Acknowledgements RCD and FB have funding from a University of Sydney postdoctoral fellowship, and the Brain Foundation (Australia). The authors thank Belinda Barton for statistical advice.
Cerebrospinal fluid neopterin in paediatric neurology: a marker of active central nervous system inflammation
Article first published online: 26 JAN 2009
© The Authors. Journal compilation © Mac Keith Press 2008
Developmental Medicine & Child Neurology
Volume 51, Issue 4, pages 317–323, April 2009
How to Cite
DALE, R. C., BRILOT, F., FAGAN, E. and EARL, J. (2009), Cerebrospinal fluid neopterin in paediatric neurology: a marker of active central nervous system inflammation. Developmental Medicine & Child Neurology, 51: 317–323. doi: 10.1111/j.1469-8749.2008.03225.x
- Issue published online: 18 MAR 2009
- Article first published online: 26 JAN 2009
- PUBLICATION DATA Accepted for publication 20th October 2008. Published online 26th January 2009.
Aim Cerebrospinal fluid (CSF) neopterin production is increased by interferon-gamma stimulation and appears to act as a marker of intrathecal immune activation. We aimed to test the usefulness of elevated CSF neopterin as a biological marker of central nervous system (CNS) inflammation.
Method We retrospectively reviewed CSF neopterin in 158 children (89 males, 69 females, mean age 4y 1mo, SD 3y 11mo, range 1mo–15y).
Results CSF neopterin levels in children with chronic static CNS disorders (n=105) were predominantly low, suggesting that inflammation is rare in these patients. We created an upper value of normal (chronic static group 95th centile 27.4nmol/l). CSF neopterin was elevated in all 10 patients with acute encephalitis and in 10 of 12 patients with other acute inflammatory CNS disorders (demyelination, post-infectious ataxia, myelitis). CSF neopterin was also significantly elevated in patients with chronic progressive disorders of inflammatory origin. Interestingly, CSF neopterin was elevated in four of six patients with chronic static disorders who were tested during a febrile exacerbation of seizures or dystonia, suggesting that intrathecal immune activation may be important in this setting.
Interpretation Neopterin has a short half-life and was useful for monitoring inflammation activity in a patient with relapsing–remitting encephalitis. CSF neopterin is a useful marker of inflammation in a broad range of acute and chronic CNS disorders, and is a significantly more sensitive marker of inflammation than CSF pleocytosis.
high-performance liquid chromatography
T-helper cell 1
Neopterin is produced by human monocyte-derived macrophages and dendritic cells on stimulation by interferon-gamma, the central cytokine in cytotoxicity mediated by T-helper cell 1 (Th1).1 Neopterin is synthesized from guanosine triphosphate and shows similarities to the metabolic pathway responsible for producing tetrahydrobiopterin. Neopterin is the oxidative product of dihydroneopterin, and the endogenous species is dihydroneopterin triphosphate. Total neopterin is increased in central nervous system (CNS) inflammation, but total biopterin is much less affected.2 Neopterin is not only a marker of an activated Th1 cellular immune system; it also has biochemical and physiological functions in host defence. Neopterin appears to promote pro-oxidative stress, which may amplify the cytotoxic effects of reactive oxygen and nitrogen species against invading pathogens.1 Persistent and inappropriate immune activation with consequent neopterin production could lead to ongoing oxidative stress.
Neopterin is therefore a direct response to Th1 cellular immune system activation and therefore a potential marker in immune-mediated and inflammatory disorders.1,3 Neopterin can be measured in urine, serum, and cerebrospinal fluid (CSF) either by radioimmunoassay4 or by high-performance liquid chromatography (HPLC). HPLC is more sensitive and more frequently used in the context of CSF neurotransmitters.5
In adult neurology, CSF neopterin is a useful marker of CNS infection and inflammation,6 particularly in assessing human immunodeficiency virus encephalopathy and its treatment.7,8 CSF neopterin is also elevated in acute infection (bacterial meningitis and viral encephalitis),6 chronic infection (subacute sclerosing panencephalitis, human T-cell lymphotropic virus type 1),9,10 and chronic inflammatory disorders (sarcoidosis and neuro-Behçets).2,11 CSF neopterin also has some uses in demyelinating disorders such as multiple sclerosis, although its sensitivity is very dependent on the activity of demyelination at the time of testing.12,13
Other than CNS infectious and inflammatory disorders, elevated CSF neopterin is present in adult neurodegenerative disorders such as advanced Parkinson,14 Alzheimer, and Huntington disease, supporting the role of inflammation in neurodegeneration.3 Elevated CSF neopterin has also been found in severe traumatic brain injury and subarachnoid haemorrhage, suggesting that inflammatory mechanisms operate after neuronal damage.15,16
In paediatric neurology, CSF neopterin is typically reported as part of CSF neurotransmitter investigations, including dopamine and serotonin metabolites and pteridines.5,17 CSF neopterin is intrathecally produced and is not elevated in infectious disease peripheral to the CNS.4,18 CSF neopterin therefore appears to be a specific marker of CNS immune activation.
In children, elevated CSF neopterin has been described in acute bacterial meningitis, viral encephalitis, and Aicardi–Goutières syndrome.11,19–21 Elevated CSF neopterin has also been noted in febrile convulsions, suggesting CNS immune activation in some fever-induced seizures.22 In addition, elevated CSF neopterin has been described in infantile encephalopathy patients who have a poor outcome.23
CSF neopterin is therefore elevated in CNS inflammation, although it cannot differentiate between infectious and inflammatory processes. However, the spectrum of paediatric neurological disorders associated with elevated CSF neopterin in a broad patient cohort has not been previously described. We therefore aimed to describe the disease categories associated with elevated CSF neopterin in an unselected population of children with acute or chronic neurological conditions.
Diagnoses were made using conventional clinical criteria and investigation. All patients from the Children’s Hospital, Westmead (Sydney, Australia), having CSF neurotransmitter analysis between January 2006 and March 2008 were included in the study. No patients who had CSF neurotransmitters measured were excluded from the cohort. The patients represent a broad cohort of patients typically seen in an acute and outpatient neurology service (Table I). However, given the investigative role of CSF neurotransmitters (measurement of homovanillic acid, 5-hydroxyindoleacetic acid, and pteridines), there is potential selection bias towards patients presenting with suspected inflammatory disorders, movement disorders, infantile epilepsies, neurodegenerative disorders, or undiagnosed disorders.
|Acute disorders||n=30||Mean 5y 1mo Range 2wk–13y||Encephalitis (n=10): encephalitis lethargica herpes simplex enterovirus rota virus chickenpox|
|Other acute inflammatory central nervous system disorders (n=12): demyelination (n=3) polio-like myelitis (n=2) post-infectious ataxia (n=2) cerebral vasculitis (n=2) opsoclonus myoclonus (n=1) inflammation, unspecified (n=1) acute necrotising encephalopathy (n=1)|
|Leukoencephalopathy (n=1) Glutaric aciduria type 1 encephalitic attack (n=1) Spinal tumour (n=1) Tics (n=1) Poisoning (n=1) Hypoxic–ischaemic encephalopathy (n=1) Unclassified (n=4)|
|Chronic progressive disorders||n=17||Mean 4y 6mo Range 2mo–15y||Inflammatory (n=6): severe immunodeficiency (severe combined immunodeficiency, hypergammaglobulinaemia M) complicated by neurodegeneration (n=3) multiple sclerosis (n=2) Rasmussen encephalitis (n=1)|
|Metabolic or genetic (n=11): pontocerebellar atrophy/cerebellar degeneration (n=2) progressive hereditary spastic paraplegia (n=2) mitochondrial, proven or suspected (n=2) metachromatic leukodystrophy (n=1) Pelizaeous Merzbacher (n=1) epileptic encephalopathy with degeneration (n=1) unclassified (n=2)|
|Febrile exacerbation of chronic static disorders||n=6||Mean 4y Range 6mo–13y||Fever-induced exacerbation of seizures in severe myoclonic epilepsy of infancy (n=2) Cortical dysplasia (n=1) Cerebral palsy and epilepsy (n=1) Fever-induced exacerbation of dystonia in dystonic cerebral palsy (n=1)|
|Chronic static disorders||n=105||Mean 3y 7mo Range 2wk–15y||Epilepsy (n=10) Epilepsy and developmental disorder (n=20) Developmental disorder (n=14) Genetic or syndromic central nervous system disorder (n=10) Cerebral palsy (n=15) Movement disorder (n=11) Cortical dysplasia (n=6) Microcephaly plus (n=3) Autism plus (n=3) Cerebrovascular disease (n=2) Unclassified or other (n=11)|
CSF neurotransmitter samples were processed from 158 patients (89 males, 69 females, mean age 4y 1mo, SD 3y 11mo range 1mo–15y, Table I). As this is a heterogeneous group, the patients were grouped according to their mode of presentation and subsequent course of illness (acute n=30, chronic progressive n=17, chronic static n=105, febrile exacerbation of chronic static n=6; Table I). The ‘febrile exacerbation of chronic static’ group all had fever due to a peripheral infection (not affecting the CNS) associated with neurological symptom exacerbation (seizures or dystonia). The patients were further subgrouped according to diagnostic categories (e.g. acute encephalitis, other acute inflammatory CNS disorders; Table I).
This retrospective study has approval from the local ethics committee at the Children’s Hospital at Westmead, Sydney. All families gave consent for CSF examination as part of their child’s routine care. Parents were not recontacted to gain consent for this retrospective review of clinical laboratory findings.
CSF handling and HPLC
CSF was sampled using an aseptic technique and collected into five specially prepared tubes for a range of tests, including neurotransmitter and pteridine analysis. The samples were immediately placed on ice and transferred directly to the –40°C freezer, where they were stored until analyzed. One of the 0.5ml fractions, which was collected into a plain tube, was used for homovanillic acid and 5-hydroxyindoleacetic acid analysis. Another 0.5ml fraction, collected into a tube containing dithioerythritol 1mg and diethylenetriamine penta-acetic acid 1mg, was used for pteridine analysis. Pteridines were measured after 20 minutes’ oxidation with 1% iodine in 2% aqueous potassium iodide solution, using HPLC with fluorescence detection at 350/450nm according to the method of Matsubara et al.24
The CSF neopterin data were compared between patient groups and subgroups. All group and subgroup data analyzed were not normally distributed. Therefore, all group and subgroup data are presented as median, interquartile range, and range. Many of the groups and subgroups had small numbers, so statistical comparison was performed only between the acute (n=30) and chronic static (n=105) groups (non-parametric two-tailed Mann–Whitney U test). SPSS version 15 (SPSS Inc., Chicago, IL, USA) was used for comparisons. To compare CSF neopterin with CSF pleocytosis in the patients with infectious or inflammatory disorders, we used the 2×2 table Fisher’s exact test (SPSS). Statistical significance was defined as p<0.05.
To create an upper limit for the normal range, we used the chronic static group 95th centile (CSF neopterin 27.4nmol/l). The proportion of patients in each group and subgroup with CSF neopterin levels of greater than 27.4nmol/l is presented in Table II. The acute group had statistically elevated CSF neopterin compared with the chronic static group (p<0.001; Table II).
|CSF neopterin nmol/l|
|Median||Range||Interquartile range||Proportion >27.4 nmol/l|
|Acute encephalitis (n=10)||94.5||43.9–494||92.48||10/10|
|Other acute inflammatory central nervous system disease (n=12)||52.4||9.5–179.3||77.4||10/12|
|Chronic progressive (n=17)||20.4||6.1–113.4||57.6||7/17|
|Chronic progressive, inflammatory (n=6)||62||17.8–113.4||64.0||4/6|
|Chronic progressive, metabolic or genetic (n=11)||15.1||6.1–95.2||20.6||3/11|
|Febrile exacerbation of chronic static (n=6)||70.8||6.9–267||81.6||4/6|
|Chronic static (n=105)||14.3||3.4–45.9||7.8||4/105|
All 10 acute encephalitis patients had an elevated CSF neopterin. The majority of children in the ‘other acute inflammatory CNS disorder’ subgroup (10/12) had an elevated CSF neopterin (Table II).
CSF neopterin versus CSF pleocytosis as a marker of brain inflammation or infection
In paediatric neurology, there are few markers of CNS immune activation available to the practising clinician. We compared CSF neopterin with CSF pleocytosis in patients with acute or chronic inflammatory or immune-mediated diseases (acute encephalitis, other acute inflammatory CNS disorders, and chronic progressive [inflammatory] disorders, n=26 tested). Twenty-two of the 26 patients had CSF neopterin levels of more than 27.4nmol/L, whereas only 12 had CSF pleocytosis of more than 5 cells/mm3 (p<0.05).
Disorders associated with elevated CSF neopterin
The diagnoses of patients with elevated CSF neopterin are presented in Table III. Those patients with CSF neopterin levels of more than 100nmol/l are highlighted.
|Acute disorders||Encephalitis: encephalitis lethargica* herpes simplex* enterovirus rota virus* chickenpox|
|Other acute inflammatory CNS: demyelination (optic neuritis and transverse myelitis) polio-like myelitis* post-infectious ataxia* cerebral vasculitis opsoclonus myoclonus*|
|Acute leukoencephalopathy* Glutaric aciduria type 1 encephalitic attack Hypoxic–ischaemic encephalopathy* Unknown*|
|Chronic progressive disorders||Inflammatory: severe immunodeficiency (severe combined immunodefiency, hypergammaglobulinaemia M) complicated by neurodegeneration Rasmussen encephalitis*|
|Metabolic or genetic: metachromatic leukodystrophy epileptic encephalopathy with neurodegeneration|
|Febrile exacerbation of chronic static disorders||Fever induced exacerbation of seizures in cortical dysplasia, cerebral palsy and epilepsy* Fever induced exacerbation of dystonia in dystonic cerebral palsy|
|Chronic static disorders||Microcephaly plus spasticity (unspecified), leukoencephalopathy (unspecified)|
A significant proportion of acute paediatric neurology disorders are infection-mediated or inflammatory in origin. However, relatively few markers of inflammation are available to the practising clinician. CSF pleocytosis, although valuable, is often normal in the setting of CNS inflammation and even infection (such as viral encephalitis). Oligoclonal bands can be used to assess the presence of two or more clones of immunoglobulin G (IgG) in the CNS and to compare CSF with blood. These bands are a potentially useful marker of CNS immune activation or infection; isoelectric focusing is the preferred method for detecting oligoclonal bands.
This is the first study examining the significance of elevated CSF neopterin in a broad paediatric neurology cohort. We aimed to define the clinical phenotypes associated with elevated CSF neopterin and to determine whether CSF neopterin is more sensitive than CSF pleocytosis as a marker of infection or inflammation. Previous investigators have shown that CSF neopterin is a sensitive marker of active inflammation and is produced by monocytes secondary to interferon species stimulation (particularly interferon-gamma and to a lesser extent interferon-alpha).1,4 CSF neopterin appears to normalize quickly once inflammation has resolved.2
As this cohort was heterogeneous, we first presented the findings according to the mode of presentation and disease course (acute, chronic progressive, chronic static, and ‘febrile exacerbation of chronic static’ disorders). CSF neopterin was elevated to a greater degree and more often in the acute group. The encephalitis group all had elevated CSF neopterin, supporting the role of interferon-gamma in these disorders. Children with other acute inflammatory CNS disorders also commonly had elevated CSF neopterin. Specifically, CSF neopterin was elevated in both patients with cerebral vasculitis (both had large-vessel vasculitis), a treatable disorder but notoriously difficult to prove definitively. Further studies with larger numbers of patients should assess whether CSF neopterin can differentiate between active cerebral vasculitis and acute thromboembolic stroke. This distinction would be important because vasculitis requires a different treatment approach. CSF neopterin was also elevated in recognized paediatric inflammatory disorders: opsoclonus myoclonus (as previously described),26 acute post-infectious cerebellar ataxia, polio-like myelitis, and acute demyelination. Interestingly, the two patients tested during multiple sclerosis remission both had normal CSF neopterin levels, further supporting the role of CSF neopterin as a marker of active inflammatory demyelination only.2 CSF neopterin was a more sensitive marker of CNS inflammation than CSF pleocytosis in the inflammatory CNS disorder subgroups.
Other acute disorders with an elevated CSF neopterin included acute leukoencephalopathy of unknown origin (suspected mitochondrial), an acute basal-ganglia ‘encephalitic attack’ during a glutaric aciduria type 1 decompensation, and acute hypoxic–ischaemic injury. These disorders are clearly heterogeneous but are all associated with rapid and severe cell injury. Recent studies in brain trauma have shown elevated neopterin (both acutely and >1y after injury), suggesting that ongoing oxidative stress and inflammation is occurring, which may contribute to continuing brain dysfunction.27,28 There is also an increasing literature devoted to inflammatory mechanisms operating in neonatal hypoxic–ischaemic encephalopathy.29
A further subgroup in the cohort associated with elevated CSF neopterin included children with chronic progressive disease of inflammatory origin. Elevated CSF neopterin was found in three patients with severe immunodeficiency complicated by neurodegeneration (fatal in one case). The mechanism of disease was either an unidentified chronic viral infection (similar to progressive multifocal leukoencephalopathy) or an immune or autoimmune neurodegeneration. The one patient with Rasmussen encephalitis also had elevated CSF neopterin. Rasmussen encephalitis is an extremely difficult diagnosis to make confidently, particularly in the early stages: CSF pleocytosis occurs rarely, and CSF neopterin may be a useful additional marker for this disabling but treatable disease.
The majority of children with metabolic or genetic disorders with a chronic progressive course had normal CSF neopterin values. The exceptions were a patient with metachromatic leukodystrophy and a patient with infantile epileptic encephalopathy with progressive atrophy and neurodegeneration. An inflammatory process has been described in some neurometabolic degenerations, particularly Batten disease (IgG deposition), metachromatic leukodystrophy (histiocytic response) and X-linked adrenoleukodystrophy (lymphocytic infiltration).30,31 Furthermore, Duarte and colleagues have recently reported elevated CSF neopterin in a subgroup of infantile epileptic encephalopathy associated with poor outcome, neurodegeneration, and significant mortality.23 Further examination of the inflammatory response in these patients may yield potential therapeutic benefits for these neurodegenerative disorders of childhood.
The CSF neopterin findings in the patients with a clinical chronic static course were mainly clustered between 5nmol/l and 25nmol/l, with a median of 14.3nmol/l. We created an upper value of normal using the 95th centile of the chronic static group (CSF neopterin 27.4nmol/l). This normal range is very similar to normal values previously reported using HPLC in children with static neurological conditions and children without neurological disease.5,18 Sawada and colleagues have shown that neonates have a slightly higher normal range, with an upper value of approximately 40nmol/l:18 this should be taken into account during interpretation of neonatal CSF neopterin. Beyond 30 days of age, Sawada et al. found the upper value of normal to be similar to our declared upper normal value.18 Therefore it would appear that patients with chronic static disorders rarely have evidence of CNS inflammation (using CSF neopterin as a marker). Our chronic static group included 30 patients with epilepsy (some with developmental delay). Seizures per se (without neurodegeneration or fever exacerbations) do not appear to result in CNS intrathecal immune activation or elevation of CSF neopterin.
The last group to be considered included six patients who had chronic static disorders but who had CSF sampling during a fever-induced exacerbation of seizures (n=5) or dystonia (n=1). None of the patients had concomitant evidence of CNS infection on culture or polymerase chain reaction. Exacerbations of seizures or movement disorders during a non-specific infectious illness are often observed in patients with non-progressive neurological disorders such as epilepsy and dystonic cerebral palsy. The mechanism of these exacerbations is probably multifactorial, but evidence is building for a role of intrathecal inflammation in some animal models of epilepsy.32,33 The finding in this report supports the previous findings of elevated CSF neopterin in febrile convulsions.22
It should be noted that, for CSF neopterin to be a practical and useful clinical marker of CNS inflammation, a rapid turnaround time for testing is required.
Neopterin is produced during intrathecal immune activation and is therefore a marker of active CNS inflammation. However, neopterin is non-specific and cannot discriminate between different infectious, inflammatory, or autoimmune CNS disorders. Inflammation occurs in the context of CNS infection, autoimmunity, demyelination, vasculitis, and some neurodegeneration or cell injury. On a simplistic level, CSF neopterin is a marker of an active or degenerative process, but not a static process. These inflammatory mechanisms are important because they are potentially modifiable with immunomodulatory therapy.