*important opinion: medRxiv It publishes preliminary scientific reports that have not been peer-reviewed and therefore should not be considered conclusive, guiding clinical practices/health-related behaviours, or treated as hard information.
In a recent study published in medRxiv* server preprint Researchers from the University of North Carolina and the National Institutes of Health have identified associations between coronavirus 2 (SARS-CoV-2) and oral antibodies against SARS-CoV-2 and coronavirus disease 2019 (symptoms) of COVID-19.
Study: Oral SARS-CoV-2 host responses predict early COVID-19 disease course. Image credit: Kittyfly/Shutterstock
background
SARS-CoV-2, the causative agent of COVID-19, multiplies in the upper respiratory tract, oral mucosa, salivary glands, and respiratory tract mucosa. The presence of angiotensin-converting enzyme 2 (ACE2) receptors and the detection of RNA of SARS-CoV-2 and virulent SARS-CoV-2 in the oral cavity indicate that SARS-CoV-2 replicates in the oral cavity. While anti-SARS-CoV-2 responses identified by the lateral flow assay (LFA) in the oral cavity are indicative of systemic immunity, oral biomarkers as indicators of the diagnosis of COVID-19 have not been explored significantly.
about studying
In this study, the researchers evaluated the detection of SARS-CoV-2 and host humoral immune responses in the oral cavity.
Throat washes and saliva samples were obtained from 47 symptomatic (n = 17) and asymptomatic (n = 30) individuals, whose diagnosis of COVID-19 was confirmed using quantitative reverse transcription polymerase chain reaction (RT-qPCR) by analysis of nasopharyngeal swabs (NP). In the asymptomatic group, 15 people seropositive for SARS-CoV-2, and the rest were negative or asymptomatic. The SARS-CoV-2 nucleocapsid (N) protein was detected using immunohistochemical assays.
Quantitative reverse transcription-polymerase chain reaction targeting the SARS-CoV-2 subunit genomic RNA (sgRNA) sequence was confirmed by Sanger sequencing, and LFA was performed to identify the binding domain of the anti-SARS-CoV-2 spike protein receptor (S). ) (RBD) Immunoglobulin G (IgG) and IgM titers. In addition, structural analysis was performed to identify molecules in the host’s saliva that are similar to the SARS-CoV-2 nucleocapsid antigen.
The severity of COVID-19 was graded using the 2019 Treatment Guidelines for Coronavirus Disease from the National Institute of Health (NIH). Full-length subgenome PCR products encoding the SARS-CoV-2 spike, core, envelope (E), or membrane (M) glycoproteins were generated from the total content of SARS-CoV-2-positive RNA in saliva. Complementary deoxyribonucleic acid (cDNA) was transfected into normal human oral keratinocytes (NOK) and 48 hours after transfection, immunoblotting analysis was performed.
The crystal structure of the terminal RNA-binding domain of SARS-CoV-2 was evaluated comparatively with publicly available structures uploaded to the Molecular Modeling Database (MMDB). Oral cavity samples containing more than 10.0 copies of RNA per RT-qPCR reaction were considered positive for SARS-CoV-2. Symptoms of COVID-19 were evaluated, including muscle pain, weakness, loss of smell, nausea, advanced age, upper respiratory symptoms, shortness of breath, cough, stuffy nose, sore throat, and secretions from the nasal cavity.
results
The average age of the study participants was 40, with an equal gender distribution. At the start of the study, immunoblot analysis confirmed the presence of SARS-CoV-2 N-antigen detected by LFA in 82.0% of throat lavage. However, only three and 17 saliva and throat lozenges samples, respectively, were positive for SARS-CoV-2 by RT-PCR. After four weeks, 60.0% and 83.0% of the saliva and throat lozenges samples, respectively, showed persistent presence of the SARS-CoV-2 nucleocapsid antigen.
Signal flow lateral analysis of SARS-CoV-2 nucleocapsid antigen among three SARS-CoV-2-negative individuals indicated a potential cross-identification of four structurally similar salivary DNA-binding protein molecules. [alignment 19 to 29 amino acid, root mean square deviation (RMSD) 1.0 to 1.5 Å]. At study entry, asymptomatic patients had subgenome-related mRNA crosses and IgG titers (94% and 100% of saliva samples and throat baths, respectively) and IgM (75% and 63% of saliva samples and throat baths, respectively). , respectively) . ).
At four weeks, anti-SARS-CoV-2 immunoglobulin G titers were maintained in 100% of saliva samples and 83% of throat rinses, and anti-IgM titers were maintained in 80% of saliva samples and 67% of throat rinses. Oral anti-SARS-CoV-2 IgG titer showed 100% correlation with RT-qPCR results analyzed by nasopharyngeal swab. The severity of fatigue, cough, presence of weakness, nausea, and upper respiratory symptoms were inversely related to anti-SARS-CoV-2 oral IgM antibody, which was greater in women than in men. Longitudinal evaluation of symptomatic COVID-19 patients indicated persistence of oral SARS-CoV-2. COVID-19 symptoms and severity have been associated with oral anti-SARS-CoV-2 antibody titers and the presence of SARS-CoV-2.
The results provide new insights into oral biomarkers to predict COVID-19 and the transmission and persistence of SARS-CoV-2. The presence of SARS-CoV-2 was detected in the oral fluids of RT-qPCR-positive individuals analyzed by nasopharyngeal swab using several RT-qPCR-based detection methods, including (1) three distinct pairs of primers targeting the framework of open read 3a (ORF3a)- or nucleocapsid-coding regions of the SARS-CoV-2 genome; (2) RNA copy number in absolute numbers; and (3) subgenomic ribonucleic acid (RNA), a biomarker of active SARS-CoV-2 replication in the initial period of symptomatic SARS-CoV-2 infection.
Overall, the results of the study showed that, critical to the transmission of SARS-CoV-2 and the development of COVID-19, the prevalence and persistence of SARS-CoV-2 in the oral cavity showed clear associations with specific symptoms of COVID-19, early on. Immunoglobulin titers, and gender of the participant at initial infection. The cross-reactivity of the nucleocapsid antigen can mimic the host cell proteins that are structurally similar.
*important opinion: medRxiv It publishes preliminary scientific reports that have not been peer-reviewed and therefore should not be considered conclusive, guiding clinical practices/health-related behaviours, or treated as hard information.
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