• 1

    Snow RW, Omumbo JA, Lowe B, Molyneux CS, Obiero JO, Palmer A, Weber MW, Pinder M, Nahlen B, Obonyo C, New-bold C, Gupta S, Marsh K, 1997. Relation between severe malaria morbidity in children and level of Plasmodium falciparum transmission in Africa. Lancet 349 :1650–1654.

    • Search Google Scholar
    • Export Citation
  • 2

    Berkley JA, Mwangi I, Ngetsa CJ, Mwarumba S, Lowe BS, Marsh K, Newton CR, 2001. Diagnosis of acute bacterial meningitis in children at a district hospital in sub-Saharan Africa. Lancet 357 :1753–1757.

    • Search Google Scholar
    • Export Citation
  • 3

    World Health Organization, 2000. Severe falciparum malaria. Trans R Soc Trop Med Hyg 94 (Suppl 1):1–74.

  • 4

    Boom R, Sol C, Weel J, Gerrits Y, de Boer M, Wertheim-van Dillen P, 1999. A highly sensitive assay for detection and quantitation of human cytomegalovirus DNA in serum and plasma by PCR and electrochemiluminescence. J Clin Microbiol 37 :1489–1497.

    • Search Google Scholar
    • Export Citation
  • 5

    Beld M, Minnaar R, Weel J, Sol C, Damen M, van der Avoort H, Wertheim-van Dillen P, van Breda A, Boom R, 2004. Highly sensitive assay for detection of enterovirus in clinical specimens by reverse transcription-PCR with an armored RNA internal control. J Clin Microbiol 42 :3059–3064.

    • Search Google Scholar
    • Export Citation
  • 6

    de Jong MD, Weel JF, Schuurman T, Wertheim-van Dillen PM, Boom R, 2000. Quantitation of varicella-zoster virus DNA in whole blood, plasma, and serum by PCR and electrochemiluminescence. J Clin Microbiol 38 :2568–2573.

    • Search Google Scholar
    • Export Citation
  • 7

    Taylor TE, Fu WJ, Carr RA, Whitten RO, Mueller JS, Fosiko NG, Lewallen S, Liomba NG, Molyneux ME, 2004. Differentiating the pathologies of cerebral malaria by postmortem parasite counts. Nat Med 10 :143–145.

    • Search Google Scholar
    • Export Citation
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ROLE OF VIRUSES IN KENYAN CHILDREN PRESENTING WITH ACUTE ENCEPHALOPATHY IN A MALARIA-ENDEMIC AREA

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  • 1 Academical Medical Centre, University of Amsterdam, Department of Medical Microbiology, Laboratory of Clinical Virology, Amsterdam, The Netherlands; The Centre for Geographic Medicine Research (Coast), Kenya Medical Research Institute, Kilifi, Kenya; Neurosciences Unit, Institute of Child Health, The Wolfson Centre, Mecklenburgh Square, London, United Kingdom

In malaria-endemic areas, it is difficult to differentiate between cerebral malaria (CM), bacterial meningitis, and viral encephalitis. We examined the cerebrospinal fluid of 49 children who fulfilled the World Health Organization’s (WHO) definition of CM and in 47 encephalopathic children, without malaria, looking for viruses with polymerase chain reaction. In the children with CM, four (9%) had evidence of Herpes simplex virus 1 in the cerebrospinal fluid, whereas in the encephalopathy group without malaria, six (12%) were positive. A significant proportion of children who fulfil the WHO clinical definition of CM may have viral encephalitis.

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.46 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:

  1. Cerebral malaria (CM): children who fulfilled the WHO definition3 (i.e., a child who is unable to localize a painful stimulus and has a peripheral asexual parasitemia and in whom bacterial meningitis and hypoglycemia were excluded as causes of the encephalopathy).

  2. Encephalopathy without evidence of malaria: children who were unable to localize a painful stimulus, in whom asexual parasites were not detected in three blood slides taken over 24 hours.

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).

In comparison with the children who fulfilled the definition of CM after exclusion of the viral infections, the children with HSV-1 encephalitis had a significantly higher hemoglobin and longer duration of hospitalization (Table 2). There were no significant differences in age, incidence of convulsions or status epilepticus, axillary temperature, weight, retinal findings on direct ophthalmoscopy, or laboratory results.

Varicella zoster viral DNA and enteroviral RNA were detected in a 1-month-old girl who presented with a history of cough before becoming unconscious and unable to localize a painful stimulus. There was no history of convulsions. She had evidence of lower respiratory tract infection. She recovered without any sequelae. Cytomegalovirus was detected in a 6-month-old boy who presented with a 7-day history of fever and cough. On admission to the hospital he was conscious and breast feeding, but had severe wasting and developed a convulsion and focal neurologic deficits. The boy was discharged without any neurologic deficits detectable.

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.

Table 1

Clinical characteristics of children studied

Clinical characteristicsGroup A (cerebral malaria)Group B (slide negative encephalopathy)
IQR, interquartile range (25th and 75th percentile).
Number of children4947
Age (months): median (IQR)22.3 (7.7, 34.9)2.8 (0.3, 21.9)
Male (%)30 (61%)32 (68%)
History
    Duration of history (days): median (IQR)3.0 (1.5, 3.0)3.0 (1.0, 3.3)
    History of convulsions (%)41 (84%)23 (49%)
    Status epilepticus: seizure > 30 minutes (%)11 (22%)6 (13%)
Examination on admission
    Axillary temperature (° C): mean (SD)38.1 (1.5)37.4 (1.2)
    Weight (kg): mean (SD)9.6 (3.7)6.8 (5.5)
    Blantyre coma score ≤ 2 (%)49 (100%)47 (100%)
    Spleen size (cm): median (IQR)0.0 (0.0, 2.5)0.0 (0.0, 0.0)
Laboratory investigations
    White blood cell count (× 109/L): median (IQR)12.0 (9.1, 20.1)15.5 (9.1, 18.7)
    Hemoglobin (g/dL): mean (SD)6.9 (2.8)12.1 (3.8)
    Platelet count (× 109/L): mean (SD)307 (126)448 (88)
    Parasitemia (× 106/L): mean (SD)28,095 (27,757)0.0 (0)
    Children without parasites (%)0 (0%)47 (100%)
    Glucose (mmol/L): mean (SD)5.0 (2.7)4.5 (2.5)
    Children with hypoglycemia (%)6 (2%)5 (11%)
Cerebrospinal fluid
    Time between admission and lumbar puncture (hours): median16.51.4
    White cell count (/mm3): median (IQR)0.0 (0.1, 4)2.0 (0.1, 16.0)
    Protein (g/dL): median (IQR)0.2 (0.1, 0.3)0.4 (0.2, 0.7)
    CSF/blood glucose ratio: mean (SD)0.8 (0.3)0.7 (0.2)
During admission
    Convulsions during admission (%)13 (27%)9 (19%)
Outcome
    Duration of hospitalization (days): median (IQR)3.0 (2.0, 5.0)6.0 (2.0, 10.0)
    Neurological deficits (%)1 (2%)2 (4%)
    Death (%)3 (6%)3 (6%)
Table 2

Characteristics HSV encephalitis and cerebral malaria

Clinical presentationHSV-1 encephalitisCerebral malariaP
* Mann-Whitney test.
† Fisher exact test.
‡ Independent samples t test, equal variances assumed.
IQR, interquartile range (25th and 75th percentile).
Number of children1045
Age (months): median (IQR)10.5 (5.4, 30.9)22.7 (8.7, 36.9)0.25*
    Male (%)50%36%0.48†
History
    Duration of history (days): median (IQR)5.5 (2.8, 10.0)3.0 (1.0, 3.0)0.60*
    History of convulsions (%)8 (80%)36 (80%)1.00†
    Number of convulsions 24 hours before admission: median (IQR)3.5 (2.0, 4.0)1.0 (1.0, 3.0)0.46*
    Status epilepticus: Seizure lasting > 30 minutes (%)2 (20%)11 (24%)0.71†
Examination
    Axillary temperature (° C): mean (SD)37.5 (1.5)38.1 (1.5)0.24‡
    Weight (kg): mean (SD)7.5 (3.7)9.8 (3.8)0.08‡
    Blantyre coma score ≤ 2 (%)10 (100%)45 (100%)1.00†
    Retinal hemorrhages0/8 (0%)4/39 (10%)1.00†
    Spleen size (cm): median (IQR)2.0 (0.0, 3.8)0.0 (0.0, 2.0)0.18*
Laboratory features
    White cell count (× 109/L): median (IQR)15.4 (11.8, 27.5)11.9 (9.1, 20.1)0.50*
    Hemogloblin (g/dL): mean (SD)9.7 (5.8)7.0 (2.8)0.03‡
    Platelet count (× 109/L): mean (SD)310 (127)378 (148)0.447‡
    Children with malaria parasitemia (%)4 (40%)45 (100%)< 0.01†
    Sodium (mmol/L): mean (SD)134.0 (7.2)134.4 (5.0)0.80‡
    Creatinine (μmol/L): median (IQR)57.0 (51.3, 75.3)58.0 (41.5, 74.5)0.69‡
    Blood glucose (mmol/L): mean (SD)4.2 (1.6)5.0 (2.8)0.34‡
    Children with hypoglycemia (blood glucose < 2.2 mol/L) (%)1 (10%)2 (4%)0.46†
Cerebrospinal fluid
    White cell count (/mm3): median (IQR)0 (0.1, 6.0)0 (0.0, 4.0)0.95*
    Protein (g/dl): median (IQR)0.2 (0.1, 0.5)0.2 (0.1, 0.3)0.33*
    CSF/blood glucose ratio: mean (SD)0.9 (0.2)0.8 (0.2)0.48‡
During admission
    Duration of fever longer than 7 days (%)1 (10%)0 (0%)0.18†
    Convulsions during admission (%)4 (40%)11 (24%)0.67†
Outcome
    Duration of hospitalization (days): median (IQR)5.5 (2.8, 10.0)3.0 (2.0, 4.5)0.05*
    Neurological deficits (%)1 (10%)0 (0%)0.17†
    Death (%)1 (10%)3 (7%)0.56†

*

Address correspondence to Christian D. Schubart, Nicolaas Witsenstraat 13, 1017 ZE, Amsterdam, The Netherlands. E-mail: cdschubart@gmail.com

Authors’ addresses: Christian Schubart, Nicolaas Witsenstraat 13, 1017 ZE, Amsterdam, The Netherlands, Telephone: 31-20-4223376, E-mail: cdschubart@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@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@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@kilifi.kemri-wellcome.org.

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.

REFERENCES

  • 1

    Snow RW, Omumbo JA, Lowe B, Molyneux CS, Obiero JO, Palmer A, Weber MW, Pinder M, Nahlen B, Obonyo C, New-bold C, Gupta S, Marsh K, 1997. Relation between severe malaria morbidity in children and level of Plasmodium falciparum transmission in Africa. Lancet 349 :1650–1654.

    • Search Google Scholar
    • Export Citation
  • 2

    Berkley JA, Mwangi I, Ngetsa CJ, Mwarumba S, Lowe BS, Marsh K, Newton CR, 2001. Diagnosis of acute bacterial meningitis in children at a district hospital in sub-Saharan Africa. Lancet 357 :1753–1757.

    • Search Google Scholar
    • Export Citation
  • 3

    World Health Organization, 2000. Severe falciparum malaria. Trans R Soc Trop Med Hyg 94 (Suppl 1):1–74.

  • 4

    Boom R, Sol C, Weel J, Gerrits Y, de Boer M, Wertheim-van Dillen P, 1999. A highly sensitive assay for detection and quantitation of human cytomegalovirus DNA in serum and plasma by PCR and electrochemiluminescence. J Clin Microbiol 37 :1489–1497.

    • Search Google Scholar
    • Export Citation
  • 5

    Beld M, Minnaar R, Weel J, Sol C, Damen M, van der Avoort H, Wertheim-van Dillen P, van Breda A, Boom R, 2004. Highly sensitive assay for detection of enterovirus in clinical specimens by reverse transcription-PCR with an armored RNA internal control. J Clin Microbiol 42 :3059–3064.

    • Search Google Scholar
    • Export Citation
  • 6

    de Jong MD, Weel JF, Schuurman T, Wertheim-van Dillen PM, Boom R, 2000. Quantitation of varicella-zoster virus DNA in whole blood, plasma, and serum by PCR and electrochemiluminescence. J Clin Microbiol 38 :2568–2573.

    • Search Google Scholar
    • Export Citation
  • 7

    Taylor TE, Fu WJ, Carr RA, Whitten RO, Mueller JS, Fosiko NG, Lewallen S, Liomba NG, Molyneux ME, 2004. Differentiating the pathologies of cerebral malaria by postmortem parasite counts. Nat Med 10 :143–145.

    • Search Google Scholar
    • Export Citation
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