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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

Open AccessPublished:September 07, 2022DOI:https://doi.org/10.1016/j.ijid.2022.09.001

      Highlights

      • COVID-19 infection can induce severe neurologic complications in children.
      • All sequences in this study belonged to the BA.2.3 lineage.
      • The sequences displayed a novel K97E mutation in the spike protein.
      • Only mutations from Japan, the United States, and Hong Kong showed a high correlation.
      • This strain appeared in April 2022 (coincided with the Omicron outbreak in Taiwan).

      Abstract

      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.

      Keywords

      Abbreviation:

      NTD (N-terminal domain)

      1. Introduction

      Since the COVID-19 pandemic outbreak, people all around the world have continued to fight it (
      • Jian MJ
      • Perng CL
      • Chung HY
      • Chang CK
      • Lin JC
      • Yeh KM
      • Chen CW
      • Hsieh SS
      • Pan PC
      • Chang HT
      • Chang FY
      • Ho CL
      • Shang HS.
      Clinical assessment of SARS-CoV-2 antigen rapid detection compared with RT-PCR assay for emerging variants at a high-throughput community testing site in Taiwan.
      ). However, as the virus evolves, more variants of concern have been reported (
      • Chung HY
      • Jian MJ
      • Chang CK
      • Lin JC
      • Yeh KM
      • Chen CW
      • Hsieh S-S
      • Hung K-S
      • Tang S-H
      • Perng C-L
      • Chang F-Y
      • Wang C-H
      • Shang H-S.
      Emergency SARS-CoV-2 variants of concern: novel multiplex real-time RT-PCR assay for rapid detection and surveillance.
      ). Patients with COVID-19 infection have also experienced neurological symptoms during the course of the infection (
      • Nordvig AS
      • Fong KT
      • Willey JZ
      • Thakur KT
      • Boehme AK
      • Vargas WS
      • Smith CJ
      • Elkind MSV.
      Potential neurologic manifestations of COVID-19.
      ). However, severe neurological complications have tended to be more common in children recently (
      • Dilber B
      • Aydın ZGG
      • Yeşilbaş O
      • Sağ E
      • Aksoy NK
      • Gündoğmuş F
      • Küçükalioğlu BP
      • Yılmaz SA
      • Demirhan YN
      • Çelik N
      • Karaca A
      • Ertem NY
      • Özdemir R
      • Aksoy Hİ
      • Öztürk EE
      • Saygın B
      • Acar FA.
      Neurological manifestations of pediatric acute COVID infections: a Single Center experience.
      ;
      • Valderas C
      • Méndez G
      • Echeverría A
      • Suarez N
      • Julio K
      • Sandoval F
      COVID-19 and neurologic manifestations: a synthesis from the child neurologist's corner.
      ).
      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 WM
      • Kwan MYW
      • Wang YL
      • Leung LK
      • Leung D
      • Chua GT
      • Ip P
      • Fong DYT
      • Wong WHS
      • Chan SHS
      • Chan JFW
      • Peiris M
      • Lau YL
      • Duque JSR.
      Severity of SARS-CoV-2 Omicron BA.2 infection in unvaccinated hospitalized children: comparison to influenza and parainfluenza infections.
      ) 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.

      2. Materials and methods

      2.1 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.

      2.2 Whole genome sequencing of SARS-CoV-2

      Ovation RNA-Seq System V2 (Nugen Technologies, San Carlos, CA, USA) was used to synthesize complementary DNA, which was then processed into a library. Whole genome sequences of the SARS-CoV-2 isolates (TSGH-70, TSGH-71, TSGH-72, TSGH-72, TSGH-73, TSGH-74, TSGH-75, TSGH-76, and TSGH-77) were obtained from RNA using the Ion TorrentTM NGS Reverse Transcription Kit (ThermoFisher Scientific). Library construction was performed according to the Ion AmpliSeqTM SARS-CoV-2 Insight Research Assay (ThermoFisher Scientific) protocol. Sequencing was performed on the Ion GeneStudio S5 Prime (ThermoFisher Scientific), according to Ion 510 & 520 & 530-Chef Kit specifications. Assembled sequences were uploaded to the Global initiative on sharing all influenza data (GISAID) database (http://www.gisaid.org/).

      2.3 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 J
      • Megill C
      • Bell SM
      • Huddleston J
      • Potter B
      • Callender C
      • Sagulenko P
      • Bedford T
      • Neher RA.
      Nextstrain: real-time tracking of pathogen evolution.
      ).

      3. Results

      3.1 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.
      Table 1Patient characteristics and laboratory findings.
      Patient NAgeSexClinical symptomsSARS-CoV-2 PCRSerumCSF
      SwabCSFLDH (U/l)

      140-271
      Ferritin (ng/ml)

      M: 30-400

      F: 13-150
      PCT (ng/ml)

      -0.05
      D-dimer (mg/l)

      -0.5
      IL-6 (pg/ml)

      <7.0
      Opening pressure

      cmH2O
      Cell count

      (<5/μl)
      Glucose

      (mg/dl)
      Total protein

      15-45 (mg/dl)
      #15yFFever, drowsiness and nonsensical babbling, GTCS, shock complicated with multiple organ failurepos.N.A.>12000>200006.95>201111N.A.N.A.N.A.N.A.
      #24yFFever, GTCS, drowsinesspos.neg.4511640.5<0.2731.426.5125187112
      #31yMFever, vomit, dizziness, GTCSpos.neg.2161880.85<0.276.517.547221
      #43yFFever, abdominal pain, vomit, GTCSpos.N.A.30086.31.51N.A.6.8N.A.N.A.N.A.N.A.
      #52y5mMFever, bilateral upward gaze with loss of consciousness and cyanotic change of lip, GTCSpos.neg.25875.21.080.62103.31556826
      CSF, cerebrospinal fluid; GTCS, generalized tonic-clonic seizure; IL-6, interleukin-6; LDH, lactate dehydrogenase; N.A., not avaliable; PCR, polymerase chain reaction; PCT, Procalcitonin; pos., positive.
      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.

      3.2 Laboratory findings

      The biochemistry assays, which reflect inflammation status, showed prominent elevation, such as procalcitonin, lactate dehydrogenase, 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.

      3.3 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 1a). 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.
      Figure 1
      Figure 1(a) Phylogenetic relationship of 21 Taiwan Omicron sequences based on Nextclade. Major clade presents that these sequences is 21L when aligned to the reference sequences (Wu-Han SARS-CoV2). Branch point BA.2.3 shows only Taiwan sequences (red line) were belonged to the new node as a potential subspecies of BA.2.3 lineage. (b) and (c): Using sequences with only K97E mutation point in spike protein shows Japan, Hong Kong, and USA are major high relationship and detection time is fit to the time of Omicron epidemic occurs in Taiwan. Red: Lineage BA.2.3. Yellow: BA.2. Pink: Unclassified lineage.
      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 1b). Moreover, these K97E mutation strains appeared at the end of March 2022, corresponding with the beginning of the Taiwan COVID-19 Omicron epidemic (Figure 1c).

      4. 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 S
      • Capasso D
      • Delre P
      • Pirone L
      • Saviano M
      • Pedone E
      • Mangiatordi GF.
      More is always better than one: the N-terminal domain of the spike protein as another emerging target for hampering the SARS-CoV-2 attachment to Host Cells.
      ) and some extensive ubiquitination events have been observed in them (
      • Stukalov A
      • Girault V
      • Grass V
      • Karayel O
      • Bergant V
      • Urban C
      • Haas DA
      • Huang Y
      • Oubraham L
      • Wang A
      • Hamad MS
      • Piras A
      • Hansen FM
      • Tanzer MC
      • Paron I
      • Zinzula L
      • Engleitner T
      • Reinecke M
      • Lavacca TM
      • Ehmann R
      • Wölfel R
      • Jores J
      • Kuster B
      • Protzer U
      • Rad R
      • Ziebuhr J
      • Thiel V
      • Scaturro P
      • Mann M
      • Pichlmair A
      Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV.
      ), 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 WT
      • Carabelli AM
      • Jackson B
      • Gupta RK
      • Thomson EC
      • Harrison EM
      • Ludden C
      • Reeve R
      • Rambaut A
      COVID-19 Genomics UK (COG-UK) Consortium, Peacock SJ, Robertson DL. SARS-CoV-2 variants, spike mutations and immune escape.
      ).
      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.

      CRediT authorship contribution statement

      Chi-Sheng Chen: Methodology, Investigation, Data curation, Writing – original draft, Visualization. Chia-Ning Chang: Resources. Chih-Fen Hu: Resources. Ming-Jr Jian: Software. Hsing-Yi Chung: Software. Chih-Kai Chang: Software. Cherng-Lih Perng: Software, Validation. Kuo-Sheng Hung: Software, Validation. Feng-Yee Chang: Supervision. Chih-Hung Wang: Supervision. Shyi-Jou Chen: Conceptualization, Data curation, Writing – review & editing. Hung-Sheng Shang: Conceptualization, Methodology, Writing – review & editing, Visualization.

      Declaration of competing interests

      The authors have no competing interests to declare.

      Acknowledgments

      The authors gratefully acknowledge both the originating and submitting laboratories for the sequence data in GISAID EpiCoV, on which the SARS-CoV-2 variants data are partially based.

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