Tonsils are major sites of prolonged sars-cov-2 infection in children

In the present study, we show that SARS-CoV-2 can infect palatine tonsils and adenoids in children without symptoms of COVID-19, with no history of recent upper airway infection. We studied 48 children undergoing tonsillectomy due to snoring/OSA or recurrent tonsillitis between October 2020 and September 2021. Briefly, nasal cytobrush (NC), nasal wash (NW) and tonsillar tissue fragments obtained at surgery were tested by RT-PCR, immunohistochemistry (IHC), flow cytometry and neutralization assay. We detected the presence of SARS-CoV-2 in at least one specimen tested in 25% of patients (20% in palatine tonsils and 16.27% in adenoids, 10.41% of NC and 6.25% of NW). Importantly, in 2 of the children there was evidence of laboratory-confirmed acute infection 2 and 5 months before surgery. IHC revealed the presence of SARS-CoV-2 nucleoprotein in epithelial surface and in lymphoid cells in both extrafollicular and follicular regions, in adenoids and palatine tonsils. Flow cytometry showed that CD20+ B lymphocytes were the most infected phenotypes by SARS-CoV-2 NP, followed by CD4+ and CD8+ T lymphocytes, and CD14+ macrophages and dendritic cells. Additionally, IF indicated that SARS-CoV-2-infected tonsillar tissues had increased expression of ACE2 and TMPRSS2. NGS sequencing demonstrated the presence of different SARS CoV-2 variants in tonsils from different tissues. SARS-CoV-2 antigen detection was not restricted to tonsils, but was also detected in nasal cells from the olfactory region. In conclusion, palatine tonsils and adenoids are sites of prolonged infection by SARS-CoV-2 in children, even without COVID-19 symptoms. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by the State of Sao Paulo Research Foundation (FAPESP) [grant numbers 2019/26119-0, 2020/07063-1], the National Research Council (CNPq) [grant number 403201/2020-9], and the Brazilian Coordination for Improvement of Superior Education Personnel (CAPES). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee of the Clinics Hospital, Ribeirao Preto Medical School, University of Sao Paulo gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.