.6 to 32.9); p < 0.001). Median duration of seizure activity was three minutes (range 0.5-600 minutes). Fifteen children (23%) had seizures that were either clinically subtle or Hi electrographic. Median time to recovery of consciousness was 20 hours (range 4-112 hours). Seventy seven per cent (50/65) of the children made a full recoveiy, 11% (7/65) died, and 12% (8/65) were left with neurological sequelae (hemiple­ 1 } gia in four, spastic quadriplegia in two, |g cognitive and speech problems in one, grand mal epilepsy in one) that were still present one in >5 - I! month after discharge (see table 2).

ELECTROPHYSIOLOGY til Background activity IP•g a O' Admission EEGs were dominated by high amplitude (>100 pv) slow wave activity with = « ; frequencies of 0.5—7 Hz. Admission recordings M a i n of very slow frequency (0.5-3 Hz) were obtained from 17/65 (26%) children, and were associated with an increased risk of death (odds ratio 25.6 (95% Cl 2.8 to 235); p = 0.004; table 3). This association remained significant despite adjustment for the possible confound­ .:5c 3 I ing effects of age, glucose, and base excess. Recordings of very slow frequency were not significantly associated with duration of coma, administration of diazepam in the 12 hours li prior to admission EEG, age, parasitaemia, >-2 glucose, or base excess (p > 0.1 for all 0 Î comparisons). Ten children had admission recordings that were asymmetric, with in­ ZZo ' ^-1II 11- creased slow wave activity over all or part of m one hemisphere. Nine of these children had a history of multiple partial motor seizures or status epilepticus in the six hour period prior to admission, and three developed neurological sequelae. iSii Ictal activity A striking feature of the EEGs in this series was I li the consistent localisation of ictal discharges over the posterior temporo-parietal regions. Over 75% of ictal or interictal discharges occurred in this region, sometimes spreading to involve both temporo-parietal regions, or one il or both cerebral hemispheres. In eight cases, II electrical seizure activity persisted for between ! li two and 140 minutes (median 45 minutes) after successful treatment of a clinical seizure with diazepam. Fifteen children had continu­ M ous electrical seizure discharges on EEG, but clinical features that were either extremely sub­ tle or not discernible (electrographic). These us I children typically presented in coma, with a X history of a prolonged partial seizure that had «oS"II either terminated spontaneously, or as a result «n nj (-3 of treatment with diazepam. Tonic eye devia­ tion, nystagmus, salivation, and hypoventila­ tion were the most distinctive clinical fea­ tures.'* The nystagmus was of large amplitude, with a slow phase of movement that crossed the midline. Respiration was shallow and irregular, “ S'--! and the children were hypoxaemic (arterial gi oxygen saturation below 80%) and hypercarbic jU I 8 .a < < z ir^ (pcO; above 6.5 kPa). EEG showed continuous

www.anhdisckild.com Crawly, Smith, Muthinji, Marsh, Kirkham

spike wave discharges over the posterior temporo-parietal region contralateral to the direction of eye deviation. The onset of generalised electrical and clini­ cal seizure activity usually followed an in­ z-p< creased frequency of bilateral, predominantly posterior, interictal spike wave discharges. Runs of up to 40 short, generalised seizures, each lasting for 0.5 to 1 minute, were observed in four children, and were followed by notable postictal flattening. Clinical features became increasingly subtle during prolonged general­ ised seizures. One child was admitted following a generalised seizure at home that had lasted for six hours. Tonic-clonic movements had ceased one hour before admission, and the child was deeply comatose, with irregular respiration, salivation, and priapism. Despite the paucity of clinical features, EEG revealed <*^ZodW u Z oop S continuous, generalised seizure activity.

Interictal epileptic activity Interictal spikes and sharp slow waves were observed on admission EEGs from three children, all with a history of prolonged (seizure activity lasting for more flian one hour) -Z partial motor status epilepticus. The discharges were bilateral and multifocal, and located pre­ dominantly in the temporo-parietal regions (table 2). All of these children developed neurological sequelae.

Episodic low amplitude events SÎ Periods of relative attenuation of background X I Jg • activity lasting three to five seconds were seen PizoZ^-;z%, on admission EEGs from five children. In two cases, these followed administration of intra­ venous diazepam 0.3 mg/kg for the treatment of seizures. Four of these children recovered normally, while one child died seven hours after admission. « Burst suppression A burst suppression pattern, consisting of .a .3 bursts of polymorphous complexes occurring synchronously over both hemispheres, and a# alternating with periods of relative quiescence, aa •o’B was observed on the initial EEGs from two in Z o o W children. One child had been hypotensive and hypoglycaemic on admission, and the other had a history of multiple, prolonged general­ ised seizures. Both children died.

Intracranial pressure monitoring and EEG Ten children had simultaneous EEG and a | intracranial pressure (IGF) monitoring. Four nj cs P U children had clinical seizures during IGF monitoring. Intracranial pressure rose by a median of 164% (range 108-285%) during generalised seizures, and by 50% (range 0-186%) during partial seizures. Goncomitant rise in mean arterial pressure meant that cerebral perfusion pressure was maintained above 50 mm Hg during clinical seizures. •Jl There was no significant rise in IGF during two I 90 minute periods of electrographic seizure activity confined to the posterior temporo­ parietal regions. At intracranial pressures of below 20 mm Hg, fluctuations in IGF appeared III--i l I1 1 1 to have no effect on the background frequency

WWW. archdischUd. com Encéphalographie and clinical features of cerebral malaria 251

The child died shortly afterwards following a cardiorespiratory arrest.

OUTCOME Serial EEGs showed a gradual increase in background fi-equency over the period from admission to recovery of consciousness. The 50 children who made a full recovery had normal EEGs one month after discharge. Children with neurological sequelae were significantly 100 nV| HFFIOOHz ICP 30 younger (p = 0.02) and had a significantly 1 sec LFF 0.5 Hz Opisthotonic MAP 92 higher creatinine (p = 0.009) compared to posturing CPP62 those who recovered fully. Admission base excess was significantly greater (p = 0.04) in children who died compared to those who recovered fully, while four of the seven children who died had sustained opisthotonic posturing during their clinical course in hospital. There were otherwise no significant differences be­ tween the three outcome groups in the admis­ sion variables, but numbers are small. Follow up EEGs fi-om children with hemiplegia showed decreased activity in the contralateral parieto-temporal region, and cerebral CT scans showed infarction in the same region. Follow up EEGs from two children with spas­ tic quadriplegia and one child with severe cog­ nitive and speech problems were of low ampli­ 100 pV I HFF 100 Hz ICP 23 tude, with a paucity of normal cerebral 1 sec LFF 0.5 Hz MAP 86 rhythms, while CT scans from these children CPP63 showed global cerebral atrophy (table 2). •idf 1 (A) EEG recording showing widespread slow wave activity in association with àotonic posturing (onset indicated with an arrow). (B) EEG recorded 10 hours later, notable attenuation o f electrical activity in the right parieto-temporal region (leads Discussion C4-Cz, T6-P4, P4~-Pz). ICP (intracranial pressure), M AP (mean arterial Traditionally, it has been assumed that seques­ m e ), and CPP (cerebral perfusion pressure) measured in mm Hg. tration of parasitised red blood cells occurs of the EEG. EEG recording from one child within the cerebral microvasculature of all with severe intracranial hypertension, however, comatose patients with falciparum malaria." showed widespread slow wave activity (fre­ Clinical and experimental work has shown, quencies of 0.5-1 Hz) in association with however, that the pathophysiology of cerebral malaria is highly complex, and that a number of episodes of opisthotonic posturing, during different pathological mechanisms may cause which ICP rose to peaks of 30 to 40 mm Hg coma.^ Reduced cerebral perfusion (secondary (fig lA). Within 10 hours, EEG showed to raised intracranial pressure and systemic attenuation of activity in the right temporo­ hypotension), severe anaemia, hypoglycaemia, parietal region (fig IB), and the right pupil and the local release of cytokines and nitric became dilated with sluggish reaction to light. oxide may all be contributory factors.^ " Over the next five hours, despite regular doses Our study suggests that an additional patho­ of mannitol 0.5 g/kg, the ICP rose to above 80 physiological factor, seizures, may play an mm Hg, the cerebral perfusion pressure fell important role in the pathogenesis of coma. below 30 mm Hg, and the entire EEG became Over one third of ihe children in this study flat and featureless. ICP rose to a peak of 125 had more than five seizures or an episode of mm Hg, and the child died following a respira­ status epilepticus during their clinical course in tory arrest. hospital. Twenty two per cent recovered Another child aged 3 years was admitted consciousness within six hours of prolonged or with a 12 hour history of opisthotonic postur­ multiple seizures, suggesting that their coma ing. Initial EEG was reactive, with a back­ may have been a postictal phenomenon, ground frequency of 2-6 Hz. ICP monitoring directly related to seizures. EEG was essential was commenced at seven hours, and the child for identifying the 15 children in whom also started continuous CFAM recording. Ini­ seizures were either clinically subtle (tonic eye tial intracranial pressures ranged fi-om 25 to 30 deviation, nystagmus, hypoventilation) or elec­ mm Hg, with peaks of 40 mm Hg during trographic. Although tonic deviation of the episodes of opisthotonus. Over the next four head and eyes commonly occurs during hours, despite inotropic support, mean arterial epileptic seizures, nystagmoid eye movements pressure remained below 75 mm Hg, and rising are an unusual ictal manifestation. The clinical intracranial pressure consequently caused a characteristics of the nystagmus in these progressive decline in cerebral perfusion pres­ patients were consistent with epileptic nystag­ sure. Subsequent infusion of mannitol was mus type two (EN-2), which typically has a accompanied by a notable deterioration in seizure focus in the temporo-parietal-occipital clinical condition, and CFAM recording cortex, contralateral to the direction of the showed a sharp drop in background frequency. nystagmus beats.'® ” This area overlaps the

zvtow. archdischUd. com Crawly, Smith, Muthinji, Marsh, Kirkham putative cortical region of visual motor sensi­ arterial pressure, suggesting that cerebral tivity (V5), which is important for the genera­ autoregulation was intact. In one case, how­ tion of ipsiversive smooth pursuit/" Subtle sei­ ever, mean arterial pressure was insufficient to zures have not been described in previous maintain adequate cerebral perfusion, despite studies of childhood cerebral malaria but are of treatment with pressor agents. Infusion of great clinical importance, as several of these mannitol was followed by clinical and electro­ children recovered consciousness within a few physiological deterioration, suggesting that the hours of anticonvulsant treatment. In a busy, integrity of the blood-brain barrier had been understaffed hospital in the developing world breached. such seizures are easily missed, yet their clinical EEGs have been used to predict prognosis in features are sufficiently distinct for them to be a number of childhood encephalopathies.’^ ’* recognised by medical or nursing staff with In this study, burst suppression or background appropriate training, without the need for activity of very slow frequency was associated EEG. with an increased risk of death. Of five children The pathogenesis of seizures in childhood with episodic low amplitude events on initial cerebral malaria is likely to be complex and EEG, four made a full recovery. In two of these multifactorial,’ but our study suggests that cer­ cases, episodic low amplitude events were ebral hypoxia may be a precipitating factor. associated with the administration of di­ EEG recordings showed that electrical seizure azepam, which may partly explain why their activity consistently arose fi-om the posterior prognosis appears better here than in other temporo-parietal region, which is a “water­ types of encephalopathy." Children with a shed” area, lying between territories supplied by history of status epilepticus prior to admission, the middle cerebral (carotid circulation) and and an initial EEG showing asymmetric back­ posterior cerebral (vertebro-basilar circulation) ground activity or continuous interictal multi­ arteries. Consequently, it is particularly vulner­ focal spike waves, had an increased incidence able to hypoxia when oxygen delivery to the of neurological sequelae. Overall, almost 70% brain is compromised as a result of sequestra­ of the children with an adverse outcome (death tion, severe anaemia, or inadequate cerebral or neurological sequelae) had one or more poor perfusion caused by hypotension or raised prognostic features on initial EEG. Although intracranial pressure." " “ Because of their this suggests that in some cases significant cer­ anatomical position close to the temporal lobes ebral pathology must have occurred prior to in the tentorial notch, the posterior cerebral hospital admission, anticonvulsant prophylaxis arteries may be particularly vulnerable to com­ given at the time of admission may prevent fur­ promise in those patients who have acute ther cerebral damage. A recent randomised, intracranial hypertension and incipient tran­ controlled study of phenobarbitone prophy­ stentorial herniation, as illustrated by the EEGs laxis in childhood cerebral malaria showed in fig 1. Raised intracranial pressure, reduced that, at a dose of 20 mg/kg, phenobarbitone cerebral perfusion pressure, and transtentorial halved seizure frequency but was associated herniation have been documented in cerebral with an unacceptable doubling of mortality.” malaria," and in other paediatric encepha­ Further studies need to be done to find a pro­ lopathies.^* By initiating the release of excito- phylactic anticonvulsant drug that is both safe toxic mediators such as glutamate or quinolinic and effective in children with cerebral malaria. acid," local hypoxia may precipitate seizures, Electrophysiological monitoring using serial which, by raising intracranial pressure and EEG and CFAM has made it possible to iden­ increasing the demand for oxygen and glucose, tify a wide variety of seizures (clinical, subtle, may exacerbate the situation further. Although and electrographic) in children with cerebral there is no evidence for failure of cerebral malaria, and has emphasised their importance autoregulation in the majority of these patients, in the pathogenesis of coma. We have been able relatively poor collateral circulation in some to correlate the electroencephalographic and patients may mean that cerebral blood flow in clinical findings from this group of uncon­ the border zone between the carotid and scious children, something that is not possible vertebro-basilar territories is insufficient to in most parts of the developed world where the compensate for increased local demand. In majority of unconscious children are venti­ these patients, a vicious cycle might then be lated. The information obtained fi-om this generated, leading to intractable partial seizures study may, therefore, provide some valuable and eventually to focal infarction (table 2). in sists into a wide variety of other paediatric Diffuse background slow wave activity on encephalopathies. EEG occurs in many conditions (metabolic, toxic, and infectious) that have a generalised This paper is published widi the permission of the Director of effect on the brain.’ " Background slow wave KEMRI. We wish to thank our clinical and nursing colleagues on the paediatric research ward at Kilifi District Hospital for activity in cerebral malaria has been described dieir skilled help with patient management. The work is in adults," " and in one previous study on chil­ supported by KEMRI and by the Wellcome Trust (040313). We are also extremely grateful to the British High Commission in dren.’" The mechanism for the increased slow Nairobi, and the Triangle Trust for additional support. KM is a wave activity seen during episodes of intracra­ Wellcome Trust Senior Research Fellow in Clinical Science (031342), and FK was supported by the Wellcome Trust nial hypertension and opisthotonus is un­ (035352). known, but may have been caused by concur­ rent hyperventilation, leading to hypocapnia 1 WHO. Malaria, 1982-1997. WMy Epidemiol Rec 1999;74; and cerebral vasoconstriction.” Intracranial 265-70. 2 Marsh K, English M , Crawley J, Peshu N. The pathogenesis pressure rose acutely during seizures, and was of severe malaria in African children. Arm Trop Med Parasir accompanied by a concurrent rise in mean tol 1996;90:395-402.

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3 English M, Marsh K. Childhood malaria—pathogenesis and 21 English M, Muambi B, Mithwani S, Marsh K. Lactic acido­ treatment. Cwrr pp«M/njfeci ZXs 1997;10:221-5. sis and oxygen debt in African children with severe 4 Molyneux ME, Taylor TE, Wirima JJ, Borgstein A. Clinical anaemia. QJM 1997;90:563-9. features and prognostic indicators in paediatric cerdiral 22 Newton CRJC, Peshu N, Kendall B, et al. Brain swelling and malaria: a study of 131 comatose Malawian children. QJM ischaemia in Kenyans with cerebral malaria. Arch Dis Child 1989;71:441-59. 1994;70:281-7. 5 Crawley J, Smith S, Kirkham F, et al. Seizures and stams 23 Newton CRJC, Kirkham FJ, Winstanley PA, et al. Intracra­ epilepticus in childhood cerebral malaria. QJM 1996;89: nial pressure in African children with cerebral malaria. 591-7. Lancet 1991;337:573-6. 6 Jaifar S, Van Hensbroek MB, Palmer A, et oL Predictors of a 24 Minns RA. T^hlems of intracranidl pressure in childhood. Lon­ fatal outcome following childhood cerebral malaria. Am J Trop Med Hyg 1997;57:20-4. don, Oxford: Blackwell Scientific Publications Ltd, 1991. 7 Brewster DR, Kwiatkowski D, White NJ. Neurological 25 Dobbie M, Crawley J, Waruiru C, et al. Cerebrospinal fluid sequelae of cerebral malaria in children. Lancet 1990;336: studies in children with cerebral malaria: an excitotoxic 1039-43. mechanism? Am J Trop Med Hyg 2000;62:284-90. 8 Bondi PS. The incidence and outcome of neurological 26 Plum F, Posner JB. The diagnosis of stupor and coma, 3rd edn. abnormalities in childhood cerebral malaria: a long-term Philadelphia: FA Davis Company, 1982. follow up of 62 survivors. Trans R Soc Trap Med Hyg 1992; 27 Stotka VL, Wenzel RP. Malaria in Viemam (1 Corps 86:17-19. Secnior): review of 214 cases including EEG patterns on 19 9 Hughes JR. EEG in dinical practice, 2nd edn. Boston: acutely ill patients. MU Med 1973;138:795-802. Butterworth-Heinemann, 1994. 28 Chen SS, Way LJ, Chen M C, Chen ER. Clinical and 10 Snow RW, Armstrong-Schellenberg JRM, Peshu N, et al. electrophysiological assessment of cerebral malaria. Kaoh- Periodicity and space-time clustering of severe childhood siungJMed Sci 1991;7:278-84. malaria on the coast of Kenya. Tram R Soc Trap Med Hyg 29 Thumasupapong S, Tin T, Sukontason K, et al. Electroen­ 1993;87:386-90. cephalography in cerebral malaria. SE Asian J Trop Med 11 WHO. Severe falciparum malaria. Thim R Soc Trop Med Hyg Public Heahh 1995;26:34r-7. 2000,-94(suppl l):l-9 0 . 30 Lemercier G, Bert J, Nouhouayi A, et al. Le 12 Newton CRJC, Crawley J, Sowumni A, et at. Intracranial neuropaludisme: aspects elecnroencephalographiques, neu­ hypertension in Africans with cerebral malaria. Arch Dis ropathologiques, problèmes physiopathologiques. Child 1997;76:219-26. Pathologie-Biologie 1969;17:459-72. 13 Hopkins A, Shorvon S, Cascino G, eds. Epilepsy, 2nd ed. 31 Kiloh LG, Mc:Comas AJ, Osselton JW, U pton ARM. Clmi- London: Chapman and Hall Medical, 1995. cal electroencephalogrc^hy, 4th edn. London: Butterworths, 14 Crawley J, English M, Waruiru C, et al. Abnormal 1981. respiratory patterns in childhood cerebral malaria. Trans R 32 Pampiglione G, Harden A. Resuscitation after cardiocircu- Soc Trop Med Hyg 1998;92:305-8. latory arrest: prognostic evaluation of early electroencepha­ 15 MacPherson GG, Warrell MJ, White NJ, et al. Human cer­ lographic findings. Lancet 1968;1:1261-5. ebral malaria. A quantitative ultrastructural analysis of 33 Kuroiwa Y, Celesia GG. Clinical significance of periodic parasitized erythrocyte sequestration. Am J Pathol 1985; EEG patterns. i4n:A Neurol 1980;37:15-20. 119:385-401. 16 White NJ, Miller KD, Marsh K, et al. Hypoglycaemia in 34 Tasker RC, Boyd S, Harden A, Matthew DJ. Monitoring in African children with severe malaria. Lancet 1987;1:708- non-traumatic coma. Part II: electroencephalography. Arch 11. Dis Child 1988;63:895-9. 17 Clark I, Rockett K, Cowden W. Proposed link between 35 Brenner RP, Schwartzman RJ, Richey ET. Prognostic cytokines, nitric oxide, and human cerebral malaria. Parasi­ s%nificance of episodic low amplitude or relatively isoelec­ tology Today 1991;7:205-7. tric EEG patterns. Dis Nerv Syst 1975;10:582-7. 18 Kaplan PW, Tusa RJ. Neurophysiologic and clinical correla­ 36 Rae-Grant AD, Strapple C, Barbour PJ. Episodic low- tions of epileptic nystagmus. Neurdogy 1993;43:2508-14. amplitude events: an under-recognised phenomenon in 19 Harris CM, Boyd S, Chong K, et a l Epilqttic nystagmus in clinical electroencephalography. J Clin Neurophysial 1991; infaacy. J Neurol Sci 1997;151:111-14. 8:203-11. 20 Lekwuwa GU, Barnes GR. Cerebral control of eye 37 Crawley J, Waruiru C, Mithwani S, et al. Effect of phénobar­ movements. 1. The relationship between cerebral lesion bital on seizure frequency and mortality in childhood sites and smooth pursuit deficits. Brain 1996;119(pt cerebral malaria: a randomised, controlled intervention 2):473-90. study. Lancet 2000;355:701-6.

www.archdischild.com Tropical M edicine and International Health VOLUME 5 N O 12 PP 855-859 DECEMBER 2000

C. Vieira e t a l . Human dirofilariasis in Colombia

infected mosquitoes. Journal o f M edical Entom ology 26, 489-490. ases: an emerging public health problem in developed countries. Levi DA, Scott LA, Hamilton RE, d’Antonio RD ÔC Swindle MM Research and R eview s in Parasitology (in press). (1983) D og heartworm infection potentiates IgE in humans. Simon F, Muro A, Cordero M Sc Martin J (1991) A seroepidemio- Journal o f A llergology and C linical Im m unology 71,115. logic survey of human dirofilariasis in Western Spain. T r o p i c a l Makiya K, Tsukamoto M & Kagei N (1987) Fifty-six cases of human M edicine and Parasitology 42,106-108. dirofilariasis reported from Japan compiles table.J o u r n a l o f Towbin H, Staehelin T Sc Gordon J (1979) Electrophoretic transfer of U O E H 9 a , 233-242. proteins from polyacrilamide gels to nitrocellulose sheets; Pro­ Milnez-de-Campos JR, Barbas CS, Filomeno FTe t a l . (1997) Human cedure and some applications. Proceedings o f the N ational pulmonary dirofilariasis: analysis of 24 cases from Sao Paulo, A cadem y o f Sciences o f the U SA 76, 4350-4354. Brazil. C h e s t 112, 729-733. Tsang VCW, Bers GE Sc Hanckock K (1985) Enzyme linked immuno- Mu§o A, Genchi C, Cordero M e t a l . (1999) Human dirofilariasis in electrotransfer blot. In: Enzym e M ediated Im m unoassay (eds TT the European Union. Parasitology Today 15,386-389. Ngo Sc HM Lenhoff), Plenum Press, New York, pp. 389-414. Perera L, Muro A, Cordero M , Villar E & Simon F (1994) Identi­ Tsuji N, Morales TH, Ozols VV e t a l . (1998) Molecular characteriza­ fication, purification and evaluation of a 22 kDa D irofilaria tion of a calcium-binding protein from the parasite D irofilaria i m m i t i s antigen for the immunodiagnosis of human pulmonary im m itis. M olecular and Biochem ical P arasitology 97, 69—79. dirofilariasis. Tropical M edicine and Parasitology 45, 249-252. Vieira C, Vzlez ID, Montoya M N e t a l . (1997) D irofilaria im m itis in Perera L, Pérez-Arellano JL, Cordero M, Simon F & c Muro A (1998) Tikuna indians and their dogs in the Colombian Amazon. A n n a l s Utility of antibodies against a 22 kDa molecule of D irofilaria o f Ttopical M edicine and Parasitology 92, 123-125. i m m i t i s in the diagnosis of human pulmonary dirofilariasis. Villanueva EJ Sc Rodriguez-Perez J (1993) Immunodiagnosis of Tropical M edicine and International H ealth 3,151—155. human dirofilariasis in Puerto Rico. A m erican Journal o f Tropical Santamaria B, Cordero M, Muro A & Simon F (1995) Evaluation of M edicine and H ygiene 48, 536-541. D irofilaria im m itis excretory/secretory products for seroepidemio- Welch J, Dobson S Sc Freeman C (1979) Distribution and diagnosis of logica studies on human dirofilariasis. P a r a s i t e 2, 269-273. dirofilariasis and toxocariasis in Australia. V eterinary Journal 55, Scherr BA ÔC Scherr GH (1983) How many humans have canine 265-274. heartworm? D iagnostics in M edicine May/June, 95-96. Yoshimura H Sc Akao N (1985) Current status of human dirofilari­ Simon F Sc Genchi C (2000) Dirofilariasis and other zoonotic filari- asis in Japan.International Journal o f Z oology 12, 53-60.

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Chloroquine is not a risk factor for seizures in childhood cerebral malaria

Jane Crawley'^ Gilbert Kokwaro^ \ David Ourna^ William Watkins^ ^ and Kevin Marsh'"*

1 K E M R J C entre for G eographic M edicine Research (C oast), K ilifi, K enya 2 N uffield D epartm ent o f C linical M edicine, U niversity o f O xford, U K 3 W ellcom e Trust Research Laboratories, N airobi, K enya 4 D epartm ent o f Pharm acology and Therapeutics, U niversity o f Liverpool, UK

Summary o b j e c t i v e s There are a number of case reports in the medical literature suggesting an a s s o c ia t io n between the ingestion of chloroquine and subsequent seizure activity. Our study was designed to investigate the rela­ tionship between blood levels of chloroquine (CQ), its metabolite desethylchloroquine (DCQ), and seizures in children admitted to hospital with cerebral malaria. M E T H O D S Serial blood levels of CQ and DCQ were measured over the first 24 h of hospital admission in children with cerebral malaria. The number and duration of all seizures was recorded, andstatistical analysis subsequently performed to determine the relationship between seizure activity and blood concentrations of CQ and DCQ. RESULTS Chloroquine was detected in 92% (100/109) of admission blood samples. 54% (59/109) of the patients had one or more seizures after admission, while 8% (9/109) had an episode of status epilepticus. Median (interquartile range) baseline concentrations of CQ and DCQ were, respectively, 169.4 gg/m l (75.1-374.9) and 352.3 |Xg/ml (81.9-580.1) for those children who had seizures after admission, compared to CQ 227.5 |xg/ml (79.4-430.2) and DCQ 364.0 (Jtg/ml (131.3-709.4) for those who did not have seizures (P > 0.5 for all comparisons). Baseline concentrations of CQ and DCQ were not significantly associated wié the occurrence of seizures lasting for 5 min or more. The nine children who had an episode of sta tu s epilepti­ cus had significantly lower median admission levels of CQ than those without status epilepticus: 75.1 pg/l (7.4-116.5) vs. 227.5 p.g/1 (85.6-441.2), P = 0.02. Multivariate logistic regression analysis, taking into account factors likely to affect the risk of seizures in hospital, failed to change the significance of these results. CONCLUSIONS These findings suggest that chloroquine does not play an important role in the aetiology of seizures in childhood cerebral malaria.

keywords malaria, chloroquine, seizures

correspondence Dr Jane Crawley, Centre for Tropical Medicine, Level 7, Nuffield Departmento f Clinical 1 Medicine, John Radcliffe Hospital, Headington, Oxford 0X3 9DU. E-mail: [email protected]; j

Seizures complicate the clinical course of more th an 301 Introduction of children admitted to hospital with falciparum m alaria Despite the relentless spread of drug-resistant Plasmodium (Waruiru et al. 1996). They occur in over 60% of cerebral falciparum, chloroquine continues to be widely used malaria cases (Crawley et al. 1996) and are associated witli throughout Africa for the treatment and prevention of an increased risk of death (Jaffaret al. 1997) and neurologi­ malaria. The drug is also used in many parts of the world as cal sequelae (Molyneux et al. 1989; Brewster et al 1990). A second-line treatment for hepatic amoebiasis and connective large proportion of patients admitted to hospital in Kenya tissue disease. A number of case reports in the medical liter­ with malaria have taken chloroquine, obtained from shops ature have described an association between seizures and the or local dispensaries, prior to attending hospital (Snow eti ingestion of chloroquine, both at therapeutic concentrations 1992; Crawley et al. 1996). The purpose of t h i s study there­ (Torrey 1968; Fish Espir 1988; Luijckx 1992) and follow­ fore was to investigate the possibility that seizures in child­ ing overdose (Cann & Verhulst 1961; Kiel 1964; Jaffe 1988; hood cerebral malaria may be caused or exacerbated by Riou et al. 1988). chloroquine or its active metabolite desethylchloroquine.

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I Crawley et al. Chloroquine and seizures in cerebral malaria

Methods water bath (37 °C) under a gentle stream of nitrogen. The residue was reconstituted in mobile phase (100 (xl) and Study site analysed by high-performance liquid chromatography, using The study was conducted at the Kenya Medical Research ultraviolet detection at 340 nm. The lower limit of detection Institute (KEMRI) unit at Kilifi, on the coast of Kenya. for both chloroquine and desethylchloroquine was 5 |xg/l.

Consent Statistical analysis

The study formed part of a randomised, double-blind, Eor analysis we used the statistical programme Stata version placebo-controlled trial of phenobarbitone prophylaxis in 5.0 (Stata Corporation, College Station, Texas). Comparisons childhood cerebral malaria (Crawley et at. 2000). Approval of normally distributed quantitative data were made by had been granted by the Kenyan National Ethical Committee. Student’s t-test, using logarithmic transformation for data Informed, written consent was obtained from first degree rel­ that had a skewed distribution. Data failing to conform to a atives in all cases. normal distribution were compared with the non-parametric Kruskal-Wallis test and Spearman’s rank correlation coeffi­ Admission procedure cient. Categorical data was compared with Fisher’s exact test. Multivariate logistic regression analysis was used to examine Children aged 9 months to 13 years were recruited if they had the effect of potential confounding variables measured at flasmodium falciparum parasitaemia and were unconscious, admission on the association between chloroquine levels and as judged by their inability to localize a painful stimulus the occurrence of seizures. Molyneux et al. 1989). Children who had pre-existing afebrile epilepsy or significant neuro-developmental prob­ lems, and those who had received treatment with phenobarbi­ Results tone or phenytoin during the current illness were excluded Samples (tom the study, as were those found on subsequent lumbar puncture to have meningitis. As soon as possible after admis­ Of 340 patients recruited to the main phenobarbitone pro­ sion, children were randomised to receive a single intramus­ phylaxis study, 170 received placebo. Sufficient blood was cular dose of phenobarbitone 20 mg/kg or identical placebo. available for measurement of CQ and DCQ on 109 (64%) of Blood was taken at baseline, 2, 4, 8,12 and 24 h for measure­ these patients. There was no significant difference in either ment of phenobarbitone, chloroquine (CQ), and the frequency or duration of seizures between patients who ilesethylchloroquine (DCQ). Analysis of the relationship had levels measured and those who did not (P > 0.1 for all Between blood CQ and DCQ concentrations and subsequent comparisons; Fisher’s exact test). Chloroquine was detected seizure activity was confined to the placebo group. in 100/109 (92%) of the baseline blood samples. Median (interquartile range) baseline concentrations were 214.9 p-g/1 (79.4-^27.3) for CQ and 349.3 jxg/1 (90.3-594.7) for DCQ. Of Treatment 80 children who had three or more CQ levels taken during Children received standard treatment for cerebral malaria their hospital course, 29 (36%) were thought to have received Crawley 1999). The number and duration of all seizures CQ in the period immediately (< 24 h) before hospital were recorded using timers pre-set to alarm at 5,15, and admission, since the concentration of CQ in their blood fell 30 min. Seizures lasting for 5 min or more were treated in a by more than 50% during the first 24 h of admission (White standardized manner, with intravenous diazepam 0.3 mg/kg et al. 1988). These children had median (IQR) baseline levels and, as second-line therapy, intramuscular paraldehyde of CQ and DCQ that were significantly higher than those 0.2 ml/kg. given chloroquine more than 24 h prior to admission (CQ 374.9 |xg/l (278.6-888.3) vs. 114.8 p.g/1 (41.0-185.8), P = 0.0001; DCQ 580.1 fxg/l (312.3-1156.2) vs. 284.1 jxg/1 Chloroquine assay (52.6-518.9), P - 0.001; Kruskal-Wallis). Chloroquine (CQ) and desethylchloroquine (DCQ) concen­ trations were measured in whole blood using a modification Seizures of the method of Patchen et al. (1983). Blood samples (100 |jl1) were extracted with a combination of methyl-tert- 54% (59/109) of the children had one or more witnessed butyl-ether and hexane (1 : 1), centrifuged (1000 X g; seizures after admission. Table 1 compares the baseline char­ 10 min), and the organic layer separated and evaporated in a acteristics of those who had seizures after admission with

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J. Crawleyet al. Chloroquine and seizures in cerebral malaria

Table I Comparison of baseline adm ission Median Seizures after admission characteristics in children with cerebral (interquartile malaria (CQ: chloroquine; range) unless stated No(n = 50) Yes (n = 59) P-value DCQ: desethylchloroquine)

Age (months) Geometric mean (95% Cl) 32.7 (28.0-38.1) 28.7 (25.1-32.7) 0.19 Seizures before admission No. (%) 39/50 (78%) 56/59 (95%) 0.01 Anticonvulsants before admission No. (%) 7/50 (14%) 17/59 (29%) 0.07 Baseline CQ (|xg/l) 227.5 (79.4-430.2) 169.4 (75.1-374.9) 0.53 Baseline DCQ (|xg/l) 364.0 (131.3-709.4) 352.3 (81.9-580.1) 0.53 AUC 0-12 CQ (|xg.hr/l) 2058.9 (1014.1-5469.4) 2057.6 (802.8-2536.8) 0.37 [n - 20) (n = 23) AUC 0-12 DCQ (tJLg.hr/1) 4534.8 (2420.3 -6416.6) 4285.2 (1985.0-5740.6) 0.53 (n = 17) (n = 22) Parasitaemia (/|xl) Geometric mean (95% Cl) 64805 (29987-140049) 42293 (22473 -79598) 0.39 Glucose (mmol/1) Geometric mean (95% Cl) 4.0 (3.0-5.3) 4.1 (3.3-5.3) 0.83 Base excess - 7 ( - 12 to-4) - 8 ( - 13 to - 3 ) 0.90

Proportions were compared by Fisher’s exact test, means by Student’s t-test, and medians by the Kruskal-Wallis test. those who did not. Children who had seizures after admis­ seizure lasting for 5 min or more, of whom nine had an sion had lower median baseline levels of CQ and DCQ than episode of status epilepticus (clinical seizure activity lasting those who did not have seizures, although the difference for 30 min or more). Median (IQR) baseline levels of CQ failed to reach statistical significance (Table 1). Children who and DCQ and the corresponding area under the concentra­ had received chloroquine less than 24 h prior to admission tion curves (AUCO-12) were not significantly associated with had a similar frequency and duration of seizures as those the occurrence of seizures lasting for 5 min or more (P >0,5 who had received it earlier (P > 0.4; Fisher’s exact). Of the for CQ, DCQ, and AUCO-12; Kruskal-Wallis). In the nine 109 children in the study, 32 (29%) had three or more children who had an episode of status epilepticus, however, seizures after admission. There was no significant correlation median baseline CQ concentrations and AUCO-12 CQ were between the total number of seizures after admission and significantly reduced (Table 2). Multivariate logistic regres­ median baseline concentrations of CQ (Spearman’s rho sion analysis, taking into account the factors likely to affect —0.089, P = 0.36) or DCQ (Spearman’s rho —0.039, the risk of seizures in hospital (Table 1), failed to change the P = 0.69). Of children with seizures, 76% (45/59) had a significance of any of the above results.

Table 2 Baseline levels of chloroquine (CQ|, Status epilepticus after admission desethylchloroquine (DCQ), and area under Median (interquartile range) N o (n - 100) Yes (n = 9) P-value the concentration curve for 0-12 h (AUC 0-12) for children with and without status epilepticus Baseline CQ (|xg/l) 227.5 (85.6-441.2) 75.1 (7.4-116.5) 0.02 Baseline DCQ (p.g/1) 375.9 (134.8-701.2) 81.9 (0-322.9) 0.07 AUC 0 -12 CQ (pg.hr/1) 2170.1 (1089.5 -4053.6) 470.3 (211.3-659.7) 0.009 (n = 40) (n = 3) AUC 0-12 DCQ (pg.hr/1) 4769.4 (2420.3 - 6416.6) 1188.7 (392.5-1985.0) 0.06 (n = 37) (n = 2)

Proportions were compared by Fisher’s exact test, means by Student’s t-test, and medians by the Kruskal-Wallis test.

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Crawley e t a l . Chloroquine and seizures in cerebral malaria

Discussion Study suggests that chloroquine is not an important determi­ nant of seizures of childhood cerebral malaria, more defini­ There are many possible explanations for the high incidence tive evidence would come from prospective longitudinal seizures in childhood cerebral malaria. Fever, known to studies. Such studies could assess the prevalence of seizures precipitate seizures in young children (Wallace 1988), is an complicating cerebral malaria in relation to changing pat­ most universal feature of cerebral malaria, although many terns of chloroquine useage. The rapid spread of chloroquine of the seizures in cerebral malaria occur at temperatures of resistance, and the urgent need to change to alternative anti- telow 38 °C (Crawley et al. 1996). Hypoglycaemia (White malarial drugs (White 1998), is likely to provide such an (tul. 1987), hyponatraemia (English et al. 1996), and cerebral opportunity in the near future. hypoxia, secondary to severe anaemia and inadequate cere- perfusion (Newton et al. 1997), may also precipitate ires. Acknowledgements Throughout Africa, chloroquine continues to be widely used This paper is published with the permission of the Director for the treatment of febrile illnesses at the community level, of KEMRI. We thank our clinical, nursing, and laboratory IS illustrated by this and by previous studies (Snow et al. colleagues at Kilifi hospital for their dedicated contribution 1992; Crawley et al. 1996). Baseline chloroquine concentra­ to patient management. The work is supported by KEMRI tions in this study were generally at the low end of the thera­ and the Wellcome Trust (040313). KM is a Wellcome Trust peutic range, and were well below concentrations (2000 p.g/1 Senior Research Fellow in clinical science (031342). iiid above) considered toxic (Riou et al. 1988). The associa­ tion between chloroquine and seizures has been described in hoth adults and children, at therapeutic concentrations References Torrey 1968; Fish & Espir 1988; Luijckx et al. 1992) and Amabeoku G (1992) Involvement of GABAergic mechanisms in after overdose (Cann & Verhulst 1961; Kiel 1964; Jaffe 1988; chloroquine-induced seizures in mice. G eneral Pharm acology 23, Riou et al. 1988). Seizures may occur within a few hours of 225-229. overdose (Cann & Verhulst 1961; Kiel 1964; Jaffe 1988), or Amabeoku GJ & Chikuni O (1993) Chloroquine-induced seizures in between one day and several weeks after the start of treat­ mice: the role of monoaminergic mechanisms. E u r o p e a n ment with therapeutic doses of chloroquine (Luijckx et al. Neuropsychopharm acology 3, 37—44. 1992). The concentration of chloroquine within the brain is Brewster DR, Kwiatkowski D & White NJ (1990) Neurological pproximately four times that of plasma (Titus 1989), and sequelae of cerebral malaria in children. L a n c e t 336,1039—1043. experimental evidence suggests that chloroquine may precipi­ Cann HM & Verhulst HL (1961) Fatal acute chloroquine poisoning tate seizures by attenuation of gamma-aminobutyric acid in children. P e d i a t r i c s 1, 95-102. Crawley J, Smith S, Kirkham F, Muthinji P, Waruiru C & Marsh K pathways (Amabeoku 1992), and by the enhancement of (1996) Seizures and status epilepticus in childhood cerebral Jopaminergic neurotransmission (Amabeoku & Chikuni malaria. Q uarterly Journal o f M edicine 89, 591-597. 1993). Crawley J, Waruiru C, Mithwani Se t a l . (2000) Effect of phénobarbi­ this study, however, there was no association between base­ tal on seizure frequency and mortality in childhood cerebral line levels or area under the concentration curve (which malaria: a randomised, controlled intervention study. L a n c e t 355, reflects both absorption and subsequent elimination) of 701-706. chloroquine or its active metabolite desethylchloroquine, and Crawley J (1999) Malaria: new challenges, new treatments.C u r r e n t the number or duration of seizures in children with cerebral P aediatrics 9,34-^1. malaria. However, baseline levels of chloroquine and English MC, Waruiru C, Lightowler C, Murphy SA, Kirigha G & Jesethylchloroquine were reduced in the small subgroup of Marsh K (1996) Hyponatraemia and dehydration in severe children who had an episode of status epilepticus. malaria. A rchives o f D isease in C hildhood 74, 201-205. Fish DR & Espir ML (1988) Convulsions associated with prophylac­ There are a number of limitations to this study. The dose and tic anti malarial drugs: implications for people with epilepsy. route of chloroquine administration were not known, and it British M edical Journal 297, 526—527. ivas only possible to obtain a rough estimate of the time of Jaffar S, Van Hensbroek MB, Palmer A, Schneider G & Greenwood B administration in 73% of the patients. Information on (1997) Predictors of a fatal outcome following childhood cerebral leizures that occurred prior to admission was based on clini­ malaria. A m erican Journal o f Tropical M edicine and H ygiene 57, cal history, which can be unreliable, and analysis was there­ 20-24. fore confined to seizures that were witnessed by clinical staff Jaffe J (1988) Childhood chloroquine poisonings- Winsconsin and in hospital. In addition, it is not known whether the reported Washington. Journal o f the A m erican M edical A ssociation 260, association between chloroquine ingestion and seizures is a 1361. dose-related or idiosyncratic phenomenon. Although this Kiel FW (1964) Chloroquine suicide. Journal o f the Am erican M edical A ssociation 190, 398—400.

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Luijckx GJ, De Krom M C & Takx-Kohlen BC (1992) Does chloro­ on the coast of Kenya. T ransactions o f the R oyal Society of quine cause seizures? Presentation of three new cases and a review Tropical M edicine and H ygiene 86, 237-239. of the literature. S e i z u r e 1, 183-185. Titus O (1989) Recent developments in the understanding of the Molyneux ME, Taylor TE, Wirima JJ&C Borgstein A (1989) Clinical pharmacokinetics and mechanisms of action of chloroquine. features and prognostic indicators in paediatric cerebral malaria: a Therapeutic D rug M onitoring 11,369-379. study of 131 comatose Malawian children. Q uarterly Journal o f Torrey EE (1968) Chloroquine seizures. Jo urn al o f the American M e d i c i n e 71, 441^59. M edical A ssociation 204,115-118. Newton CRJC, Crawley J, Sowumni Ae t a l . (1997) Intracranial Wallace S (1988) T he C hild w ith Febrile Seizures. Wright, hypertension in Africans with cerebral malaria. A rchives o f London. D isease in C hildhood 76, 219-226. Waruiru CM, Newton CR, Forster D e t a l . (1996) Epileptic seizures Patchen EC, Mount DC, Schwartz IK & c Churchill EC (1983) and malaria in Kenyan children. T ra n sa ctio n s o f the Royal Sociei, Analysis of hlter-paper absorbed, finger-stick blood samples for o f Tropical M edicine and H ygiene 90,152-155. chloroquine and its major metabolite using high performance liq­ White NJ, Miller KD, Churchill ECe t a l . (1988) Chloroquine treat- ^ uid chromatography with fluorescence detection. J o u r n a l o f ment of severe malaria in children: pharmacokinetics, toxicity,and! C hrom atography 278, 81-89. new dosage recommendations. N e w E ngland Journal of Medicine Riou B, Barriot P, Rimailho A & Baud FJ (1988) Treatment of severe 319,1493-1500. chloroquine poisoning. N ew England Journal o f M edicine 318, White NJ, Miller KD, Marsh Ke t a l . (1987) Hypoglycaemia in 1—6 . African children with severe malaria. L a n c e t 1,708-711. Snow RW, Peshu N, Forster D, Mwenesi H & Marsh K (1992) The White NJ (1998) Drug resistance in malaria.B ritish M edical llulletif- role of shops in the treatment and prevention of childhood malaria 54,703-715.

864 © 2 0 0 0 B lackw ell Science lli ARTICLES

et of phénobarbital on seizure frequency and mortality in Idhood cerebral malaria: a randomised, controlled ervention study

ïWey, Catherine Waruiru, Sadik Mithwani, Isiah Mwangi, William Watkins, David Duma, Peter Winstanley, ÉfPeto, Kevin Marsh

nary Introduction Seizures commonly complicate cerebral Every year, more than one million children in sub- Siiaand are associated with an increased risk of death Saharan Africa die or are disabled as a result of cerebral IjBimlogical sequelae. We undertook a randomised malaria.' Seizures complicate a high proportion of cases a to assess the efficacy of intramuscular phénobarbital and are associated with an increased risk of death^ and mting seizures in childhood cerebral malaria. neurological sequelae.T he most common sequelae are hemiplegia, spastic quadriplegia, visual impairment, and Ws Children with cerebral malaria admitted to one epilepsy,but a much wider range of motor and cognitive Kilifi, Kenya, were randomly assigned a single impairments occurs. If anticonvulsant prophylaxis can iKiscuiar dose of phénobarbital (20 mg/kg) or identical lower the frequency of seizures complicating cerebral Clinical tolerance was assessed at the start of the malaria, this benefit might in turn reduce the risk of with particular reference to respiratory depression and death and neurological sequelae, and have an important jsision. Seizures were timed and recorded, and impact on the educational potential of children in sub- 8(1 in a standard way. Plasma phénobarbital Saharan Africa. iBitrations were measured. Analyses were by intention Phénobarbital has been used as an anticonvulsant for many years, and is highly effective in the treatment of both partial and generalised seizures.® It is cheap and, Ifs440 children with cerebral malaria were admitted unlike most anticonvulsant drugs used for seizure «hospital; 100 were not recruited to the study. Of the prophylaxis, widely available throughout Africa. It can g 340, 170 received phénobarbital and 170 be given by intramuscular injection, which is a further The drug was adequately absorbed and well advantage, since intravenous therapy is not possible in I. Seizure frequency was significantly lower in the many health facilities throughout the continent. The rbital group than in the placebo group (18 [11%] vs loading dose recommended for children is 10-20 '27%] children had three or more seizures of any mg/kg.’’® hn: odds ratio 0-32 [95% 01 0 18-0 58]) but mortality There have been two previous studies of phénobarbital doubled (30 [18%] vs 14 [8%] deaths; 2 39 prophylaxis in cerebral malaria. In a small, randomised M'64]). The frequency of respiratory arrest was study on adults in Thailand, three of 24 patients treated fin the phénobarbital group than in the placebo with a single dose of phénobarbital (3-5 mg/kg) had (.and mortality was greatly increased in children who seizures after admission to hospital, compared with 13 of phénobarbital plus three or more doses of 24 given placebo." However, in a pharmacokinetic study lça(n(odds ratio 31 7 [1-2-814]). on Kenyan children with cerebral malaria,'" phénobarbital 10 mg/kg did not produce blood fetation In children with cerebral malaria, concentrations (15 mg/L and above) known to provide rtiarbital 20 mg/kg provides highly effective seizure effective prophylaxis against febrile seizures." '^ Iflaxis but is associated with an unacceptable We undertook a randomised, placebo-controlled study in mortality. Use of this dose cannot, therefore, be to assess whether a single intramuscular dose of mended. phénobarbital (20 mg/kg) given on admission to Kenyan «2000; 355: 701-06 children with cerebral malaria could lower the frequency of seizures complicating the clinical course in hospital. ^mentary page 671 The safety and clinical tolerance of this dose were assessed at the start of the trial.

iledlcal Research Institute/Wellcome Trust Centre for Methods Ifiic Medicine Research (Coast), Kilifi, Kenya Study participants ÉyMRCP, C Waruiru mb, S Mithwani mb, I Mwangi mb, The study took place at the Kenya Medical Research Institute llbfsh FRCP); Nuffield Department of Clinical Medicine, (KEMRI) unit, at Kilifi District Hospital, on the coast of ally of Oxford, Oxford, UK(J Crawley, I Peto Dphii, Kenya. The hospital serves a predominantly rural population, Wh); Wellcome Trust Research Laboratories, Nairobi, and about 3000 children are admitted to the 35-bed paediatric iWWatkins PhD, D Ouma bsc); and Department of ward each year. Malaria transmission (of which over 95% is Mogy and Therapeutics, University of Liverpool, Liverpool, Plasm odium falciparum ) occurs throughout the year, with peak [Mins, P Winstanley frcp) transmission after the rainy seasons of April to May and pndence to:Dr Jane Crawley, Centre for Tropical Medicine, October to November.'’ «Department of Clinical Medicine, John Radcliffe Hospital, The study was approved by the Kenyan National Ethical Igon. Oxford 0X3 9DU, UK Committee. Informed, written consent was obtained from first- [email protected]) degree relatives in all cases.

IJNCET • Vol 355 • February 26, 2000 701 ARTICLES

injection. The intravenous route was chosen because à 440 children admitted infusion could have been stopped if any adverse events In with cerebral malaria occurred. As in the main study, the clinical investigators wa unaware of which patients had received phénobarbital. P* 100 not recruited respiratory rate, blood pressure, and transcutaneous 63 consent refused saturation were measured at baseline and every 30 min for il) 23 death imminent Blood was taken at the same times for measurement 14 insufficient staff phénobarbital concentration, and every 1 h for venous gas a available lactate measurements. The trial was then unmasked for liq 23 patients only, and the phénobarbital and placebo gi 340 randomised compared in terms of all clinical and biochemical findings. Baseline blood samples were taken for quantitative pi count, full blood count, measurement of glucose, eleci and phénobarbital concentrations, blood gases, and bii 170 assigned 170 assigned culture. Further blood samples for phénobarbital measure! phénobarbital placebo were taken from all patients at 1 h, 2 h, 4 h, 8 h, 12 h, 21 36 h, and 48 h. Parasite counts were repeated every 8 h discharge, death, or clearance of parasitaemia. Si 8 met criteria 3 met criteria intracranial hypertension is a feature of cerebral malaa,' for exclusion for exclusion 3 0 died 14 died lumbar puncture was delayed until the neurological statu 9 lost to 12 lost to the child had improved, or was done post mortem for those follow-up follow-up died. Cerebrospinal fluid was assessed by microscopy culture, and all samples with a white-cell count of above 10 per ptL were screened for antigens of H aem ophilus infh 170 analysed for 1 7 0 analysed for Streptococcus pneum oniae. primary endpoint primary endpoint Children received antimalarial chemotherapy with a loi (seizures) (seizures) dose of intravenous quinine dihydrochloride (15 mg/kg), subsequent doses of 10 mg/kg every 12 h.'’ All children Figure 1: Trial profile treated with intravenous benzylpenicillin (60 mg/kg every Eligible children were aged 9 months to 13 years and had and chloramphenicol (25 mg/kg every 6 h) until the results cerebral malaria defined as unrousable coma (inability to lumbar puncture were available. Intravenous fluids and localise a painful stimulus: Blantyre score 3 or less’) in the were given as clinically indicated.'® Children with ted! presence of Plasm odium falciparum parasitaemia, detected by temperatures above 38 5°C were treated with paracei light microscopy. Children who had had seizures before (15 mg/kg per rectum every 6 h). Hypoglycaemia admission were assessed a minimum of 30 min after the end of glucose below 2 2 m mol/L) was corrected with seizure activity. We excluded children who had pre-existing intravenous bolus of 0 5 g/kg dextrose. The number afebrile epilepsy or significant neurodevelopmental problems, duration o f all seizures were recorded by means of timers those who had received treatment with phénobarbital or to alarm at 5 min, 15 min, and 30 min. Seizures lasting phenytoin during the current illness, and those found on min or longer were treated with diazepam 0 3 mg/kg, given lumbar puncture to have meningitis. an intravenous injection over 2 min. After two diazepam had been given without improvement, intrami Design and procedures paraldehyde 0 2 mL/kg was used as second-line Anticonvulsants were administered at 5 min, 15 min, 30 We calculated, assuming 90% power and a significance level of and 45 min if seizure activity persisted. Phenytoin 20 mgkg 5%, that a sample size of 320 children would be required to infused intravenously over 20 min if a child had received detect a 50% reduction (from 30% to 15%) in seizures doses o f both diazepam and paraldehyde without resolution, occurring before recovery of consciousness in the had experienced six or more seizures within 2 h. phenobarbital-treated group. The seizure categories judged to Vital signs (temperature, pulse, respiratory be of particular clinical importance were: three or more transcutaneous oxygen saturation), Blantyre coma score, seizures of any duration; any seizure lasting for 5 min or longer; and any episode of status epilepticus (defined as seizure activity blood glucose (BM Stix, Roche Diagnostics, Lewes, lasting for 30 min or longer, or six or more seizures within a Sussex, U K ) were m onitored every 4 h. Oxygen was available period of 2 h). required, but there were no facilities for artificial ventilation. By means of a sequentially numbered register and as soon as All patients were neurologically examined at the time possible after admission, children were randomly assigned a discharge from hospital. All patients were asked to single intramuscular injection of phénobarbital 20 mg/kg 3 months later for full neurodevelopmental assessment. (200 g/L), or the same volume (OT mL/kg) placebo. The Serial blood sam ples were taken from all patients placebo, 90% propylene glycol (the vehicle for parenteral phénobarbital assay. W hole-blood phénobarbital con preparations of phénobarbital), had the same viscosity and were measured in Nairobi, Kenya, by reverse-] colour as the phénobarbital preparation. Numbered 5 mL performance liquid chromatography.*" Patients in whom ampoules of phénobarbital and placebo were prepared by the phénobarbital was detected in the blood samples taken at 1 pharmacy department of Torbay Hospital, Torbay, UK. The 2 h, and 4 h were assumed to have received placebo, code identifying drug and placebo was kept at Torbay Hospital, assignation was verified at the end of the study when the and therefore none of the clinical or scientific staff involved in had been broken. W e derived pharmacokinetic v the study knew which patients had received phénobarbital. (maxim um concentration, time to maximum com Since previous work’ had shown an increased frequency of and area under the concentration/time curve [AUQ.J) seizures among younger children with cerebral malaria, every ten profiles on patients who had received randomisation was stratified into two age-groups (24 months or Since there was very little variability in the data, wiii| younger and above 24 months). significant change in the mean derived pharmacokinetic The clinical tolerance of phénobarbital 20 mg/kg was as numbers increased, we decided that profiles on 50 pati assessed at the start of the trial. 23 children were given the would provide a good representation of the phénobarbital study drug (phénobarbital or placebo) by constant-rate as a whole. In this way, we avoided the cost and time ofassat intravenous infusion over 4 h instead of by intramuscular samples from all 170 children who received phénobarbital.

702 T H E LA NC ET • Vol 355 • February ARTICLES

[| Placebo (n=170) Phénobarbital (n=170) Repeat analysis, without these cases, did not affect the outcome results. 35 4 (32 4-38-5) 35 4 (32-7-38-0) The placebo and phénobarbital groups were well [_ 93 (55%)/77 (45%) 91 (54%)/79 (46%) matched in terms of most clinical and laboratory ■ligatures variables (table 1). 28% of the children (49 placebo, ms before admission 148 (87%) 133 (78%) 45 phénobarbital), had previously had a febrile (kpticus before admission* 82 (48%) 63 (37%) #1 duration of coma before 5 (2-10) 5 (2-10) illness complicated by seizures. During the current Bi(h) illness, a significantly higher proportion of children |toma scoret assigned placebo had had seizures before admission 45 (26%) 38 (22%) 3 125 (74%) 132 (78%) (table 1; p=0 04). The difference in the proportions l^erature (°C) 38-9 (38-8-39-1) 38-8 (38-6-38-9) of children who had been given a dose of diazepam leatspermin) 149 (146-152) 148 (144-152) during the 6 h before admission was not, however, lujrate (breaths per min)$ 41 (39-43) 42 (40-44) tm distress 50 (29%) 49 (29%) significant (43 [25%] placebo, 32 [19%] phénobarbital; p=0T 9). Iwy variables P ) parasitaemia (per p,L) 79 620 (6448-351120) 89 600 (4294-422 400) 5obifi(g/dL) 7-2 (6-8-7 5) 7-2 (6-9-7-6) Clinical tolerance #hmol/L) 135 (134-136) 134 (133-135) Among the 23 children (ten phénobarbital, 13 placebo) P l potassium (mmol/L) 4-3 (3-9-4-8) 4-3 (3-8-4-9) assessed for clinical tolerance, baseline clinical and SP) urea (mmol/L) 4-2 (3-1-6-7) 4-9 (3-2-7-7) |»(|imoi/L)t 63-8 (58 9-69-1) 64-5 (59-6-69-7) biochemical characteristics were similar in both groups Jmoi/L) 5-4 (4-9-5-9) 5-3 (4-8-5-8) (p>0 40 for all comparisons). There were no significant 7-30 (7-28-7-32) 7-31 (7-28-7-33) differences between the groups in the mean change P) pCO, (kPa) 4-1 (32-4-8) 3-8 (3-0-4-4) Bate (mmol/L) 15-6 (14-8-16-5) 15-1 (14-2-15-9) between baseline and 5 h or baseline and maximum or -9-2 (-10-3 to -8-2) -9-5 (-10-6 to -8-4) minimum values for any of the variables measured #nol/L|* 3-7 (3-44-2) 4-0 (3 5-4-5) (p=0 07 for fall in lactate 0-5 h and maximum rise in ïîinean (95% Cl) or number (%) unless otherwise stated, pH, p>0 10 for all other comparisons). teseizures (“too many to count") or any seizure lasting >30 min. 3coma. 5=full consciousness, Pharmacokinetics c mean. el: Admission clinical and laboratory features by Whole-blood concentrations of phénobarbital obtained ent group by intramuscular injection and intravenous infusion of 20 mg/kg are shown in figure 2. Intramuscular injection (n=50) resulted in a median maximum concentration of were carried out with Stata version 5.0. We used 25 6 mg/L (IQR 23 6-30 2). Median time to maximum nt’s t test for comparisons of normally distributed concentration was 4 0 h (2 0-12 0). Concentrations ative data, with logarithmic transformation if the were maintained above 15 mg/L, which is within the ibution was skewed, the non-parametric Mann-Whitney range (10-30 mg/L) thought to provide effective seizure ■ data without a normal distribution, and Fisher’s exact prophylaxis'® for at least 48 h. The mean AUC 0 .1 2 after I for categorical data. Outcome data were analysed by intramuscular injection was 250 mg h L ' (95% Cl non to treat. Multivariate logistic regression analysis was 232 3- 268-2). Maximum phénobarbital concentrations li to examine the relation between admission characteristics were slightly lower in eight children who died than in 42 lijpbsequent outcome. Recovery times in the two groups compared by survival analysis and the log-rank survivors (median 25 1 [range 15 3-33-1] vs 26-6 tional hazards) and Peto-Peto-Wilcoxon” (non- [10 0-44-9] mg/L). There were no significant differences dona 1 hazards) tests for equality of survivor functions, in pharmacokinetic variables between survivors and planned interim analysis was undertaken by an those who died (p>0-4 for all comparisons), but ndent statistician in January, 1997, when 170 patients numbers are small. dbcen recruited. The results of the analysis were reviewed by For technical reasons, we could obtain it trial monitor, but were not made available to the clinical pharmacokinetic profiles on only four of the ten children igators. There was a significantly (p<0 05) lower rate of and neurological sequelae among the phénobarbital 30-1 I than in the placebo group. However, there was also a

tad towards increased mortality in the phénobarbital group, 20 - gh it did not reach statistical significance (p=0T2). The itor therefore advised continuation of the trial, on the mption that the unexpected mortality difference was likely

Ibc a chance fluctuation in numbers, and would attenuate if 10 - ■trial continued. Recruitment was ended once the calculated iplesize had been achieved.

lilts o. o Intramuscular iticipants • Intravenous ween June, 1995, and January, 1998, 440 children ^omet the criteria for entry to the study were admitted : research ward (figure 1). 100 were not recruited, remaining 340 children were randomly assigned 0 10 20 30 40 50 tient, and all are included in the main analysis. 11 Time (h) Iren were recruited to the study but were Figure 2: Whole-blood phénobarbital concentrations according kquently (before the study code was broken) found to mode of administration I have characteristics that met criteria for exclusion. Error bars=l SD.

HEIANCET • Vol 355 • February 26, 2000 703 ARTICLES who had received phénobarbital by intravenous infusion. 1-0-, The mean AUCg ;; after intravenous infusion was 196-2 0 Phénobarbital • Placebo mg h L' (95% Cl 154-1-238-4). The difference in (/) 0-8 AUCo . , 2 between the intravenous and intramuscular O3 routes of administration was of borderline statistical % significance (p=0-05). o 0 - 6 4 ■H rib Outcomes .9 0-4- # 213 (63%) of the 340 patients achieved parasite e Ik clearance before discharge or death. The placebo and R 2 bt phénobarbital groups did not differ in the median time qI 0-2 to parasite clearance (40-0 [IQR 3T5-52-8] vs 45-7 [30-6-63-0] h; p=0-21). There was no difference p=0-39 between the groups in the time taken to recover consciousness (the ability to localise a painful stimulus;’ 0 2 0 4 0 6 0 8 0 figure 3, p=0-39, log-rank test). Number at risk Phénobarbital provided effective seizure prophylaxis, Phénobarbital 170 75 35 17 3 Placebo 170 72 35 12 7 decreasing the frequency and duration of seizures by over 50% (table 2). Multivariate logistic regression analysis, with allowance for the slight excess of children in the placebo group who had a history of seizures before admission, did not affect this finding. Significantly fewer children assigned phénobarbital than of those assigned pi placebo subsequently required treatment with phenytoin .> 0-9 0 (14/170 [8-2%] 27/170 [15-9%], p=0-04). Mortality among children assigned phénobarbital was 0 -8 5 - more than double that of the placebo group (table 2). Of the 44 children who died, 30 were in the phénobarbital I 0 -8 0 - group, compared with 14 in the placebo group (odds ratio 2-39 [95% Cl T28-4-46], p=0-01). Use of 0 -7 5 - multivariate logistic regression analysis showed that this p = 0 - 0 1 difference could not be accounted for by minor 0-70^ imbalances in risk factors between the groups. Median 4 0 60 time from drug administration to death was 22-5 h Time (hours) (range 0-4-92-7) for children in the phénobarbital group and 24-2 h (0-4-66-7) for those in the placebo group Number at risk (p=0-72). Kaplan-Meier curves, comparing overall Phénobarbital 170 Placebo 170 survival in the two groups, are shown in figure 3. Children who died were, on admission, more deeply comatose and significantly more dehydrated, acidotic, Figure 3:Kaplan-Meier plots of coma resolution andoverall P and hypoglycaemic than those who survived (p<0-01 for survival Note that the scales on both axes differ between the two parts of the all variables). Among children who died, mean figure. admission respiratory rate was higher in the placebo group than in the phénobarbital group (57 [95% Cl during their clinical course in hospital; of these 30 diei 50-64] vs 47 [41-52], p=0-03). However, respiratory Of the 33 who had a respiratory arrest, 22 had rec rate at 4 h (median time to maximum phénobarbital phénobarbital and 11 placebo (odds ratio 2-1 [9Î concentration) was similar for the two groups (47 TO-4-5], p=0-05). In analyses according to [41-43] vs 48 [43-53], p=0-76). There were no other diazepam dose (in four categories; this total incluM significant differences between the groups in admission doses given in the 6 h before admission, plus all i clinical or laboratory characteristics. given in hospital), the odds of death for children treattl 33 children had a respiratory arrest (cessation of with phénobarbital rose from 0-2 for those given few breathing in the presence of normal cardiac function) than three doses of diazepam to 1-7 for those giventhrtc’

Placebo Phénobarbital Unadjusted analyses Adjusted analyses (n=170) (n=170) Odds ratio (95% Cl) P Odds ratio (95% Cl) P J Seizures Three or more of any duration 46 (27%) 18 (11%) 0 32 (0-18-0 58) <0-001 0-34 (0-19-0-62)* Any lasting 5 min or longer 43 (25%) 20 (12%) 0-39 (0-22-0-70) 0-002 0-42 (0-24-0-76)* o-owl Any episode of status epllepticusf 23 (14%) 9(5%) 0-36 (0-16-0-78) 0-01 0-38 (0-17-0-85)* 0-02 \ Death 14 (8%) 30 (18%) 2-39 (1-28-4-64) 0-01 2-49 (1-19-5-23)$ 0-02 J Neurological sequelae ill At discharge 33/156 (21%) 18/140 (13%) 0-55 (0-30-1-02) 0-06 0-56 (0-30-1-05)* 0-07 3 months after discharge 15/144 (10%) 9/131 (7%) 0-63 (0-27-1-47) 0-39 0-69 (0-29-1-65)» 0-40 H * Adjusted for seizures before admission. fLasting >30 min or more than six within 2 h. ^Adjusted for factors associated with increased mortality (Blantyre score, respiratory distress, base excess, glucose, urea, creatinine). Table 2: Clinical outcome

704 T H E L A N C E T • Vol 355 • February 26,201 ARTICLES

I P doses Placebo Phénobarbital Odds ratio (95% Cl) treatment, so increasing the attendant morbidity and 13/150 (9%) 25/162 (15%) 1 9 (0 9-3 9) 0 07 mortality. PS 1/20(5%) 5/8(62%) 31-7 (1-2-814) 0-001 In this study, the unacceptable price of effective k3: Mortality in phénobarbital and placebo groups, seizure prophylaxis was the doubling of mortality in the wiling to number of doses of diazepam phénobarbital group. The most plausible explanation is acre doses (p=0-004 for unequal odds). The relation that phenobarbital-induced respiratory depression ra death and the interaction between drug group precipitated respiratory arrest in a group of children who ioobarbital or placebo) and diazepam category were critically dependent on their respiratory drive (and m than three doses/ three or more doses) was then for whom ventilatory support was not available). This mned by logistic regression and the likelihood ratio hypothesis is supported by the findings that respiratory L Diazepam category alone was not significantly arrest was more likely in the phénobarbital group, and idated with an increased risk of death (odds ratio that mortality was higher among children treated with g B [0 07-4-48], p=0-5). The interaction between both phénobarbital and several doses of diazepam. This içam category and drug group (phénobarbital or was, however, a post-hoc analysis on a very small was, however, associated with a greatly number of patients (of the 16 “excess deaths” in the rased risk of death (16-5 [1-3-215], p=0-03). The phénobarbital group, only five of these children received d ratio test, comparing the logistic regression three or more doses of diazepam), and should, therefore, with and without the interaction term, was be interpreted with caution. Children who continue to icant (p=0-015) and, after adjustment for the have seizures despite phénobarbital prophylaxis traction between drug group and diazepam category, represent a subgroup with particularly refractory effect of phénobarbital on death became non- cerebral pathology, and may be unusually susceptible to further sedation. The apparent reduction in neurological nfficant (odds ratio 1-92 [0-94-3-91], p=0-07). Table sequelae among children treated with phénobarbital may Smpares the difference in mortality between the reflect the higher mortality in that group; those who died Dobarbital and placebo groups after fewer than three might, had they survived, have had a high frequency of three or more doses of diazepam. The excess sequelae. naiitv’ in the phénobarbital group may be partly Intravenous diazepam is the drug of first choice for the Winted for by the interaction between phénobarbital immediate treatment of status epilepticus, because of its multiple (three or more) doses of diazepam. This ease of administration and rapid onset of action.® This jtstion is consistent with the hypothesis that the drug is highly lipid soluble and is rapidly distributed to ied mortality is due to respiratory depression. cerebral tissue and lipid stores. After repeated (17%) children had neurological sequelae at administration, redistribution ceases as tissue stores rge from hospital. Although the proportion of equilibrate, and further doses result in high, persistent Mren with sequelae was lower in the phénobarbital concentrations within the brain, and the consequent risk sip than in the placebo group (table 2), the difference of sudden respiratory arrest and hypotension. In I of borderline significance (p=0-06). Of 275 children contrast, two controlled studies have shown that s3 months after discharge, 24 (9%) had persistent phénobarbital, is highly effective in the treatment of (iielae (table 2). Of these, six children (one status epilepticus, with no associated increase in Bobarbital, five placebo) who were blind, with spastic respiratory depression or h y p o te n sio n .A retrospective aiiriplegia and global developmental delay, were review of children with refractory status epilepticus, who [ed to have severe sequelae. The remaining 18 had been treated with phénobarbital (30-120 mg/kg), (eight phénobarbital, ten placebo) had one or showed a striking lack of respiratory depression. K of hemiplegia, epilepsy, ataxia, visual impairment, Published case reports suggest that the combination of ijed speech. phénobarbital and diazepam may result in respiratory depression and hypotension,^®-^^ and this study icussion documents an increase in mortality resulting from create many reasons why anticonvulsant prophylaxis concomitant use of the two drugs. to be beneficial in childhood cerebral malaria, Other drug interactions should be considered, since all (re is clinical and experimental evidence that long- children were treated with quinine, benzylpenicillin, Biion, uncontrolled seizure activity can damage the chloramphenicol, and, in most cases, paracetamol. Seizures complicating cerebral malaria are Winstanley and colleagues'® found that, in children with 1 with an increased risk of neurological severe malaria, intravenous quinine did not affect the lelae,^’ and survivors can have substantial cognitive disposition of intramuscular phénobarbital. We know of no evidence that benzylpenicillin, chloramphenicol, or içerimental evidence suggests that phénobarbital paracetamol may interfere with the action of prevent structural damage resulting from phénobarbital. moiled seizure activity within the developing Is there a dose of phénobarbital that is both safe and We found that a single intramuscular dose of effective in childhood cerebral malaria? Phénobarbital is Bobarbital was effective in preventing seizures in said to provide effective seizure prophylaxis at plasma with cerebral malaria. We emphasise, however, concentrations of between 10 mg/L and 30 mg/L. clinical conditions of this study, with rapid Plasma concentrations may, however, vary widely meat and close observation of all children with between individuals given the same dose,^® a finding ares, are very different from those that prevail in confirmed in this study. In addition, phénobarbital has a ly hospitals throughout Africa. Inadequate staffing pKa of 7-2, and changes in body pH therefore affect the ilpaucity of drugs and equipment mean that children distribution and excretion of the drug. If the pH of the It have seizures for many hours without adequate blood is lower than that of the brain, the gradient will

IIAVCET • Vol 355 • February 26, 2000 705 ARTICLES favour movement of phénobarbital into the brain. 9 White NJ, Phillips RE, Looareesuwan S, Chanthavanich P, Status epilepticus disrupts the blood-brain barrier, Warrell D A. Single-dose phenobarbitone prevents convulsiffl#® cerebral malaria. Lancet 1988; ii: 64-66. increasing cerebral uptake of phénobarbital even 10 Winstanley PA, Newton CRJC, Pasvol G, et al. Prophylactic further.’®’^' The relation between plasma concentration phenobarbitone in young children with severe falciparum malaiii: and effect in cerebral malaria could be different from pharmacokinetics and clinical effects. B rJ Clin Pharmacol that in other disorders without generalised cerebral 149-54. 11 Faero O, Kastrup KW, Nielsen EL, Melchior JC, ThomI pathology. Effective anticonvulsant prophylaxis might Successful prophylaxis of febrile convulsions with phenobarbitij result from plasma concentrations of phénobarbital Epilepsia 1979; 13: 279-85. generally judged subtherapeutic.^^ Phénobarbital 3-5 12 Pearce JL, Sharman JR, Forster RM. Phénobarbital in the acute management of febrile convulsions. Pediatrics 1977; 60: 569-72. mg/kg provided effective seizure prophylaxis in adults 13 Snow RW, Armstrong-Schellenberg JRM, Peshu N, et al, Perioiiiil with cerebral malaria.® Phénobarbital 10 mg/kg, in a and space-time clustering of severe childhood malaria on Ée co« larger study, might prove effective in childhood cerebral Kenya. Trans R Soc Trop Med Hyg 1993; 87: 386-90. malaria, despite blood concentrations below the 14 Newton CRJC, Crawley J, Sowumni A, et al. Intracranial hypertension in Africans with cerebral malaria. Arch Dis Child IW) “therapeutic” cut-off of 15 mg/L.'® 76: 219-26. Other therapeutic options, such as magnesium 15 W instanley PA, M beru EK, Watkins WM, Murphy SA, LoweB, sulphate,” should be explored in children with cerebral Marsh K. Towards optimal regimens of parenteral quinine for yi® African children with cerebral malaria: unbound quinine malaria. Once a drug that is both safe and effective has concentrations following a simple loading dose regimen. TramRSt been found, the next step would be to establish, in a Trop M ed H yg 1994; 88: 577-80. sufficiently large study, whether seizure prophylaxis can 16 Crawley J. Malaria; new challenges, new treatments.CunPmàt lower the frequency of neurological sequelae 1999; 9: 34-41. 17 Peto R, Peto J. Asymptotically efficient rank invariant test complicating this devastating disease. procedures. J R Star Soc 1972; 135: 185-207. Contributors 18 Dodson WE. Antiepileptic drug utilization in pediatric patients. Jane Crawley was responsible for trial design and coordination, Epilepsia 1984; 25: 132-39. clinical care of patients, data reduction, and statistical analysis. 19 M eldrum BS, Vigouroux RA, Brierley JB. Systemic factors and Catherine Waruiru, Sadik Mithwani, and Isiah Mwangi were responsible epileptic brain damage. Arch Neurol 1973; 29: 82-87. for clinical care of patients and data collection. William Watkins was 20 Stafstrom CE, Chronopoulos A, Thurber S, Thompson ]L responsible for phénobarbital assays and calculation of derived H olm es GL. Age-dependent cognitive and behavioral deficits afe pharmacokinetic data. David Ouma was responsible for phénobarbital kainic acid seizures. Epilepsia 1993; 34: 420-32. assays. Peter Wynstanley was involved in trial design, pharmacokinetics, 21 Sutula T , Cavazos J, Golarai G. Alteration of long-lasting strucim and clinical tolerance. Timothy Peto was trial monitor and gave and functional effects of kainic acid in the hippocampus by brief statistical advice. Kevin Marsh was involved in trial design, clinical care treatment with phénobarbital. J Neurosci 1992; 12 : 4 1 73-87. of patients, and data collection. Jane Crawley prepared the paper, which 22 Mikati MA, Holmes GL, Chronopoulos A, et al. PhenobarbW was then critically appraised by all the other investigators. modifies seizure-related brain injury in the developing brain, dw Acknowledgments Neurol 1994; 36: 425-33. We thank the Director of KEMRI for permission to publish this paper; 23 Shaner M D , McCurdy SA, Herring MO, Gabor AJ. Treatment of the nurses on the paediatric research ward at Kilifi Hospital, Boneface status epilepticus: a prospective comparison of diazepam and Muambi, Steven Wale, Gladys Binns, Anderson Kahindi, Naomi Lewa, phenytoin versus phénobarbital and and optional phenytoin. Neurology 1988; 38: 202-07. Elizabeth Mwangi, Mercy Mwabonje, Susan Mwangi, Simon Kenga, Janet Mwaro, Judith Peshu, Ann Muhoro, and our ward assistant, 24 Treiman DM , Meyers PD , Walton NY, et al. A comparison of fosr Connie Dama for their skilled help; our clinical colleagues, in particular treatments for generalized convulsive status epilepticus. N En^J Michael Yung and Norbert Peshu; the laboratory and computing staff of M ed 1998; 339: 792-98. KEMRI Clinical Research Centre; Nicholas Day and Catherine 25 Crawford TO , Mitchell WG, Fishman LS, Snodgrass SR. Veiy-lii|| Maitland for statistical advice, and Nicholas White for stimulating and dose phénobarbital for refractory status epilepticus in children. helpful discussions. The work is supported by KEMRI and the Neurology 1988; 38: 1035-40. Wellcome Trust (040313). KM is a Wellcome Trust Senior Research 26 Prensky AL, Raff M C, Moore MJ, Schwab RS. Intravenous Fellow in Clinical Science (031342). diazepam in the treatment of prolonged seizure activity. NEni] M ed 1967; 276: 779-84. 27 Sawyer GT, Webster D D , Schut LJ. Treatment of uncontrolled' References seizure activity with diazepam. JAMA 1968; 203: 111-16. 1 WHO. World malaria situation in 1994. Wkly Epidemiol Rec 1997; 28 Treiman DM, Gunawan S, Walton NY, Meyers PD. Serum 72: 269-90. concentrations of antiepileptic drugs following intravenous administration for the treatment of status epilepticus. 2 Jaffar S, Van Hensbroek MB, Palmer A, Schneider G, Greenwood B. 33 (suppl 3): 3-4. Predictors of a fatal outcome following childhood cerebral malaria. 29 Sim on RP, Copeland JR, Benowitz NL, Jacob P, BronsteinJBm A m J Trap M ed H yg 1997; 57: 20-24. phénobarbital uptake during prolonged status epilepticus. JW 3 Molyneux ME, Taylor TE, Wirima JJ, Borgstein A. Clinical features Blood Flow M etab 1987; 7: 783-88. and prognostic indicators in paediatric cerebral malaria: a study of 30 Ramsay RE, H am m ond EJ, Perchalski RJ, Wilder BJ. Brain upub 131 comatose Malawian children. QJM 1989; 71: 441-59. of phenytoin, phénobarbital, and diazepam. Arch Nmoll^l%]h\ 4 Brewster DR, Kwiatkowski D, White NJ. Neurological sequelae of 5 3 5 -3 9 . cerebral malaria in children. Lancet 1990; 336: 1039-43. 31 Brzakovic B, Pokrajac M, Dzoljic E, Le vie Z, Varagic VM. 5 Crawley J, Smith S, Kirkham F, Muthinji P, Waruiru C, Marsh K. Cerebrospinal fluid and plasma pharmacokinetics of phenobaibml Seizures and status epilepticus in childhood cerebral malaria. QJM after intravenous administration to patients with status epileprics( 1996; 89: 591-97. Clin Drug Invest 1997; 14: 307-13. 6 Shorvon S. Status epilepticus: its clinical features and treatment in 32 Goldberg MA, MacIntyre HB. Barbiturates in the treatment of children and adults. Cambridge: Cambridge University Press, 1994. status epilepticus. In: Delgado-Escueta AY, Wasterlain CG, 7 Rylance GW. Treatment of epilepsy and febrile convulsions in Treim an D M , Porter RJ, eds. Advances in neurology. New York: children. Lancet 1990; 336: 488-91. Raven Press, 1983: 499-503. 8 Bone RC. Treatment of convulsive status epilepticus: 33 Duley L. Which anticonvulsant for women with eclampsia? recommendations of the Epilepsy Foundation of America’s working Evidence fi-om the Collaborative Eclampsia Trial. Lam\995;ilit group on status epilepticus. JAMA 1993; 270: 854-59. 1455-63.

706 T H E LANC ET • Vol 355 • Febmary26,20l| TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (1998) 92, 305-308 305

Abnormal respiratory patterns in childhood cerebral malaria

Jane Crawley^»^, Michael E nglish^C atherine Waruiru^, Isiah Mwangi^ and Kevin Marsh^’^ ^Kenya Me­ dical Research Institute (Clinical Research Centre), Kilifi Unit, Kilifi, Kenya; ^Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK

Abstract O f 295 children with cerebral malaria, 117 (40%) had an abnormal respiratory pattern; 15 children ex­ hibited more than one pattern during their clinical course. Four distinct patterns were seen, (i) Deep breathing (80 children); this was associated with severe metabolic acidosis, and resolved following treat­ ment with intravenous fluids and/or blood, (ii) Hypoventilation with nystagmus and salivation (18 children); simultaneous electroencephalographic recording revealed continuous electrical seizure activity, demonstrating that these children were in subtle status epilepticus; anticonvulsant treatment resulted in return to normal of blood gases and recovery of consciousness, (iii) Hyperventilation with extensor pos­ turing (20 children), which was associated with varying degrees of intracranial hypertension, (iv) Periodic respiration (14 children); all had clinical features suggestive of transtentorial herniation, and died follow­ ing a respiratory arrest. Abnormal respiratory patterns can alert the clinician to complications of cerebral malaria that require treatment. Recognition of these patterns and rapid initiation of appropriate support­ ive therapy may help to reduce the high mortality rate of this disease.

Keywords: malaria, cerebral malaria,Plasm odium falciparum , respiratory patterns, children, Kenya

Introduction urement of transcutaneous oxygen saturation, central Cerebral malaria, a febrile encephalopathy caused by venous pressure (ENGLISH et a l, 1996b), electroen­ Plasmodium falciparum, has a mortality rate of between cephalography (EEG; 14-channel M edele^ 1A94 ma­ 10 and 40%, with 10% of survivors being left with per­ chine) (C rawley et al., 1996), cerebral function manent neurological sequelae. The pathophysiology is monitoring (CFAM, channel EEG, Medaid Ltd.) and, complex and, in addition to coma, children may present with selected children, intracranial pressure monitoring with seizures, which are often prolonged and multiple using a subarachnoid catheter (model 110-4B, Camino (Mo l y n e u x et a l, 1989; Brew ster et a l, 1990; Laboratories) (NEWTON et a l, 1997). All studies were Craw ley et a l, 1996; War u ir u et a l, 1996), anaemia, carried out with the approval of the KEMRI Scientific metabolic acidosis (TAYLOR et a l, 1993; ENGLISH et a l, Co-ordinating Committee, and with written informed 1997), and hypoglycaemia (WHITE et a l, 1983; MARSH consent from parents. et a l, 1995). Most deaths occur within the first 12 h af­ ter admission, highlighting the need for rapid assess­ R esults ment and treatment. In many African hospitals, Between January 1993 and September 1995, 295 however, diagnostic facilities are extremely limited, and children were admitted with cerebral malaria. One hun­ clinical signs may provide the only indication of under­ dred and seventeen (40%) had an abnormal respiratory lying pathophysiology requiring treatment. As a result of pattern, of whom 15 had more than one pattern during a series of research studies, we have come to recognize 4 their clinical course. The 4 patterns were (i) acidotic or distinct respiratory patterns and their associated patho­ deep breathing (Kussmaul’s respiration), (ii) hypoventi­ physiology which have proved helpful in the clinical lation with associated nystagmus, (iii) hyperventilation management of children with cerebral malaria. with extensor posturing, and (iv) periodic respiration.

M ethods Deep breathing The studies were conducted at the Kenya Medical Deep breathing, defined as a persistent increase in the Research Institute (KEMRI) Research Unit at Kilifi Dis­ depth and rate of respiration, was observed in 80 chil­ trict Hospital on the Kenyan coast. Three thousand to dren. It is dealt with only briefly here, since details have 4000 children are admitted each year to the 36 beds already been published (ENGLISH er a/., 1996a, 1996b). general paediatric ward, while children with severe ma­ Deep breathing was associated with severe metabolic laria (WHO, 1990) are transferred to the 6 beds paedi­ acidosis (base excess -12 or more), with a sensitivity of atric high dependency unit. Cerebral malaria was 91% and a specificity of 83% (ENGLISH et a l, 1996a). defined as an inability to localize pain (MOLYNEUX et These children had higher mean blood lactate, urea and a l, 1989) in the presence of P. falciparum parasitaemia. creatinine levels and osmolality, and lower mean pH Clinical assessment included close observation of respi­ and haemoglobin level, than those without deep breath­ ratory pattern and rate, depth of coma, posture, and fo­ ing (En g l ish et a l, 1997), suggesting that hypovolae- cal neurological signs. Blood was taken for parasite mia, anaemia, and renal impairment were important count, full blood count, determination of glucose, elec­ contributing factors. Central venous pressure was nor­ trolytes, lactate and venous blood gas levels, and blood mal (<6 cm of water) in 24 of these children (ENGLISH culture. Children received standard antimalarial treat­ et a l, 1996b), and rapid transfusion (over 1-4 h) of 20 ment with intravenous quinine or intramuscular arte- mL/kg of normal saline or blood (for those with a hae­ mether, as part of an open randomized study comparing moglobin level <5 g/dL) was followed by a dramatic fall the efficacy of the 2 drugs (MURPHY et a l, 1996). Ben­ in lactate level and clinical improvement in the majority zyl penicillin and chloramphenicol were administered in of patients. Of the 80 children in this group, 80% went standard doses until meningitis was excluded by the re­ on to make a full recovery, and 20% died. sults of lumbar puncture. Intravenous fluids, blood, dextrose, and anticonvulsant drugs were given as clini­ Hypoventilation with associated nystagmus cally indicated. Investigations that have been carried out during the course of research studies include the meas- Eighteen children presented in deep coma with shal­ low, irregular respiration. Transcutaneous oxygen satu­ ration was below 90% in all children, while the median Address for correspondence: Dr Jane Crawley, KEMRI (CRC), PCO 2 of 15 children who had concurrent blood gas Kilifi Unit, Box 230, Kilifi, Kenya; e-mail kemriklf@africaon- analysis done was 7 0 kPa (range 5-5-11T kPa). Other line.co.ke important clinical features were bilateral consensual eye 306 JANE CRAWLEYETAL deviation with nystagmus, and salivation. Eight children went ICP monitoring had severe intracranial hyperten­ had simultaneous electrophysiological recordings made sion (ICP> 40 mmHg for at least 15 min, in association by EEG or CFAM, and all showed electrical evidence of with CPP <40 mmHg). Although intravenous mannitol continuous seizure activity (CRAWLEY et al., 1996). Al­ (0-5-0-75 ^kg over 20 min) was given to 7 children, though electroencephalographs from 5 children re­ only 2 received more than 4 doses, the other 5 receiving vealed continuous unilateral spike-wave discharges in one or 2 doses just before death. All children died (see the parieto-temporal region, sometimes spreading below) following respiratory arrest in the presence of a throughout the hemisphere, there was either no mani­ normal pulse and absent pupillary responses, features festation in the contralateral limbs, or minimal, inter­ suggestive of transtentorial herniation. mittent clonic movements of a digit, eyebrow, or angle of the mouth. CFAM confirmed continuous seizure ac­ Periodic respiration tivity in these patients, and demonstrated seizure activi­ Fourteen children (including 8, described above, who ty in a further 3 patients who did not have EEG. These had sustained opisthotonic posturing) had a respiratory children were therefore in subtle status epilepticus. Sei­ pattern that changed abruptly a median of 12 h after ad­ zure activity resolved spontaneously in one patient and, mission. Breathing became shallow and irregular, with with the others, anticonvulsant treatment was followed intermittent gasping. Pupillary responses were slugpsh by an improvement in respiratory effort and blood gas­ and the oculocephalic response absent. Nine received es. Children recovered consciousness, as judged by their one or more doses of mannitol, but all went on to be­ ability to localize a painful stimulus, a median of 6 h come apnoeic without initial cardiac arrest. Despite at­ (range 2-70) after anticonvulsant treatment. Two chil­ tempted resuscitation, all these children died. dren, both of whom had prolonged partial seizures, had mild hemiplegia on discharge. One child, who was pro­ foundly anaemic and acidotic on admission, subse­ Discussion quently died (see below). Fifteen children made a full This series of children with cerebral malaria illustrat­ recovery. ed how anomalies in respiratory pattern can provide the Two cases were of particular interest. One child of 5 clinician with valuable information on pathophysiology months was hypoventilating on admission despite pro­ that may result in a beneficial change of treatment. The found metabolic acidosis (base excess -30). Following findings are of particular relevance to hospitals in the ru­ treatment with diazepam, the child developed deep, ral tropics, where diagnostic facilities are limited. rapid respiration, and the PCO2 fell firom 7 6 to 2 6 kPa, In humans, respiration is under dual control from providing appropriate partial compensation for the met­ metabolic and neural influences. Metabolic respiratory abolic acidosis. Another child, of 51 months, had been control is directed principally at maintaining normal unconscious for 4 h following a prolonged generalized oxygenation and acid-base balance, and is regulated by seizure at home. There was no clonic activity, and the respiratory ‘centres’ in the lower brain stem. Neural child was deeply unconscious with shallow irregular res­ control originates from prepontine structures lying piration, eye deviation, nystagmus, salivation, and pria­ mainly at forebrain level. The 2 controlling systems are pism. EEG revealed generalized electrical status, which integrated largely in the lower brainstem, and each sys­ was unresponsive to 4 doses of diazepam and loading tem projects by distinct descending pathways to the spi­ doses of phenytoin and phenobarbitone. Seizure activity nal respiratory motor neurones. The presence of this was finally abolished after 2 intravenous doses of thio- dual control mechanism means that diseases causing pentome 3 mg/kg, and the child recovered conscious­ coma commonly induce respiratory abnormalities that ness 6 h later. can reflect either the pathological anatomy or chemistry Full clinical details of these children can be obtained of the illness (PLUM & PoSN E R , 1982). The pathologi­ from the first author. cal hallmark of cerebral malaria is sequestration of para­ sitized red blood cells in the cerebral microvasculature Hyperventilation with extensor posturing (MACPHERSON et al., 1985). Neuronal damage may re­ Twenty children had episodes of hyperventilation (in­ sult from interference with microcirculatory flow and creases in rate and/or depth of respiration), associated consequent hypoxia, intracranial hypertension second­ with extensor posturing. The posturing varied in severi­ ary to increased cerebral blood volume (NEWTON et al, ty from intermittent increases in extensor tone (decere­ 1997), or the local release of toxic mediators such as cy­ brate rigidity) through to full-blown opistiiotonic tokines (G r a u et al., 1989; KWIATKOWSKI et al, 1990; posturing (arms extended, adducted, and hyperpronat- W h i t e & Ho, 1992), nitric oxide (C l a r k & Rock ­ ed, legs extended, feet plantar flexed). Six children had e t t , 1996), and excitotoxic neurotransmitters (R. Sur­ concomitant arterial blood gas levels which were sugges­ tees, personal communication). Prolonged, multiple tive of primary hyperventilation in the absence of meta­ seizures are a common complicating factor (C rawley bolic acidosis, pH being 7-48 or above, median PCO 2 et a l, 1996). Abnormalities in respiratory pattern may 2-9 kPa (range T3-3-9), median PO2 16-5 kPa (range therefore arise either as a direct result of the diffuse en­ 13-6-19-5), and median base excess -2 (range -7 to ze­ cephalopathy, or secondary to metabolic disturbances ro). (anaemia, acidosis, hypoglycaemia) that complicate the In 13 of these children, hyperventilation and postur­ disease process. ing were intermittent. Four children had concurrent Deep breathing is the commonest respiratory abnor­ EEG, which showed no evidence of seizure activity, mality observed in children with severe malaria in coast­ while monitoring of intracranial pressure (ICP) in 2 al Kenya. It is a clinical sign with good inter-observer children (NEW TON et ah, 1997) demonstrated mild in­ agreement amongst clinicians who regularly care for tracranial hypertension, the ICP being between 10 and children with severe malaria (ENGLISH et al, 1995) and 20 mmHg with cerebral perfusion pressures (CCP) is reliably associated with metabolic acidosis (ENGLISH above 50 mmHg. No child received treatment with et a l, 1996a) which may be caused by anaemia, dehy­ mannitol. One child, with a history of status epilepticus, dration, or a combination of both. Detection may alert was left with mild hemiplegia, one went on to develop the clinician to the need for urgent treatment w ith blood sustained opisthotonic posturing (see below), and 11 transfusion (ENGLISH et a l, 1996b) and/or intravenous made a full recovery. fluids. Seven children in this group had severe, sustained Hypoventilation leading to hypoxia and hypercapnia opisthotonic posturing and hyperventilation on admis­ is a potentially life-threatening situation which is easily sion, while one child developed it while in hospital. overlooked in many clinical settings. When diazepam There was no electrical evidence of seizure activity on has been given before admission it is tempting to at­ EEG in 7 of these children, but 2 children who under­ tribute the ensuing hypoventilation to drug-related res­ ABNORMAL RESPIRATORY PATTERNS IN MALARIA 307

piratory depression. However, our experience suggests Acknowledgements that hypoventilation in a child with cerebral malaria is This paper is published with the permission of the Director more likely to be caused by continuing subtle seizure ac­ o f KEMRI. We thank our clinical, nursing, and laboratory col­ tivity than by diazepam. It is appropriate, therefore, to leagues at Kilifi H ospital for their dedicated contribution to pa­ tient management. The research work is supported by KEMRI give further diazepam or another anticonvulsant drug. and the Wellcome Trust. We are grateful to the British High Nystagmus and salivation are important clinical features Commission in Nairobi and the Triangle Trust for additional that suggest continued seizure activity, while clonic ac­ support. K. M. is a Wellcome Trust Senior Research Fellow in tivity may be minimal or absent. Subtle seizure activity clinical science (031342). has been described in the late stages of prolonged gen­ eralized status epilepticus (T reim an et al., 1984), in References comatose adults (Lowenstein & Amtnoff, 1992), Aicardi, J. (1986).E pilepsy in C hildren. N ew York: Raven Press. and following cerebral anoxia (SiMON & AMINOFF, Aicardi, J. & Chevrie, J. J. (1983). Consequences of status ep­ 1986), and attacks of nystagmus tiiat are of epileptic or­ ilepticus in infants and children. A dvances in Neurology, 34, igin may occur without other seizure manifestations 115-125. Brewster, D. R., Kwiatkowski, D. & White, N. J. (1990). Neu­ (W hite, 1971). The physiological basis for the diminu­ rological sequelae of cerebral malaria in children. Lancet, tion in motor activity with prolonged seizures is unclear, 336,I039-I043. though biochemical alterations at a synaptic or cellular Clark, I. A. & Rockett, K. A. (1996). N itric O xide and Parasitic level, or sedative drug therapy, may all contribute D i s e a s e . London: Academic Press. (Shorvon, 1994). There is a large clinical and experi­ Corsellis, J. A. N. & Bruton, C. J. (1983). Neuropathology of mental literature (MELDRUM et a l, 1973; Aicardi & status epilepticus in humans. Advances in Neurology, 34, Chevrie, 1983; Corselus & Bruton, 1983; 129-139. S ta fs tr o m et a l, 1993) to support the hypothesis that Crawley, J., Smith, S., Kirkham, F., Muthinji, P., Waruiru, C. prolonged seizure activity can damage the brain, causing & Marsh, K. (1996). Seizures and status epilepticus in child­ hood cerebral malaria. Q uarterly Journal of M edicine, 89, deficits in both motor and cognitive function. Both in­ 5 9 1 -597. creased cerebral metabolism and hypercapnia cause an English, M., Murphy, S., Mwangi, I., Crawley, J., Peshu, N. & increase in cerebral blood flow and therefore cerebral Marsh, K. (1995). Interobserver variation in respiratory blood volume (MlNNS, 1991), so exacerbating the in­ signs of severe malaria. A rchives o f D isease in C hildhood, 72, tracranial hypertension that can complicate cerebral 3 3 4-336. malaria (NEWTON et a l, 1997). Epileptic inhibition of English, M ., Waruiru, C., Amukoye, E., Murphy, S., Crawley, respiration prevented one child in this series from com­ J., Mwangi, I., Peshu, N. & Marsh, K. (1996a). Deep pensating for profound metabolic acidosis. Subtle sei­ breathing reflects acidosis and is associated with poor prog­ nosis in children with severe malaria and respiratory distress. zure activity is, by definition, easily missed, yet the Am erican Journal of Tropical M edicine and Hygiene, 55, majority of the patients in this series recovered con­ 5 2 1 -524. sciousness within 6 h of anticonvulsant treatment, and English, M., Waruiru, C. & Marsh, K. (1996b). Transfusion went on to make a full recovery. Subtle seizure activity for respiratory distress in life threatening childhood malaria. should be considered with any child who fails to recover Am erican Journal of Tropical M edicine and Hygiene, 55, consciousness and is hypoventilating after a prolonged 5 2 5 -530. seizure: more rather than less anticonvulsant therapy is English, M., Sauerwein, R., Waruiru, C., Mosobo, M ., Obiero, often the answer. J., Lowe, B. & Marsh, K. (1997). Acidosis in severe child­ hood malaria. Q uarterly Journal o f M edicine, 90, 263-270. There is a number of possible explanations for the hy­ Grau, G. E., Taylor, T. E., Molyneux, M. E., Wirima, J. J., Vas- perventilation seen in association with abnormal postur­ salli. P., Hommel, M. & Lambert, P. H. (1989). Tumor ing in children with cerebral malaria. Tonic seizures can necrosis factor and disease severity in children with falci­ induce abnormal contraction of the limbs in association parum malaria. 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Sus­ Lowenstein, D. H. & Aminoff, M. J. (1992). Clinical and EEG tained deep, rapid respiration can result from dysfunc­ features of status epilepticus in comatose patients. N e u r o l o g y , tion of the rostral brainstem between the levels of low 42,100-104. midbrain and mid pons (PLUM & POSNER, 1982), and Macpherson, G. G., Warrell, M. J., White, N. J., Looareesu­ may occur during the course of transtentorial hernia­ wan, S. & Warrell, D. A. (1985). Human cerebral malaria: a quantitative ultrastructural analysis of parasitized erythro­ tion. As herniation progresses, hyperventilation may cyte sequestration. Am erican Journal of Pathology, 119, give way to periodic or ataxic respiration. Extensor pos­ 385-401. turing can, in addition, result from a number of patho­ Marsh, K., Forster, D., Waruiru, C., Mwangi, 1., Winstanley, logical processes such as anoxia or hypoglycaemia that M., Marsh, V., Newton, C., Winstanley, P., Warn, P., Peshu, depress relatively selectively the function of the deep di­ N., Pasvol, G. & Snow, R. (1995). Indicators of life-threat­ encephalon and forebrain and which are potentially re­ ening malaria in African children. N ew England Journal of versible. Hyperventilation in association with extensor M e d i c i n e , 332, 1399-1404. posturing in a child with cerebral malaria should there­ Meldrum, B. S.,Vigouroux, R. A. & Brierley, J. B. (1973). Sys­ temic factors and epileptic brain damage. A rchives o f N eurol­ fore prompt the clinician to check for anaemia and hy­ o g y , 2 9 , 82 -8 7 . poglycaemia and, having corrected these as necessary, Mendelow, A. D., Teasdale, G. 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Seizures and status epilepticus in childhood cerebral malaria

). CRAWLEY^'^ S. SMITHS F. KIRKHAM", P. MUTHINJI^, C. WARUIRU' and K. MARSH' " From the ^ KEMRI Clinical Research Centre, Kilifi, Kenya, ^Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK, ^Institute o f Neurology, London, UK,'^Institute of Child Health, London, UK, ^EEG Department, Kenyatta National Hospital, Nairobi, Kenya.

Received 5 February 1996 and in revised form 15 April 1996

Summary

Prolonged, multiple seizures complicate a high pro­ seizures were partial motor, 34% generalized tonic- portion of cases of childhood cerebral malaria, and clonic, and 14% partial with secondary generaliza­ several studies have shown an association between tion. In 22%, coma appeared to be due to a pro­ these and neurological sequelae. We prospectively longed postictal state. Ten children had subtle motor studied 65 patients (38 female) admitted to Kilifi seizures. Posterior parieto-temporal discharges were Hospital in 1994. Electroencephalographic record­ the most common EEG finding. Seven children died, ings (EEGs) were made at 12-hourly intervals, with eight developed neurological sequelae, and 50 continuous recordings made on a cerebral function (77%) recovered fully. Status epilepticus was associ­ analysing monitor (CFAM). Survivors were seen ated with the development of neurological sequelae. one month after discharge. Cerebral computerized Prolonged, multiple seizures may play an important tomography was performed on children with neuro­ part in the pathogenesis of coma in childhood cereb­ logical sequelae. Sixty-two percent of patients had ral malaria, and are likely to contribute to both the seizures following admission, of whom half had an morbidity and mortality of this disease. episode of status epilepticus. Fifty-two percent of

Introduction Malaria is a major cause of morbidity and mortality the local release of toxic mediators such as cytokines, in sub-Saharan Africa, causing the death of over one nitric oxide and excitotoxic neurotransmitters/'*® million children each year/ Approximately 1% of Patients present with a diffuse encephalopathy, and clinical infections with Plasmodium falciparum result seizures, which are often prolonged and multiple, in severe disease, of which one of the most serious occur in up to 80% of cases/'"'*^ Uncontrolled forms is cerebral malaria,’'^ with a mortality of seizure activity can damage the brain by aggravating 10-40% /'* Although most survivors make a full hypoxia, hypoglycaemia and intracranial hyperten­ recovery, neurological sequelae (hemiplegia, speech sion,’® and several studies have suggested an associ­ problems, cortical blindness, epilepsy) occur in ation between status epilepticus and neurological 5-15% /'^ sequelae in childhood cerebral malaria/'® Although the cause of coma in cerebral malaria Despite their high prevalence and potential is not known, the essential pathological feature is pathogenic importance, there have been no detailed sequestration of parasitized red blood cells in the studies of seizures in cerebral malaria. Here cerebral microvasculature/ Neuronal damage may we describe the clinical and electrophysio­ result from interference with microcirculatory flow logical spectrum of seizures in childhood cerebral and consequent hypoxia, intracranial hypertension malaria in an area of Kenya where malaria is secondary to increased cerebral blood volume,® or endemic.

Address correspondence to Dr j. Crawley, KEMRI, Clinical Research Centre Kilifi Unit, Box 230, Kilifi, Kenya © Oxford University Press 1996 592 J. C rawley et s l\.

Methods arial treatment with intravenous quinine 20 mg/kg loading dose and 10 mg/kg 8-hourly, or intramuscu­ Study site lar artemether 3.2 mg/kg loading dose and 1.6 mg/kg every 24 h, as part bf a multicentre study comparing The study was conducted on the 5-bed KEMRI the efficacy of quinine and artemether in the treat­ paediatric research ward at Kilifi District Hospital, ment of childhood cerebral malaria. Intravenous Kiiifi, Kenya, between January and September 1994. benzyl penicillin and chloramphenicol were adminis­ The characteristics of malaria transmission and the tered until the results of lumbar puncture were population from which patients were drawn have known. Intravenous fluids and blood were given as been described previously.’"*^ clinically indicated. Seizures lasting more than 5 min were treated with intravenous diazepam 0.3 mg/kg. Patients Recurrent (> 3 ) seizures were treated with a loading Children aged 9 months and above were eligible for dose of intravenous phenytoin 18 mg/kg and, if that enrolment in the study if they fulfilled the World failed, with intramuscular phenobarbitone 18 mg/kg. Health Organisation (WHO) definition of cerebral Children with continuous seizure activity lasting for malaria, namely unrousable coma not attributable to 30 min or more were considered to have status any other cause in the presence of asexual epilepticus.’® Plasmodium falciparum parasitaemia.’^ To fulfil the definition of cerebral malaria, coma had to persist Follow-up for at least 1 h after a seizure and/or after the All survivors were seen one month after discharge administration of diazepam. Depth of coma was for neurological examination and repeat EEC. quantified using the Blantyre coma score,^ in which Cerebral computerized tomography (CT) was per­ a coma score of 5 denotes full consciousness, and formed on all children with neurological sequelae. 0 complete absence of any response to painful stimulus. One hundred and ten children who fulfilled this definition of cerebral malaria were admitted Analysis during the study period. Sixty-five (60%) were EEGs were analysed by SS who knew the age of recruited, since for technical reasons it was only each child, but was blind to any other clinical possible to study two children at a time. information. Statistical analysis was carried out using SPSS (version 5.0; 1992). Analysis of variance and Clinical investigations and management Student's t test were used to compare means of normally distributed data. Data not conforming to a A clinical history and complete physical examination normal distribution were compared by analysis of was performed on all children. Blood was taken for variance after logarithmic transformation, or by the baseline assessment of parasite count, full blood Mann-Whitney U test. Proportions were compared count, urea and electrolytes, glucose, lactate, by the ^ test. blood gas, and plasma chloroquine level. Electro- encephalographic (EEC) recordings were made on a 14-channel Medelec 1A94 EEC machine. Silver/ silver chloride electrodes were fixed with Elefix and Results tape to the child's shaved head, and the International Sixty-five children were studied, of whom 38 were 10-20 system was used for electrode placement. female. Ages ranged from 9 months to 11 years Recordings were taken within 6 h of admission, and (median 30 months). All had previously normal at 12-h intervals until recovery of consciousness development. Children were unconscious for (Blantyre coma score 5). Continuous recordings using between 1 and 72 h (median 7 h) prior to admission. a CFAM (cerebral function analysing monitor, Medaid In over half the cases, onset of coma coincided with Ltd) were obtained from children unconscious for the onset of epileptic seizures. more than 24 h. Any unusual neurological signs or witnessed seizure activity were recorded on video. On admission In deeply comatose children, intracranial pressure was monitored using a subarachnoid catheter Presenting clinical features and investigations are (Camino 110-4B). Intracranial hypertension is a fea­ shown in Tables 1 and 2. Seventy-five percent of the ture of childhood cerebral malaria® and, to reduce children were deeply unconscious on admission, the risk of transtentorial herniation, lumbar puncture with a Blantyre coma score of 2 or less. Thirteen was performed once the clinical condition of the children (20%) had received diazepam on or up to child had improved, or was done post-mortem in 6 h prior to admission, in doses of between those who died. Children received standard anti mal­ 0.2-1 mg/kg. In five cases, diazepam had been given Seizures and cerebral malaria 593

Table 1 Clinical comparison of children with and without seizures after admission (n = 65)

No seizures after Partial seizures Generalized admission (n=25) (n=28) seizures (n = 12)

Age (months)* 38.5 (29.9-47.1) 26.7 (20.6-32.9) 64.0 (44.7-83.5) Past history of febrile seizures 2 (8%) 5 (18%) 2 (17%) Pre-admission Any seizures 15 (60%) 22 (78%) 7 (58%) Status epilepticus 11(44%) 13(46%) 3 (25%) Admission coma score** 2 (2-3) 2 (1-4) 2 (0-4) Admission temperature (°C) 38.8 (38.3-39.3) 38.4 (38.0-38.8) 39.4 (38.4-40.5) Status epilepticus after admission 14 (50%) 4 (33%) Neurological sequelae 3(12%) 4(14%) 1 (8%) Death 4 (16%) 2 (7%) 1 (8%)

Data expressed as frequency (proportion) or mean (95% Cl). *p<0.001 for comparison between the three groups. For all other variables there was no statistically significant difference at the 5% level. ** Admission coma score expressed as median (range).

Table 2 Laboratory parameters on admission: comparison of children with and without seizures after admission (n = 65)

No seizures post-admission Seizures post-admission (n = 25) (n = 40)

Parasitaemia (per fil)* 41523 (15306-112645)* 44846 (22315-90129)* Haemoglobin (g/dl) 7.0 (6.1-7.9) 6.8 (5.8-7.7) Na (mmol/l) 137 (133-140) 136 (134-137) K (mmol/l) 4.4 (4.0-4.8) 4.4 (4.1-4.7) Urea (mmol/l)* 4.8 (3.2-7.2)* 4.7 (3.7-6.0)* Creatinine (p.mol/1)* 68 (54-86)* 54 (45-66)* Glucose (mmol/l) 4.4 (3.5-5.3) 5.9 (4.6-7.2) Corrected calcium (mmol/l) 2.05 (1.95-2.15) 2.10 (2.02-2.18) pH 7.36 (7.32-7.40) 7.32 (7.28-7.36) Base excess -7.3 (-10.7 to -0.9) -7.2 (-9.7 to -4.7) Lactate (mmol/l)* 3.3 (2.4-4.7)* 4.0 (3.1-5.0)* Osmolality (mOsmol/kg) 289 (279-299) 289 (284-294) Chloroquine (ng/ml of free base)** 80 (0-1570)** 43 (0-902)** Data expressed as means (95% Cl). * Geometric mean (95% Cl). ** Median (range); n = 54, of whom 38 (70%) had detectable levels of plasma chloroquine. There were no statistically significant differences at the 5% level between the two groups for any of the above variables.

intramuscularly at a health centre. Fourteen children Three children had both partial and generalized (22%) had multiple seizures or status epilepticus on seizures during their clinical course. Partial seizures or immediately prior to recruitment, but had regained were slightly (64%), but not significantly, more consciousness within 6 h of cessation of seizure common on the right side of the body. Only 42% activity. of the 310 seizures documented were associated with a rectal temperature of 38 °C or above. Two Clinical course were associated with hypoglycaemia. Ten children (25% of those with seizures after admission), with a Forty children (62%) had between one and more median age of 26 months (range 9-60), had seizures than 20 clinical seizures (median five), of duration that were clinically very subtle. In nine children 0.5-600 min (median 3 min). Eighteen children these consisted of nystagmoid eye movements, saliva­ (28%) had one or more episodes of status epilepticus. tion, and shallow, irregular respiration with concur­ Seventy-six percent of all seizures occurred within rent hypoxaemia (arterial oxygen saturation below 24 h of admission. Fifty-two percent of the seizures 80%) and hypercarbia (pC02>6.5 kPa). Although were partial motor, 14% partial with secondary EEGs revealed continuous unilateral spike-wave dis­ generalization, and 34% generalized tonic-clonic. charges in the parieto-temporal region, sometimes 594 J. Crawley et a\. spreading to involve the whole of that hemisphere, generalized ictal discharges. All children who made there were either no manifestations in the contralat­ a full recovery had normal EEGs at one month eral limbs, or intermittent minimal clonic movements follow-up. of a digit, eyebrow, or mouth. One child was admitted in deep coma, with irregular respiration, Outcome priapism, and salivation, following 6 h of generalized status epilepticus at home. EEC revealed generalized Fifty children (77%) made a full recovery, seven seizure activity, yet there were no other clinical (11%) children died, and eight (12%) had persistent accompaniments. neurological sequelae one month after discharge. Twenty-six children received between one and Among survivors, median time to localize a painful five doses of intravenous diazepam, 0.3 mg/kg. In stimulus was 21.5 h (range 4-111 h). Of the eight the majority of cases, cessation of clinical seizures children with sequelae, four had a hemiplegia, two occurred within 5 min of drug administration. In had spastic quadriplegia, one had severe cognitive eight children, electrographic seizure activity per­ and speech problems, and one had epilepsy. The sisted for between 2 and 140 min despite cessation children with sequelae were significantly (p=0.02) of the clinical seizure with treatment. Intravenous younger (mean age 20.4 months, 95% Cl 6.2-34.5 phenytoin 18 mg/kg was given to 24 children, of months) than those who recovered fully (39.8 whom 63% had no further seizures. Two out of four months, 95% Cl 33.2-46.3 months). Six (75%) had status epilepticus before or after admission, as did children who received intramuscular phenobarbitone 27 (54%) normal survivors. Two children who also had no further seizure activity. In one child, developed a spastic quadriplegia had severe hyper- intravenous thiopentone 4 mg/kg was effective at natraemic dehydration on admission. There were stopping generalized status epilepticus that had been otherwise no significant differences in clinical or unresponsive to three doses of diazepam and laboratory parameters between those with sequelae 18 mg/kg of both phenytoin and phenobarbitone. and normal survivors, but numbers are small. Seven Intracranial pressure (ICP) was monitored in 10 of the eight children with sequelae had CT scans at children, of whom four had a total of 31 (range one month follow-up and, with the exception of the 1-16) seizures during the course of monitoring. Both scan from the child with epilepsy, all showed abnor­ generalized and partial seizures caused a rise in malities. Scans from 3/4 children with hemiplegia intracranial pressure, the median rise in ICP revealed an area of infarction in the contralateral ( -P164%, range 108-285) being greater during 18 posterior parieto-temporal region (Figure 2). During generalized seizures than during 13 partial seizures their clinical course in hospital, two of these children (-f 50%, range 0-186). The magnitude of the rise had developed partial status epilepticus with elec­ was not affected by seizure duration. In all cases, a trical discharges arising from the same area. One concurrent rise in mean arterial pressure meant that child with partial motor status epilepticus and sub­ cerebral perfusion pressure was adequately main­ sequent hemiplegia failed to attend for CT scan. CT tained above 50 mmHg. Generally, intracranial pres­ scans on the two children with spastic quadriplegia sure fell at the end of each seizure, but remained and one with profound cognitive and speech prob­ high (above 30 mmHg) for 2 min following the lems showed generalized cerebral atrophy. Follow- cessation of one generalized seizure. One period of up EEGs from children with residual hemiplegia electrographic seizure activity with no associated showed low-amplltude slow wave activity in the clinical manifestations was not associated with a contralateral parieto-temporal region, while EEGs significant rise in intracranial pressure. from children with spastic quadriplegia were fea­ tureless and of low amplitude. Electrophysiology Two (29%) of the seven children who died had status epilepticus during their clinical course. Those Two hundred and seventy EEC and 30 CFAM record­ who died were significantly more acidotic on admis­ ings were made. During coma, the EEC was domin­ sion ( p values 0.04 and 0.01 for pH and base excess, ated by high-amplitude slow waves (4 Hz and respectively) than those who survived. One had below). Fifteen of 28 children with partial seizures Haemophilus influenzae septicaemia (but normal had an EEC recorded during a clinical seizure. In all cerebrospinal fluid) and three had one or more cases, ictal spike-wave discharges arose from the episodes of hypoglycaemia. Laboratory parameters posterior parieto-temporal region (Figure 1 ). In eight were otherwise unremarkable. of these cases there was considerable disparity between clinical and electrical seizure activity, with electrical activity spreading to involve all of one Discussion or both hemispheres despite the clinical seizure remaining partial. A further eight recordings from The WHO definition of cerebral malaria attempts to seven children with generalized seizures showed delineate a homogenous encephalopathic syndrome Seizures and cerebral malaria 595

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Figure 1. Continuous electrographic discharge over the left posterior temporal/occipital region. Clinical manifestations of the seizure were confined to nystagmus and eye deviation to the right. associated with high levels of morbidity and mortal­ important point here is that the ictal features would ity.’^ Our data suggest that, in a proportion of cases, be easily missed unless specifically looked for, yet recurrent seizures play an important role in the uncontrolled seizure activity can damage the pathogenesis of coma. Eighty-five percent of children brain.In creased cerebral metabolism and hyper- in this series had at least one seizure during their capnia both cause an increase in cerebral blood clinical course, with the onset of coma associated flow and therefore cerebral blood volume,^® so with seizures in over half the cases. For the 22% potentially exacerbating the intracranial hypertension who regained consciousness within 6 h of admission, that can complicate cerebral malaria.® Subtle seizures coma appeared to result from prolonged or repeated are an easily treated cause of coma, and five of seizures, and these children are likely to represent a these children regained consciousness within 6 h of different pathophysiological entity from those with treatment with intravenous diazepam. Most children prolonged coma. Following a prolonged seizure, the with cerebral malaria are admitted to busy, under­ brain enters a phase of 'cortical exhaustion', with staffed hospitals in the developing world, where depletion of adenosine triphosphate, glucose, oxygen subtle seizures are likely to be missed unless health and the accumulation of lactic acid.’^ In the postictal personnel are trained in their detection. phase the EEC may be flattened, or show diffuse What are the possible causes of seizures in cereb­ slow wave activity, the duration of the postictal ral malaria? Fever is known to precipitate seizures phase being increased after prolonged or multiple in young children.^’ However, more than half the seizures. seizures documented after admission occurred This study has also shown that in a proportion of when the rectal temperature was below 38 °C. children with cerebral malaria, coma is due to Hypoglycaemia and electrolyte imbalance can cause continuing subtle seizure activity which is likely to seizures, yet only two seizures were associated with go undetected, but which is responsive to anticon­ a blood glucose <2.2 mmol/l. It is likely that hyper- vulsant drugs. Ten children (25% of those with natraemic dehydration was partly responsible for the seizures on or after admission) had seizures with status epilepticus and subsequent spastic quadriple­ minimal clinical manifestations, a recognized feature gia that developed in two children. Although mild of prolonged generalized status epilepticus.’^’^ The hyponatraemia and hypocalcaemia occurred in a 596 J. Crawley et al.

of hypoxia, or by initiating the release of excitotoxic mediators such as glutamate or quinolinic acid.^"^'^^ However, whilst sequestration appears to be a global phenomenon, the majority of patients in this study had partial seizures. Electrographically, these seizures were associated with ictal spike-wave activity in the posterior parieto-temporal region. This is a 'water­ shed' area, lying between territories supplied by the posterior and middle cerebral arteries. It is therefore particularly vulnerable to ischaemia when oxygen delivery to the brain is compromised as a possible result of severe anaemia, or inadequate cerebral blood flow due to hypotension, raised intracranial pressure or impaired autoregulation. CT scans on two children with partial seizures and subsequent hemiplegia showed infarction in this area. Although it is difficult to know whether the seizures caused infarction or vice versa, it is clear that uncontrolled seizure activity, with the concomitant increased demand for oxygen and glucose, is likely to exacer­ bate the situation. Several studies of cerebral malaria^'^ have shown an association between status epilepticus and neuro­ logical sequelae, which occur in 5-15% of survivors. There is a large clinical and experimental literat- 8 ,19,26,27 support the hypothesis that prolonged seizure activity can damage the brain, causing defi­ cits in both motor and cognitive function. The hippocampus, which plays an important role in short- and long-term memory, is particularly prone Figure 2. Cerebral CT scan obtained one month after to seizure-induced damage.^®'^^ The gross neurolog­ discharge, showing extensive areas of infarction (num­ ical sequelae described in this and other studies of bered 1 and 2) in the left posterior temporal/occipital region, with associated cerebral atrophy. This 9-month- cerebral malaria are likely to represent one end of a old child had multiple right partial motor seizures (includ­ spectrum of a much wider range of handicaps. Many ing several episodes of status epilepticus) during her 'normal' survivors of cerebral malaria may have clinical course, and subsequently developed a dense right significant cognitive problems, with serious implica­ hemiplegia. tions for their subsequent educational potential. Can anticonvulsant prophylaxis reduce the incid­ ence of status epilepticus complicating cerebral mal­ proportion of children in this study, neither were aria? An ideal candidate drug would need to be significantly associated with seizures. Very high doses cheap, safe, effective, and preferably administered (plasma levels >2000 ng/ml) of chloroquine are by the intramuscular route. Phenobarbitone fulfils known to cause seizures,but even low prophylactic these criteria, yet its role in seizure prophylaxis in doses can exacerbate seizures in an individual with cerebral malaria is unclear, since two small published a personal or family history of epilepsy.It is difficult studies have produced somewhat conflicting to draw any firm conclusions from the chloroquine results.®”'®’ If prophylactic phenobarbitone can data presented in this study, since numbers are small reduce the incidence of status epilepticus complicat­ and the timing, dose, and route of chloroquine ing cerebral malaria, it is possible that this will also administration are unknown. Four out of five have an impact on the incidence of subsequent (80%) children with plasma chloroquine levels of neurological sequelae. A definitive study of intramus­ > 4 0 0 ng/ml had seizures after admission, as did cular phenobarbitone would therefore seem to be an nine (56%) out of 16 children with undetectable important next step. chloroquine levels. The characteristic histopathological feature of cerebral malaria is intense sequestration of parasit­ ized red blood cells in the cerebral microvasculat- Acknowledgements ure.^ This suggests that local interference with blood This paper is published with the permission of the flow could lead to seizures, either directly as a result Director of KEMRI. We wish to thank our clinical, SEIZURES IN CHILDHOOD CEREBRAL MALARIA

A thesis submitted to the University of London for the degree of Doctor of Medicine