Study design and subjects
This was a retrospective observational clinical study. It included seven children who were admitted to the pediatric intensive care unit at Kobe Children’s Hospital, Japan, between 2016 and 2017 because of SICF, and diagnosed finally as AE or FS, with residual samples stored more than once within 72 h after onset. We obtained clinical course-related information and imaging data of each patient from our clinical database and medical charts. Control samples were obtained from eight children who were diagnosed without epilepsy on examining the cause of developmental delay while they were physically healthy. All serum samples from the patients in the disease group were collected when clinicians determined that it was clinically necessary. Samples from patients in both disease and control groups were collected and frozen. We measured cytokine levels in 29 samples collected from the disease group and eight samples from the control group. The protocol and procedures of this retrospective observational study were approved by the Ethics Committees of Kobe University and Hyogo Prefectural Kobe Children’s Hospital. All experiments were performed in accordance with the relevant guidelines and regulations of these institutions and the Code of Ethics of the World Medical Association (Declaration of Helsinki). Informed consent was obtained from the patient’s parents in written form.
Selection and measurement of cytokines
Cytokine profiling was performed using the Bio-Plex suspension array system and cytokine Human 27-Plex Panel (Bio-Rad Laboratories, Tokyo, Japan) according to the manufacturer’s instructions. We analyzed 16 cytokines: IL-1β, IL-1 receptor agonist (IL-1RA), IL-4, IL-5, IL-6, IL-8, IL-10, IL-17, eotaxin, fibroblast growth factor (FGF), granulocyte colony-stimulating factor (GCSF), interferon gamma (IFN-γ), interferon-inducible protein-10 (IP-10), macrophage chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), and platelet-derived growth factor-bb (PDFG-bb). The following 11 cytokines with levels below the sensitivity of the tests conducted in patients of the disease group were excluded: IL-2; IL-7; IL-9; IL-12; IL-13; IL-15; granulocyte/macrophage colony-stimulating factor; vascular endothelial growth factor; MIP-1β regulated upon activation, normal T-cell expressed and secreted; and tumor necrosis factor alpha. The levels in all samples were measured in duplicate to improve accuracy. For statistical purposes, cytokine levels (pg/mL) below the lower limit of quantification were reported as the lower limit of quantification per analyte. In addition, cytokine levels above the upper limit of quantification were reported as the upper limit of quantification per analyte.
Evaluation and statistical analysis
We evaluated the time course of the changes in the levels of 16 cytokines 72 h after onset according to the syndromes. We also compared the maximum levels of all types of cytokines at 0–23 h (less than 24 h) and 24–47 h after onset using the Mann–Whitney U test in six patients, with cytokines measured during both these time periods. Moreover, we investigated the relationship between cytokine levels and interventions targeted at IL-6. All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria) .
Clinical definitions and management
According to the criteria determined in our previous studies, we defined onset time as the time at which the initial manifestation of neurological symptoms, including convulsions or impaired consciousness, was first recognized by family members or other persons [1, 6, 18]. We defined FS as a seizure accompanied by fever (temperature ≥ 100.4 °F or 38 °C by any method), without central nervous system infection, that occurs in infants and children from 6 months to 14 years of age [19, 20]. We also defined AE as an impairment in consciousness of acute onset, with severity of Japan Coma Scale 20 or Glasgow Coma Scale < 11, and with duration of 24 h or longer according to the Guidelines for the diagnosis and treatment of acute encephalopathy in childhood.
. Neurological sequelae were defined as the worsening of the Pediatric Cerebral Performance Category (PCPC) score . HSES and AESD were diagnosed in accordance with previous reports [22, 23].
These patients were examined using laboratory investigations such as microbiological cultures of blood and cerebrospinal fluid, rapid assays for influenza virus, respiratory syncytial virus, and rotavirus, and real-time polymerase chain reaction for human herpes virus type 6 and 7; computed tomography imaging; magnetic resonance imaging; and electroencephalography (EEG). Clinical examinations and therapies were performed when clinicians determined that they were clinically necessary. Targeted temperature management (TTM) was performed according to a previous report . High-dose steroid (HDS) treatment consisted of 30 mg/kg/day of methylprednisolone (maximum 1000 mg/day) for three continuous days.