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In malaria-endemic areas, the diagnosis of falciparum malaria is one of exclusion, because up to 70% of the children in the community may have parasitemia and yet be asymptomatic.1 Thus, if a patient presents with a febrile illness, this can only be attributed to malaria after exclusion of other causes. This is particularly important in children presenting with impaired level of consciousness. Bacterial meningitis cannot be excluded by clinical examination, but can be excluded by the examination and culture of the cerebrospinal fluid (CSF).2 However, in resource-poor countries, exclusion of viral encephalitis is more problematic. Little information is available on prevalence and manifestation of the neurotropic viruses in Africa. The contribution of viral pathogens to the encephalopathy syndrome usually attributed to Plasmodium falciparum has not yet been assessed. We looked for evidence of herpesviruses and enteroviruses in the children who fulfilled the World Health Organization’s (WHO) criteria for cerebral malaria.3
Polymerase chain reaction (PCR) was performed to detect DNA of herpes viruses and RNA of enteroviruses in CSF. The microbiological examination and measurement of glucose and protein were as described previously.2 The residual CSF was stored at –20°C within an hour of sampling and later at –80°C until PCR analysis was performed. DNA of the samples was isolated according to the Boom-method.4 Positive controls (viral DNA) and negative controls (calf-thymus DNA) were also included. Primers for the enteroviruses and the herpes viruses were selected according to well-established techniques.4–6 The primer pair sequences and amplification conditions are available on request. The purified PCR products were used for hybridization and were measured by electrochemoiluminescence using the M8 system (M-Series; IGEN, Oxfordshire, U.K.) with streptavidin coated magnetic beads (Dynabeads; Dynal, Oslo, Norway).
We studied 96 children admitted to Kilifi District Hospital (KDH) between 1999 and 2001. KDH is situated in the rural coast of Kenya, in a malaria endemic area, where malaria is the most important cause of childhood morbidity and most frequent cause of admission.
In patients who were unconscious (unable to localize a painful stimulus), a lumbar puncture was performed to exclude central nervous system infections. Examination of the ocular fundus was carried out on admission. Patients with proven or probable bacterial meningitis after blood culture, glucose measurement, or antigen detection were excluded from the study.2
We looked for viruses in two clinically defined groups:
All children were given intravenous antibiotics (benzylpenicillin and chloramphenicol) until the results of the CSF culture were reported, and intravenous quinine if they had asexual P. falciparum parasites on their peripheral smear or until three blood slides were negative. Anti-viral medication (e.g. acyclovir) was not available. The Kenya National Ethics committee approved this study.
The clinical details on the children are shown in Table 1
. The PCR procedure was successful for all subjects. Herpes simplex virus type 1 (HSV-1) DNA was detected in the CSF of four (9%) children with a clinical diagnosis of CM (group A). No other viral DNA was identified in this group. In children with slide-negative encephalopathy (group B), eight (17%) were positive for viral DNA, six (12%) for HSV-1, one for cytomegalovirus, and one for varicella zoster virus and enteroviral DNA. There was no statistical difference in any of the clinical features between the patients with HSV who had a positive slide (N = 4) compared with those who had a negative slide (N = 6).
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). There were no significant differences in age, incidence of convulsions or status epilepticus, axillary temperature, weight, retinal findings on direct ophthalmoscopy, or laboratory results.
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We found that 9% of children who fulfilled the WHO criteria for the diagnosis of CM had evidence of HSV-1 within their CSF. There were no useful clinical features that distinguished between these conditions. In particular, there was no cut-off for age, platelet count, or CSF white cell count that could be used to distinguish between these two conditions.
In malaria-endemic areas, the diagnosis of falciparum malaria is one of exclusion, because many children in the community may have parasitemia and yet be asymptomatic.1 A recent post-mortem study found that 23% of the children who fulfilled the WHO definition of CM died of other causes.7 Although virology was not reported in this study, none of the children had pathologic features of encephalitis. Only features of malaria retinopathy were associated with sequestration of parasites in the brain. We were only able to examine the fundi with direct ophthalmoscopy; more thorough examination with indirect ophthalmoscopy may detect differences between HSV-1 encephalitis and CM.
We found that HSV-1 was the most common cause of viral encephalitis in this study. Although we did not look for flaviviruses in this study, a previous study of the CSF from 25 encephalopathic children admitted to KDH did not detect any RNA of 68 known flaviviruses, including West Nile and dengue viruses (L. Dunster, personal communication).
This study suggests that a significant proportion of children who fulfill the WHO definition of CM may have other causes, including viral encephalitis, and this may confound studies on pathophysiology and neurocognitive sequelae. If these results are confirmed by further studies, the research on CM should be reinterpreted with these constraints.
Received May 5, 2006. Accepted for publication July 25, 2006.
Acknowledgments: We thank the technical staff of the microbiology laboratory department of the KEMRI/Wellcome Trust Research Unit and the Department of Clinical Virology of the Academic Medical Centre Amsterdam, especially Tony Kazungu, Brett Lowe, and Alex van Breda. Research support has been provided by Stichting Harald Quintus Bosz, Nationaal Epilepsie Fonds, Stichting Bekker-La Bastide-Fonds, Hersenstichting Nederland, Schuurman Schimmel-van Outeren Stichting, and Fonds van Beuningen van Heilsdingen. Professor CRJC Newton is funded by the Wellcome Trust (070114). None of the study sponsors had any influence in the study design, the collection of data, analysis, or interpretation of data.
* Address correspondence to Christian D. Schubart, Nicolaas Witsenstraat 13, 1017 ZE, Amsterdam, The Netherlands. E-mail: cdschubart{at}gmail.com 
Authors’ addresses: Christian Schubart, Nicolaas Witsenstraat 13, 1017 ZE, Amsterdam, The Netherlands, Telephone: 31-20-4223376, E-mail: cdschubart{at}gmail.com. Neema Mturi, KEMRI/Wellcome Trust Collaborative Programme, PO Box 230, Kilifi 80108, Kenya, Telephone: 254-41-525043/525446, Fax: 254-41-522390. Marcel Beld, Academic Medical Center, Department of Clinical Virology, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands, Telephone: 31-20-5665472, Fax: 31-20-6974005, E-mail: m.beld{at}amc.uva.nl. Pauline Wertheim, Academic Medical Center, Department of Clinical Virology, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands, Telphone: 31-20-5665472, Fax: 31-20-6974005, E-mail: p.m.wertheim{at}amc.uva.nl. Charles Newton, KEMRI/Wellcome Trust Collaborative Programme, PO Box 230, Kilifi 80108, Kenya, Telephone: 254-41-525043/525446, Fax: 254-41-522390, E-mail: cnewton{at}kilifi.kemri-wellcome.org.
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