key: cord-0720719-vy50n169 authors: Marques, Barbara Bruno Fagundes; Guimarães, Taísa Coelho; Fischer, Ricardo Guimarães; Tinoco, Justine Monteiro Monnerat; Pires, Fábio Ramoa; Lima Junior, Josué da Costa; Stevens, Roy H.; Tinoco, Eduardo Muniz Barretto title: Morphological alterations in tongue epithelial cells infected by SARS‐CoV‐2: A case–control study date: 2021-08-09 journal: Oral Dis DOI: 10.1111/odi.13988 sha: af8d303a8c3e378defaad64d1ad95d90a8cc029c doc_id: 720719 cord_uid: vy50n169 OBJECTIVE: The aim of the present case–control study was to evaluate the morphological aspects of the epithelial cells from the dorsum of the tongue and the expression of the SARS‐CoV‐2 Spike protein in these cells, in patients with and without COVID‐19 infection. METHODS: 24 individuals with at least one symptom of COVID‐19 were recruited among inpatients from Hospital Universitário Pedro Ernesto (Rio de Janeiro, Brazil). 14 patients who tested positive for COVID‐19 by RT‐PCR were included in the case group, and 10 patients who tested negative were included in the control group. Cytological smears from the dorsum of the tongue were obtained from all patients and analyzed using immunohistochemistry directed against SARS‐CoV‐2‐Spike protein. Morphological changes in epithelial cells were analyzed using light microscopy. RESULTS: Immunohistochemistry showed that 71% of the COVID‐19 patients presented epithelial cells positive for the presence of the SARS‐CoV‐2 Spike protein, and all cells coming from patients in the control group were negative. Cytological analysis showed significant differences when comparing epithelial cells from COVID‐19‐positive and COVID‐19‐negative patients. CONCLUSION: COVID‐19 may generate dimensional changes in tongue epithelial cells; however, further studies are necessary to understand how this happens. infection and have been suggested as a manifestation of the disease (Riad et al., 2020) . The aim of the present study was to evaluate the morphological aspects of the epithelial cells from the dorsum of the tongue and the expression of the SARS-CoV-2 Spike protein in these cells, in patients with and without COVID-19 infection. Reporting of this case-control study follows the STROBE guidelines (Elm et al., 2007) . The study was approved by the Ethical Committee from Pedro Ernesto University Hospital (CAAE-31222820.6.0000.5259), all individuals included in the study gave a written consent to participate, and the study was performed in accordance with the Declaration of Helsinki (World Medical Association, 2013). Sample size calculation using EPI Info™ software (Centers for Disease Control and Prevention, Atlanta, GA, U.S.) was based on the following estimates: α error of 0.05; β error of 0.20 (80% power). Frequency of SARS-CoV-2-Spike protein expression in COVID-19+ patients was arbitrarily set at 90% and in control group at 25%. Assuming an unmatched case-control study design with groups of equal size, these estimates into the equation yield a sample size of 20 individuals (10 per group). Twenty-four individuals were recruited among inpatients from Hospital Universitário Pedro Ernesto (Rio de Janeiro, Brazil). All of them had at least one symptom of COVID-19 (fever, coughing, headache, sore throat, diarrhea, or loss of taste/smell). Fourteen of these participants tested positive for COVID-19 by RT-PCR assay (kit SARS-CoV-2 Bio-Manguinhos, Rio de Janeiro, Brazil) and were included in the case group, and 10 of the participants tested negative and were included in the control group. A cytological smear containing superficial epithelial cells from the dorsum of the tongue by scraping with a spatula was obtained from all patients. The cells were distended in six histological glass slides for each patient and fixed in 95º alcohol. Smears were stained with hematoxylin and eosin and Papanicolaou, and one histological slide from each patient was submitted to immunohistochemistry directed against SARS-CoV-2 Spike protein (mouse monoclonal antibody anti-SARS-CoV-2 Spike protein, clone 1A9, Invitrogen, Massachusetts, US, dilution 1:200) through the immunoperoxidase technique followed by diaminobenzidine as chromogen and counterstaining with Carazzi hematoxylin. The hematoxylin and eosin and Papanicolaou-stained smears were analyzed by two examiners (BM and TG) in high power using light microscopy, and two desquamate epithelial cells from each patient were measured in four planes: maximum cell diameter, maximum perpendicular to this measure, maximum diameter of the nucleus, and maximum perpendicular to this measure. All measures were performed by using the Image J software (NIH, Bethesda, MD, USA). All immunohistochemical slides were analyzed by a single oral pathologist (FRP) and interpreted as positive or negative for the presence of SARS-CoV-2 Spike protein. Data were analyzed using Statistical Package for Social Sciences (SPSS Statistics 20, IBM Corporation, Armonk, NY, USA). After verifying that the data were not normally distributed, nonparametric Mann-Whitney (U) test was used. Significance level was set at 0.05. An intraclass correlation coefficient to determine the agreement between the two examiners (BM and TG) on the four measured plans was calculated and ranged from 0.97 to 0.99. Cytological analysis showed significant differences in all measures when comparing epithelial desquamate cells derived from COVID-19-positive and COVID-19-negative patients (Table 2) . Immunohistochemistry showed that 10 out 14 (71%) In this study, morphological changes in epithelial cells were particularly associated with positivity of RT-PCR assay in nasopharyngeal swabs, as this method is the gold standard for diagnosis of SARS-CoV-2 infection and is a much more sensitive detection technique than immunohistochemistry. It is possible that the lower sensitivity of immunohistochemistry assay may have influenced the results. Expression of ACE2, furin, and TMPRSS2 has been demonstrated in tongue epithelial cells, which reinforces the perspective that the oral mucosa, and specifically this anatomical region, is an access route to SARS-CoV-2 infection . It has been suggested that these features would explain the mechanism of dysgeusia in these patients and also that removing the tongue coating would reduce the risk of virus exposure (Sakaguchi et al., 2020) . The results of this study showed that the presence of SARS- previously demonstrated (Parada et al., 2021) . In this study, ethnic background, gender, proportion of smokers, and distribution of symptoms were similar in both groups. However, the mean age of the control group was lower (34.9 yrs) than in the case group (55,4 yrs), which may have act as a confounder. There In conclusion, COVID-19 may generate dimensional changes in tongue epithelial cells; however, further studies are necessary to understand how this happens. This study received financial support from Rio de Janeiro Foundation for Research Support (FAPERJ, E-26/202.810/2018) . All authors gave their final approval and agreed to be accountable for all aspects of the work. All authors reported no conflict of interest. Pulmonary pathology of ARDS in COVID-19: a pathological review for clinicians Gene expression for ACE2 (ENSG00000130234.10). 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