*Important note: medRxiv It publishes preliminary scientific reports that have not been peer-reviewed and therefore should not be considered conclusive, directing clinical practice/health-related behaviour, or treated as hard information.In a recent study published in medRxiv* server preprint Researchers at the University of North Carolina and the National Institutes of Health have identified associations between coronavirus disease-CoV-2 (SARS-CoV-2) and oral antibodies against SARS-CoV-2 and coronavirus disease 2019 (COVID-19) symptoms.
Stady: 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 SARS-CoV-2 RNA and virulent SARS-CoV-2 in the oral cavity indicate that SARS-CoV-2 replicates in the oral cavity. While specific anti-SARS-CoV-2 anti-SARS-CoV-2 lateral flow assay (LFA) responses in the oral cavity suggest 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 the host’s humoral immune responses in the oral cavity.
Throat washes and saliva samples were obtained from 47 symptomatic (n = 17) and asymptomatic (n = 30) individuals, the diagnosis of COVID-19 confirmed using quantitative reverse transcription polymerase chain reaction (RT-qPCR) by nasopharyngeal (NP) analysis . ) wipes. In the asymptomatic group, 15 individuals seropositive for SARS-CoV-2, and the remainder were negative or asymptomatic. The SARS-CoV-2 nucleocapsid (N) protein was detected using immunohistochemical assays.
Quantitative reverse transcriptase-polymerase chain reaction targeting the SARS-CoV-2 genomic RNA (sgRNA) sequence was confirmed by Sanger sequencing, and LFA was performed to identify the receptor-binding domain of the anti-SARS-CoV-2 spike (S) protein (RBD). ) Immunoglobulin G (IgG) and IgM titers. In addition, structural analysis was performed to identify molecules in host saliva similar to SARS-CoV-2 nucleocapsid. antigen.
The severity of COVID-19 was graded using the National Institutes of Health (NIH) Coronavirus Disease Treatment Guidelines. Subgenome full-length PCR products generated for SARS-CoV-2 spike, nucleocapsid, envelope (E), or membrane (M) glycoproteins of SARS-CoV-2 – total positive-sense RNA content in saliva . Complementary deoxyribonucleic acid (cDNA) was transfected into normal human oral keratinocytes (NOK), and 48 hours after infection, immunoblot analysis was performed.
The crystal structure of antisense nucleocapsid N-terminal binding RNA was evaluated comparatively with publicly available structures uploaded to the Molecular Modeling Database (MMDB). Oral cavity samples with more than 10.0 copies of RNA per RT-qPCR reaction were considered positive for SARS-CoV-2. Symptoms of COVID-19 diseaseincluding muscle pain, weakness, loss of appetite, nausea, sneezing, upper respiratory tract symptoms, shortness of breath, cough, nasal congestion, sore throat, and secretions from the nasal cavity.
results
The average age of the study participants was 40 years, with an equal distribution between the sexes. At study initiation, immunoblotting 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 wash samples, respectively, tested 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 the presence of the SARS-CoV-2 Nucleocapsid antigen.
A lateral flow assay signal of SARS-CoV-2 nucleocapsid antigen among three SARS-CoV-2-negative individuals indicated potential cross-identification of four structurally similar salivary RNA-binding protein molecules. [alignment 19 to 29 amino acid, root mean square deviation (RMSD) 1.0 to 1.5 Å]. At study initiation, asymptomatic patients with subgenome-associated mRNA crosses showed IgG titers (94% and 100% of saliva and throat wash samples, respectively) and IgM titers (75% and 63% of saliva and throat wash samples, 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. An anti-SARS-CoV-2 oral IgG titer showed 100% binding with Nasopharyngeal swab– Analysis of RT-qPCR results. The severity of fatigue, cough, and the presence of weakness, nausea, and upper respiratory symptoms were inversely related to the anti-SARS-CoV-2 oral immunoglobulin titer, which was more significant among women than men. Longitudinal evaluation of symptomatic COVID-19 patients indicated persistence of oral SARS-CoV-2. Symptoms and severity of COVID-19 correlate with anti-SARS-CoV-2 oral 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. SARS-CoV-2 has been detected in oral fluids from RT-qPCR-positive individuals. Nasopharyngeal swabs were analyzed using multiple RT-qPCR-based detection methods including (1) three distinct pairs of primers targeting the open reading frame. 3a (ORF3a)-, or nucleocapsid-coding regions of the SARS-CoV-2 genome; (ii) the numbers of RNA copies in absolute numbers; and (3) subgenomic ribonucleic acid (RNA), a biomarker for 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 course 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 immunoglobulin titers, and The gender of the participant during the initial infection. The nucleocapsid antigen cross-reactivity may represent mimicry of structurally similar host cell proteins.
*Important note: medRxiv It publishes preliminary scientific reports that have not been peer-reviewed and therefore should not be considered conclusive, directing clinical practice/health-related behaviour, or treated as hard information.
