Role of cerebrospinal fluid tau protein levels as a biomarker of brain injury in pediatric status epilepticus

Abstract Background Various biomarkers have been studied for predicting etiology and outcome in status epilepticus (SE); cerebrospinal fluid (CSF) total tau (t-tau) protein levels is foremost among them. Only handful of studies are available regarding role of t-tau in childhood SE. Methodology This prospective study was conducted in a tertiary care center of Northern India in children 6 months −12 years of age. The Cases were patients with convulsive status epilepticus (CSE) whereas Controls were patients without SE and normal CSF. The t-tau levels were done in CSF of both the groups. The outcome was assessed by GOS-E Peds score. Results A total of 50 (62% males) cases and 15 (67% males) controls were enrolled in the study. SE was generalized in 78% cases whereas 14% had refractory SE. Most common etiology of CSE was acute symptomatic (52%), followed by prolonged febrile seizure (24%), remote symptomatic group (14%), unknown etiology (8%) and progressive disorder (2%). Case fatality rate was 10%. Poor outcome was seen in 30% cases. Median (IQR) CSF t-tau levels was significantly lower 2.6 × 103 (0.5–9.4 × 103) pg/ml in cases vs 10.6 × 103 (6.0–14.2 × 103) pg/ml in controls (p = 0.004). There was no significant correlation seen between type, duration, etiology and response to antiepileptic drugs of SE with CSF t-tau levels. Also, no significant correlation of poor sensorium, outcome of SE and critical care needs with CSF t-tau levels was noted. Conclusion CSF t-tau is not a useful diagnostic or prognostic biomarker in pediatric SE.


Introduction
Convulsive status epilepticus (CSE) is a neurological emergency with case fatality rate of 3%-30% and high morbidity in childhood [1][2][3]. It can lead to various acute and chronic complications. Brain atrophy and chronic encephalopathy occur in 6%-15% patients with status epilepticus (SE), these changes presumably result from diffuse cortical injury. SE can lead to cognitive defects and epilepsy [4]. The development of focal neurological deficits after SE has been reported in 9%-11% of children [5]. Literature says that neurological sequelae are usually caused by the underlying condition rather than seizures, this statement is supported by the fact that acute symptomatic SE have more chances of developing neurological sequelae as compared to prolonged febrile seizures and cryptogenic SE [6].
Like many other diseases, biomarkers could be an important field of research in SE. They could be used to identify etiology, risk factors for developing refractory status and poor outcome. Thus they could be immensely helpful in diagnosis, management and prognostication. These biomarkers include clinical, demographic, electrophysiological, biochemical and structural data. The heterogeneity of SE etiology and semiology renders development of prognostic markers challenging. Currently, prognostication in SE is limited to a few clinical scores. Future research is being directed to integrate clinical, genetic, metabolic, inflammatory, and structural biomarkers into prognostication models for SE outcome [7]. The common blood/cerebrospinal fluid (CSF) biomarkers on which extensive research is being done are CSF lactate, CSF and serum albumin ratio, micro RNAs (MiRNAs), Enolase-2 (NSE), Proguanulin (PGRN), Glial fibrillary acid protein (GFAP) and CSF tau levels [7][8][9].
Tau protein (tau) is a microtubule-associated phosphorylated protein primarily located in neuronal axons where it promotes microtubule assembly and stability [10]. T-tau is considered as a good biomarker for brain injury in various conditions and literature suggests it to be useful in status epilepticus also. In a study done on adult patients, higher t-tau levels were found in refractory SE as compared to SE responsive to antiepileptic drugs (AEDs). Further, tau levels positively correlated with SE duration and a higher risk of subsequent epilepsy and sequelae [11]. Another researcher found low CSF tau levels in patients with seizures compared to controls [12]. We could not find any study on pediatric SE evaluating role of t-tau despite it being a very common condition and having significant impact on the developing brain. We planned this study to see association of CSF t-tau levels with SE characteristics and outcome at discharge in pediatric patients.

Study design and population
This prospective single centre case-control study was conducted at the Government Medical College and Hospital, a tertiary care hospital of Northern India situated in the city of Chandigarh between January 2019 to November 2020. The study was conducted after approval by the institute's ethics committee (Ethics approval no GMC/IEC/2018/242). All study subjects were enrolled after obtaining written informed consent from either parent.

Inclusion and exclusion criteria
A convenient sample size of 50 cases and 15 controls was taken. The eligibility criteria of age for both the groups was taken as 6 months-12 years. Cases were all eligible patients in pediatric emergency ward and pediatric intensive care unit with CSE lasting for more than 30 min or 2 or more tonic clonic generalized/focal with secondary generalization seizures with no regaining of consciousness in between. The patients with recent history of head trauma or having any contraindication for lumbar puncture or parents denying consent were excluded. Controls were all eligible patients in whom lumbar puncture was done to rule out intracranial infection (headache, suspected meningitis etc.) but CSF studies came out normal. The patients with history of seizures or head injury, evidence of intracranial tumour or brain abscess on neuroimaging, having contraindication for lumbar puncture or parents denying consent were excluded.

Study procedure and data collection
All potential cases and controls were screened for eligibility ( Figure 1). The patients were enrolled consecutively from pediatric emergency ward and intensive care unit. The demographic and clinical data like age, sex, presenting complaints, duration of CSE, previous episodes of CSE if any, treatment taken from outside, family history of seizures, neuro-development history, findings of physical examination, investigations, treatment details, response to antiepileptic drugs (AEDs) and critical care needs were noted on a pretested proforma by the study investigator. The detailed neurological examination was done at the time of admission and discharge. Assessment of consciousness level was done by Glasgow coma scale (GCS) in older children and modified Glasgow coma scale in younger children. For classifying protein energy malnutrition, standard WHO charts were used [13]. The investigations for determining the etiology were done as per standard protocol. Other investigations to determine the etiology such as Computed Tomography (CT), M a g n e t i c R e s o n a n c e I m a g i n g ( M R I ) , Electroencephalogram (EEG), laboratory tests to determine infections, tandem mass spectrometry (TMS) etc. were done accordingly.

CSF t-tau
The lumbar puncture was done only once within 24-78 h of presentation to the hospital. The first 2 ml of CSF was used for routine clinical tests and the subsequent 0.5 ml for the present study. CSF sample for total tau (t-tau) protein was stored at −80 °C. CSF t-tau levels were measured in pg/ml, determined by Sandwich ELISA technique using Ray Bio human tau ELISA kits. The assay was performed according to the protocol supplied with the kit, and CSF tau concentrations of the samples were estimated from standard curves made for each assay. In samples with t-tau values showing < min, we assumed a CSF tau concentration of 50 pg/ml for calculation of the median.
CSE was managed as per standard protocol. Etiology was assigned according to clinical and investigational data as per ILAE criteria (2015) into Known and Unknown groups [14]. The known etiology was further subdivided into: Acute symptomatic, Remote Symptomatic, Prolonged febrile convulsion and Progressive disorders. Unknown etiology was assigned where no diagnosis could be made despite all investigations.
The requirement for mechanical ventilation, ionotropic support and management for raised intracranial pressure was termed as need for critical care support.

Assessment of outcome
At the time of discharge, thorough neurological examination was done and outcome was graded as per Glasgow outcome scale-extended pediatric version (GOS-E Peds) [15]. It is an 8 item instrument designed to provide a developmentally appropriate measure of outcome in children. This measure assesses functional independence inside and outside of the home, capacity for work/school, participation in social and leisure activities, and family and peer interactions/psychological problems. The 8 categories of outcome (from highest score of 8 to lowest score of 1) are: Dead, Vegetative State (unable to follow simple motor commands or communicate), Lower Severe Disability (always needs support in the home), upper Severe Disability (sometimes needs support inside the home or always needs support outside of the home), Lower Moderate Disability (self-contained school program, unable to participate in social activities, or daily intolerable psychosocial difficulties), upper Moderate Disability (reduced academic capacity, significant decrease in social/leisure participation, or frequent/weekly psychosocial difficulties), Lower Good Recovery (slightly reduced social/leisure participation, occasional psychosocial difficulties, or any other persisting TBI symptoms), and upper Good Recovery (no identifiable difficulties related to the injury that affect daily life). We included upper good and lower good as good outcome and upper moderate, lower moderate, upper severe, lower severe, vegetative state and death as poor outcome.
Primary outcome measures were kept as median (IQR) CSF t-tau levels in patients and controls, correlation of CSF t-tau levels with assigned etiology and duration of CSE and immediate outcome in both the groups.

Statistical analysis
CSF t-tau levels were expressed as median (IQR) pg/ml. Student's t-test and Mann-Whitney u-test were used for comparison of normally distributed and non-parametric data, respectively, between groups. ANOVA and Kruskal-Wallis tests were used for comparison of normally distributed and non-parametric data, respectively, between multiple groups. Spearman's correlation coefficient was used to correlate variables in the groups studied. Frequency tables were compared with Pearson Chi-square test and Fisher's exact test when appropriate. Correlational analysis was performed to study the relationship between CSE duration, etiology and outcome. Statistical calculations were carried out using SPSS window software, version 22.

Clinicodemographic characteristics
A total of 50 (62% males) cases and 15 (67% males) were enrolled in the study (Table 1). Both the groups were comparable in terms of clinicodemographic profile except for the past history of seizures, where 19% cases and none of the controls had history of seizures in the past (p = 0.012). Fever was present in high proportion of cases (76%) and controls (60%) (p = 0.376).
The cases presented with significant poorer sensorium with mean (SD) GCS of 12.54 (2.98) as compared to controls who had mean (SD) GCS of 14.67 (1.29) (p = 0.009). Twenty-three (46%) cases presented with GCS of <12 whereas only one control had GCS < 12 (p = 0.043). Eleven (22%) cases had respiratory failure and 7 (14%) had circulatory shock. The neurological abnormalities like raised intracranial pressure, pupillary and muscle tone abnormalities were seen only in cases and the difference was statistically significant (p < 0.05) ( Table 1). The statistically significant p values are shown in bold.

Laboratory investigations
The cases had significantly lower mean (SD) haemoglobin of 10.2 (1.43) gm/dL as compared to 11.3 (1.54) gm/dL in controls (p = 0.008) (Supplementary table). Hypoalbuminemia was seen in 10 cases and none of the controls (p = 0.050). Blood and urine cultures showed growth in one case each in cases and none in controls (p = 0.000).

Etiology
The etiology of SE was assigned as acute symptomatic in 26 (52%), out of which acute central nervous system (CNS) infections (n = 15) were the most common etiology (Table 4). Seven (14%) patients had remote symptomatic etiology, 24% prolonged febrile and 1 case had progressive disorder (Adrenoleukodystrophy). Four patients were assigned unknown etiology of SE.

Outcome
Immediate outcome was graded as survived or died. Five patients among cases and one control died (Table  1). Outcome was calculated using the GOS-E Ped score. Thirty-five of 50 (70%) cases and 14/15 (93%) controls had good outcome (p = 0.09). Major causes of death were raised intracranial pressure, sepsis with disseminated intravascular coagulation (DIC), septic shock and respiratory failure among cases and one death in control group occurred due to septic shock.

Correlation of CSF t-tau with various variables
We further analysed association of various clinicodemographic factors with t-tau (Table 6). We found no correlation of CSF t-tau with age, gender, nutritional status, developmental delay, past history of seizures, poor sensorium, need for critical care support or outcome at discharge. Patients with history of birth asphyxia had significantly lower CSF t-tau levels as compared to those without birth asphyxia (p = 0.049).

Correlation of CSF t-tau in SE cases with various variables
There was no correlation of CSF tau levels seen with type of seizures (Generalized vs Focal) (p = 0.267), duration of status (p = 0.725), CSF abnormalities, neuroimaging findings (p = 0.618), etiology of SE (p = 0.968) or number of antiepileptics used (p = 0.393) ( Table 7). Patients with good outcome had a CSF t-tau median (IQR) of 3 × 103 (0.7-11 × 10 3 ) and those with poor outcome had CSF t-tau of 8 × 10 3 (0.5-14 × 10 3 ) according to the GOS-Ped score. There was no statistically significant difference between the two groups (p = 0.122).

Discussion
This study is first of its kind in prospective assessment of children with SE and CSF t-tau levels. It was planned to evaluate if CSF t-tau helped as a diagnostic or prognostic tool in paediatric SE. Since t-tau is considered a good biomarker of neuronal damage we hypothesized that it would increase in refractory SE and status caused by inflammatory disorders like meningitis/ encephalitis and, further since disability after SE is caused by neuronal damage, patient with poor outcome would have higher tau levels. The children with CSE (n = 50) and controls (n = 15) between the age of 6 months −12 years of age were enrolled. Mean (SD) CSF t-tau was significantly elevated (p = 0.004) in controls compared to cases. There was no significant correlation seen between CSF t-tau and type, duration, etiology of SE and AED responsiveness. Further, no significant correlation was noted between CSF t-tau and poor GCS, outcome at discharge and critical care needs. Only a handful of studies have evaluated CSF tau levels in patients with seizures, and results are varying. Majority have evaluated impact of seizures on tau levels whereas only one study on adult patients (15-79 years old) is dedicated to the effect of SE on tau levels [11,12,16]. In this cohort study, Monti et al. included only cases of SE excluding acute structural brain damage e.g. CNS infections to avoid the confounding factors [11]. However, cases with idiopathic or genetic epilepsy were also not included. This retrospective study supported positive correlation of CSF t-tau with type, duration, severity and outcome of SE. Higher CSF t-tau levels were seen in refractory and super refractory SE compared to seizures controlled by AEDs; authors hypothesized that SE cases classified as responsive to AEDs did not induce tau pathology and neuronal damage. They also found that patients with higher CSF t-tau had higher risk of developing disability and chronic epilepsy at 6 months follow up suggesting CSF t-tau as a biomarker of SE severity and prognosis. Our study included only children till 12 years of age and we included all types of SE, 30% cases were of CNS infection. CSF t-tau was significantly higher in controls compared to cases, thus it argues against a major effect of SE on t-tau release. T-tau values showed great variability, very low values (50 pg/ml) to very high levels (15.9 × 10 3 pg/ml). There was no correlation seen between CSF tau levels and type, duration and etiology of SE, type of AED received, neuroimaging and EEG findings. Although t-tau levels were higher in FSE cases it was not statistically significant. Likewise patients who received 4 antiepileptics (BZD, Phenytoin, Valproate and Levetiracetam) had higher t-tau levels but not significantly higher than in patients who were controlled on lesser AEDs.
Palmio et al. studied t-tau levels in adult patients after epileptic seizures. Only 17% patients had SE. All patients with epilepsy of unknown origin and controls had normal t-tau level whereas 31% patients with symptomatic seizures had abnormal t-tau levels [16]. Further they found no association of number of seizures or SE with levels of t-tau. In our study no relation with etiology was seen.
In a multicentre study, Shahim et al. included 607 children <16 years of age for cerebrospinal fluid brain injury biomarkers, out of them 117 patients were of epilepsy (4 patients of SE) [17]. Elevated CSF t-tau was seen in epilepsy, infectious and inflammatory CNS disorders and progressive encephalopathy as  compared to controls. In SE, t-tau levels were higher than focal and primary generalized epilepsy group. The authors also noticed highest biomarker concentration in SE suggesting deleterious effect of SE on neuronal tissues [17]. However in another study in adults with seizures by Shahim et al., t-tau levels were lower in seizure patients as compared to controls [12]. Acute symptomatic causes were excluded and 91% patients had epilepsy/seizures of unknown origin. Thirty-six percent patients had repetitive seizures and 8% had non-convulsive SE. No difference in CSF t-tau levels between seizure subgroups was noted. The authors found no difference in CSF t-tau levels in patients with or without MRI abnormalities; however since acute symptomatic cases were excluded from the study, MRI changes were most probably due to old insult. The authors hypothesised that seizure activity may disturb normal turnover and release of t-tau, a protective mechanism of CSF tau in seizures was also suggested. Our study also has similar findings with higher t-tau levels in controls. On subgroup analysis of SE patients in our study, we found no significant difference between CSF tau levels in various etiologies (p value 0.96) and abnormal neuroimaging findings (p value 0.33). In a study on adult patients with various neurological disorders, Sussumuth et al. found significant elevation of t-tau levels in patients with meningoencephalitis and cerebral haemorrhage, suggesting correlation of tau protein with brain parenchymal damage [10]. The studies on children with cerebral malaria found that CSF t-tau is a good biomarker of brain parenchymal damage and long term neurologic/cognitive impairment [18,19]. We had no patient of intracranial haemorrhage or cerebral malaria in our study group and no significant difference between CNS infections and other etiologies of SE was found.
In our study we did not find statistically significant association of t-tau with time interval between CSF sample collection and SE onset. Palmio et al. reported similar findings [16]. Higher t-tau levels were found in patients in whom CSF samples were collected during general anaesthesia with propofol, thus leading to the conclusion that general anaesthetics might have a role in inducing tau pathology or alternatively use of anaesthetic drugs itself implied the intrinsic severity of SE [11]. We did not use propofol anaesthesia but midazolam infusion was used in 2 cases, but in these cases tau levels were surprisingly very low (Table 7).
In control group, we found high t-tau levels, highest levels were seen in cases of bronchopneumonia and post varicella ataxia. These patients had lumbar puncture for either excessive irritability or lethargy. In one DKA patient, GCS was poor but it improved after DKA resolution, the CSF was normal in all aspects except for high tau protein. One patient of severe acute malnutrition died but the cause was septic shock and not neurological reason. We could not find any specific correlation with etiology in control group also.
The main limitations of our study are the small sample size in both the groups, which reduces the power to detect significant effects. Since it is unethical to do lumbar puncture in normal children, we could only include children who required lumbar puncture, so the raised CSF tau in controls could be due to their underlying conditions. Another limitation is that CSF tau levels could be done only once in every child thus we do not know the pattern of change in tau levels over time.
We found very high levels of t-tau in our study as compared to other studies done with similar technique, which raises the possibility of ethnic variations in tau protein levels, further studies are required for it.

Conclusion
To summarize, CSF t-tau is neither a good diagnostic or prognostic biomarker in pediatric SE.

Disclosure statement
No potential conflict of interest was reported by the author(s).