Critical pediatric neurological illness associated with COVID-19 (Omicron BA.2.3.7 variant) infection in Taiwan: immunological assessment and viral genome analysis in tertiary medical center

Objectives Since April 2022, another wave of the Omicron epidemic has struck Taiwanese society, and children with severe neurological complications have been reported frequently. A few cases even developed acute fulminant encephalitis. To investigate the possible causes of the increased incidence of such complications in Taiwan, we reviewed several cases of pediatric patients with severe neurological symptoms. Methods We collected the medical records of pediatric patients with COVID-19 infection who presented with severe neurological symptoms. The COVID-19 infection was diagnosed by nasal swab reverse transcriptase-polymerase chain reaction. The remaining samples were sent for whole genome sequencing and spike (S) protein amino acid variation mapping. Results The increase of several inflammatory markers was observed in all patients included in this study. However, none of the cerebrospinal fluid samples tested positive for SARS-CoV-2. The result of whole genome sequencing showed that all the sequences belonged to the lineage BA.2.3.7. However, the sequences had a K97E mutation in the S protein that differed from other BA.2.3.7 lineage strains, which was located at the S protein N-terminal domain. Conclusion The new mutation in the S protein, which had not previously been observed but was discovered in this study, potentially explains the sudden increase in incidence of extremely adverse neurological symptoms in pediatric patients.


Introduction
Since the COVID-19 pandemic outbreak, people all around the world have continued to fight it . However, as the virus evolves, more variants of concern have been reported . Patients with COVID-19 infection have also experienced neurological symptoms during the course of the infection ( Nordvig et al. , 2021 )  have tended to be more common in children recently ( Dilber et al. , 2021 ;Valderas et al. , 2022 ). In Taiwan, another wave of the pandemic began in April 2022. Among the reported pediatric cases, several patients presented with severe symptoms, including seizures, meningeal symptoms, and encephalopathy. These complications were also reported in Hong Kong ( Tso et al. , 2022 ) and Japan over the previous Omicron epidemic. However, studies in the reports lacked viral genome sequencing data. Therefore, we investigated the pediatric patients with similar complications admitted to our hospital during the outbreak, and we attempted to elucidate commonalities between them.

Study design and clinical specimens
We included the medical records and clinical samples of five patients with COVID-19 who were admitted to Tri-Service General Hospital, Taipei City, Taiwan, for suspected meningoencephalitis in May 2022. Participants were confirmed as positive for COVID-19 infection by reverse transcriptase-polymerase chain reaction, using nasopharyngeal swabs.

Mapping amino acid variation in spike (S) protein and phylogenetic relationship analysis
A total of 12 highly similar Taiwanese COVID-19 Omicron sequences identified by GISAID (including the three uploaded TSGH sequences from this study) were collected. CoV-GLUE ( http:// cov-glue.cvr.gla.ac.uk ) was used to investigate amino acid variations in the three strains from our study and to identify S protein mutations. To investigate the amino acid variations in the S region of these Taiwanese strains and classify pathogen evolution relationships, further sequence analyses were executed using Nextclade ( http://clades.nextstrain.org/ ) with Augur developed by Nextstrain ( Hadfield et al. , 2018 ).

Patient characteristics
The age of the participants ranged from 1 to 5 years (median = 3 years). All patients presented with neurological symptoms ( Table 1 ) within 1-2 days after the onset of respiratory symptoms and febrile episodes.
Patient #1 ultimately passed away due to acute cerebral edema with brain stem compression and multiple organ failure. All other patients recovered fully and were discharged after a median of 6 days (4-9 days) stay in the hospital.

Laboratory findings
The biochemistry assays, which reflect inflammation status, showed prominent elevation, such as procalcitonin, lactate de- hydrogenase, and ferritin. Elevation of interleukin-6 was seen in three patients, which appeared to correlate with disease severity and intensive care unit length of the stay. Three patients had cerebrospinal fluid samples, and all were negative for SARS-CoV-2 polymerase chain reaction.

Whole genome sequencing and S protein amino acid variation mapping
Nextclade showed that the phylogenetic relationships of all the sequences were clustered as a new node different from other BA.2.3.7 lineage strains ( Figure 1 a). Surprisingly, all sequences in this subgroup displayed the S protein K97E mutation. Mutation diversity events in this group also indicated the same results in K97E, and this missense mutation point was in the N-terminal domain (NTD) region of the S protein, which was identified in host immune issues.
It is also interesting that when the S protein K97E mutation information was used in GISAID mapping, only Japan, Hong Kong, and the United States showed high correction ( Figure 1 b). Moreover, these K97E mutation strains appeared at the end of March 2022, corresponding with the beginning of the Taiwan COVID-19 Omicron epidemic ( Figure 1 c).

Discussion
Based on our patients' findings, the increased cytokine and inflammation markers, although having negative polymerase chain reaction results in cerebrospinal fluid, can lead to the conclusion that the causes of these severe neurological symptoms might be related to hyperimmune states rather than a direct viral invasion of the central nervous system.
Our analysis of the eight TSGH sequences indicated that a new Omicron BA.2.3.7 subspecies with special S protein K97E might have been produced in Taiwan, and these sequences are similar to Japan, the United States, and Hong Kong.
The location of K97 is between two beta sheets and the edge of the druggable cavity region of S protein NTD domain ( Di Gaetano et al. , 2021 ) and some extensive ubiquitination events have been observed in them ( Stukalov et al. , 2021 ), which might have an effect on immune regulation. Furthermore, mutations of the NTD region in S protein that allow for immune evasion have also been reported ( Harvey et al. , 2021 ). Therefore, the K97E mutation, which has not been observed in Taiwan previously, potentially explains the sudden increase in the incidence of severe neurological symptoms in pediatric patients due to its possible effect on immune regulation.

Funding
This work was supported by the Tri-Service General Hospital, Taipei, Taiwan, R.O.C. (grant number: TSGH-D-111086 and TSGH-C04-111033). The funders had no role in study design, data collection, and interpretation or the decision to submit the work for publication.

Ethical approval statement
This study was approved by the institutional review board of Tri-Service General Hospital (TSGHIRB number: C202005041) and registered on April 14, 2022.

Declaration of competing interests
The authors have no competing interests to declare.