Background and objective From this April, there was a COVID-19 outbreak in Taiwan. The first fatal case of pediatric COVID-19 encephalitis was reported on April 19, 2022 and fatal fulminant cerebral edema in other 4 children with COVID-19 encephalitis was reported within 1 month from Taiwan CDC registry. To date, around 700,000 children got COVID-19 recently. Several children developed MIS-C (multi-system inflammatory syndrome in children)-related shock about 2-6 weeks after COVID-19. Since both COVID-19 associated encephalopathy/ encephalitis and MIS-C are life-threatening, it is urgent to delineate its prognostic biomarker, host genetic factors, immunopathogenesis and viral pathogenesis.

Methods Pediatricians will enroll cases of both COVID-19 associated encephalopathy/ encephalitis and MIS-C from several hospitals and medical centers. Their clinical manifestations, lab findings, severity and outcomes will be collected. Clinical assessment of all the systems will be performed. Blood, nasopharyngeal swab and stool will be collected at acute, subacute and convalescent stages for whole exome sequencing, immunopathogenesis including chemokine/cytokine, T/B lymphocyte subset, SARS-CoV2 specific Ab/T/B cell, T and B cell repertoire, viral pathogenesis including multiple viral detection, persistence of fecal SARS-COVID-2 as well as respiratory and gut microbiota. We will establish the animal models for COVID-19 associated encephalopathy/encephalitis and MIS-C, based on the K18-hACE2 or R26R-AGP mouse models established in NTU animal center. Moreover, specific viral or host factors involved in regulating the pathogenesis and immune responses can be investigated, to optimize the protocol for further improvement of the animal models and also to help identify the putative therapeutic targets.

Expected results We will delineate the clinical and laboratory characteristics of COVID-19 associated encephalopathy and encephalitis, the role of immune, virology, genetics mechanism in pathophysiology, and will optimize the treatment algorithm based on the result of this study. We also expect that the important biomarkers and risk factors associated with clinical outcome and severity, the immunopathogenesis of MIS-C, host genetic factors and the viral pathogenesis and microbiota associated with MIS-C will be found.

Background for COVID-19 associated encephalopathy/encephalitis in children The COVID-19 primarily cause the respiratory diseases, such as croup, bronchiolitis, or pneumonia, but it also can affect the nervous system. According to the data of 1,695 children and adolescents, from March 15, 2020, to December 15, 2020, hospitalized for COVID-19 in 61 hospitals in the United States, 365 (22%) were found to have neurological symptoms. The majority children (322 children, 88%) had transient neurological symptoms. Unfortunately, 43 of them (12%) developed severe life-threatening conditions, including severe encephalopathy (15 children), ischemia or hemorrhagic stroke (12 children), acute central nervous system infection or acute disseminated encephalomyelitis (ADEM) (8 children), acute fulminant cerebral edema (4 children), and Guillain-Barré syndrome (GBS) (4 children). Of the 43 children, 11 patients (26%) died, and another 40% were discharged from the hospital with new neurological sequelae. In Asia, Hong Kong also experienced the Omicron pandemic in March. 171 (14.9%) of 1147 children hospitalized for Omicron were found to have neurological manifestations. The most common was the febrile seizure (11.60%), but there were also 5 children (0.44%) who developed coronavirus-related encephalopathy or encephalitis. Finally, two of them died of neurological causes: one with encephalopathy and the other with fulminant cerebral oedema.

Although the clinical course of acute encephalitis caused by COVID-19 in children is relatively mild in United States and European, the clinical course seemed to be more severe and fulminant in Taiwan. There are five cases of COVID-19 associated encephalopathy and encephalitis in Hong Kong, but the data available for reference is limited. Besides, the clinical course and pathophysiology of children with COVID-19 associated encephalopathy and encephalitis in Taiwan were unknown, it urgently need a clinically oriented, integrated research project.

Clinical research projects for COVID-19 associated encephalopathy/encephalitis in children The aim of this project is

1. To analyze the clinical course and classify the stage of COVID-19 associated encephalopathy and encephalitis in children 2. To find the clinical predictor, biomarker, and imaging characteristic in critical case of COVID-19 associated encephalopathy and encephalitis in children 3. To explore the immune mechanism of COVID-19 associated encephalopathy and encephalitis in children 4. To explore the role of genetics in COVID-19 associated encephalopathy and encephalitis in children 5.To Explore the role of viral variants and co-infection in COVID-19 associated encephalopathy and encephalitis in children 6. To establish proper animal models for studying the pathogenesis and therapeutics for COVID-19 associated encephalopathy/encephalitis

Method: case planning for COVID-19 associated encephalopathy/encephalitis in children Active surveillance will be performed nationally to identify children and adolescents (age≤18 years) with COVID-19 related illness hospitalized from July 01, 2022, to February 28, 2023. The data will registry to the public database hold by NTUH and CGMH.

Estimated including patient number:

110 children (critical group: 30 children; non-critical group:80 children) Including criteria

1. Age less than 18 years old.

2. A positive SARS-CoV-2 test result (reverse transcriptase-polymerase chain reaction and/or antibody)。

3. Hospitalized children.

4. Clinical diagnostic criteria for encephalitis.

   Major criteria:

   1. . Altered mental status greater than 24 hours without alternative cause identified Minor criteria: need at least 2 minor criteria for encephalitis
   1. Fever
   2. Seizures
   3. Focal neurologic signs
   4. CSF: pleocytosis
   5. EEG: abnormal slow background or epileptiform discharge
   6. Neuroimaging: abnormal brain inflammation on MRI *****Major+2 minor: possible encephalitis; Major+3 minor: probable encephalitis; Brain biopsy: confirmed encephalitis Exclusion criteria

   1) Age more than 18 years old 2) Previous history of encephalopathy, acute encephalopathy caused by other etiology, not COVID-19, development delay, autism, ADHD, epilepsy and febrile seizure 3) Non-hospitalized children Critical case was defined as children who admitted to pediatric intensive care unit. And Non-critical case was defined as children who admitted to general ward.

   Classification of COVID-19 associated encephalopathy and encephalitis Children will be classified as the following four diagnoses: 1. Encephalopathy (MERS, ANEC, ASED); 2. Acute encephalitis; 3. ADEM; 4. Fulminant cerebral edema. The classification will be adjudicated and discussed by neurology and critical care experts on the NTUH and CGMH study team (W.T.L, J.J.L, and K.L.L.)

   Method: Research content for COVID-19 associated encephalopathy/encephalitis in children Clinical manifestations and laboratory/imaging data collection This is a prospective observational study that does not involve clinical treatment. We propose a schedule for specimen collection /examination in clinical care. The routine ICU laboratory test on day 1, 2,3 7 include CBC/DC, PT/APTT, Fibrinogen, d-dimer, AST/ALT, BUN/Cr, Troponin-I, CPK, BNP or NT-Pro-BNP, CK-MB, Na/K/Cl/Ca/P/Mg, cholesterol, TG, CRP, PCT, IL-6, Ferritin, LDH. We also collect the serum before immunotherapy (such as IVIG, IL-6 antagonist (Tocilizumab) or methylprednisolone pulse therapy) and PMBC (peripheral blood mononuclear cell). Besides, throat swab, Filmarray NP panel (depends on the situation of each hospital) will be also arranged. If lumbar puncture will be performed, routine CSF survey will include routine (WBC)/biochemistry (TP, sugar, lactate)/culture/Filmarray ME panel and 1 ml of CSF will be also reverse. In term of examination, EEG, brain CT and/or MRI will also encourage and depend on the situation of each hospital. Besides, we will also perform the 2D echo and EKG for evaluation of cardiac function of these patients.

   We will perform systematic data collection, including past history (such as preterm, congenital heart disease, chronic lung disease, obesity and DM), the evolution of the disease course, the results of routine clinical tests and examinations and outcome. The disease course includes the worse vital signs of every day, time to start use of antivirus drug, immunotherapy, inotropic agent, ventilator, anticonvulsant and IICP management. Outcomes will be determined at hospital discharge. The primary outcome is mortality. The secondary outcome is ICU stay, duration of hospitalization and neurologic outcome. Neurologic deficits are defined as gross impairment in motor, cognitive, or speech and language functions as well as epilepsy.

   Background for MIS-C From this April, there was a big COVID-19 outbreak in Taiwan. Infection with COVID-19 was laboratory-confirmed in 3,803,049 cases and about 20% of the cases were children, so around 700,000 children got COVID-19 recently in Taiwan. Several children developed MIS-C (multi-system inflammatory syndrome in children)-related shock about 1 month after COVID-19. There would be about 200 MIS-C in Taiwan if the incidence of MIS-C per 1,000,000 COVID-19 infections is around 300 according to the recent reports. Since MIS-C is life-threatening, it is urgent to delineate its prognostic biomarker, host genetic factors, immunopathogenesis and viral pathogenesis.

   The study flow chart of MIS-C is the following:

   (1). TPIDA Clinical core for Case enrollment, data and sample collection The clinical study will be conducted by Taiwan Pediatric Infectious Disease Alliance (TPIDA), a collaborative consortium of pediatric infectious disease departments in tertiary medical centers in Taiwan.

   A. Pediatricians of TPIDA will enroll 100 to 200 cases of MIS-C at National Taiwan University Children Hospital, Chang Gung Memorial Hospital, National Taiwan University Hospital Hsin-Chu Branch and Yun-Lin Branch, Chi-Mei Medical Center, National Cheng Gung University Hospital and MacKay Children Hospital after the written informed consent is obtained from their parents or guardians. The following 6 criteria for MIS-C have to be met: age 0 to 19 years, fever for ≥3 days, clinical signs of multisystem involvement (at least 2 systems), elevated markers of inflammation (e.g., CRP, procalcitonin or ferritin), evidence of SARS-CoV-2 infection and no other obvious microbial cause of inflammation.

   B. Clinical severity The severity of MIS-C will be dependent on the degree of inflammation, heart involvement and the presence of shock. Mild MIS-C is defined as without shock, moderate as shock with VIS score less than 10, and severe as shock with VIS score equal or over 10. [Vasoactive-Inotropic Score (VIS) = Dopamine dose (μg/kg/min) + Dobutamine dose (μg/kg/min) + 10 x Milrinone dose (μg/kg/min) + 100 x Epinephrine dose (μg/kg/min) + 100 x Norepinephrine dose (μg/kg/min) + 10,000 x Vasopressin dose (units/kg/min)].

   C. Clinical assessment and data collection Demographics, BMI, underlying medical diseases, past history of hospitalization and COVID-19 infection, past history or family history of Kawasaki disease, COVID-19 vaccination status, and their current clinical manifestations such as fever, mucocutaneous manifestations, lymphadenopathy, gastrointestinal symptoms, activity, appetite, urine output, treatment including intravenous immunoglobulin (IVIG), steroid, immunomodulators, inotropic agents, antibiotics and outcome will be collected.

   D. Sample collection For the subsequent workup of viral pathogenesis, host genetic factors and immunopathogenesis, the following specimens will be collected at different stages.

   E. Control group Age and gender matched healthy control children or mild COVID-19 cases without MIS-C will be also included for further comparison.

   F. Retrospective and prospective case review The clinical manifestations, lab findings, severity, treatment and outcomes of the MIS-C cases, either retrospectively collected or prospectively enrolled, will be collected and analyzed to find the important biomarkers and risk factors associated with their clinical severity and outcomes.

Inclusion Criteria:

1. Age less than 18 years old.
2. A positive SARS-CoV-2 test result (reverse transcriptase-polymerase chain reaction and/or antibody)。
3. Hospitalized children.
4. Clinical diagnostic criteria for encephalitis.

Major criteria:

1). Altered mental status greater than 24 hours without alternative cause identified Minor criteria: need at least 2 minor criteria for encephalitis

1. Fever
2. Seizures
3. Focal neurologic signs
4. CSF: pleocytosis
5. EEG: abnormal slow background or epileptiform discharge
6. Neuroimaging: abnormal brain inflammation on MRI *****Major+2 minor: possible encephalitis; Major+3 minor: probable encephalitis; Brain biopsy: confirmed encephalitis

The following 6 criteria for MIS-C have to be met: age 0 to 19 years, fever for ≥3 days, clinical signs of multisystem involvement (at least 2 systems), elevated markers of inflammation (e.g., CRP, procalcitonin or ferritin), evidence of SARS-CoV-2 infection and no other obvious microbial cause of inflammation.

Exclusion Criteria:

1. Age more than 18 years old
2. Previous history of encephalopathy, acute encephalopathy caused by other etiology, not COVID-19, development delay, autism, ADHD, epilepsy and febrile seizure
3. Non-hospitalized children

Active surveillance will be performed nationally to identify children and adolescents (age≤18 years) with COVID-19 related illness hospitalized from July 01, 2022, to February 28, 2023. The data will registry to the public database hold by NTUH and CGMH.